Organic compounds

Abstract

The present disclosure relates to the use of phosphodiesterase 1 (PDE1) inhibitors, alone or in combination with immune checkpoint inhibitor therapy, for the treatment of breast cancer, including for promoting anti-tumor immunity and reducing side effects (i.e., inflammation-related adverse events) associated with checkpoint inhibitor therapy.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 269,209, filed March 11, 2022, and U.S. Provisional Application No. 63 / 479,938, filed January 13, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Field of Disclosure The field relates to the use of phosphodiesterase 1 (PDE1) inhibitors, alone or in combination with immune checkpoint inhibitor therapy, for the treatment of breast cancer, including promoting anti-tumor immunity and reducing side effects (i.e., inflammation-related adverse events) associated with checkpoint inhibitor therapy. [Background technology]

[0003] Background of the disclosure Breast cancer is a cancer that originates from breast tissue. Approximately 10-15% of breast cancers are classified as triple-negative breast cancer (TNBC). TNBC is a complex, aggressive subtype of breast cancer that lacks estrogen receptors, progesterone receptors, and HER2. TNBC is one of the most difficult breast cancers to treat because it does not respond to drugs that target estrogen receptors, progesterone receptors, or HER2. Therefore, there is a significant need for better treatment options for TNBC.

[0004] Immunotherapy, including checkpoint inhibitors, has revolutionized cancer treatment and has been effective in many breast cancer patients. Immunotherapy helps the patient's immune system prevent the growth of cancer. As part of its normal function, the immune system detects and destroys abnormal cells and can also prevent or suppress the growth of many cancers. However, cancer cells have ways of evading the immune response. Immune activation is primarily mediated by T cells and is regulated by stimulatory, costimulatory and inhibitory (checkpoint) signals. When T cells encounter self-cells, there are important receptor-ligand interactions that suppress activation so that the immune cells do not attack the body's normal cells. Although cancer cells have genetic and epigenetic changes that can result in antigen expression that can trigger immune activation, cancer cells can also utilize immune checkpoint interactions such as PD-1 / PD-L1 and CTLA4 / B7-1 / B7-2 to inactivate immune cells and prevent the immune system from destroying the cancer. Immune checkpoint inhibitors are effective in many patients suffering from various types of cancer because they allow the cancer to be destroyed by the patient's own immune system. Unfortunately, some patients fail to benefit from these therapies, and the development of resistance can lead to cancer progression in patients who demonstrate a primary clinical response. Thus, resistance to checkpoint inhibitor (e.g., PD-1 / PD-L) blockade remains a significant challenge, hindering their more widespread application.

[0005] Furthermore, adverse events associated with immunotherapy may limit the use of checkpoint blockade therapy and lead to severe adverse outcomes. Blocking immune checkpoints may allow the immune system to attack normal tissues. This results in inflammatory conditions such as dermatitis, colitis, arthritis, nephritis, myositis, polymyalgia-like syndrome, and cytokine release syndrome (CRS), which is caused by the massive and rapid release of cytokines into the blood from immune cells affected by immunotherapy. These side effects can be very severe and sometimes fatal. Thus, immune checkpoint blockade may be limited by the occurrence of immunotherapy-related adverse events, despite the considerable benefits it brings to cancer patients.

[0006] Although 11 families of phosphodiesterases (PDEs) have been identified, only family I PDEs, Ca2+ / calmodulin-activated Ca2+ / calmodulin-dependent phosphodiesterases (CaM-PDEs), have been shown to mediate calcium-dependent cyclic nucleotide (e.g., cGMP and cAMP) signaling pathways. The three known CaM-PDE genes, PDE1A, PDE1B and PDE1C, are all expressed in central nervous system tissues. PDE1A is expressed in the brain, lung and heart. PDE1B is primarily expressed in the central nervous system, but has also been detected in monocytes and neutrophils and has been shown to be involved in the inflammatory response of these cells. PDE1C is expressed in olfactory epithelium, cerebellar granule cells, striatum, heart, vascular smooth muscle and tumor cells. PDE1C has been shown to be a major regulator of smooth muscle proliferation in human smooth muscle. Cyclic nucleotide phosphodiesterases downregulate intracellular cAMP and cGMP signaling by hydrolyzing these cyclic nucleotides to their respective 5'-monophosphates (5'AMP and 5'GMP), which are inactive from the perspective of intracellular signaling pathways. Both cAMP and cGMP are central intracellular second messengers and play a role in regulating many cellular functions. PDE1A and PDE1B preferentially hydrolyze cGMP over cAMP, whereas PDE1C has approximately equal hydrolysis of cGMP and cAMP.

[0007] There is a substantial need for novel therapeutic approaches to complement and enhance checkpoint inhibitor therapy, and safe and selective strategies to mitigate the severe side effects (i.e., inflammation-related adverse events) associated with checkpoint inhibitor therapy are needed. Summary of the Invention

[0008] Disclosure Summary In one aspect, the present disclosure provides a method for treating breast cancer, comprising administering a pharma- tically acceptable amount of a PDE1 inhibitor alone or in combination with a pharma-tically acceptable amount of an immune checkpoint inhibitor to a subject in need of the treatment. In some embodiments, the breast cancer is triple-negative breast cancer (TNBR), which is estrogen receptor negative, progesterone receptor negative and HER2 negative. In certain embodiments, the TNBC is high-risk early stage TNBC. In another embodiment, the treatment is an adjuvant therapy after surgical removal of the TNBC. In yet another embodiment, the subject has locally recurrent, unresectable or metastatic TNBC, whose tumor expresses PD-L1, e.g., a combined positive score (CPS) of ≥ 1, as determined by an FDA-approved test, where CPS is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the number of viable tumor cells multiplied by 100.

[0009] In some embodiments, the immune checkpoint inhibitor is selected from one or more of CTLA-4, PD-1 and / or PD-L1 inhibitors. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, such as an anti-PD-1 antibody. In some embodiments, the PDE1 inhibitor is a PDE1 inhibitor represented by formula I, Ia, II, III, IV, V and / or VI as set forth below, in free form or in pharma- ceutically acceptable salt form. In some embodiments, the PDE1 inhibitor is compound A, in free form or in pharma-ceutically acceptable salt form, or compound B, in free form or in pharma-ceutically acceptable salt form: [ka] [ka] It is.

[0010] In another aspect, the disclosure provides a method of preventing or ameliorating a disease, disorder, or adverse effects resulting from administration of immune checkpoint inhibitor therapy to a subject suffering from breast cancer, comprising reducing the amount of checkpoint inhibitor administered to the subject, and administering to the subject a pharma- ceutically acceptable amount of a PDE1 inhibitor in combination with immune checkpoint inhibitor therapy.

[0011] In another aspect, the disclosure provides a pharmaceutical combination therapy comprising a pharma- ceutically acceptable amount of a PDE1 inhibitor and a pharma- ceutically acceptable amount of an immune checkpoint inhibitor, for use in a method for the treatment of breast cancer or for the prevention or amelioration of a disease, disorder, or adverse effects resulting from the administration of checkpoint inhibitor therapy. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the mean volume of E0771 tumors from mice treated with Compound A at 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound A (900 ppm) + anti-PD1 (10 mg / kg). n=6-7 / group. *P<0.05, ns indicates no statistical difference.

[0013] [Diagram 2] FIG. 2 shows individual growth curves (volume) of E0771 tumors from mice treated with 900 ppm of dietary Compound A, anti-PD1 (10 mg / kg), or combination therapy with Compound A (900 ppm) + anti-PD1 (10 mg / kg).

[0014] [Diagram 3] Figure 3 shows the mean tumor weight of E0771 tumors on the day of harvest from mice treated with Compound A at 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound A (900 ppm) + anti-PD1 (10 mg / kg). n=6-7 / group. *P<0.05.

