RU2811918C2 - Methods of treating cancer diseases and tumors using pde1 inhibitors - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: group of inventions discloses a method of treating glioma, including administering a pharmaceutically acceptable amount of a PDE1 inhibitor to a subject in need thereof, where the PDE1 inhibitor is selected from any of the following compounds in free form or in the form of a pharmaceutically acceptable salt. A method of inhibiting cytokine release syndrome is also disclosed.

EFFECT: technical result is the implementation of the purpose of the claimed methods.

6 cl, 5 tbl, 7 dwg, 3 ex

Description

Cross reference to related applications

This application claims the priority and benefit of U.S. Provisional Application Serial No. 62/676,638, which was filed May 25, 2018, and U.S. Provisional Application Serial No. 62/688,641, which was filed June 22, 2018, the contents of which are incorporated herein in their entirety. application via link.

Field to which the invention relates

[0001] The field relates to phosphodiesterase 1 (PDE1) inhibitors useful for the treatment of certain types of cancer and tumors, such as tumors of the central nervous system. This field also relates to the administration of phosphodiesterase 1 (PDE1) inhibitors for the treatment of certain types of cancers and tumors, such as tumors of the central nervous system, or for the treatment of related conditions characterized by increased expression of PDE1.

Prerequisites for creating the invention

[0002] Glioblastoma is the most common malignant brain tumor in adults, accounting for more than half of all primary brain tumors with an incidence of 2 to 3 per 100,000 in Western developed countries. The prognosis is almost always poor: the average survival rate is 6-9 months, and the five-year survival rate is less than 3%. Brodbelt A, et al.; (UK) National Cancer Information Network Brain Tumor Group. Glioblastoma in England: 2007-2011. Eur. J. Cancer. Mar 2015; 51(4):533-42.

[0003] Glioblastoma is an aggressive tumor that is characterized by rapid growth and invasion of healthy brain. The effectiveness of existing treatments is compromised by the need to preserve normal brain function during surgery, the inherent resistance of tumors to radiation therapy, and the inability of many drugs to cross the blood-brain barrier. There have been no significant improvements for adult patients with glioblastoma over the past three decades, as subsequent phase III trials of targeted therapies failed to show improved survival (Touat M, et al., Glioblastoma targeted therapy: updated approaches from recent biological insights. Ann. Oncol. 2017 Jul 1; 28(7):1457-1472), and temozolamide (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, radiation therapy, 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.

[0004] There is currently a huge unmet need for an effective treatment for central nervous system tumors such as glioblastoma multiforme. Improved therapeutic compositions and methods for treating such conditions are urgently needed.

[0005] Eleven families of phosphodiesterases (PDEs) have been identified, but only the PDEs in Family I, Ca2+/calmodulin-dependent phosphodiesterases (CaM-PDEs), which are activated by Ca2+/calmodulin and have been shown to mediate calcium and cyclic nucleotide signaling pathways (e.g. , cGMP and cAMP). The three known CaM-PDE genes, PDE1A, PDE1B, and PDE1C, are all expressed in central nervous system tissue. PDE1A is expressed in the brain, lungs and heart. PDE1B is predominantly expressed in the central nervous system, but is also found in monocytes and neutrophils and has been shown to be involved in the inflammatory responses of these cells. PDE1C is expressed in the olfactory epithelium, cerebellar granule cells, striatum, heart, and vascular smooth muscle. PDE1C has been demonstrated to be a major regulator of smooth muscle proliferation in human smooth muscle.

[0006] Cyclic nucleotide phosphodiesterases inhibit intracellular cAMP and cGMP signaling by hydrolyzing these cyclic nucleotides to their corresponding 5'-monophosphates (5'AMP and 5'GMP), which are inactive in intracellular signaling pathways. Both cAMP and cGMP are central intracellular second messengers and they play a role in the regulation of various cellular functions. PDE1A and PDE1B preferentially hydrolyze cGMP over cAMP, while PDE1C exhibits approximately equal hydrolysis of cGMP and cAMP.

[0007] With regard to PDE1C in particular, recent evidence indicates that PDE1C is a proliferation-related gene because it is expressed exclusively 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). Additionally, there have been anecdotal reports of PDE1C being expressed along with other PDE subtypes 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 stimulatory G protein alpha, Q227L Gsalpha. Exp Mol Med 2001;33:37-45) and osteosarcoma (Ahlström 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).

[0008] Previous studies have shown that inhibition of PDE1 (specifically the PDE1B isoform) 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 five of six human glioblastoma cell lines from the National Cancer Institute exhibit high expression of PDE1 (predominantly PDE1C) and only minor expression of PDE4. 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 cells expressing human malignant melanoma associated antigen (MAA), and growth is inhibited by the nonselective PDE1 inhibitor vinpocetine. 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 show significant overexpression of PDE1C in 20% of glioblastoma compared to normal human brain, siRNA-mediated silencing of PDE1C inhibits proliferation (45-50%) and invasion (40-60%) in cultured patient-derived glioblastoma cells. Rowther FB, et al., Cyclic nucleotide phosphodiesterase-1C (PDE1C) drives cell proliferation, migration and invasion in glioblastoma multiforme cells in vitro. Mol Carcinog. Mar 2016; 55(3):268-79.

[0009] Tumor-promoting cell proliferation, migration, tissue invasion and inflammation are considered essential characteristics of cancer development. Each of these processes is time-dependent, variable and complex and involves multiple signal transduction pathways. Multitargeted drugs are believed to provide greater benefit than that observed with single-target therapies, have acceptable tolerability profiles, and are active against a wider range of tumor types. The regulation of cyclic nucleotide signaling is accordingly viewed as a collection of multiple constituent pathways involved in various aspects of tumor cell function. Impaired cAMP generation has been described in various cancer pathologies. Attempts to directly regulate cyclic nucleotides in cancer cells, although antiproliferative, turned out to be nonproductive due to high cytotoxicity. New, safer and selective strategies for cAMP modulation in cancer cells are needed.

The essence of the invention

[0010] The inventors have previously shown that inhibition of PDE1 activity using the compounds disclosed herein can safely 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 some diseases. More recently, we showed that PDE1 inhibitors interfere with cell migration of microglia and monocytes. Recent evidence shows that PDE1, especially the PDE1C isoform, is overexpressed in experimental tumor models such as melanoma, neuroblastoma, and osteosarcoma. Additionally, focal genomic dominance of PDE1C has been demonstrated in glioblastoma multiforme (GBM) cells. Genomic amplification of PDE1C is associated with increased expression in GBM-derived cell cultures and is important for controlling cell proliferation, migration, and invasion in cancer cells.

[0011] 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 in PDE1 inhibitor molecules) or enzymatic activity. These cancer cells also exhibit low levels of cAMP, which can be increased by PDE1 inhibitors. Such characteristics can be treated with PDE-1 inhibitors alone or in combination with chemotherapeutic agents, gene therapies and/or immunological approaches. Inhibition of PDE1 induces apoptotic cell death, prevents migration, limits metastasis, and reduces inflammation. Thus, PDE1 inhibitors have synergistic effects with chemotherapeutic and immunological approaches.

[0012] In addition, many gene therapy and antibody approaches are known to cause cytokine release syndrome (CRS). CRS is caused by a large and rapid release of cytokines into the blood by immune cells targeted by immunotherapy. PDE1 has previously been shown to reduce the expression of the inflammatory cytokines IL1β, TNFα and Ccl2. See international patent publication WO2018/049147. PDE1 inhibition is useful in these cases prophylactically, and a PDE1 inhibitor can be administered along with corticosteroids and antihistamines to prevent CRS.

[0013] Without being limited by theory, it can be assumed that impaired cAMP (or cGMP) generation occurs due to overexpression of PDE1 isoforms in various cancer pathologies. Inhibition of selective PDE1 isoforms, which increases intracellular cAMP (and/or cGMP) levels, induces apoptosis and cell cycle arrest in a wide range of tumor cells and regulates the tumor microenvironment by preventing cell migration, inflammation and tissue invasion. Therefore, the development and clinical use of inhibitors specific for individual PDE1 and its isoforms, especially PDE1c, may selectively restore normal intracellular signaling, providing antitumor therapy with reduced side effects.

[0014] The present disclosure relates to the use of a PDE1 inhibitor for the treatment of cancer or tumor, including, for example, carcinomas, melanomas and astrocytomas. In a preferred embodiment, the PDE1 inhibitor is selective. Previous studies have demonstrated that PDE1 (i.e., PDE1C) is significantly overexpressed in patients with glioblastoma compared to healthy patients (i.e., those without glioblastoma). siRNA-mediated silencing of PDE1C has been shown to inhibit proliferation and invasion in patient-derived glioblastoma cell cultures. Without being limited to any theory, inhibition of PDE1, such as PDE1C, may be effective in therapeutic intervention for certain types of cancer or tumors, such as glioblastoma.

[0015] In particular, the treatment of brain tumors requires compounds that have the ability to cross the blood-brain barrier. The compounds of the present invention are potent PDE1 inhibitors. In particular, the compounds disclosed herein exhibit high selectivity for both isoforms, PDE1A and PDE1C, and are able to penetrate the blood-brain barrier.

[0016] Thus, in various embodiments, the present invention provides methods for treating a condition selected from cancer or tumor, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor disclosed herein to a subject in need thereof. In some embodiments, the cancer or tumor is a glioma, leukemia, melanoma, neuroblastoma, or osteosarcoma. In some embodiments, the cancer or tumor is an astrocytoma, such as glioblastoma multiforme. In some embodiments, the PDE1 inhibitor is administered in combination with an anticancer agent.

[0017] In various embodiments, the present invention provides methods for inhibiting tumor cell proliferation, migration and/or invasion, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor disclosed herein to a subject in need thereof. In some embodiments, the cancer or tumor is a glioma, leukemia, melanoma, neuroblastoma, or osteosarcoma. In some embodiments, the cancer or tumor is an astrocytoma, such as glioblastoma multiforme. In some embodiments, the PDE1 inhibitor is administered in combination with an anticancer agent.

[0018] In various embodiments, provided herein is a method of treating glioma, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor to a subject in need thereof. In some embodiments, the cancer or tumor is an astrocytoma, such as glioblastoma multiforme. In some embodiments, the PDE1 inhibitor is administered in combination with an anticancer agent.

[0019] In another aspect, the present invention also includes a PDE1 inhibitor of formula I, Ia, II, III and/or IV, described below, in free or salt form. In a preferred embodiment, the PDE1 inhibitor is a selective PDE1 inhibitor. In another embodiment, the invention also provides a pharmaceutical composition comprising a PDE1 inhibitor in free form or in the form of a pharmaceutically acceptable salt in admixture with a pharmaceutically acceptable carrier.

[0020] In various embodiments, the present invention provides combination therapies comprising a PDE1 inhibitor of formula I, Ia, II, III and/or IV described below, in free or salt form, and an antitumor agent. Combination therapy can be used in combination with any of the methods described in this application. In some embodiments, the antineoplastic agent is administered concomitantly with, before, or after administration of the PDE1 inhibitor.

[0021] In various embodiments, the present invention also provides pharmaceutical compositions containing the compounds of the present invention prepared using conventional diluents or excipients and methods known in the art. Thus, oral dosage forms may include tablets, capsules, solutions, suspensions, and the like.

[0022] In various embodiments, the present invention also provides PDE1 inhibitors according to Formula I, Ia, II, III and/or IV described below in free or salt form for use in treating a condition selected from cancer or tumor , inhibiting proliferation, migration and/or invasion of tumor cells, or treating glioma.

Brief description of drawings

[0023] FIG. Figure 1 shows PDE1C expression in short-term cultures of glioblastoma multiforme (GBM) cells.

[0024] FIG. Figure 2 shows changes in PDE1C expression in fresh frozen GBM biopsy specimens presented in comparison with normal human astrocytes.

[0025] FIG. 3 shows cellular proliferation of the studied GBM cell cultures after siRNA-mediated depletion of PDE1C.

[0026] FIG. 4 shows a comparison of cell migration across a polycarbonate membrane of cells modified to specifically target PDE1C and non-targeted cells.

[0027] FIG. 5 shows a comparison of cell invasion through an ECM-coated polycarbonate membrane of cells modified to specifically target PDE1C and non-targeted cells.