[0015] [Figure 4] Figures 4A and B show flow cytometry analysis of E0771 tumors in mice treated with compound A at 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with compound A (900 ppm) + anti-PD1 (10 mg / kg). Figure 4A shows the relative proportions of CD45 cells and macrophages in E0771 tumors. Figure 4B shows the M1 / M2 ratio of macrophages in E0771 tumors. n=6-7 / group. t-test; *P<0.05.

[0016] [Diagram 5] Figure 5 shows flow cytometry analysis of E0771 tumors in mice treated with Compound A at 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound A (900 ppm) + anti-PD1 (10 mg / kg). Figure 5 shows the relative proportions of T cells, CD8+ cells, CD4+ cells, and NK cells in E0771 tumors. n=6-7 / group. t-test; *P<0.05.

[0017] [Figure 6] Figure 6 shows gene expression comparison volcano plots of Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors and (control) groups. The volcano plots show that 48 genes are downregulated and 136 genes are upregulated in Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors (fold change < -1.5 or > 1.5; P < 0.05).

[0018] [Figure 7] FIG. 7 shows pathways enriched in differentially expressed genes in Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors (top), and transcriptional regulators associated with genes that were up- or down-regulated in Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors (bottom).

[0019] [Figure 8]Figure 8 shows the mean volume of 4T1 tumors from mice treated with Compound A at 300 ppm or 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound A (300 ppm or 900 ppm) + anti-PD1 (10 mg / kg). n=7-8 / group. *P<0.05; **P<0.01; and ***P<0.001, ns indicates no statistical difference.

[0020] [Figure 9] FIG. 9 shows individual growth curves (volume) of 4T1 tumors from mice treated with 300 ppm or 900 ppm of dietary Compound A, anti-PD1 (10 mg / kg), or combination treatment with Compound A (300 ppm or 900 ppm) + anti-PD1 (10 mg / kg).

[0021] [Figure 10] Figure 10 shows the average tumor weight of 4T1 tumors on the day of harvest from mice treated with Compound A at 300 ppm or 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound A (300 ppm or 900 ppm) + anti-PD1 (10 mg / kg). n=7-8 / group. *P<0.05; **P<0.01; and ***P<0.001, ns indicates no statistical difference.

[0022] [Figure 11] Figure 11 shows survival curves of mice treated with Compound A at 300 ppm or 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound A (300 ppm or 900 ppm) + anti-PD1 (10 mg / kg). n=7-8 / group.

[0023] [Figure 12]Figure 12 shows the mean E0771 tumor volumes from mice treated with 100 ppm, 300 ppm or 900 ppm of compound B in the diet, anti-PD1 (1 mg / kg), or combination treatment with compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (1 mg / kg). n=5-6 / group. *P<0.05, ns indicates no statistical difference.

[0024] [Figure 13] FIG. 13 shows individual growth curves (volume) of E0771 tumors from mice treated with 100 ppm, 300 ppm or 900 ppm of dietary Compound B, anti-PD1 (1 mg / kg), or combination treatment with Compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (1 mg / kg).

[0025] [Figure 14] Figure 14 shows the average tumor weight of E0771 tumors on the day of harvest from mice treated with Compound B at 100 ppm, 300 ppm or 900 ppm in the diet, anti-PD1 (1 mg / kg), or combination treatment with Compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (1 mg / kg). n=5-6 / group. *P<0.05; **P<0.01.

[0026] [Figure 15] Figure 15 shows the average volume of 4T1 tumors from mice treated with 100 ppm, 300 ppm, or 900 ppm of compound B in the diet, anti-PD1 (10 mg / kg), or combination treatment with compound B (100 ppm, 300 ppm, or 900 ppm) + anti-PD1 (10 mg / kg). n=7-9 / group. *P<0.05; **P<0.01; and ***P<0.001, ns indicates no statistical difference.

[0027] [Figure 16]FIG. 16 shows individual growth curves (volume) of 4T1 tumors from mice treated with 100 ppm, 300 ppm or 900 ppm of dietary Compound B, anti-PD1 (10 mg / kg), or combination treatment with Compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (10 mg / kg).

[0028] [Figure 17] Figure 17 shows the average tumor weight of 4T1 tumors on the day of harvest from mice treated with Compound B at 100 ppm, 300 ppm or 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (10 mg / kg). n=7-9 / group. *P<0.05; **P<0.01; and ***P<0.001, ns indicates no statistical difference.

[0029] [Figure 18] Figures 18A and B show flow cytometry analysis of 4T1 tumors in mice treated with Compound B at 900 ppm in the diet, anti-PD1 (10 mg / kg), or combination treatment with Compound B (900 ppm) + anti-PD1 (10 mg / kg). Figure 18A shows the relative proportions of CD45 cells and macrophages in 4T1 tumors. Figure 18B shows the M1 / M2 ratio of macrophages in 4T1 tumors. n=6-7 / group. t-test; *P<0.05.

[0030] [Figure 19] Figure 19 shows flow cytometry analysis of 4T1 tumors in mice treated with 900 ppm of compound B in the diet, anti-PD1 (10 mg / kg), or combination treatment with compound B (900 ppm) + anti-PD1 (10 mg / kg). Figure 19 shows the relative proportions of various T cells in 4T1 tumors. n=6-7 / group. t-test; *P<0.05.

[0031] [Figure 20]Figure 20 shows a volcano plot of gene expression comparison of Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors and (control) groups. The volcano plot shows that 281 genes are downregulated and 708 genes are upregulated in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors (fold change < -1.5 or > 1.5; P < 0.05).

[0032] [Figure 21] FIG. 21 shows pathways enriched in differentially expressed genes in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors (FIG. 21A), and transcriptional regulators in genes up- or down-regulated in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors (FIG. 21B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Disclosure The inventors have previously shown that inhibition of PDE1 activity with the disclosed compounds can safely restore cAMP function in a wide range of pathologies, including models of neurodegeneration and neuroinflammation, heart failure, pulmonary hypertension and peripheral inflammation, as well as in humans with certain diseases. More recently, the inventors have shown that PDE1 inhibitors regulate immune cell (microglia and macrophages) function by altering cell migration and the levels of key cytokines (mainly CCL2 and TNF-α). Recent evidence shows that PDE1, particularly the PDE1C isoform, is overexpressed in experimental tumor models such as melanoma, neuroblastoma, renal cell carcinoma, colon carcinoma and osteosarcoma. Furthermore, focal genomic over representation of PDE1C in glioblastoma multiforme (GBM) cells has been demonstrated. Genomic gain of PDE1C is associated with increased expression in GBM-derived cell cultures and is essential for promoting cell proliferation, migration and invasion in cancer cells.

[0034] Many types of cancer cells overexpress PDE1 activity, identified through a variety of biomarkers such as increased RNA expression, DNA copy number, PDE1 binding (PET or radioisotope retention of PDE1 inhibitor molecules) or enzyme activity. These cancer cells also exhibit low levels of cAMP, which can be increased by PDE1 inhibitors.

[0035] In the present invention, it has been found that PDE-1 inhibitors, administered alone or in combination with immune checkpoint inhibitor therapy, promote antitumor immunity and result in breast cancer growth inhibition. It has been found that PDE-1 inhibitors, alone or in combination with sub-effective amounts of anti-PD-1 antibodies, can inhibit breast cancer growth in a mouse model of triple-negative breast cancer (TNBC). In contrast, tumor growth in mice treated with anti-PD-1 antibodies alone is comparable to isotype controls. In addition, it has been found that the combination treatment shifts the polarization of macrophages in the tumor microenvironment to a more inflammatory phenotype. Without being bound by any theory, it is believed that PDE-1 inhibitors affect macrophage infiltration and polarization, thereby promoting antitumor immunity. The synergistic ability of PDE-1 inhibitors and immune checkpoint inhibitors to alter the tumor microenvironment and inhibit tumor growth may provide a means to extend the utility of immune checkpoint inhibitors to treatment-resistant tumors such as TNBC. Furthermore, this synergistic ability of a PDE-1 inhibitor in combination with a sub-effective immune checkpoint inhibitor may provide a means to reduce the dose of checkpoint inhibitor administered to a subject suffering from breast cancer, thereby mitigating the adverse effects resulting from the administration of checkpoint inhibitor therapy to the subject.

[0036] Compounds for use in the disclosed methods In one embodiment, the PDE1 inhibitors for use in the methods of treatment and prevention described herein are selective PDE1 inhibitors.

[0037] PDE1 Inhibitors In one embodiment, the PDE1 inhibitors for use in the methods of treatment and prevention described herein have the formula I: [ka] [In the formula, (i) R1 is H or C 1-4 alkyl (e.g., methyl); (ii) R4 is H or C 1-4 alkyl, and R and R are independently H or C 1-4 alkyl (e.g., R2 and R3 are both methyl, or R2 is H and R3 is isopropyl), aryl, heteroaryl, (optionally hetero)arylalkoxy, or (optionally hetero)arylalkyl; or R2 is H and R3 and R4 together form a dimethylene, trimethylene or tetramethylene bridge (preferably, R3 and R4 together have a cis configuration, e.g., the carbons bearing R3 and R4 have the R and S configurations, respectively); (iii) R5 is a substituted heteroarylalkyl, e.g., substituted with haloalkyl; or R5 is attached to one of the nitrogens of the pyrazolo moiety of formula I and has formula A: [ka] (Wherein, X, Y and Z are independently N or C; R8, R9, R 11 and R 12 are independently H or halogen (e.g., Cl or F), and R 10is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl optionally substituted with halogen (e.g., pyridyl (e.g., pyrid-2-yl), or thiadiazolyl (e.g., 1,2,3-thiazol-4-yl)), diazolyl, triazolyl, tetrazolyl, arylcarbonyl (e.g., benzoyl), alkylsulfonyl (e.g., methylsulfonyl), heteroarylcarbonyl, or alkoxycarbonyl; provided that when X, Y, or Z is nitrogen, then R, R, or R, respectively, are each independently substituted with halogen. 10 does not exist) The part indicated by; (iv) R6 is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g., phenylamino), heteroarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-(arylalkyl)amino (e.g., N-phenyl-N-(1,1'-biphen-4-ylmethyl)amino); (v) n is 0 or 1; (vi) If n is 1, A is -C(R 13 R 14 )-where R 13 and R 14 are independently H or C 1-4 alkyl, aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl. It is a compound represented by the formula:

[0038] In another embodiment, the PDE1 inhibitor for use in the methods of treatment and prevention described herein has the formula 1a: [ka] [In the formula, (i) R2 and R5 are independently H or hydroxy and R3 and R4 together form a trimethylene or tetramethylene bridge [preferably the carbons bearing R3 and R4 have the R and S configurations, respectively]; or R2 and R3 are each methyl and R4 and R5 are each H; or R2, R4 and R5 are H and R3 is isopropyl [preferably the carbon bearing R3 has the R configuration]; (ii) R6 is phenylamino (optionally halo-substituted), benzylamino (optionally halo-substituted), C 1-4 Alkyl, or C 1-4 alkylsulfide; for example, phenylamino or 4-fluorophenylamino; (iii) R 10 is C 1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, (optionally halo- or hydroxy-substituted) phenyl, (optionally halo- or hydroxy-substituted) pyridyl (e.g., 6-fluoropyrid-2-yl), or thiadiazolyl (e.g., 1,2,3-thiazol-4-yl); (iv) X and Y are independently C or N. It is.

[0039] In another embodiment, the PDE1 inhibitors for use in the methods of treatment and prevention described herein have the formula II: [ka] (i) X is C 1-6 alkylene (e.g., methylene, ethylene or prop-2-yn-1-ylene); (ii) Y is a single bond, alkynylene (e.g., -C≡C-), arylene (e.g., phenylene) or heteroarylene (e.g., pyridylene); (iii) Z is H, aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, e.g., pyrid-2-yl), halo (e.g., F, Br, Cl), haloC 1-6 Alkyl (e.g., trifluoromethyl), -C(O)-R 1 , -N(R 2 )(R 3 ), or C, which may contain at least one atom selected from the group consisting of N or O 3-7 cycloalkyl (e.g., 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 (e.g., -OCH3); (v) R 2 and R 3 are independently H or C 1-6 is alkyl; (vi) R 4 and R 5 are independently H, C 1-6 alkyl, or one or more halo (e.g., fluorophenyl, e.g., 4-fluorophenyl), hydroxy (e.g., hydroxyphenyl, e.g., 4-hydroxyphenyl or 2-hydroxyphenyl) or C 1-6 aryl (e.g., phenyl) optionally substituted with alkoxy; (vii) X, Y and Z are independently one or more halo (e.g., F, Cl or Br), C 1-6 Alkyl (e.g., methyl), haloC 1-6 For example, Z may be substituted with one or more halo (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), haloC 1-6 Alkyl (e.g., 5-trifluoromethylpyrid-2-yl) or C 1-6-heteroaryl, e.g., pyridyl, optionally substituted with alkyl (e.g., 5-methylpyrid-2-yl); or Z is aryl, e.g., phenyl, optionally substituted with one or more halo (e.g., 4-fluorophenyl). It is a compound represented by the formula:

[0040] In yet another embodiment, the PDE1 inhibitors for use in the methods of treatment and prevention described herein have the formula III: [ka] [In the formula, (i) R1 is H or C 1-4 alkyl (e.g., methyl or ethyl); (ii) R2 and R3 are independently H or C 1-6 alkyl (e.g., methyl or ethyl); (iii) R4 is H or C 1-4 alkyl (e.g., methyl or ethyl); (iv) R5 is independently -C(=O)-C 1-6 Alkyl (e.g., -C(=O)-CH3) and C 1-6 -aryl (e.g., phenyl) optionally substituted with one or more groups selected from hydroxyalkyl (e.g., 1-hydroxyethyl); (v) R6 and R7 are independently H or C 1-6 Aryl (e.g., phenyl) optionally substituted with one or more groups selected from alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), such as unsubstituted phenyl or phenyl substituted with one or more halogen (e.g., F) or one or more C 1-6 Phenyl or one C substituted with alkyl and one or more halogens 1-6 phenyl substituted with alkyl and one halogen, for example 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3-methylphenyl; (vi) n is 1, 2, 3 or 4. It is.

[0041] In yet another embodiment, the PDE1 inhibitor for use in the methods of treatment and prevention described herein is a compound represented by formula IV, in free or salt form: [ka] [In the formula, (i) R1 is C 1-4 alkyl (e.g., methyl or ethyl), or -NH(R2), where R2 is phenyl optionally substituted with halo (e.g., fluoro), such as 4-fluorophenyl; (ii) X, Y and Z are independently N or C; (iii) R3, R4 and R5 are independently H or C 1-4 alkyl (e.g., methyl); or R3 is H and R4 and R5 together form a trimethylene bridge (preferably, R4 and R5 together have a cis configuration, e.g., the carbons bearing R4 and R5 have the R and S configurations, respectively); (iv) R6, R7 and R8 are independently H, C 1-4 Alkyl (e.g., methyl), hydroxy-substituted pyrid-2-yl; or -S(O)2-NH2 and; (v) with the proviso that when X, Y and / or Z are N, then R6, R7 and / or R8 are absent, respectively; and when X, Y and Z are all C, then at least one of R6, R7 or R8 is -S(O)2-NH2, or pyrid-2-yl substituted with hydroxy. It is.

[0042] In another embodiment, the PDE1 inhibitor for use in the methods described herein has the formula V: [ka] [In the formula, (i) R1 is -NH(R4), where R4 is phenyl optionally substituted with halo (e.g., fluoro), such as 4-fluorophenyl; (ii) R2 is H or C 1-6 alkyl (e.g., methyl, isobutyl, or neopentyl); (iii) R3 is -SO2NH2 or -COOH. It is.