[0028] FIG. 6 shows a decrease in the number of cells that migrated to the wound area in cells modified to specifically target PDE1C compared to non-targeted cells in three cultures (IN1472, IN1760 and U251MG).

[0029] FIG. 7 shows PDE activity in several GBM cell lines measured in both the presence and absence of the Ca2+/calmodulin system.

Detailed Description of the Invention

Compounds for use in the methods of the invention

[0030] In one embodiment, PDE1 inhibitors for use in the methods of treatment and prevention described herein are selective PDE1 inhibitors.

PDE1 inhibitors

[0031] In one embodiment, the invention provides that PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are compounds of formula I:

Formula I

Where

(i) R ₁ represents H or C _1-4 alkyl (eg methyl);

(ii) R ₄ is H or C _1-4 alkyl, and R ₂ and R ₃ are independently H or C _1-4 alkyl

(eg, R ₂ and R ₃ are both methyl, or R ₂ is H and R ₃ is isopropyl), aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl; or

R ₂ is H, and R ₃ and R ₄ together form a di-, tri- or tetramethylene bridge

(previously, where R ₃ and R ₄ together have a cis configuration, for example, where the carbon atoms bearing R ₃ and R ₄ have the configurations R and S, respectively);

(iii) R ₅ is substituted heteroarylalkyl, for example substituted haloalkyl;

or R ₅ is attached to one of the nitrogen atoms on the pyrazole moiety of formula I and is a moiety of formula A

Formula A

where X, Y and Z are independently N or C, and _R8 , _R9 , _R11 and _R12 are independently H or halogen (eg Cl or F), and _R10 is halogen, alkyl, cycloalkyl, haloalkyl (eg, trifluoromethyl), aryl (eg, phenyl), heteroaryl (eg, pyridyl (eg, pyrid-2-yl) optionally substituted with halogen, or thiadiazolyl (eg, 1,2,3-thiadiazol-4-yl)) , diazolyl, triazolyl, tetrazolyl, arylcarbonyl (eg benzoyl), alkylsulfonyl (eg methylsulfonyl), heteroarylcarbonyl or alkoxycarbonyl; with the proviso that when X, Y, or Z is nitrogen, _R8 , _R9 , or _R10 , respectively, is absent; And

(iv) R ₆ is H, alkyl, aryl, heteroaryl, arylalkyl (eg benzyl), arylamino (eg phenylamino), heteroarylamino, N, N-dialkylamino, N, N-diarylamino or N-aryl-N-( arylalkyl)amino (eg N-phenyl-N-(1,1'-biphen-4-ylmethyl)amino); And

(v) n=0 or 1;

(vi) when n=1, A is -C(R ₁₃ R ₁₄ )-

where R ₁₃ and R ₁₄ independently represent H or C _1-4 alkyl, aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl;

in free, salt or prodrug form, including its enantiomers, diastereoisomers and racemates.

[0032] In another embodiment, the invention provides that PDE1 inhibitors for use in the methods described herein are represented by Formula 1a:

Formula Ia

Where

(i) R ₂ and R ₅ independently represent H or hydroxy, and R ₃ and R ₄ together form a tri- or tetra-methylene bridge [prev. with carbon atoms bearing R ₃ and R ₄ having the configuration R and S, respectively]; or R ₂ and R ₃ are each methyl, and R ₄ and R ₅ are each H; or R ₂ , R ₄ and R ₅ are H and R ₃ is isopropyl [prev. the carbon atom bearing R ₃ has the R configuration];

(ii) R ₆ is (optionally halogen-substituted) phenylamino, (optionally halogen-substituted) benzylamino, C _1-4 alkyl or C _1-4 alkyl sulfide; for example, phenylamino or 4-fluorophenylamino;

(iii) R ₁₀ is C _1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, (optionally halogen- or hydroxy-substituted) phenyl, (optionally halogen- or hydroxy-substituted) pyridyl (e.g., 6-fluoropyride- 2-yl) or thiadiazolyl (eg 1,2,3-thiadiazol-4-yl); And

X and Y are independently C or N,

in free form, pharmaceutically acceptable salt or prodrug form, including its enantiomers, diastereoisomers and racemates.

[0033] In another embodiment, the invention provides that PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are compounds of formula II:

Formula II

(i) X is C _1-6 alkylene (eg methylene, ethylene or prop-2-yn-1-ylene);

(ii) Y is a single bond, alkynylene (eg -C≡C-), arylene (eg phenylene) or heteroarylene (eg pyridylene);

(iii) Z is H, aryl (eg phenyl), heteroaryl (eg pyridyl, eg pyrid-2-yl), halogen (eg F, Br, Cl), haloC _1-6 alkyl (eg trifluoromethyl ), -C(O)-R ¹ , -N(R ² )(R ³ ) or C _3-7 cycloalkyl, optionally containing at least one atom selected from the group consisting of N or O (for example, cyclopentyl, cyclohexyl, tetrahydro-2H-pyran-4-yl or morpholinyl);

(iv) R ¹ is C _1-6 alkyl, haloC _1-6 alkyl, -OH or -OC _1-6 alkyl (eg -OCH ₃ );

(v) R ² and R ³ are independently H or C _1-6 alkyl;

(vi) R ⁴ and R ⁵ are independently H, C _1-6 alkyl or aryl (e.g. phenyl), optionally substituted with one or more halogens (e.g. fluorophenyl, e.g. 4-fluorophenyl), hydroxy (e.g. hydroxyphenyl, for example, 4-hydroxyphenyl or 2-hydroxyphenyl) or C _1-6 alkoxy;

(vii) wherein X, Y and Z are independently and optionally substituted with one or more halogens (eg F, Cl or Br), C _1-6 alkyl (eg methyl), halogen C _1-6 alkyl (eg trifluoromethyl), e.g. , Z represents heteroaryl, e.g. pyridyl, substituted with one or more halogens (e.g. 6-fluoropyrid-2-yl, 5-fluoropyrid-2-yl, 6-fluoropyrid-2-yl, 3-fluoropyrid-2-yl, 4-fluoropyrid-2-yl, 4,6-dichloropyrid-2-yl), _haloC1-6 alkyl (e.g. 5-trifluoromethylpyrid-2-yl) or _C1-6 alkyl (e.g. 5-methylpyrid-2 -yl), or Z is aryl, for example phenyl, substituted with one or more halogens (for example, 4-fluorophenyl),

in free, salt or prodrug form.

[0034] In yet another embodiment, the invention provides that PDE1 inhibitors for use in the methods of treatment and prevention described herein are represented by Formula III:

Formula III

Where

(i) R ₁ represents H or C _1-4 alkyl (eg methyl or ethyl);

(ii) R ₂ and R ₃ are independently H or C _1-6 alkyl (eg, methyl or ethyl);

(iii) R ₄ represents H or C _1-4 alkyl (eg methyl or ethyl);

(iv) R ₅ is aryl (eg phenyl) optionally substituted with one or more groups independently selected from -C(=O)-C _1-6 alkyl (eg -C(=O)-CH ₃ ) and C _1-6 -hydroxyalkyl (eg 1-hydroxyethyl);

(v) R ₆ and R ₇ are independently H or aryl (eg phenyl) optionally substituted with one or more groups independently selected from C _1-6 alkyl (eg methyl or ethyl) and halogen (eg F or Cl ), for example unsubstituted phenyl or phenyl substituted with one or more halogens (for example, F), or phenyl substituted with one or more C _1-6 alkyls and one or more halogens, or phenyl substituted with one C _1-6 alkyl and one halogen , for example 4-fluorophenyl, or 3,4-difluorophenyl, or 4-fluoro-3-methylphenyl; And

(vi) n has the value 1, 2, 3 or 4,

in free form or in salt form.

[0035] In yet another embodiment, the invention provides that PDE1 inhibitors for use in the methods of treatment and prevention described herein are represented by Formula IV

Formula IV

in free form or in salt form, where

(i) R ₁ is C _1-4 alkyl (eg methyl or ethyl) or -NH(R ₂ ), where R ₂ is phenyl, optionally substituted with halogen (eg fluorine), eg 4-fluorophenyl;

(ii) X, Y and Z are independently, N or C;

(iii) R₃, R₄ and R₅ independently represent H or C_1-4alkyl (eg methyl); or R₃ represents H, and R₄And R₅together form a trimethylene bridge (presumably, where R₄And R₅together have a cis configuration, for example where the carbon atoms bearing R₄And R₅, have configurations R and S, respectively),

(iv) R ₆ , R ₇ and R ₈ are independently:

H,

C _1-4 alkyl (eg methyl),

pyrid-2-yl substituted with a hydroxy group, or

-S(O) ₂ -NH ₂ ;

(v) Provided that when X, Y and/or Z are N, then R ₆ , R ₇ and/or R ₈ , respectively, are absent; and when X, Y and Z are all C, then at least one of R ₆ , R ₇ or R ₈ is -S(O) ₂ -NH ₂ or pyrid-2-yl substituted with a hydroxy group.

[0036] In one embodiment, the invention provides administration of a PDE1 inhibitor for the treatment or prevention of inflammation or inflammation-related disease or disorder, wherein the inhibitor is a compound according to the following formula:

in free form or in the form of a pharmaceutically acceptable salt.

[0037] In yet another embodiment, the invention provides administration of a PDE1 inhibitor for the treatment or prevention of inflammation or inflammation-related disease or disorder, wherein the inhibitor is a compound according to the following formula:

in free form or in the form of a pharmaceutically acceptable salt.

[0038] In yet another embodiment, the invention provides administration of a PDE1 inhibitor for the treatment or prevention of inflammation or inflammation-related disease or disorder, wherein the inhibitor is a compound according to the following formula:

in free form or in the form of a pharmaceutically acceptable salt.

[0039] In yet another embodiment, the invention provides administration of a PDE1 inhibitor for the treatment or prevention of inflammation or inflammation-related disease or disorder, wherein the inhibitor is a compound according to the following formula:

in free form or in the form of a pharmaceutically acceptable salt.

[0040] In yet another embodiment, the invention provides administration of a PDE1 inhibitor for the treatment or prevention of inflammation or inflammation-related disease or disorder, wherein the inhibitor is a compound according to the following formula:

in free form or in the form of a pharmaceutically acceptable salt.

[0041] In one embodiment, selective PDE1 inhibitors of any of the preceding formulas (e.g., formulas I, Ia, II, III and/or IV) are compounds that inhibit phosphodiesterase-mediated (e.g., PDE1-mediated, especially PDE1B-mediated ) cGMP hydrolysis, for example, preferred compounds have an IC ₅₀ value of less than 1 µM, preferably less than 500 nM, preferably less than 50 nM and preferably less than 5 nM in a PDE assay based on affinity for the immobilized metal, in free or salt form.

[0042] In other embodiments, the invention provides administration of a PDE1 inhibitor for treating a condition selected from cancer or tumor; to inhibit proliferation, migration and/or invasion of tumor cells; and/or for the treatment of glioma, wherein the inhibitor is a compound according to the following formula:

[0043] Other examples of PDE1 inhibitors suitable for use in the methods and treatments discussed herein can be found in international publication WO2006133261A2; US Patent 8273750; US Patent 9000001; US Patent 9624230; International publication WO2009075784A1; US Patent 8273751; US Patent 8829008; US Patent 9403836; International publication WO2014151409A1, US Patent 9073936; US Patent 9598426; US Patent 9556186; US Publication 2017/0231994A1, International Publication WO2016022893A1, and US Publication 2017/0226117A1, each of which is incorporated by reference in its entirety.

[0044] Other examples of PDE1 inhibitors suitable for use in the methods and treatments discussed herein can be found in International Publication WO2018007249A1; US Publications 2018/0000786; International publication WO2015118097A1; US Patent 9718832; International publication WO2015091805A1; US Patent 9701665; US Publications 2015/0175584A1; US Publications 2017/0267664A1; International publication WO2016055618A1; US Publications 2017/0298072A1; International publication WO2016170064A1; US Publications 2016/0311831A1; International publication WO2015150254A1; US Publications 2017/0022186A1; International publication WO2016174188A1; US Publications 2016/0318939A1; US Publications 2017/0291903A1; International publication WO2018073251A1; International publication WO2017178350A1; and US Publications 2017/0291901A1; each of which is incorporated by reference in its entirety. In any situation where statements in any documents incorporated by reference conflict or are inconsistent with any statements made in this disclosure, it should be understood that the statements in this disclosure control.