[0043] In another embodiment, the PDE1 inhibitor for use in the methods described herein has the formula VI: [ka] [In the formula, (i) R1 is -NH(R4), where R4 is phenyl optionally substituted with halo (e.g., fluoro), such as 4-fluorophenyl; (ii) R2 is H or C 1-6 alkyl (e.g., methyl or ethyl); (iii) R3 is H, halogen (e.g., bromo), C 1-6 alkyl (e.g., methyl), aryl optionally substituted with halogen (e.g., 4-fluorophenyl), heteroaryl optionally substituted with halogen (e.g., 6-fluoropyrid-2-yl or pyrid-2-yl), or acyl (e.g., acetyl). It is.

[0044] In one embodiment, the disclosure relates to the administration of a PDE1 inhibitor (e.g., a compound according to Formula I, Ia, II, III, IV, V, and / or VI) for use in the methods described herein, wherein the inhibitor is [ka] [ka] [ka] [ka] The present invention provides a compound according to the method of the present invention,

[0045] In one embodiment, the invention relates to the administration of a PDE1 inhibitor for the treatment or prevention described herein, wherein the inhibitor is in free form or in pharma- ceutical acceptable salt form: [ka] The present invention provides a compound according to the method of the present invention,

[0046] In yet another embodiment, the present invention relates to the administration of a PDE1 inhibitor for the treatment or prevention described herein, wherein the inhibitor is in free form or in pharma- ceutical acceptable salt form: [ka] The present invention provides a compound according to the method of the present invention,

[0047] In yet another embodiment, the present invention relates to the administration of a PDE1 inhibitor for the treatment or prevention described herein, wherein the inhibitor is in free form or in pharma- ceutical acceptable salt form: [ka] The present invention provides a compound according to the method of the present invention,

[0048] In yet another embodiment, the present invention relates to the administration of a PDE1 inhibitor for the treatment or prevention described herein, wherein the inhibitor is in free form or in pharma- ceutical acceptable salt form: [ka] The present invention provides a compound according to the method of the present invention,

[0049] In one embodiment, a selective PDE1 inhibitor according to any of the above formulas (e.g., Formula I, Ia, II, III, IV, V, and / or VI) is a compound that inhibits phosphodiesterase-mediated (e.g., PDE1-mediated, particularly PDE1B-mediated) hydrolysis of cGMP, e.g., preferred compounds have an IC of less than 1 μM, preferably less than 500 nM, preferably less than 50 nM, preferably less than 5 nM, in the immobilized metal affinity particle reagent PDE assay, in free or salt form. 50 has.

[0050] In another embodiment, the present invention relates to the administration of a PDE1 inhibitor for the treatment or prevention described herein, wherein the inhibitor is in free form or in pharmacologic form, [ka] The present invention provides a compound according to the method of the present invention,

[0051] Further examples of PDE1 inhibitors suitable for use in the methods and treatments described herein include those disclosed in WO2006133261A2; U.S. Patent No. 8,273,750; U.S. Patent No. 9,000,001; U.S. Patent No. 9,624,230; WO2009075784A1; U.S. Patent No. 8,273,751; U.S. Patent No. 8,829,008; U.S. Patent No. 9,403,836 ... Publication No. 2014151409 A1, U.S. Patent No. 9,073,936; U.S. Patent No. 9,598,426; U.S. Patent No. 9,556,186; U.S. Patent Application Publication No. 2017 / 0231994 A1, WO 2016022893 A1, and U.S. Patent Application Publication No. 2017 / 0226117 A1, each of which is incorporated herein by reference in its entirety.

[0052] Further examples of PDE1 inhibitors suitable for use in the methods and treatments described herein include those described in WO 2018007249 A1; U.S. Patent Application Publication No. 2018 / 0000786; WO 2015118097 A1; U.S. Patent No. 9,718,832; WO 2015091805 A1; U.S. Patent No. 9,701,665; U.S. Patent Application Publication No. 2015 / 0175584 A1; U.S. Patent Application Publication No. 2017 / 0267664 A1; WO 2016 055618 A1;U.S. Patent Application Publication No. 2017 / 0298072 A1;WO Publication No. 2016170064 A1;U.S. Patent Application Publication No. 2016 / 0311831 A1;WO Publication No. 2015150254 A1;U.S. Patent Application Publication No. 2017 / 0022186 A1;WO Publication No. 2016174188 A1;U.S. Patent Application Publication No. 2016 / 0318939 A1;U.S. Patent Application Publication No. 2017 / 0291903 A1;WO Publication No. 2018073251 A1;WO Publication No. 2017178350 A1; U.S. Patent Application Publication No. 2017 / 0291901 A1; WO 2018 / 115067; U.S. Patent Application Publication No. 2018 / 0179200 A; U.S. Patent Application Publication No. 20160318910 A1; U.S. Patent No. 9,868,741; WO 2017 / 139186 A1; WO 2016 / 040083; U.S. Patent Application Publication No. 2017 / 0240532; WO 2016033776 A1; U.S. Patent Application Publication No. No. 2017 / 0233373; WO 2015130568; WO 2014159012; U.S. Patent No. 9,034,864; U.S. Patent No. 9,266,859; WO 2009085917; U.S. Patent No. 8,084,261; WO 2018039052; U.S. Patent Application Publication No. 20180062729; and WO 2019027783 (each of which is incorporated herein by reference in its entirety). In any situation where the statements of the references incorporated herein by reference contradict or are inconsistent with the statements of this disclosure, the statements of this disclosure shall be understood to control.

[0053] As used herein, unless otherwise specified or clear from the context, the following terms have the following meanings: As used herein, a "selective PDE1 inhibitor" refers to a PDE1 inhibitor that has at least 100-fold selectivity for inhibiting PDE1 over inhibiting other PDE isoforms. b. "Alkyl," as used herein, refers to a saturated or unsaturated hydrocarbon moiety, preferably saturated, preferably having 1 to 6 carbon atoms, which may be straight or branched, and which may be mono-, di-, or tri-substituted, for example, with halogen (e.g., chloro or fluoro), hydroxy, or carboxy. c. As used herein, "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon moiety, preferably saturated, preferably containing 3 to 9 carbon atoms, at least some of which form a monocyclic or bicyclic or bridged ring structure, and which may be substituted, for example, with halogen (e.g., chloro or fluoro), hydroxy or carboxy. Cycloalkyl may contain one or more atoms selected from N and O and / or S, and the cycloalkyl may be a heterocycloalkyl. d. "Heterocycloalkyl" means, unless otherwise specified, a saturated or unsaturated non-aromatic hydrocarbon moiety, preferably saturated, preferably containing 3 to 9 carbon atoms, at least a portion of which form a non-aromatic monocyclic or bicyclic or bridged hypercyclic structure, in which at least one carbon atom is replaced by N, O or S, and the heterocycloalkyl may be optionally substituted, for example, by halogen (e.g., chloro or fluoro), hydroxy or carboxy. e. As used herein, "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon, preferably phenyl, optionally substituted with, for example, alkyl (e.g., methyl), halogen (e.g., chloro or fluoro), haloalkyl (e.g., trifluoromethyl), hydroxy, carboxy, or further aryl or heteroaryl (e.g., biphenyl or pyridylphenyl). f. As used herein, "heteroaryl" refers to an aromatic moiety in which one or more of the atoms forming the aromatic ring is sulfur or nitrogen rather than carbon, e.g., pyridyl or thiadiazolyl, which may be optionally substituted, e.g., with alkyl, halogen, haloalkyl, hydroxy or carboxy.

[0054] The compounds of the present disclosure, such as the PDE1 inhibitors described herein, can be in free form or salt form, for example, as acid addition salt.In this specification, unless otherwise specified, phrases such as "compounds of the present disclosure" are understood to include the compounds in any form, for example, free or acid addition salt form, or, if the compound contains an acidic substituent, base addition salt form.The compounds of the present disclosure are intended to be used as pharmaceuticals, and therefore pharma-ceutically acceptable salts are preferred.Salts that are not suitable for pharmaceutical use are also included, for example, because they may be useful for isolating or purifying the free compounds of the present disclosure or their pharma-ceutically acceptable salts.