[0045] Unless otherwise stated or clear from the context, the following terms as used herein have the following meanings:

(a) “Selective PDE1 inhibitor” as used herein refers to a PDE1 inhibitor with at least 100-fold selectivity for inhibition of PDE1 over inhibition of any other PDE isoform.

(b) “Alkyl” as used herein means a saturated or unsaturated hydrocarbon group, preferably saturated, preferably containing from one to six carbon atoms, which may be linear or branched and may optionally be mono-, di- or tri-substituted, for example with halogen (for example, chlorine or fluorine), hydroxy or carboxy.

(c) “Cycloalkyl” as used herein is a saturated or unsaturated non-aromatic hydrocarbon group, preferably saturated, preferably containing from three to nine carbon atoms, at least some of which form a non-aromatic mono- or bicyclic or bridged cyclic structure, and which may be optionally substituted, for example, with halogen (eg chlorine or fluorine), hydroxy or carboxy. When the cycloalkyl optionally contains one or more atoms selected from N, O and/or S, said cycloalkyl may also be heterocycloalkyl.

(d) “Heterocycloalkyl” is, unless otherwise specified, a saturated or unsaturated non-aromatic hydrocarbon group, preferably saturated, preferably containing from three to nine carbon atoms, at least some of which form a non-aromatic mono- or bicyclic or bridged cyclic structure, wherein at least one carbon atom is substituted with N, O or S, wherein heterocycloalkyl may be optionally substituted, for example, with halogen (eg chlorine or fluorine), hydroxy or carboxy.

(e) “Aryl” as used herein is a mono- or bicyclic aromatic hydrocarbon, preferably phenyl, optionally substituted with, for example, alkyl (e.g. methyl), halogen (e.g. chlorine or fluorine), haloalkyl (e.g. trifluoromethyl), hydroxy, carboxy or additional aryl or heteroaryl (eg biphenyl or pyridylphenyl).

(f) “Heteroaryl” as used herein means an aromatic group in which one or more of the atoms making up the aromatic ring is sulfur or nitrogen rather than carbon, for example pyridyl or thiadiazolyl, which may be optionally substituted, for example, with alkyl , halogen, haloalkyl, hydroxy or carboxy.

[0046] The compounds of the present invention, for example, PDE1 inhibitors as described herein, may exist in free form or in salt form, for example, as acid addition salts. In this specification, unless otherwise indicated, terms such as “Compounds of the present invention” are to be understood to include the compounds in any form, for example, free form or acid addition salt form, or, when the compounds contain acid substituents, in the form base addition salt. The compounds of the present invention are intended for use as pharmaceuticals, so pharmaceutically acceptable salts are preferred. Salts not suitable for pharmaceutical use may be useful, for example, for isolating or purifying the free compounds of the invention or their pharmaceutically acceptable salts, and are therefore also included.

[0047] The compounds of the present invention may, in some cases, also exist in the form of prodrugs. A prodrug form is a compound that is converted in the body into a compound of the present invention. For example, when the compounds of the present invention contain hydroxy or carboxy substituents, these substituents can form physiologically hydrolyzable and acceptable esters. In the context of this application, “physiologically hydrolyzable and acceptable ester” means esters of the compounds of the present invention that are hydrolyzed under physiological conditions to form acids (in the case of compounds of the invention that have hydroxy substituents) or alcohols (in the case of compounds of the invention that have carboxy substituents), which themselves are physiologically acceptable at the doses intended for administration. Therefore, if the compound of the present invention contains a hydroxy group, for example, Compound-OH, the acyl ether prodrug of such compound, i.e., Compound-OC(O)-C _1-4 alkyl, can be hydrolyzed in the body to form a physiologically hydrolyzable alcohol ( Compound-OH) on one side and an acid on the other (for example, HOC(O)-C _1-4 alkyl). Alternatively, if the compound of the present invention contains a carboxylic acid, for example, Compound-C(O)OH, the acid ester prodrug of such compound, Compound-C(O)OC _1-4 alkyl, can be hydrolyzed to form Compound- C(O)OH and HO-C _1-4 alkyl. It should be understood that the term thus covers conventional pharmaceutical forms of prodrugs.

[0048] In another embodiment, the invention also provides a pharmaceutical composition comprising a PDE1 inhibitor in combination with an antitumor agent, each in free form or in pharmaceutically acceptable salt form, admixed with a pharmaceutically acceptable carrier. The term “combination”, as used herein, covers the simultaneous, sequential or coincidental administration of a PDE1 inhibitor and an antitumor agent. In another embodiment, the invention provides a pharmaceutical composition containing such a compound. In some embodiments, the combination of a PDE1 inhibitor and an antineoplastic agent allows the antineoplastic agent to be administered at a lower dose than would be effective if administered as a single monotherapy.

Methods for using the compounds of the present invention

[0049] In another embodiment, the present application provides a method (Method 1) of treating a condition selected from cancer or tumor, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II, III and /or IV) to the subject in need.

1.1 Method 1, wherein the cancer or tumor has increased expression of PDE1 compared to normal cells of the same tissue type as the cancer or tumor cells.

1.2 Any preceding method where the cancer or tumor is characterized by increased expression of PDE1C compared to normal cells of the same tissue type as the cancer or tumor cells.

1.3 Any of the foregoing methods wherein cancer cells exhibit one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding (e.g., PET or radioisotope retention in PDE1 inhibitor molecules), or PDE1 enzymatic activity (e.g., as measured in an enzymatic assay or as reflected by low levels of cAMP in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as cancer cells.

1.4 Any previous method where the condition is a tumor.

1.5 Any preceding method where the tumor is selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, gliomas (eg, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymomas, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal tumor (PNET), schwannoma, adenomas (eg, basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibromyomas (fibrous histiocytoma), fibromas, hemangiomas, lipomas (eg, angiolipomas, myelolipomas, fibrolipomas, spindle cell lipomas, hibernoma, atypical lipomas), myxomas, osteomas, preleukemia, radomyomas, papillomas, seborrheic keratosis, cutaneous adnexal tumors, liver adenomas, renal tubular adenomas, bile duct adenomas, transitional -cell papillomas, chorionaden, ganglionuroma, meningomes, neuromumomes, neurofibromes, C-cell hyperplasia, pheochromocytoma, insulinomas, gastrinomas, carcynoids, chemodectoma, palaglyglioma, nevus, actinic keratosis, shamey uterus, metaplasia (for example, metaplasia, metaplasia (for example, metaplasia, metaplasia, metaplasia, metaplasia. light), leukoplakia, hemangiomas, lymphangiomas, carcinomas (eg, squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, germinal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma prominent gland, bronchial carcinoma, carcinoma oat cell carcinoma, islet cell carcinoma, malignant carcinoid), sarcoma (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), bl astomas (for example, medulloblastomas and glioblastomas, types of brain tumor, retinoblastoma, retinal tumor, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant cutaneous adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, tumor from Merkel cells, phyllodes cystosarcomas or Wilms tumor.

1.6 Any preceding method where the condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

1.7 Any preceding method where the condition is a glioma (eg, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, or mixed gliomas, eg, oligoastrocytomas).

1.8 Method 1.6, where the glioma is an astrocytoma (eg, glioblastoma multiforme).

1.9 Any preceding method where the condition is glioblastoma multiforme.

1.10 Any of methods 1-1.2, where the condition is cancer.

1.11 Any of methods 1-1.2 or 1.10, where the condition is leukemia.

1.12 Method 1.11, wherein the leukemia is lymphocytic leukemia or myelogenous leukemia.

1.13 Any preceding method further comprising the step of administering an antitumor agent to the patient.

1.14 Method 1.13, where the antitumor agent is administered simultaneously with the PDE1 inhibitor.

1.15 Method 1.13, wherein the antitumor agent is administered before the PDE1 inhibitor is administered.

1.16 Method 1.13, wherein the antitumor agent is administered after administration of the PDE1 inhibitor.

1.17 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent is administered with radiation therapy or chemotherapy.

1.18 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered simultaneously with radiation therapy or chemotherapy.

1.19 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered before radiation therapy or chemotherapy.

1.20 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered after radiation therapy or chemotherapy.

1.21 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy and/or immunological treatment.

1.22 Any of the preceding methods, wherein administration of a PDE1 inhibitor is effective to induce in cancer or tumor cells one or more of the following: apoptotic cell death, inhibition of migration, inhibition of metastasis, and/or reduction of inflammation.

1.23 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy, immunological treatment, corticosteroid and/or antihistamine.

1.24 Any of the preceding methods where the cancer or tumor is mediated by PDE.

1.25 Any of the preceding methods where the cancer or tumor is mediated by PDE1.

1.26 Any of the preceding methods where the cancer or tumor is mediated by PDE1C.

1.27 Any of the preceding methods where the cancer or tumor is characterized by loss of calcium/calmodulin control.

1.28 Any of the foregoing methods where the PDE1 enzymes in cancer or tumor cells have undergone a structural change that alters the calcium/calmodulin control of PDE1 activity.

[0050] The present invention also provides a PDE1 inhibitor for use in a method of treating a condition selected from cancer or tumor, for example, for use in any of Methods 1 and following.

[0051] The present invention also provides the use of a PDE1 inhibitor for the preparation of a medicament for use in a method of treating a condition selected from cancer or a tumor, for example, a medicament for use in any of Methods 1 and below.

[0052] The present invention also provides a pharmaceutical composition comprising a PDE1 inhibitor, for example, any of the compounds of formula I, Ia, II, III and/or IV, for use in any of methods 1 and following.

[0053] In another embodiment, the present application provides a method (Method 2) of inhibiting the proliferation, migration and/or invasion of cancer or tumor cells, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II, III and/or IV) to the subject in need.

2.1 Method 2, where the method is intended to inhibit the proliferation of cancer or tumor cells.

2.2 Any preceding method wherein cancer or tumor cells have increased expression of PDE1 compared to normal cells of the same tissue type as the cancer or tumor cells.

2.3 Any preceding method wherein cancer or tumor cells have increased expression of PDE1C compared to normal cells of the same tissue type as the cancer or tumor cells.

2.4 Any of the foregoing methods wherein cancer cells exhibit one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding (e.g., PET or radioisotope retention in PDE1 inhibitor molecules), or PDE1 enzymatic activity (e.g., as measured in an enzymatic assay or as reflected by low levels of cAMP in cancer cells or subcellular domain (eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as cancer cells.

2.5 Any previous method where the condition is a tumor.

2.6 Any preceding method where the tumor is selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, gliomas (eg, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymomas, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal tumor (PNET), schwannoma, adenomas (eg, basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibromyomas (fibrous histiocytoma), fibromas, hemangiomas, lipomas (eg, angiolipomas, myelolipomas, fibrolipomas, spindle cell lipomas, hibernoma, atypical lipomas), myxomas, osteomas, preleukemia, radomyomas, papillomas, seborrheic keratosis, cutaneous adnexal tumors, liver adenomas, renal tubular adenomas, bile duct adenomas, transitional -cell papillomas, chorionaden, ganglionuroma, meningomes, neuromumomes, neurofibromes, C-cell hyperplasia, pheochromocytomas, insulinomas, gastrinomas, carcynoids, chemodectoma, palaglioma, nevus, actinic keratosis, shamey uterus, metaplasia (for example, metaplasia, metaplasia, metaplasia (for example, metaplasia, metaplasia, metaplasia. light), leukoplakia, hemangiomas, lymphangiomas, carcinomas (eg, squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, germinal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma prominent gland, bronchial carcinoma, carcinoma oat cell carcinoma, islet cell carcinoma, malignant carcinoid), sarcoma (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), bl astomas (for example, medulloblastomas and glioblastomas, types of brain tumor, retinoblastoma, retinal tumor, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant cutaneous adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, tumor from Merkel cells, phyllodes cystosarcomas or Wilms tumor.

2.7 Any preceding method where the condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

2.8 Any preceding method where the condition is a glioma (eg, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, or mixed gliomas, eg, oligoastrocytomas).

2.9 Method 2.5, where the glioma is an astrocytoma (eg, glioblastoma multiforme).

2.10 Any preceding method where the condition is glioblastoma multiforme.

2.11 Any of methods 2-2.3, where the condition is cancer.

2.12 Any of methods 2-2.3 or 2.11, where the condition is leukemia.

2.13 Method 2.11, wherein the leukemia is lymphocytic leukemia or myelogenous leukemia.

2.14 Any preceding method further comprising the step of administering an antitumor agent to the patient.

2.15 Method 2.14, where the antitumor agent is administered simultaneously with the PDE1 inhibitor.

2.16 Method 2.14, where the antitumor agent is administered before the PDE1 inhibitor is administered.

2.17 Method 2.14, where the antitumor agent is administered after administration of the PDE1 inhibitor.

2.18 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent is administered with radiation therapy or chemotherapy.

2.19 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered simultaneously with radiation therapy or chemotherapy.

2.20 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered before radiation therapy or chemotherapy.

2.21 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent are administered after radiation therapy or chemotherapy.

2.22 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy and/or immunological treatment.

2.23 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy, immunological treatment, corticosteroid and/or antihistamine.

2.24 Any of the preceding methods where the cancer or tumor is mediated by PDE.

2.25 Any of the preceding methods where the cancer or tumor is mediated by PDE1.

2.26 Any of the preceding methods where the cancer or tumor is mediated by PDE1C.

2.27 Any of the preceding methods where the cancer or tumor is characterized by loss of calcium/calmodulin control.

2.28 Any of the preceding methods where the PDE1 enzymes in cancer or tumor cells have undergone a structural change that alters the calcium/calmodulin control of PDE1 activity.

2.29 Any of the preceding methods, further comprising the step of assessing in the subject the degree of calcium/calmodulin sensitivity in expressed PDE1, restoration of cyclic nucleotide levels, expression of PDE1 RNA, or mutation of the PDE1 gene.

[0054] The present invention also provides a PDE1 inhibitor for use in a method for inhibiting proliferation, migration and/or invasion of cancer or tumor cells, for example, for use in any of Methods 2 and below.

[0055] The present invention also provides the use of a PDE1 inhibitor for the preparation of a medicament for use in a method of inhibiting the proliferation, migration and/or invasion of cancer or tumor cells, for example, a medicament for use in any of Methods 2 and below.

[0056] The present invention also provides a pharmaceutical composition comprising a PDE1 inhibitor, for example, any of the compounds of formula I, Ia, II, III and/or IV, for use in any of methods 2 and following.

[0057] In another embodiment, the present application provides a method (Method 3) for treating glioma, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II, III and/or IV) to a patient in need of this subject.

3.1 Method 3, where the glioma is characterized by increased expression of PDE1 compared to normal cells of the same tissue type as the affected glial cells.

3.2 Any preceding method where the glioma is characterized by increased expression of PDE1C compared to normal cells of the same tissue type as the affected glial cells.

3.3 Any of the foregoing methods wherein the glioma is characterized by one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding (eg, PET or radioisotope retention in PDE1 inhibitor molecules), or PDE1 enzymatic activity (eg, as measured in an enzyme assay or as reflected in low levels of cAMP in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as cancer cells.

3.4 Any preceding method where the glioma is an astrocytoma (eg, glioblastoma multiforme).

3.5 Any preceding method where the condition is glioblastoma multiforme.

3.6 Any preceding method further comprising the step of administering an antitumor agent to the patient.

3.7 Method 3.6, where the antitumor agent is administered simultaneously with the PDE1 inhibitor.

3.8 Method 3.6, where the antitumor agent is administered before the PDE1 inhibitor is administered.

3.9 Method 3.6, where the antitumor agent is administered after administration of the PDE1 inhibitor.

3.10 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent are administered with radiation therapy or chemotherapy.

3.11 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered simultaneously with radiation therapy or chemotherapy.

3.12 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered before radiation therapy or chemotherapy.

3.13 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered after radiation therapy or chemotherapy.

3.14 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy and/or immunological treatment.

3.15 Any of the preceding methods, wherein administration of a PDE1 inhibitor is effective in inducing one or more of the following in glioma: apoptotic cell death, inhibition of migration, inhibition of metastasis, and/or reduction of inflammation.

3.16 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy, immunological treatment, corticosteroid and/or antihistamine.

3.17 Any of the previous methods where the glioma is mediated by PDE.

3.18 Any of the previous methods where glioma is mediated by PDE1.

3.19 Any of the previous methods where glioma is mediated by PDE1C.

3.20 Any of the preceding methods where the glioma is characterized by loss of calcium/calmodulin control.

3.21 Any of the preceding methods where the PDE1 enzymes in glioma cells have undergone a structural change that alters calcium/calmodulin control of PDE1 activity.

[0058] The present invention also provides a PDE1 inhibitor for use in a method for treating glioma, for example, for use in any of methods 3 and following.

[0059] The present invention also provides the use of a PDE1 inhibitor for the preparation of a drug for use in a method of treating glioma, for example, a drug for use in any of methods 3 and below.

[0060] The present invention also provides a pharmaceutical composition comprising a PDE1 inhibitor, for example, any of the compounds of formula I, Ia, II, III and/or IV, for use in any of methods 4 and following.

[0061] In another embodiment, the present application provides a method (Method 4) of treating cancer in a patient where the cancer cells have increased PDE1 activity or expression, for example, where the cancer cells exhibit one or more of increased PDE1 RNA expression, DNA copy number, binding PDE1 (eg, PET or radioisotope retention in PDE1 inhibitor molecules) or PDE1 enzymatic activity (eg, as measured in an enzyme assay or as reflected by low levels of cAMP in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared with normal cells of the same tissue type as cancer cells, comprising administering to the patient an effective amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II, III and/or IV) alone or in combination with chemotherapeutic agents, gene therapeutic agents and/or immunological methods for treating a subject in need thereof.

4.1 Method 4, wherein the method is intended to inhibit the proliferation of cancerous or tumor cells.

4.2 Any preceding method wherein cancer or tumor cells have increased expression or activity of PDE1 compared to normal cells of the same tissue type as the cancer or tumor cells.

4.3 Any preceding method wherein cancer or tumor cells have increased expression of PDE1C compared to normal cells of the same tissue type as the cancer or tumor cells.

4.4 Any of the foregoing methods wherein cancer cells exhibit one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding (e.g., PET or radioisotope retention in PDE1 inhibitor molecules), or PDE1 enzymatic activity (e.g., as measured in an enzymatic assay or as reflected by low levels of cAMP in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as cancer cells.

4.5 Any previous method where the condition is a tumor.

4.6 Any preceding method where the tumor is selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, gliomas (eg, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymomas, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal tumor (PNET), schwannoma, adenomas (eg, basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibromyomas (fibrous histiocytoma), fibromas, hemangiomas, lipomas (eg, angiolipomas, myelolipomas, fibrolipomas, spindle cell lipomas, hibernoma, atypical lipomas), myxomas, osteomas, preleukemia, radomyomas, papillomas, seborrheic keratosis, cutaneous adnexal tumors, liver adenomas, renal tubular adenomas, bile duct adenomas, transitional -cell papillomas, chorionaden, ganglionuroma, meningomes, neuromumomes, neurofibromes, C-cell hyperplasia, pheochromocytomas, insulinomas, gastrinomas, carcynoids, chemodectoma, palaglioma, nevus, actinic keratosis, shamey uterus, metaplasia (for example, metaplasia, metaplasia, metaplasia (for example, metaplasia, metaplasia, metaplasia. light), leukoplakia, hemangiomas, lymphangiomas, carcinomas (eg, squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, germinal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma prominent gland, bronchial carcinoma, carcinoma oat cell carcinoma, islet cell carcinoma, malignant carcinoid), sarcoma (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), bl astomas (for example, medulloblastomas and glioblastomas, types of brain tumor, retinoblastoma, retinal tumor, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant cutaneous adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, tumor from Merkel cells, phyllodes cystosarcomas or Wilms tumor.

4.7 Any preceding method where the condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

4.8 Any preceding method where the condition is a glioma (eg, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, or mixed gliomas, eg, oligoastrocytomas).

4.9 Method 4.8, wherein the glioma is an astrocytoma (eg, glioblastoma multiforme).

4.10 Any preceding method where the condition is glioblastoma multiforme.

4.11 Any of methods 4-4.3, where the condition is cancer.

4.12 Any of methods 4-4.3 or 4.11, where the condition is leukemia.

4.13 Method 4.12, wherein the leukemia is lymphocytic leukemia or myelogenous leukemia.

4.14 Any preceding method further comprising the step of administering an antitumor agent to the patient.

4.15 Method 4.14, where the antitumor agent is administered simultaneously with the PDE1 inhibitor.

4.16 Method 4.14, where the antitumor agent is administered before the PDE1 inhibitor is administered.

4.17 Method 4.14, where the antitumor agent is administered after administration of the PDE1 inhibitor.

4.18 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent is administered with radiation therapy or chemotherapy.

4.19 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered simultaneously with radiation therapy or chemotherapy.

4.20 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered before radiation therapy or chemotherapy.

4.21 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent are administered after radiation therapy or chemotherapy.

4.22 Any of the preceding methods, wherein administration of a PDE1 inhibitor is effective to induce in cancer cells one or more of the following: apoptotic cell death, inhibition of migration, inhibition of metastasis, and/or reduction of inflammation.

4.23 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy, immunological treatment, corticosteroid and/or antihistamine.

4.24 Any of the preceding methods where the cancer or tumor is mediated by PDE.

4.25 Any of the preceding methods where the cancer or tumor is mediated by PDE1.

4.26 Any of the preceding methods where the cancer or tumor is mediated by PDE1C.

4.27 Any of the preceding methods where the cancer is characterized by loss of calcium/calmodulin control.

4.28 Any of the preceding methods where the PDE1 enzymes in cancer cells have undergone a structural change that alters calcium/calmodulin control of PDE1 activity.

[0062] The present invention also provides a PDE1 inhibitor for use in a method of treating cancer in a patient where the cancer cells have increased PDE1 activity or expression, for example, where the cancer cells exhibit one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding ( e.g., PET or radioisotope retention in PDE1 inhibitor molecules) or PDE1 enzymatic activity (e.g., as measured in an enzymatic assay or as reflected by low levels of cAMP in cancer cells or subcellular domain, e.g., microtubule domains, cancer cells) compared with normal cells the same tissue type as the cancer cells, for example, for use in any of methods 4 and following.

[0063] The present invention also provides the use of a PDE1 inhibitor for the preparation of a medicament for use in a method of treating cancer in a patient where the cancer cells have increased PDE1 activity or expression, for example, where the cancer cells exhibit one or more of increased PDE1 RNA copy number expression DNA, PDE1 binding (eg, PET or radioisotope retention in PDE1 inhibitor molecules), loss of calcium/calmodulin control, the enzyme becoming constitutively active, or PDE1 enzymatic activity (eg, as measured in an enzyme assay or as reflected by low cAMP levels in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as the cancer cells, eg drug for use in any of methods 4 and following.

[0064] The present invention also provides a pharmaceutical composition containing a PDE1 inhibitor, for example, any of the compounds of formula I, Ia, II, III and/or IV, for use in any of methods 4 and following.

[0065] In another embodiment, the present application provides a method (Method 5) of inhibiting cytokine release syndrome, comprising administering an effective amount of a PDE1 inhibitor to a patient in need thereof, for example, where the patient suffers from cancer and is receiving one or more of a chemotherapy treatment, an immunological treatment, gene therapy and/or antibody therapy (including antibodies directed to cancer antigens and/or antibodies to immune checkpoint targets), and where the method optionally further includes administering corticosteroids and/or antihistamines to the patient.

5.1 Method 5, where the patient is suffering from cancer.

5.2 Any preceding method wherein cancer or tumor cells have increased expression or activity of PDE1 compared to normal cells of the same tissue type as the cancer or tumor cells.

5.3 Any preceding method wherein cancer or tumor cells have increased expression of PDE1C compared to normal cells of the same tissue type as the cancer or tumor cells.

5.4 Any of methods 5.1-5.3 where the cancer cells exhibit one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding (e.g., PET or radioisotope retention in PDE1 inhibitor molecules), or PDE1 enzymatic activity (e.g., as measured by enzymatic analysis or as reflected by low levels of cAMP in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as cancer cells.