[0055] The compounds of the present disclosure may exist in prodrug form in some cases. A prodrug form is a compound that converts to a compound of the present disclosure in the body. For example, when a compound of the present disclosure contains hydroxy or carboxy substituents, these substituents may form physiologically hydrolyzable and acceptable esters. As used herein, "physiologically hydrolyzable and acceptable esters" refers to esters of compounds of the present disclosure that are hydrolyzable under physiological conditions to produce an acid (in the case of a compound of the present disclosure having a hydroxy substituent) or an alcohol (in the case of a compound of the present disclosure having a carboxy substituent) that is itself physiologically acceptable in the administered dose. Thus, when a compound of the present disclosure contains a hydroxy group, for example the compound -OH, an acyl ester prodrug of such a compound, i.e. the compound Compound -OC(O)-C, is an acyl ester prodrug of the compound Compound -OC(O)-C. 1-4 Alkyl groups can be hydrolyzed in the body to form physiologically hydrolyzable alcohols (compounds -OH) on the one hand, and acids (e.g., HOC(O)-C 1-4Alternatively, when a compound of the present disclosure contains a carboxylic acid, for example, Compound -C(O)OH, an acid ester prodrug of such a compound, Compound -C(O)OC, can be formed. 1-4 Alkylalkyls can be hydrolyzed to give the compounds -C(O)OH and HO-C 1-4 As will be appreciated, the term encompasses conventional pharmaceutical prodrug forms.

[0056] In another embodiment, the present disclosure further provides a pharmaceutical composition comprising a PDE1 inhibitor in combination with an immune checkpoint inhibitor, each in free form or in pharma- ceutically acceptable salt form, in admixture with a pharma- ceutically acceptable carrier. The term "combination" as used herein includes simultaneous, sequential, or contemporaneous administration of a PDE1 inhibitor and an immune checkpoint inhibitor. In some embodiments, the combination of a PDE1 inhibitor and an immune checkpoint inhibitor allows the immune checkpoint inhibitor to be administered at a lower dose than would be effective if administered as the only monotherapy.

[0057] Methods of Using the Disclosed Compounds In one embodiment, the present application provides a method for treating breast cancer (Method 1), comprising administering to a subject in need of such treatment a pharma- ceutically acceptable amount of a PDE1 inhibitor (e.g., a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V and / or VI), alone or in combination with a pharma- ceutically acceptable amount of an immune checkpoint inhibitor.

[0058] 1.1 Method 1, wherein the breast cancer has elevated expression of PDE1.

[0059] 1.2 Any of the above methods, wherein the breast cancer expresses PD-L1 and has a combined positive score (CPS) of > 1 as determined, e.g., by an FDA approved test (where CPS is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the increase in the number of viable tumor cells multiplied by 100).

[0060] 1.3 Any of the above methods, wherein the breast cancer is triple-negative breast cancer (TNBC), which is estrogen receptor negative, progesterone receptor negative and HER2 negative.

[0061] 1.4 The TNBC is high-risk early stage TNBC, method 1.3.

[0062] 1.5 Method 1.3, where the treatment is adjuvant therapy following surgery.

[0063] 1.6 The subject has locally recurrent unresectable or metastatic TNBC whose tumor expresses PD-L1, e.g., has a combined positive score (CPS) of > 1 as determined by an FDA approved test (where CPS is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the number of viable tumor cells multiplied by 100), Method 1.3.

[0064] 1.7 Any of the above methods, wherein administration to a subject of a pharma- ceutically acceptable amount of a PDE1 inhibitor in combination with a pharma-ceutically acceptable amount of an immune checkpoint inhibitor increases the M1 / M2 ratio of macrophages in the tumor microenvironment.

[0065] 1.8 Any of the above methods, wherein administration to a subject of a pharma- ceutically acceptable amount of an immune checkpoint inhibitor alone (i.e., without in combination with a PDE1 inhibitor) is not effective in treating breast cancer, e.g., does not inhibit the growth of breast cancer.

[0066] 1.9 Any of the above methods, wherein administration to a subject of a pharma- ceutically acceptable amount of a PDE1 inhibitor alone (i.e., without in combination with a checkpoint inhibitor) is not effective in treating breast cancer, e.g., does not inhibit the growth of breast cancer.

[0067] 1.10 Any of the above methods, wherein the immune checkpoint inhibitor is selected from one or more of an inhibitor of CTLA-4, PD-1 and / or PD-L1.

[0068] 1.11 Any of the above methods, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, and the immune checkpoint inhibitor may be an anti-PD-1 antibody, and the antibody may be monoclonal or polyclonal.

[0069] 1.12 Any of the above methods, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1, and the immune checkpoint inhibitor may be an anti-PD-L1 antibody, and the antibody may be monoclonal or polyclonal.

[0070] 1.13 Any of the above methods, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, and the immune checkpoint inhibitor may be an anti-CTLA-4 antibody, and the antibody may be monoclonal or polyclonal.

[0071] 1.14 Any of the above methods, wherein the immune checkpoint inhibitor comprises one or more members selected from nivolumab, pembrolizumab, cemiplimab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

[0072] 1.15 Any of the above methods, wherein the subject is suffering from a systemic inflammatory response, a gastrointestinal inflammation-related disorder, an endocrine inflammation-related disorder, a skin inflammation-related disorder, an ocular inflammation-related disorder, a neurological inflammation-related disorder, a hematological inflammation-related disorder, a genitourinary inflammation-related disorder, a respiratory inflammation-related disorder, a musculoskeletal inflammation-related disorder, a cardiac inflammation-related disorder, or a defined systemic inflammation-related disorder, or a hematological inflammation-related disorder, a genitourinary inflammation-related disorder, a respiratory inflammation-related disorder, a musculoskeletal inflammation-related disorder, a cardiac inflammation-related disorder, or a defined systemic inflammation-related disorder.

[0073] 1.16 Any of the above methods, wherein the subject in need of said treatment has previously been administered an immune checkpoint inhibitor therapy, and the pharma- ceutically acceptable amount of the immune checkpoint inhibitor administered to the subject may be lower than the amount of the immune checkpoint inhibitor administered in the prior immune checkpoint inhibitor therapy.

[0074] 1.17 Any of the above methods, wherein the subject in need of said treatment is suffering from a disease, disorder, or adverse effects from immune checkpoint inhibitor therapy.

[0075] 1.18 Any of the above methods, wherein the subject is suffering from an inflammation-associated disorder resulting from immune checkpoint inhibitor therapy.

[0076] 1.19 Any of the above methods, wherein the subject is suffering from or at risk of suffering from an immune checkpoint inhibitor therapy side effect, e.g., the side effect is selected from a systemic inflammatory response, a gastrointestinal inflammation-related disorder, an endocrine inflammation-related disorder, a skin inflammation-related disorder, an ocular inflammation-related disorder, a neurological inflammation-related disorder, a hematological inflammation-related disorder, a genitourinary inflammation-related disorder, a respiratory inflammation-related disorder, a musculoskeletal inflammation-related disorder, a cardiac inflammation-related disorder, or a specific systemic inflammation-related disorder.

[0077] 1.20 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a gastrointestinal inflammation-related disorder, e.g., selected from colitis, enterocolitis, colitis complicated by intestinal perforation, hepatitis, and pancreatitis.

[0078] 1.21 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, which side effect is an endocrine inflammation-related disorder, e.g., selected from hypophysitis (e.g., manifested as panhypopituitarism), thyrotoxicosis, hypothyroidism, syndrome of inappropriate antidiuretic hormone secretion, central adrenal insufficiency, primary adrenal insufficiency, and diabetes mellitus.