5.5 Any previous method where the patient suffers from a tumor.

5.6 Any preceding method where the patient suffers from a tumor selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, gliomas (eg, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal tumor (PNET), schwannoma, adenomas (eg, basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibroids (fibrous histiocytomas ), fibromas, hemangiomas, lipomas (for example, angiolipomas, myelolipomas, fibrolipomas, spindle cell lipomas, hibernomas, atypical lipomas), myxomas, osteomas, preleukemia, radomyomas, papillomas, seborrheic keratosis, cutaneous adnexal tumors, liver adenomas, renal tubular adenomas, adenomas bile duct, transitional cell papilloma, chorionadenoma, ganglioneuroma, meningoma, neurilemmoma, neurofibroma, C-cell hyperplasia, pheochromocytoma, insulinoma, gastrinoma, carcinoids, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia (eg, metaplasia of the lung th ), leukoplakia, hemangiomas, lymphangiomas, carcinomas (eg, squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, germinal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoma, oat cell carcinoma, islet cell carcinoma, malignant carcinoid), sarcoma (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyo sarcomas, neurofibrosarcomas), blastomas (for example, medulloblastoma and glioblastoma, types of brain tumor, retinoblastoma, retinal tumor, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant cutaneous adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma ohms , melanoma, Merkel cell tumor, phyllodes cystosarcoma or Wilms tumor.

5.7 Any previous method where the patient suffers from glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

5.8 Any previous method where the patient suffers from a glioma (eg ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma or mixed gliomas such as oligoastrocytomas).

5.9 Method 5.8, where the patient suffers from an astrocytoma (eg glioblastoma multiforme).

5.10 Any previous method where the patient suffers from glioblastoma multiforme.

5.11 Any previous method where the patient is suffering from cancer.

5.12 Any previous method where the patient suffers from leukemia.

5.13 Method 5.12, wherein the leukemia is lymphocytic leukemia or myelogenous leukemia.

5.14 Any preceding method further comprising the step of administering an antineoplastic agent to the patient.

5.15 Method 5.14, where the antitumor agent is administered simultaneously with the PDE1 inhibitor.

5.16 Method 5.14, where the antitumor agent is administered before the PDE1 inhibitor is administered.

5.17 Method 5.14, where the antitumor agent is administered after administration of the PDE1 inhibitor.

5.18 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent is administered with radiation therapy or chemotherapy.

5.19 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered simultaneously with radiation therapy or chemotherapy.

5.20 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered before radiation therapy or chemotherapy.

5.21 Any of the preceding methods, wherein a PDE1 inhibitor and optionally an antitumor agent is administered after radiation therapy or chemotherapy.

5.22 Any of the preceding methods, wherein the above method, wherein administration of a PDE1 inhibitor is effective for inducing in cancer cells one or more of the following: apoptotic cell death, inhibition of migration, inhibition of metastasis, and/or reduction of inflammation.

5.23 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy, immunological treatment, corticosteroid and/or antihistamine.

5.24 Any of the methods 5.1-5.23, where the cancer or tumor is mediated by PDE.

5.25 Any of the methods 5.1-5.24, where the cancer or tumor is mediated by PDE1.

5.26 Any of the methods 5.1-5.25, where the cancer or tumor is mediated by PDE1C.

5.27 Any of methods 5.1-5.26, where the cancer or tumor is characterized by loss of calcium/calmodulin control.

5.28 Any of methods 5.1-5.26 wherein the PDE1 enzymes in cancer or tumor cells have undergone a structural change that alters calcium/calmodulin control of PDE1 activity.

[0066] The present invention also provides a PDE1 inhibitor for use in a method of inhibiting cytokine release syndrome, for example, for use in any of methods 5 and following.

[0067] The present invention also provides the use of a PDE1 inhibitor for the preparation of a drug for use in a method of inhibiting cytokine release syndrome, for example, a drug for use in any of methods 5 and following.

[0068] The present invention also provides a pharmaceutical composition comprising a PDE1 inhibitor, for example, any of the compounds of formula I, Ia, II, III and/or IV, for use in any of methods 5 and following.

[0069] In another embodiment, the present application provides a method (Method 6) of treating a cancer or tumor characterized by constitutively active PDE1, the method comprising administering an effective amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II , III and/or IV) to the patient in need.

6.1 Method 6, where PDE1 in cancer or tumor cells became independent of Ca2+/calmodulin mediation.

6.2 Method 6 or 6.1, where PDE1 is PDE1A.

6.3 Method 6 or 6.1, where PDE1 is PDE1B.

6.4 Method 6 or 6.1, where PDE1 is PDE1C.

6.5 Any preceding method where the cancer or tumor is characterized by increased expression of PDE1 compared to normal cells of the same tissue type as the cancer or tumor cells.

6.6 Any preceding method where the cancer or tumor is characterized by increased expression of PDE1C compared to normal cells of the same tissue type as the cancer or tumor cells.

6.7 Any of the foregoing methods wherein the cancer or tumor is characterized by one or more of increased PDE1 RNA expression, DNA copy number, PDE1 binding (e.g., PET or radioisotope retention in PDE1 inhibitor molecules), or PDE1 enzymatic activity (e.g., as measured in an enzymatic assay or as reflected by low levels of cAMP in cancer cells or subcellular domain, eg microtubule domains, cancer cells) compared to normal cells of the same tissue type as cancer cells.

6.8 Any preceding method where the condition is a tumor.

6.9 Any preceding method where the tumor is selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma (eg, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal tumor (PNET), schwannoma, adenomas (eg, basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibromyomas (fibrous histiocytoma), fibromas, hemangiomas, lipomas (eg, angiolipomas, myelolipomas, fibrolipomas, spindle cell lipomas, hibernoma, atypical lipomas), myxomas, osteomas, preleukemia, radomyomas, papillomas, seborrheic keratosis, cutaneous adnexal tumors, liver adenomas, renal tubular adenomas, bile duct adenomas, transitional -cell papillomas, chorionaden, ganglionuroma, meningomes, neuromumomes, neurofibromes, C-cell hyperplasia, pheochromocytomas, insulinomas, gastrinomas, carcynoids, chemodectoma, palaglioma, nevus, actinic keratosis, shamey uterus, metaplasia (for example, metaplasia, metaplasia, metaplasia (for example, metaplasia, metaplasia, metaplasia. light), leukoplakia, hemangiomas, lymphangiomas, carcinomas (eg, squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, germinal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma prominent gland, bronchial carcinoma, carcinoma oat cell carcinoma, islet cell carcinoma, malignant carcinoid), sarcoma (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), bl astomas (for example, medulloblastomas and glioblastomas, types of brain tumor, retinoblastoma, retinal tumor, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant cutaneous adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, tumor from Merkel cells, phyllodes cystosarcomas or Wilms tumor.

6.10 Any preceding method where the condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

6.11 Any preceding method where the condition is a glioma (eg, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, or mixed gliomas, eg, oligoastrocytomas).

6.12 The preceding method, where the glioma is an astrocytoma (eg, glioblastoma multiforme).

6.13 Any preceding method where the condition is glioblastoma multiforme.

6.14 Any preceding method where the condition is glioblastoma multiforme characterized by the presence of constitutively active PDE1 (eg, PDE1C).

6.15 Any of methods 6-6.7, where the condition is cancer.

6.16 Any of methods 6-6.7 or 6.15, where the condition is leukemia.

6.17 The preceding method, where the leukemia is lymphocytic leukemia or myelogenous leukemia.

6.18 Any preceding method further comprising the step of administering an antineoplastic agent to the patient.

6.19 Method 6.18, where the antitumor agent is administered simultaneously with the PDE1 inhibitor.

6.20 Method 6.18, where the antitumor agent is administered before the PDE1 inhibitor is administered.

6.21 Method 6.18, wherein the antitumor agent is administered after administration of the PDE1 inhibitor.

6.22 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent is administered with radiation therapy or chemotherapy.

6.23 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered simultaneously with radiation therapy or chemotherapy.

6.24 Any of the preceding methods, wherein the PDE1 inhibitor and, optionally, an antitumor agent are administered before radiation therapy or chemotherapy.

6.25 Any of the preceding methods, wherein the PDE1 inhibitor and optionally an antitumor agent are administered after radiation therapy or chemotherapy.

6.26 Any of the preceding methods wherein the PDE1 inhibitor is administered together with an antineoplastic agent, chemotherapy, gene therapy and/or immunological treatment.

6.27 Any of the preceding methods where the cancer or tumor is mediated by PDE.

6.28 Any of the preceding methods where the cancer or tumor is mediated by PDE1.

6.29 Any of the preceding methods where the cancer or tumor is mediated by PDE1C.

6.30 Any of the preceding methods where the cancer or tumor is characterized by loss of calcium/calmodulin control.

6.31 Any of the preceding methods where the PDE1 enzymes in cancer or tumor cells have undergone a structural change that alters the calcium/calmodulin control of PDE1 activity.

[0070] The present invention also provides a PDE1 inhibitor for use in a method of treating a cancer or tumor characterized by constitutively active PDE1, for example, for use in any of methods 6 and following.

[0071] The present invention also provides the use of a PDE1 inhibitor for the preparation of a medicament for use in a method of treating a cancer or tumor characterized by constitutively active PDE1, for example, a medicament for use in any of methods 6 and below.

[0072] The present invention also provides a pharmaceutical composition comprising a PDE1 inhibitor, for example, any of the compounds of formula I, Ia, II, III and/or IV, for use in any of methods 6 and following.

[0073] In some embodiments, the pharmaceutical compositions are administered in combination with one or more antineoplastic drugs, for example drugs that are known to be effective in treating or eliminating various types of cancer and/or tumors. Non-limiting examples of anticancer drugs include Abemaciclib, Abiraterone acetate, Abitrexate (Methotrexate), Abraxane (an albumin-stabilized nanoparticle formulation of paclitaxel), ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab vedotin), ADE, Ado -Tuzumab emanzin, Adriamicin (Doksorubicin Hydrochloride), Atathaniba Dimaleat, Athenler (Everolymus), Akinzeo (inaccurate and Palosetron Hydrochloride), Aldar (Imikvimod), Aldexleikin, Alecenes (Alektinib), Alemtuzumab, Ailimab, Ailimab, Ailimaba, Ailimaba, Ailimaba, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailimab, Ailima, Ailima, Ailima, Ailimois. Yetreksed Dinatri), Alikopa ( Copanlisib hydrochloride), Alkeran injection (Melphalan hydrochloride), Alkeran tablets (Melphalan), Aloxi (Palonosetron hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia ( Pamidronate disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranone (Nelarabine), Arsenic trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene siloleucel, Axitinib, Azacitidine, Bavencio (Aveluma) b ), BEACOPP, Becenum (Carmustine), Beleodac (Belinostat), Belinostat, Bendamustine hydrochloride, BEP, Besponsa (Inotuzumab ozogamicin), Bevacizumab, Bexarotene, Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometics (Cabozantinib-S-malate), Cabozantinib-S-malate, CAF, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil - for topical use), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin hydrochloride ), Cervarix (recombinant bivalent HPV vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clorafex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometrik ( Cabozantinib-S-malate), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyphos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine liposomal, Cytosar -U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin hydrochloride, Daunorubicin hydrochloride and Cytarabine liposomal, Decitabine, Sodium Defibrotide, Defitelio (Sodium Defibrotide ) , Degarelix, Denileukin diftitox, Denosumab, DepoCyt (Cytarabine liposomal), Dexamethesone, Dexrazoxane hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin hydrochloride liposomal), Doxorubicin hydrochloride, Doxorubicin hydrochloride liposomal, Dox-SL (Doxorubicin hydrochloride liposomal) posomal), DTIC-Dome ( Dacarbazine), Durvalumab, Efudix (Fluorouracil - topical), Elitek (Rasburicase), Ellens (Epirubicin hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag olamine, Emend (Aprepitant), Emplicity (Elotuzumab), Enasidenib mesylate, Enzalutamide , Epirubicin hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulina mesylate, Erivedge (Vismodegib), Erlotinib hydrochloride, Erwinase (Erwinia chrysanthemi Asparaginase), Etiol (Amifostine), Etopophos (Etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (Doxorubicin hydrochloride liposomal), Everolimus, Evista (Raloxifene hydrochloride), Evomela (Melphalan hydrochloride), Exemestane, 5-FU (Fluorouracil injection), 5-FU (Fluorouracil - topical), Fareston (Toremifene), Faridak (Panobinostat), Faslodex (Fulvestrant) , FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine phosphate), Fludarabine phosphate, Fluoroplex (Fluorouracil - for topical use), Fluorouracil injection, Fluorouracil - for topical use, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate) , FOLFIRI, FOLFIRI-Bevacizumab, FOLFIRI-Cetuximab, Folfirinox, Folfox, Folotin (Pralatrexate), FU-LV, Fulvestrant, Gardasil (tetravalent recombinant HPV vaccine), Gardasil 9 (ninevalent recombinant HPV vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuzumab ozogamicin, Gemzar (Gemcitabine hydrochloride), Gilotrif (Afatinib dimaleate), Gleevec (Imatinib mesylate), Gliadel (Carmustine implant), Gliadel wafer (Carmustine implant), Glucarpidase , Gozerelina acetate, Halaven (Eribulina mesylate), Gemangiol (Propranolol hydrochloride), Herceptin (Trastuzumab), Human papillomavirus vaccine, bivalent, recombinant; The vaccine against human papillomavirus is nine-valent, recombinant; The vaccine against human papillomavirus is tetravalent, recombinant; Hycamtin (Topotecan hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritutomab tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib hydrochloride), Idamycin (Idarubicin hydrochloride), Idarubicin hydrochloride, Idelalisib, Aidhifa (Enasidenib) mesylate ), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imligik (Talimogen laherparepvec), Inlyta (Axitinib), Inotuzumab ozogamicin, Interferon alpha-2b recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant interferon alpha-2b), Ipilimumab, Iressa (Gefitinib), Irinotecan hydrochloride, Irinotecan hydrochloride liposomal, Istodax (Romidepsin), Ixabepilone, Ixazomib citrate, Ixempra (Ixabepilone ), Jakavi (Ruxolitinib phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (Raloxifene hydrochloride), Kepivanse (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide acetate, Lapatinib ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib mesylate, Lenvima (Lenvatinib mesylate), Letrozole, Leucovorin calcium, Leukeran (Chlorambucil), Leuprorelin acetate, Leustatin (Cladribine), Levulan (Aminolevulinic acid), Linfolysin (Chlorambucil), Lipodox (Doxorubicin hydrochloride liposomal), Lomustine, Lonsurf (Trifluridine and Tipiracil hydrochloride), Lupron (Leuprorelin acetate), Lupron Depot (Leuprorelin acetate), Lupron Depot-Ped (Leuprorelin acetate), Lynparza (Olaparib), Markibo (Vincristine sulfate liposomal), Matulan (procarbazine hydrochloride), Mechlorethamine hydrochloride, Megestrol acetate, Mekinist (Trametinib), Melphalan, Melphalan hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Metazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone hydrochloride, Mitozitrex (Mitomycin C), MOPP, Mozobail (Plerixafor), Mustargen (Mechlorethamine hydrochloride), Mutamycin (Mitomycin C), Mileran ( Busulfan), Milosar (Azacitidine), Mylotarg (Gemtuzumab ozogamicin), Nanoparticle-based Paclitaxel (Albumin-stabilized nanoparticle formulation of paclitaxel), Navelbine (Vinorelbine tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib maleate, Nerlinx (Neratinib maleate), Netupitant and Palonosetron hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib citrate), Niraparib tosylate monohydrate, Nivolumab, Nolvadex (Tamoxifen citrate), Enplate ( Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine mepesuccinate, Oncaspar (Pegaspagaza), Ondansetron hydrochloride, Onivide (Irinotecan hydrochloride liposomal), Ontak (Denileukin diftitox), Opdivo (Nivolumab), OPPA , Osimertinib, Oxaliplatin, Paclitaxel, Albumin stabilized nanoparticle formulation of paclitaxel, PAD, Palbociclib, Palifermin, Palonosetron hydrochloride, Palonosetron hydrochloride and Netupitant, Pamidronate disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib hydrochloride, PCV, PEB , Pegaspargase, Pegfilgrastim, Peginterferon alfa-2b, PEG-Intron (Peginterferon alfa-2b), Pembrolizumab, Pemetrexed disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst ( Pomalidomide), Ponatinib hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag olamine), Propranolol hydrochloride, Provenge (Sipuleucel-T), Purinetol (Mercaptopurine), Purixan (Mercaptopurine), Radium-223 dichloride, Raloxifene hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant bivalent human papillomavirus (HPV) vaccine, Recombinant nine-valent human papillomavirus (HPV) vaccine, Recombinant quadrivalent human papillomavirus (HPV) vaccine human papillomavirus (HPV), Recombinant interferon alpha-2b, Regorafenib, Relistor (Methylnaltrexone bromide), R-EPOCH, Revlimid (Lenalidomide), Revmatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hitsela (Rituximab) and human Hyaluronidase), Rituximab, Rituximab and Human Hyaluronidase, Rolapitant hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin hydrochloride), Rubraca (Rucaparib camzilate), Rucaparib camzilate, Ruxolitinib phosphate, Rydapt (Midostaurin), intrapleural aerosol Sclerosol (Talc), Siltuximab , Sipuleucel-T, Somatulin Depot (Lanreotide acetate), Sonidegib, Sorafenib tosylate, Sprycel (Dasatinib), STANFORD V, Sterile talc powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib malate, Sutent (Sunitinib malate) , Silatron (Peginterferon alfa-2b), Silvant (Siltuximab), Sinribo (Omacetaxine mepesuccinate), Tabloid (Tioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene laherparepvec, Tamoxifen citrate, Tarabine PFS (Cytarabine) , Tarceva (Erlotinib hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalidomide (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolac (Fluorouracil - topical), Topotecan hydrochloride, Toremifene, Torisel (Temsirolimus), Totect (Dexrazoxane hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine hydrochloride), Trifluridine and Tipiracil hydrochloride, Trizenox (Arsenic trioxide), Tykerb (Lapatinib ditosylate), Unituxin (Dinutuximab), Uridine triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine sulfate) , Velcade (Bortezomib), Velsar (Vinblastine sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzeneo (Abemaciclib), Viadur (Leuprorelin acetate), Vidaza (Azacitidine), Vinblastine sulfate, Vincasar PFS (Vincristine sulfate), Vincristine sulfate, Vincristine sulfate liposomal, Vinorelbine tartrate, VIP, Vismodegib, Vistogard (Uridine triacetate), Voraxaz (Glucarpidase), Vorinostat, Votrient (Pazopanib hydrochloride), Vixeos (Daunorubicin hydrochloride and Cytarabine liposomal), Wellcovorin (Leucovorin calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Exjiva (Denosumab), Xofigo (Radia-223 dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Escarta (Axicabtagene siloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (Filgrastim), Zejula (Niraparib tosylate monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritutomab tiuxetan), Zinecard (Dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (Ondansetron hydrochloride), Zoladex (Goserelin acetate), Zoledronic acid, Zolinza ( Vorinostat), Zometa (Zoledronic acid), Zydelig (Idelalisib), Zykadia (Ceritinib), Zytiga (Abiraterone acetate).

[0074] As used herein, the term “antitumor agent” refers to any chemical agents or drugs effective in preventing or inhibiting the formation or growth of cancer or tumors. Antineoplastic agents discussed herein may include alkylating agents, antimetabolites, natural products, hormones and/or antibodies. Treatment of tumors or cancer may include limiting the proliferation, migration and/or invasion of cancer or tumor cells in the body or limiting symptoms associated with the cancer or tumor. In the context of the present application, anticancer agents should be understood as encompassing anticancer agents or synonymous with anticancer agents.

Methods for preparing the compounds of the present invention

[0075] The PDE1 inhibitors of the present invention and their pharmaceutically acceptable salts can be prepared using the methods described and illustrated in US 8273750, US 2006/0173878, US 8273751, US 2010/0273753, US 8697710, US 8664207, US 8633180, US 853 6159 , US 2012/0136013, US 2011/0281832, US 2013/0085123, US 2013/0324565, US 2013/0338124, US 2013/0331363, WO 2012/171016 and WO 2013/192556, as well as in ways similar to them, and in ways well-known in the field of chemistry. Such methods include, but are not limited to, the methods described below. If they are not commercially available, the starting materials for these methods can be obtained using procedures selected from the field of chemistry, using methods similar or analogous to methods for the synthesis of known compounds.

[0076] Various PDE1 inhibitors and their starting materials can be prepared using the methods described in US 2008-0188492 A1, US 2010-0173878 A1, US 2010-0273754 A1, US 2010-0273753 A1, WO 2010/065153, WO 2010 /065151, WO 2010/065151, WO 2010/065149, WO 2010/065147, WO 2010/065152, WO 2011/153129, WO 2011/133224, WO 2011/153135, WO 2011/153 136, WO 2011/153138. All documents cited herein are incorporated by reference in their entirety.

[0077] Other PDE1 inhibitors and related methods are disclosed in US Provisional Application No. 62/833481, which is incorporated herein by reference in its entirety. Additional related PDE1 inhibitors and corresponding methods are disclosed in international publication WO2018/049417, which is incorporated herein by reference in its entirety.

[0078] The compounds of the present invention include their enantiomers, diastereomers and racemates, as well as their polymorphs, hydrates, solvates and complexes. Some individual compounds within the scope of this disclosure may contain double bonds. The representation of double bonds in the present disclosure is intended to include both the E and Z isomers of the double bond. In addition, some compounds within the scope of the present disclosure may contain one or more centers of asymmetry. The present invention includes the use of any of the optically pure stereoisomers, as well as any combination of stereoisomers.

[0079] It is also intended that the compounds of the invention include their stable and unstable isotopes. Stable isotopes are non-radioactive isotopes that contain one extra neutron compared to common nuclides of the same species (i.e. element). It is expected that the activity of compounds containing such isotopes will be maintained, and such a compound will also be useful for measuring the pharmacokinetics of non-isotopic analogues. For example, a hydrogen atom at a certain position in the compounds of the invention may be replaced by deuterium (a stable isotope that is not radioactive). Examples of known stable isotopes include, but are not limited to, deuterium, ¹³ C, ¹⁵ N, ¹⁸ O. Alternatively, unstable isotopes, which are radioactive isotopes that contain additional neutrons compared to common nuclides of the same kind (i.e. e. element), for example, ¹²³ I, ¹³¹ I, ¹²⁵ I, ¹¹ C, ¹⁸ F, can replace the corresponding common species I, C and F. Another example of a useful isotope of a compound of the present invention is the isotope ¹¹ C. These radioisotopes are useful for radiation imaging and/or pharmacokinetic studies of the compounds of the present invention.

[0080] Melting points are not adjusted, and (dec.) means “decomposition.” Temperatures are in degrees Celsius (°C); unless otherwise stated, procedures are performed at room or ambient temperature, i.e. at temperatures in the range of 18-25°C. Chromatography means flash chromatography on silica gel; Thin layer chromatography (TLC) is carried out on silica gel plates. NMR data are presented as delta values of the main diagnostic protons expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Use common abbreviations for the waveform. Coupling constants (J) are given in Hz. For mass spectra (MS), the lowest mass major ion is reported for molecules in which isotopic resolution results in multiple mass spectral peaks. Compositions of solvent mixtures are presented as volume percentages or volume ratios. In cases of complex NMR spectra, only diagnostic signals are indicated.

[0081] The words “treating” and “treating” should be understood, respectively, to cover the treatment or alleviation of symptoms of a disease, as well as treatment of the cause of a disease.

[0082] For methods of treatment, the word “effective amount” is intended to mean a therapeutically effective amount for treating a particular disease or disorder.

[0083] The term “patient” includes a human patient or a non-human (ie, animal) patient. In a particular embodiment, the disclosure covers both human and non-human animal. In another embodiment, the disclosure covers a non-human animal. In another embodiment, the term includes a person.

[0084] The term “comprising” in the context of this disclosure is intended to be non-limiting and does not exclude additional, not listed elements or method steps.

[0085] Dosages used in the practice of the present invention will, of course, vary depending, for example, on the particular disease or condition being treated, the particular compounds of the present invention used, the route of administration and the desired therapy. The compounds of the present invention can be administered by any suitable route, including oral, parenteral, transdermal or inhalation, but oral administration is preferred. In general, satisfactory results, for example for the treatment of diseases as indicated above, should be obtained when administered orally in doses of the order of about 0.01 to 2.0 mg/kg. In larger mammals, such as humans, the indicated daily oral dose of both PDE1 inhibitors will suitably range from about 0.50 to 300 mg, which is conveniently administered once or in divided doses 2-4 times daily or sustained release form. Thus, unit dosage forms for oral administration may include, for example, from about 0.2 to 150 or 300 mg, for example, from about 0.2 or 2.0 to 10, 25, 50, 75, 100, 150 or 200 mg compounds of the present invention, together with a pharmaceutically acceptable diluent or carrier thereof.