[0079] 1.22 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, the side effect being a skin inflammation-related disorder, e.g., selected from rash, pruritus, vitiligo, dermatitis, Sweet's syndrome, drug rash, leukoderma, delayed hypersensitivity reaction, alopecia universalis, Grover's disease, pyoderma gangrenosum, toxic epidermal necrolysis, chronic noncaseating granuloma, bullous pemphigoid, and psoriasis.

[0080] 1.23 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is an ocular inflammation-related disorder, e.g., selected from uveitis, conjunctivitis, orbital inflammation, Graves' ophthalmopathy, choroidal neovascularization, optic neuropathy, keratitis, and retinopathy.

[0081] 1.24 Any of the above methods, wherein the subject is suffering from an adverse effect of immune checkpoint inhibitor therapy, the adverse effect being a neurological inflammation-related disorder, e.g., selected from encephalopathy, Guillain-Barré syndrome, polyradiculopathy, symmetric multifocal neuropathy, transverse myelitis, necrotizing myelopathy, myasthenia gravis, phrenic nerve paralysis, immune-related meningitis, meningoradiculoneuritis, peripheral neuropathy, autoimmune inner ear disease, multiple sclerosis, and inflammatory enteric neuropathy.

[0082] 1.25 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a hematological inflammation-related disorder, e.g., selected from thrombocytopenia, pancytopenia, neutropenia, eosinophilia, pure red blood cell aplasia, acquired hemophilia A, and disseminated intravascular coagulation.

[0083] 1.26 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a genitourinary inflammatory associated disorder, e.g., selected from renal failure, acute / granulomatous interstitial nephritis, acute tubular necrosis, and lymphocytic vasculitis (e.g., uterine lymphocytic vasculitis).

[0084] 1.27 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, and the side effect is a respiratory inflammation-related disorder, e.g., selected from pneumonitis and acute respiratory distress.

[0085] 1.28 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a musculoskeletal inflammatory associated disorder, e.g., selected from polyarthritis, arthralgia, myalgia, chronic granulomatous inflammation of the rectus abdominis muscle, and rhabdomyolysis.

[0086] 1.29 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, and the side effect is a cardiac inflammation-related disorder, e.g., selected from precarditis and takotsubo-like syndrome.

[0087] 1.30 Any of the above methods, wherein the subject is suffering from a side effect of immune checkpoint inhibitor therapy, the side effect being a systemic inflammation-related disorder, e.g., selected from pulmonary sarcoidosis, cutaneous and pulmonary sarcoidosis, polymyalgia rheumatica, giant cell arteritis, muscular sarcoidosis, neurological and pulmonary sarcoidosis, celiac disease, lupus nephritis, dermatomyositis, autoimmune inflammatory myopathy, and Vogt-Koyanagi-like syndrome.

[0088] 1.31 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered before, after, or in conjunction with radiation therapy or chemotherapy.

[0089] 1.32 Any of the above methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered concurrently with radiation therapy or chemotherapy.

[0090] 1.33 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered prior to radiation therapy or chemotherapy.

[0091] 1.34 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered following radiation therapy or chemotherapy.

[0092] 1.35 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered in conjunction with an additional anti-tumor agent, chemotherapeutic treatment, gene therapy treatment and / or immunological treatment.

[0093] 1.36 The PDE1 inhibitor is a PDE1 inhibitor according to formula I, Ia, II, III, IV, V and / or VI, or in free form or in pharma- ceutical acceptable salt form, [ka] [ka] Any of the above methods, wherein the compound is according to

[0094] 1.37 Any of the above methods, wherein the PDE1 inhibitor is a PDE1 inhibitor according to formula Ia.

[0095] 1.38 The PDE1 inhibitor is, in free form or in pharma- ceutical acceptable salt form, [ka] Any of the above methods, wherein

[0096] 1.39 The PDE1 inhibitor is in free form or in pharma- ceutical acceptable salt form, e.g., in monophosphate form, [ka] Any of the above methods, wherein

[0097] 1.40 Any of the above methods further comprising administering to said subject a pharma- ceutically acceptable amount of a beta-blocker.

[0098] 1.41 Any of the above methods, wherein the subject is a human.

[0099] In another embodiment, the disclosure provides a PDE1 inhibitor, alone or in combination with an immune checkpoint inhibitor, for use in the treatment of breast cancer, such as for use in any of Method 1.

[0100] In another embodiment, the present application provides a method for preventing or ameliorating a disease, disorder, or adverse effect resulting from administration of an immune checkpoint inhibitor to a subject suffering from breast cancer, comprising reducing the amount of checkpoint inhibitor administered to the subject, and administering to the subject a pharma- ceutically acceptable amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and / or VI) in combination with immune checkpoint inhibitor therapy (Method 2).

[0101] 2.1 Method 2, wherein the breast cancer has elevated expression of PDE1.

[0102] 2.2 Any of the above methods, wherein the breast cancer expresses PD-L1 and has a combined positive score (CPS) of > 1 as determined, e.g., by an FDA approved test (where CPS is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the increase in the number of viable tumor cells multiplied by 100).

[0103] 2.3 Any of the above methods, wherein the breast cancer is triple negative breast cancer (TNBC).

[0104] 2.4 The TNBC is high-risk early stage TNBC, method 2.3.

[0105] 2.5. Immune checkpoint inhibitor therapy is adjuvant therapy following surgery, Method 2.3.

[0106] 2.6 The subject has locally recurrent unresectable or metastatic TNBC whose tumor expresses PD-L1, e.g., has a combined positive score (CPS) of > 1 as determined by an FDA approved test (where CPS is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the number of viable tumor cells multiplied by 100), Method 2.3.

[0107] 2.7 Any of the above methods, wherein the reduced amount of the immune checkpoint inhibitor, when administered to a subject alone (i.e., without combination with a PDE1 inhibitor), is not effective in treating breast cancer, e.g., administration of the reduced amount of the immune checkpoint inhibitor alone (i.e., without combination with a PDE1 inhibitor) to a subject does not inhibit the growth of breast cancer.

[0108] 2.8 Any of the above methods, wherein administering to a subject a pharma- ceutically acceptable amount of a PDE1 inhibitor in combination with said reduced amount of an immune checkpoint inhibitor increases the M1 / M2 ratio of macrophages in the tumor microenvironment.

[0109] 2.9 Any of the above methods, wherein the immune checkpoint inhibitor is selected from one or more of an inhibitor of CTLA-4, PD-1 and / or PD-L1.

[0110] 2.10 Any of the above methods, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, and the immune checkpoint inhibitor may be an anti-PD-1 antibody, and the antibody may be monoclonal or polyclonal.

[0111] 2.11 Any of the above methods, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1, and the immune checkpoint inhibitor may be an anti-PD-L1 antibody, and the antibody may be monoclonal or polyclonal.

[0112] 2.12 Any of the above methods, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, and the immune checkpoint inhibitor may be an anti-CTLA-4 antibody, and the antibody may be monoclonal or polyclonal.

[0113] 2.13 Any of the above methods wherein the immune checkpoint inhibitor comprises one or more members selected from nivolumab, pembrolizumab, cemiplimab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

[0114] 2.14 Any of the above methods, wherein the subject is suffering from an inflammation-associated disorder resulting from immune checkpoint inhibitor therapy.

[0115] 2.15 Any of the above methods, wherein the subject is suffering from a systemic inflammatory response, a gastrointestinal inflammation-related disorder, an endocrine inflammation-related disorder, a skin inflammation-related disorder, an ocular inflammation-related disorder, a neurological inflammation-related disorder, a hematological inflammation-related disorder, a genitourinary inflammation-related disorder, a respiratory inflammation-related disorder, a musculoskeletal inflammation-related disorder, a cardiac inflammation-related disorder, or a specific systemic inflammation-related disorder.

[0116] 2.16 Method 2.15, wherein the gastrointestinal inflammation-related disorder is selected from colitis, enterocolitis, and colitis complicated by intestinal perforation, hepatitis, and pancreatitis.