[0086] The compounds of the present invention, especially for use or administration in any of methods 1-6 and following, can be administered in higher doses if necessary for the treatment of cancer or tumor, for example, glioblastoma multiforme. It is contemplated that the administration of the PDE1 inhibitor for such a method may range from about 50 mg to 1000 mg per day. For example, a patient being administered a PDE1 inhibitor for a condition in accordance with any of Methods 1-6 et seq. may be administered a PDE1 inhibitor in accordance with Formula I, Ia, II, III, or IV in an amount of 50 mg to 1000 mg per day. 50 mg - 900 mg per day, 50 mg - 800 mg per day, 50 mg - 700 mg per day, 50 mg - 600 mg per day, 50 mg - 500 mg per day, 50 mg - 400 mg per day, 50 mg - 350 mg per day, 50 mg - 300 mg per day, 50 mg - 250 mg per day, 50 mg - 200 mg per day, 50 mg - 150 mg per day or 50 mg - 100 mg per day.

[0087] The compounds of the present invention can be administered by any suitable route, including orally, parenterally (intravenously, intramuscularly or subcutaneously) or transdermally, but is preferably administered orally. In some embodiments, the compounds of the invention, eg in a depot formulation, are preferably administered parenterally, eg by injection.

[0088] The compounds of the present invention and the pharmaceutical compositions of the present invention can be used in combination with one or more additional therapeutic agents, especially at lower doses than when using the individual agents as monotherapy, to enhance the therapeutic activity of the combination agents without causing undesirable effects. side effects usually observed with traditional monotherapy. Therefore, the compounds of the present invention can be administered simultaneously, separately, sequentially or at the same time with other agents useful for treating the disease. In another example, side effects can be reduced or minimized by administering a compound of the present invention in combination with one or more additional therapeutic agents in free or salt form, wherein the doses of (i) the second therapeutic agent(s), or (ii) and of a compound of the present invention and a second therapeutic agent is lower than when the agent/compound is administered as monotherapy. By way of non-limiting example, such additional therapeutic agents may include ACE inhibitors, angiotensin II receptor antagonists, calcium channel blockers, etc.

[0089] The term “concurrently”, as used therapeutically, means administering two or more active ingredients at or about the same time by the same route of administration.

[0090] The term “separately”, as related to therapeutic use, means the administration of two or more active ingredients at or about the same time by different routes of administration.

[0091] Pharmaceutical compositions comprising the compounds of the present invention can be prepared using conventional diluents or excipients and methods known in the medical field. Thus, oral dosage forms may include tablets, capsules, solutions, suspensions, and the like.

EXAMPLES

[0092] The data shown below in Examples 1-4 was published in F. Rowther, et al. 2016, Cyclic nucleotide phosphodiesterase-1C (PDE1C) drives cell proliferation, migration and invasion in glioblastoma multiforme cells in vitro. Molecular Carcinogenesis. 55(3):268-79.

COMPARATIVE EXAMPLE 1: PDE1C Expression in Glioblastoma Multiforme Tissue

[0093] Short-term cell cultures were established from adult glioblastoma multiforme (GBM) biopsy tissue as previously described in Lewandowicz GM, et al., Chemosensitivity in childhood brain tumors in vitro: Evidence of differential sensitivity to lomustine (CCNU) and vincristine. Eur J Cancer 2000; 36:1955. Samples were maintained in Hams F10 [Invitrogen, UK] containing 10% fetal bovine serum in a 37°C CO ₂ -free incubator. Normal human astrocytes (NHA) [Lonza, UK] and the established U251-MG cell line were also included. Permitted GBM biopsy specimens were obtained from Brain Tumor NW Research Tissue Bank, Preston, UK. Histological evaluation of GBM tissues was performed in situ and tumors were classified according to the 2007 WHO classification scheme. Control samples of normal human brain, including the entire fetal cerebellum and corpus callosum, were also used to determine PDE1C expression.

[0094] DNA was extracted from GBM cultures using the QIAmp DNA Mini Kit [Qiagen, UK]. Total RNA was extracted from short-term GBM cultures (10 ⁶ cells) or from approximately 5 mg of snap-frozen biopsy tissue using the RNeasy mini kit [Qiagen, UK]. DNA and total RNA were quantified using NanoDrop ND-2000 [Thermo Scientific, UK]. cDNA was synthesized from total RNA (500 ng) using QuantiTect Reverse Transcription [Qiagen, UK].

[0095] Quantitative real-time PCR was performed from 20 ng/well (96-well) GBM-derived cultures using a PDE1C TaqMan copy number assay with a FAM reporter (Hs01364699_cn [Life Technologies, UK]. Probe locations were selected in the same locations in the genome where aCGH detected a focal gain. Taqman analysis for TERT was used [Life Technologies, UK] as an endogenous control (VIC reporter) and PDE1C copy number was normalized to its levels. Reference DNA was prepared by mixing equal amounts of male and female genomic DNA [Promega, UK] and used as a normal control {carrying diploid copies of PDE1C for analysis} qPCR amplifications were performed in triplicate in three independent experiments to obtain accurate copy number data.

[0096] For expression analysis, qPCR was performed using the Solaris QPCR PDE1C Gene Expression Assay with FAM Reporter [Thermo Scientific, UK] on an ABI 7500 Sequence Detection System [Applied Biosystems, UK]. 2 μl of cDNA was amplified in a 20 ml reaction volume using 1x Solaris Low ROX qPCR Master Mix [Thermo Scientific, UK], and amplifications were performed in triplicate. PDE1C expression levels were normalized to GAPDH, and relative expression was determined using the ^2ΔΔCT method. Control normal brain samples (total brain RNA, human cerebellum and corpus callosum) were obtained from Clontech [France].

[0097] The results of the qPCR analysis are summarized in Table 1 below.

Table 1
qPCR analysis of PDE1C copy number Sample PDE1C copy number (qPCR) PDE1C status IN1461 3.31 ± 0.070 addition IN1951 2.62 ± 0.210 addition IN1528 3.67 ± 0.315 addition IN1682 3.61 ± 0.025 addition IN1752 2.64 ± 0.261 addition IN1612 3.03 ± 0.178 addition U251-MG 2.92 ± 0.060 addition IN2132 2.91 ± 0.216 addition IN1760 3.03 ± 0.036 addition IN1265 3.75 ± 0.053 addition IN2093 3.19 ± 0.259 addition IN1979 2.66 ± 0.286 addition IN1472 4.78 ± 0.077 addition IN859 3.18 ± 0.080 addition IN2045 (Control culture) 2.30 ± 0.136 diploid Reference DNA 2.00 ± 0.114 diploid

[0098] As shown above, qPCR analysis confirms the addition of PDE1C in short-term cultures of GBM cells. Expression of PDE1C in short-term cultures using qPCR analysis and the relationship between genomic overrepresentation and PDE1C expression was evident (Figure 1), as increased expression (compared to normal human astrocytes) was present in all genomic overrepresentation cell cultures tested. with the exception of control culture IN2045. Compared to the control culture, significantly increased expression was observed in each short-term culture by 23- to 360-fold (P<0.0001).

COMPARATIVE EXAMPLE 2: Determination of the role of PDE1C in GBM proliferation

[0099] SiRNA transfections were facilitated using DharmaFECT-1 transfection reagent in serum containing medium, and transfection efficiency was assessed using cyclophilin-B siRNA positive control [Thermo Scientific, UK]. Cultures were transfected in parallel with a non-target pool of siRNA at the same concentration. Non-targeting siRNA and PDE1C-specific siRNA are abbreviated as siNT and siPD. Cells were plated at 40–50% confluency and incubated for 48 hours before transfection. At the end of the initial 48-hour incubation, cells were transfected and incubated for 4 days. On day 5, cells were trypsinized, reseeded into a fresh flask, and allowed to adhere overnight. Cells were retransfected on day 6 (using either siNT or siPD) and incubated for an additional four days before harvesting for further experiments. siNT and siPD cells were allowed to grow for 72 hours and a proliferation assay was performed using SRB as described in Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 2006; 1:1112-1116. Vinpocetine was dissolved in DMSO and a serial dilution dose range from 500 μM to 0.977 μM was used.

[00100] As shown in FIG. 3, after termination of transfection, the ability of cells to proliferate was assessed. Regardless of the initial level of PDE1C expression, PDE1C depletion consistently reduced proliferation by 45–54% compared with PDE1C-expressing cells (P < 0.05) in all cultures except IN2045. cAMP and cGMP activities were also assessed after PDE1C depletion. It was observed that an increase in both cAMP and cGMP activity (P<0.05) was found in siPD cells compared to siNT cells, with the exception of the IN2045 control culture.

COMPARATIVE EXAMPLE 3: Determining the Role of PDE1C in GBM Cell Migration and Invasion

[00101] The migration and invasion potential of siNT and siPD was assessed using the CytoSelect 24-well format migration and invasion assay [Cell Biolabs, Inc., USA]. 20,000 siNT or siPD cells were seeded on a polycarbonate membrane (8 mM) in serum-free medium, and serum-containing medium was used as a chemoattractant. At the end of the incubation time, the migrated cells were removed and the non-migrating cells on the membrane were stained using crystal violet and quantified by colorimetry. Polycarbonate membranes precoated with extracellular matrix (ECM) proteins [Cell Biolabs, Inc., USA] were used for invasion assays.

[00102] Cell migration was also assessed using the CytoSelect 24-well wound healing assay [Cell Biolabs, Inc., USA] following the manufacturer's instructions. % Migration was calculated using the formula:

[(cells migrated to the wound area)/(total number of cells on the slide)] × 100,

[00103] Migration into siNT was taken as 100%. For vinpocetine, treatment was continued throughout the study period.

[00104] As illustrated in FIG. 4 and 5, siPD cells showed reduced ability to both migrate (through the polycarbonate membrane) and penetrate (through the ECM-coated polycarbonate membrane) compared to siNT cells. Migrated and invaded cells were quantified, and a 40-60% reduction in migratory and invasion potential was noted in siPD cells compared to siNT. In IN1472 and IN1760 cultures, the reduction in cell migration and invasion was statistically significant (P<0.05). It is interesting to note that the relative degree of inhibition of migration and invasion was very similar in each of the individual crops. As with proliferation, there was no inhibition of migration or invasion in siPD IN2045 cells. The results of the Boyden chamber assay were confirmed in the wound healing assay (Figure 6), in which the number of cells that migrated to the wound site was significantly reduced in siPD cells compared to siNT cells in three cultures (IN1472, IN1760 and U251MG) . These data indicate that PDE1C also acts upstream of migratory and invasive pathways in GBM cells.

COMPARATIVE EXAMPLE 4: Identification of PDE1C Downstream Effectors

[00105] For genome-wide expression analysis, 1 mg of total RNA was reverse transcribed using the MessageAmpTM IIBiotin Enhanced Kit [Ambion; Life Technologies, UK] and hybridized with the human genome U133 Plus 2.0 [Affymetrix, UK]. Microarrays were washed in a GeneChip Fluidics Station 450 and scanned using a GeneChip Scanner 3000 7G [Affymetrix, UK]. Microarrays were prepared in triplicate for siNT and siPD treatments.

[00106] The raw microarray data from the Affymetrix CEL files for each of the siNT and siPD biological replicates was normalized using a standard protocol. Microarray background, Q values, and average intensity were within acceptable ranges for all microarrays. Statistical comparisons and data visualization were performed using GeneSpring GX12 (Agilent Technologies, UK). First, a ≥2-fold change in expression levels was detected in siPD cells compared to siNT, followed by the identification of statistically significant genes using one-way ANOVA (P < 0.05) and the Benjamini-Hochberg test to control for false positive rate. with multiple checks. Significant biological, technological networks, gene ontology (GO) processes and disease markers affected by PDE1C depletion were identified using MetacoreTM [GeneGo, Thompson Reuters, USA]. The cBIO canver genomics portal was used to examine the expression status of genes downregulated by PDE1C in The Cancer Genome Atlas(TCGA) GBM cohorts and correlated with patient outcome.