[0117] 2.17 Method 2.15, wherein the endocrine inflammation-related disorder is selected from hypophysitis (e.g., manifesting as panhypopituitarism), thyrotoxicosis, hypothyroidism, syndrome of inappropriate secretion of antidiuretic hormone, central adrenal insufficiency, primary adrenal insufficiency, and diabetes mellitus.

[0118] 2.18 Method 2.15, wherein the skin inflammation-related disorder is selected from rash, pruritus, vitiligo vulgaris, dermatitis, Sweet's syndrome, drug rash, leukoderma, delayed hypersensitivity reaction, alopecia universalis, Grover's disease, pyoderma gangrenosum, toxic epidermal necrolysis, chronic noncaseating granuloma, bullous pemphigoid, and psoriasis.

[0119] 2.19 Method 2.15, wherein the ocular inflammation-related disorder is selected from uveitis, conjunctivitis, orbititis, Graves' ophthalmopathy, choroidal neovascularization, optic neuropathy, keratitis, and retinopathy.

[0120] 2.20 Method 2.15, wherein the neurological inflammation-related disorder is selected from encephalopathy, Guillain-Barré syndrome, polyradiculopathy, symmetric multifocal neuropathy, transverse myelitis, necrotizing myelopathy, myasthenia gravis, phrenic nerve paralysis, immune-related meningitis, meningoradiculitis, peripheral neuropathy, autoimmune inner ear disease, multiple sclerosis, and inflammatory enteric neuropathy.

[0121] 2.21 Method 2.15, wherein the hematological inflammation-related disorder is selected from thrombocytopenia, pancytopenia, neutropenia, eosinophilia, pure red cell aplasia, acquired hemophilia A, and disseminated intravascular coagulation.

[0122] 2.22 Method 2.15, wherein the genitourinary inflammatory-related disorder is selected from renal failure, acute / granulomatous interstitial nephritis, acute tubular necrosis, and lymphocytic vasculitis (e.g., uterine lymphocytic vasculitis).

[0123] 2.23 Method 2.15, wherein the respiratory inflammation-related disorder is selected from pneumonitis and acute respiratory distress.

[0124] 2.24 Method 2.15, wherein the musculoskeletal inflammation-related disorder is selected from polyarthritis, arthralgia, myalgia, chronic granulomatous inflammation of the rectus abdominis muscle, and rhabdomyolysis.

[0125] 2.25 Method 2.15, wherein the cardiac inflammation-related disorder is selected from pericarditis and takotsubo-like syndrome.

[0126] 2.26 Method 2.15, wherein the specific systemic inflammation-related disorder is selected from pulmonary sarcoidosis, cutaneous and pulmonary sarcoidosis, polymyalgia rheumatica, giant cell arteritis, muscular sarcoidosis, neurological and pulmonary sarcoidosis, celiac disease, lupus nephritis, dermatomyositis, autoimmune inflammatory myopathy, and Vogt-Koyanagi-like syndrome.

[0127] 2.27 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered in conjunction with radiation therapy or chemotherapy.

[0128] 2.28 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered simultaneously with radiation therapy or chemotherapy.

[0129] 2.29 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered prior to radiation therapy or chemotherapy.

[0130] 2.30 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered after radiation therapy or chemotherapy.

[0131] 2.31 Any of the above methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered together with an additional anti-tumor agent, chemotherapeutic treatment, gene therapy treatment and / or immunological treatment.

[0132] 2.32 The PDE1 inhibitor is a PDE1 inhibitor according to formula I, Ia, II, III, IV, V and / or VI, or in free form or in pharma- ceutical acceptable salt form, [ka] [ka] Any of the above methods, wherein the compound is according to

[0133] 2.33 Any of the above methods, wherein the PDE1 inhibitor is a PDE1 inhibitor according to formula Ia.

[0134] 2.34 The PDE1 inhibitor is, in free form or in pharma- ceutical acceptable salt form, [ka] Any of the above methods, wherein

[0135] 2.35 The PDE1 inhibitor is in free form or in the form of a pharma- ceutical acceptable salt, e.g., in the form of a monophosphate. [ka] Any of the above methods, wherein

[0136] 2.36 Any of the above methods further comprising administering to said subject a pharma- ceutically acceptable amount of a beta-blocker.

[0137] 2.37 Any of the above methods, wherein the subject is a human.

[0138] In another embodiment, the disclosure provides a PDE1 inhibitor (e.g., a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V and / or VI) for use in preventing or alleviating a disease, disorder, or adverse effects resulting from administration of checkpoint inhibitor therapy, such as for use in any of Method 2.

[0139] Combination therapy with PDE1 inhibitors In the present invention, the PDE1 inhibitor may be administered in combination with an immune checkpoint inhibitor. The combination therapy may be achieved by administering a single composition or pharmacological formulation comprising a PDE1 inhibitor and an immune checkpoint inhibitor, or by administering two different compositions or formulations separately, simultaneously, or sequentially, one composition comprising a PDE1 inhibitor and the other composition comprising an immune checkpoint inhibitor. Treatment with one inhibitor may precede or follow administration of the other inhibitor by an interval ranging from minutes to weeks. In embodiments where the other inhibitor is administered separately on the cells, it is generally ensured that no significant time has elapsed between each delivery so that the PDE1 inhibitor and the immune checkpoint inhibitor can still exert their beneficial combined effect on the cells. In some embodiments, it is typically contemplated that both inhibitors will be contacted with the cells within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, with a delay of only about 12 hours being most preferred. However, in some circumstances it may be desirable to extend the treatment period significantly, such that several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between individual administrations.

[0140] It is also conceivable that it may be desirable to administer either the PDE1 inhibitor or the immune checkpoint inhibitor more than once. In this regard, various combinations may be employed. By way of example, when the PDE1 inhibitor is "A" and the immune checkpoint inhibitor is "B", the following permutations based on a total of three and four administrations are exemplified: [ka]

[0141] Thus, in various embodiments, the present disclosure also provides pharmaceutical combination [combination 1] therapies comprising a pharma- ceutically acceptable amount of a PDE1 inhibitor (e.g., a compound according to Formula I, Ia, II, III, IV, V, and / or VI) and a pharma- ceutically acceptable amount of an immune checkpoint inhibitor for administration in a method for treating breast cancer, such as according to any of Methods 1, or for the prevention or amelioration of a disease, disorder, or adverse effect resulting from administration of a checkpoint inhibitor therapy, such as according to any of Methods 2. For example, the present disclosure provides the following combinations:

[0142] 1.1 Combination 1, in which the PDE1 inhibitor and the checkpoint inhibitor are in combination with or associated with a pharma- ceutically acceptable diluent or carrier, in a single dosage form, e.g., a tablet or capsule.

[0143] 1.2 Combination 1, in which the PDE1 inhibitor and the checkpoint inhibitor are in a single package, e.g., with instructions for simultaneous or sequential administration.

[0144] 1.3 The PDE1 inhibitor is a PDE1 inhibitor according to formula I, Ia, II, III, IV, V and / or VI, or in free form or in pharma- ceutical acceptable salt form, [ka] [ka] Any of the above combinations, wherein the compound is according to

[0145] 1.4 Any of the above combinations, wherein the PDE1 inhibitor is a PDE1 inhibitor according to formula Ia.

[0146] 1.5 The PDE1 inhibitor is, in free form or in pharma- ceutical acceptable salt form, [ka] Any of the above combinations, wherein

[0147] 1.6 The PDE1 inhibitor is, in free form or in pharma- ceutical acceptable salt form, [ka] Any of the above combinations, wherein

[0148] 1.7 Any of the above combinations wherein the immune checkpoint inhibitor is selected from one or more of an inhibitor of CTLA-4, PD-1 and / or PD-L1.

[0149] 1.8 Any of the above combinations, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, and wherein the immune checkpoint inhibitor may be an anti-PD-1 antibody, and wherein the antibody may be monoclonal or polyclonal.