[00107] Genome-wide expression analysis was performed on IN1472 and IN1760 cultures, where the effect of PDE1C depletion on proliferation, invasion and migration was most significant (Figures 2C, 4B, 4D and 4F). Considering differential expression based on fold change of ≥2-fold expression and statistical significance of P < 0.05 between siNT and siPD cells, 565 transcripts (465 genes) were identified. For IN1760, 411 differentially expressed genes were found, of which 146 were up-regulated and 265 were down-regulated. In IN1472, 49 genes were up-regulated and 5 were down-regulated in siPD cells. Successful knockdown of PDE1C was also established in the expression biochip, as it was a 3- and 13-fold knockdown for IN1472 and IN1760, respectively. This analysis also established that the PDE1C siRNA pool used in the current study is highly specific for PDE1C, as the authors did not detect any changes in the expression of other PDEs.

[00108] In IN1760-siPD cells, commonly down-regulated genes that showed 3- to 13-fold downregulation included IL8, PDE1C, KCTD4, PLD5, CXCL6, IL11, B3GALT2, INHBE, OPCML, ELAVL2, and EXT1 (Additional File A). Similarly, SPARCL1, APOE, CFI, COMP, FABP3, CLU, FAM107A, PTGDS, CHI3L1, MAN1C1, TDO2, and QPRT showed 4-8-fold upregulation in IN1760-siPD cells. In contrast, in IN1472, PDE1C was the only gene that showed more than 3-fold downregulation in siPD cells (Additional File B). TNFSF15 was also the only gene that showed >3-fold increase in IN1472-siPD cells. Four genes were differentially expressed in both IN1760-siPD and IN1472-siPD cells, including APOE (positively regulated), HSD11B1 (positively regulated), ST6GALNAC5 (negatively regulated), and PDE1C (negatively regulated).

[00109] PDE1C depletion has been shown to alter genes involved in important cellular pathways in GBM cells. Genes involved in the most significant pathways affected by PDE1C knockdown were identified as shown below in Tables 2-4.

table 2
Pathways involving cell-matrix interactions, ECM remodeling, and platelet-endothelium-leukocyte interactions Negatively regulated EDN1, IL1B, IL6, CCL2, TFPI2, THBS1, KAL1, ITGB1, IL8, SERPINE1, COL3A1 Positively regulated ENG, SERPING1, GPR56, COL4A4, THBD, CXCL5, IL6ST, KDR, CD9 Table 3
Pathways involving cell cycle (mitosis) and proliferation control genes Negatively regulated NOX4, INHBA, IL18, CDC6, IL6, SMYD2, THBS1, CTNNB1, ITGB1, GNAQ, ADAMTS1, ETS2, IL8, WNTSA, MORC3, BDNF, BRIP1, RERG, SMAD4, BCL2, INHBE, CD274 Positively regulated HLA-DRA, RARRES3, COX2, PLA2G16, AZGP1, HLA-DRB1, IFITM1, GPNMB, ENG, NDRG2, CDH5, HMOX1, IFI30, AGT, SCG2, INHBA, APOE, FABP3, IGFBP2, ITGA7, COL4A4, EREG, CD9, KRIT1 Table 4
Pathways involving glioma-specific genes Negatively regulated microRNA 30c-2, PTPRD, CDK5R1, ADAMTS6, DKK3, CALD1, MKI67, VIM, PEG3, SLC7A, KIF3B, MYLK, EPG5, ELAVL2, HMMR, KIF14 Positively regulated MAFB, CHI3L1, CTSL1, TNXA, TNXB, SOD3, TIMP4, RTN1, CYGB, TNFRSF1B, MGP, MATN2, LAMA4, ABCC3, MDK, SERPINI1

[00110] Differentially expressed genes as a consequence of PDE1C depletion were found to be highly correlated with the gene expression profiles of the TCGA GBM cohorts. In the combined list of differential gene expression of siPD IN1760 and IN1472 cells, 32% of PDE1C-mediated up-regulated genes were down-regulated in the TCGA cohorts, and 53% of PDE1C-mediated down-regulated genes were up-regulated in the TCGA cohorts. In this list of TCGA genes, upregulation of at least one or more query genes, i.e. IL8, CXCL2, FOSB, NFE2L3, SUB1, SORBS2, WNT5A and MMP1 were associated with worse patient outcome (P=0.0165), and PDE1C knockdown resulted in downregulation of their expression. Therefore, although PDE1C expression itself is not associated with outcome, altered expression of its downstream effectors as a consequence of PDE1C depletion has the potential to improve survival.

EXAMPLE 1: Determination of the ability of compounds 1 and 2 to inhibit GBM proliferation, invasion and migration

[00111] GBM cell cultures were prepared from adult human glioblastoma multiforme (GBM) biopsy tissue as described in Comparative Examples 2 and 3. Compounds 1 and 2 were administered to the cultures to evaluate their effect on GBM proliferation, invasion and migration. Compounds 1 and 2 are:

[00112] After obtaining cell cultures as discussed above in Comparative Example 2, the ability of cells treated with Compound 1 or Compound 2 to proliferate was assessed. Successful compound trials will show reduced proliferation compared to PDE1C-expressing cells in all cultures except controls. Increased activity of both cAMP and cGMP will be detected in siPD cells compared to siNT cells, with the exception of the control culture.

[00113] The ability of cells treated with Compound 1 or Compound 2 to migrate and proliferate was also assessed. Successful compound trials will show reduced migration and/or proliferation compared to control cultures.

EXAMPLE 2: Effect of PDE1 inhibitors on various GBM cell lines.

[00114] Seven different cell lines derived from GBM biopsies were obtained and prepared in short-term granular culture media, each containing 3-4 million cells respectively. To prepare for use in the IMAP assay for PDE activity, cell pellets were washed once with TBS (150 nM NaCl, 50 mM Tris-HCl, pH 7.4). The pellets were then centrifuged and the supernatant was removed. Cells were lysed in 0.15 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl ₂ , protease inhibitor mixture). The resulting solution was sonicated twice for 30 seconds and then centrifuged at 15,000 rpm for 15 minutes at 4ºC. The supernatant was transferred to a new vessel and immediately frozen. The cells were then analyzed for PDE1 expression and sensitivity to inhibition by compounds 1 and 2 as determined in Example 1. The results are presented in Table 3 below.

Table 3
PDE1 Expression and Sensitivity to Compounds 1 and 2 in Different Cell Populations Patient-Derived GBM Cell Lines PDE1C expression (fold change compared to normal brain) ID ₅₀ Compounds 1 (µM) ID ₅₀ Compounds 2 (µM) UWLV301 75 5.4 4.73 IN1951 64 2.04 1.03 UWLV304 63 6.4 2.87 IN1528 55 26.5 9.45 U251MG 45 17.8 32.0 IN859 23 34.9 9.77 IN2045 5 18.4 12.0

[00115] As shown above, PDE1C levels were increased in all cell lines, with some cell lines showing a 75-fold increase in PDE1C expression. Both compound 1 and compound 2 showed equal potency in all cell lines tested.

[00116] Further studies were carried out to test the effect of Compounds 1 and 2 on the proliferation and survival of GBM cells. The proliferation of each cell population was tested against compounds 1 and 2 at concentrations of 10 μM and 20 μM. Results regarding changes in cell population were observed and recorded daily for 8 days. In all cases, the treated cell lines proliferated more slowly compared to untreated samples. In UWLV301, UWLV304, IN1951, U251MG, and IN2045, GBM-treated cell populations showed an overall decrease by day 8.

[00117] Thus, both compounds 1 and 2 showed high effectiveness in slowing down or eliminating the proliferation of GBM cell lines.

EXAMPLE 3: Analysis of GBM cell lines for PDE1 activity, including calcium dependence and correlation

[00118] Cell pellets used in Example 2 were analyzed to measure calcium-dependent PDE activity as a means to assess the relative presence of full-length versus truncated enzymes. The truncated enzymes lack the N-terminal regulatory domain and thus become constitutively active in this assay. Assays were prepared according to the protocol of Example 2.

[00119] Analysis of PDE1 activity in cell lines is shown in FIG. 7. As shown, the UWLV301 and UWLV304 cell lines showed highly active PDE in both the presence and absence of Ca2+/calmodulin. This shows that PDE activity in these cell lines is independent of calcium. PDE activity using cAMP as substrate was not affected by the absence of Ca2+/calmodulin in these two cell lines. On the other hand, some of the cell lines, such as IN1528 and U251MG, showed much higher levels of activity in the presence of Ca2+/calmodulin than in its absence.

[00120] This suggests that progression of GBM may cause constitutive activation of PDE1 in brain cells. To investigate this point, additional tests were carried out and are summarized in Table 4 below. As shown, various cell lines were tested in the presence and absence of Ca2+/calmodulin, as well as in the presence of EGTA, a calcium chelator, at various concentrations. These tests confirmed that the UWLV301 and UWLV304 cell lines were constitutively active, i.e. that they remain active without regulation by Ca2+/calmodulin.

Table 4
Percentage of PDE activity in different cell lines Activity (% compared to baseline +CA/CaM) +Ca/CaM -Ca/CaM -Ca/CaM/ 10µM EGTA -Ca/CaM/ 40µM EGTA IN1528 100 43.18 50.08 40.02 IN1951 100 74.16 67.77 59.37 U251MG 100 37.7 39.99 23.69 UWLV301 100 119.73 116.45 98.9 UWLV304 100 96.63 91.55 70.13

[00121] Additional tests were performed to confirm the PDE isoform predominantly associated with GBM cell proliferation. The inhibition constant (K _i ) values for Compound 1 against various PDE enzyme families are presented in Table 5 below.

Table 5
Inhibition constant values for compound 1 against PDE PDE target K _i (µM) PDEx/PDE1A ratio PDE1A 0.000034 1 PDE1B 0.00038 11.5 PDE1C 0.000037 1.1 PDE1 (bovine brain) 0.000058 1.7 hPDE2A >10 29,411 hPDE3B 3.1 93,939 hPDE4D 0.033 970 r-bovine PDE5A 0.63 19,090 bovine retina PDE6 0.32 9696 hPDE7B 0.36 10,909 hPDE8A 3 90,909 hPDE9A >10 294.117 hPDE10A 1.8 54,545 hPDE11A 1.3 39,393

[00122] It has previously been observed that increased expression of PDE1C is associated with GBM cells. The results showed that the IC ₅₀ for inhibition of PDE activity in the UWLV301 and UWLV304 cell lines for Compound 1 closely matches the K _i value obtained for purified PDE1C, supporting the idea that the detected PDE activity in these lines is due to PDE1C.

[00123] Without being limited by theory, it is believed that PDE1C has been structurally altered to cause constitutive activation of the UWLV301 or UWLV304 cell lines. Upon further examination, neither UWLV301 nor UWLV304 showed any detectable full-length PDE1C. During the trial, this was supported by evidence as antibodies to the N-terminus of PDE1C could no longer detect PDE1C in those cells that had lost calcium sensitivity. On the other hand, IN1528 and U251MG cell lines, which both remain regulated by the Ca2+/calmodulin system, showed detectable full-length PDE1C. Thus, these trials suggest that GBM cancer cells may induce dysregulation and structural changes of PDE1C.

[00124] Alternative combinations and variations of the presented examples will become apparent based on the disclosure. It is not possible to provide specific examples for all of the many possible variations of the described embodiments, but such combinations and variations may be claims ultimately claimed.

Claims (10)

1. A method of treating glioma, comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor to a subject in need thereof, wherein the PDE1 inhibitor is selected from any of the following: or in free form or in the form of a pharmaceutically acceptable salt. 2. The method according to claim 1, where the glioma is glioblastoma multiforme. 3. The method according to claim 1 or 2, wherein the PDE1 inhibitor is administered in combination with an antitumor agent. 4. A method of inhibiting cytokine release syndrome, comprising administering an effective amount of a PDE1 inhibitor to a patient suffering from glioma and receiving one or more of chemotherapy treatment, immunological treatment, gene therapy and/or antibody therapy, wherein the PDE1 inhibitor is selected from any of the following: or in free form or in the form of a pharmaceutically acceptable salt. 5. The method according to claim 4, where the glioma is glioblastoma multiforme. 6. The method according to claim 4 or 5, wherein the PDE1 inhibitor is administered in combination with an antitumor agent.

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