[0150] 1.9 Any of the above combinations, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1, and wherein the immune checkpoint inhibitor may be an anti-PD-L1 antibody, and which antibody may be monoclonal or polyclonal.

[0151] 1.10 Any of the above combinations, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, and wherein the immune checkpoint inhibitor may be an anti-CTLA-4 antibody, and wherein the antibody may be monoclonal or polyclonal.

[0152] 1.11 Any of the above combinations where the immune checkpoint inhibitor includes one or more members selected from nivolumab, pembrolizumab, cemiplimab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

[0153] 1.12 Any of the above combinations wherein the checkpoint inhibitor is administered simultaneously with the PDE1 inhibitor.

[0154] 1.13 Any of the above combinations, wherein the checkpoint inhibitor is administered prior to the PDE1 inhibitor.

[0155] 1.14 Any of the above combinations, wherein the checkpoint inhibitor is administered after the PDE1 inhibitor.

[0156] 1.15 Any of the above combinations, wherein the subject in need of said treatment has previously or concomitantly been administered checkpoint inhibitor therapy.

[0157] 1.16 Any of the above combinations further comprising a pharma- ceutically acceptable amount of a beta-blocker.

[0158] In some embodiments, the pharmaceutical composition is administered in combination with one or more additional antitumor drugs, such as drugs known to be effective in treating or eliminating cancer and / or tumors. Non-limiting examples of antitumor drugs include abemaciclib, abiraterone acetate, avitrexate (methotrexate), abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab , Alimta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alumbrig (brigatinib), Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia, Pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), Arsenic trioxide, Arzera (ofatumumab), Asparaginase Erwinia chrysanthemi), atezolizumab, Avastin (bevacizumab), avelumab, axicabtagene ciloleucel, axitinib, azacitidine, BAVENCIO (avelumab), BEACOPP, Becenum (carmustine), Beleodaq (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,CABOMETYX (cabozantinib-S-malate), CABOZANTINIB-S-MALATE, CAF, CALQUENS (acalabrutinib), CAMPATH (alemtuzumab), CAMPTOSAR (irinotecan hydrochloride), capecitabine, CAPOX, Carac (topical fluorouracil), carboplatin, CARBOPLATIN-TAXOL, carfilzomib, CARMBRIS (carmustine), carmustine, carmustine implant, CASODEX (bicalutamide), CEM, ceritinib, CERBIDIN (daunorubicin hydrochloride), CERVIX (genetic Recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chlorar (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide Sufamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darazalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomal, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox , denosumab, Depocyte (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (topical fluorouracil), Erythec (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine,Emend (aprepitant), Empliti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Elibage (vismodegib), erlotinib hydrochloride, Erwinase (blackleg asparaginase), Ethiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), Evome la (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical), Fairston (toremifene), Faridak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil topical), fluorouracil injection, fluorouracil topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), Forfili, Forfili-bevacizumab, Forfili-cetuximab, Forfirinox, Forfox, Forlotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV pentavalent 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, goserelin acetate, Halaven (eribulin mesylate), Hemangiol (propranolol hydrochloride), Herceptin (trastuzumab), recombinant HPV bivalent vaccine, recombinant HPV 9-valent vaccine, recombinant HPV 4-valent vaccine, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib),Ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (talimogene laherparepvec), Inlyta (axitinib), Inotuzumab Buozogamicin, recombinant interferon alpha-2b, interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2b), ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib salt), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxif ene (raloxifene hydrochloride), Kepivans (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), leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), (vincristine sulfate liposomal), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposomal), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot Ped (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposomal), Matulane (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, Mitozytrex (Mitomycin C), MOPP, Mozovir (Plerixafor), Mustargen (Mechlorethamine hydrochloride), Mutamycin (Mitomycin C), Myleran (Bus) rufan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron ( Nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepecate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal), Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (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, Portraza (necitumumab), pralatrexate, prednisone, procarba, dsin hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopagulamine), propranolol hydrochloride, Provenzi (cypressel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HP V) Bivalent vaccine, recombinant human papillomavirus (HPV) 9-valent vaccine, recombinant human papillomavirus (HPV) 4-valent vaccine, recombinant interferon alpha-2b, regorafenib, Relistol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and hyaluronidase human), rituximab, rituximab and hyaluronidase human, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, Rydapt (midostaurin), Sclerosol Intrapleural Aerosol (talc), siltuximab, sipuleucel-T, somatuline depot (lanreotide acetate), sonidegib, sorafenib tosylate, sprycel (dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepecate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TarabinePFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (topical fluorouracil), topotecan hydrochloride, toremifene, Toricel (temsirolimus), Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and and tipiracil hydrochloride, Trisenox (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, Veneclesta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, VincasarPFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab) , Yescarta (axicabtagene ciloleucel), Yondelis (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zedula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0159] In some embodiments, PDE1 inhibitor and immune checkpoint inhibitor are administered in combination with one or more β-blockers.A non-exhaustive list of such β-blockers includes various β-adrenergic receptor antagonists, also called β-blockers, which are currently used clinically to eliminate the harmful chronic myocardial stimulation caused by failing heart. Preferred beta-adrenergic receptor antagonists include metoprolol, metoprolol succinate, carvedilol, atenolol, propranolol, acebutolol, acebutolol HCL, betaxolol, betaxolol HCL, nadolol, talinolol, bisoprolol, bisoprolol hemifumarate, carteolol, carteolol HCL, esmolol, esmolol HCL, labetalol, labetalol HCL, metoprolol, metoprolol succinate, metoprolol tartrate, nadolol, penbutolol, penbutolol sulfate, pindolol, propranolol, propranolol HCL, sotalol, sotalol HCL, timolol, and timolol hydrogen maleate salt, or a pharma- ceutically acceptable salt thereof. According to the present invention, the β-adrenergic receptor antagonists can be administered in the daily doses clinically accepted for such drugs. For example, a suitable daily dose for metoprolol as the tartrate or succinate is about 100-200 mg, and for carvedilol about 5-50 mg, depending on the condition to be treated, the route of administration, and the age, weight and condition of the patient.

[0160] As used herein, the term "anti-tumor agent" is understood to refer to any chemical agent or drug that is effective in preventing or inhibiting the formation or growth of cancer or tumors. Anti-tumor agents described herein may include alkylating agents, antimetabolites, natural products, hormones, and / or antibodies. Treating tumors or cancer may include limiting the proliferation, migration, and / or invasion of cancerous or neoplastic cells in the body, or limiting symptoms associated with said cancer or tumor. As used herein, anti-tumor agents are understood to include anti-cancer agents and are otherwise synonymous with anti-cancer agents.

[0161] Methods for making the compounds of the present disclosure The PDE1 inhibitors and pharma- ceutically acceptable salts thereof of the present disclosure are described in U.S. Patent No. 8,273,750, U.S. Patent Application Publication No. 2006 / 0173878, U.S. Patent No. 8,273,751, U.S. Patent Application Publication No. 2010 / 0273753, U.S. Patent No. 8,697,710, U.S. Patent No. 8,664,207, U.S. Patent No. 8,633,180, U.S. Patent No. 8,536,159, U.S. Patent Application Publication No. 2012 / 0136013, U.S. Patent Application The starting materials for these processes may be prepared by using the methods described or exemplified in US Patent Publication No. 2011 / 0281832, US Patent Publication No. 2013 / 0085123, US Patent Publication No. 2013 / 0324565, US Patent Publication No. 2013 / 0338124, US Patent Publication No. 2013 / 0331363, WO Publication No. 2012 / 171016 and WO Publication No. 2013 / 192556, and similar methods and methods known in the chemical arts. Such methods include, but are not limited to, the methods described below. The starting materials for these processes, if not commercially available, may be prepared by procedures selected from the chemical arts, using techniques similar or similar to the synthesis of known compounds.

[0162] Various PDE1 inhibitors and starting materials thereof are disclosed in U.S. Patent Application Publication No. 2008-0188492 A1, U.S. Patent Application Publication No. 2010-0173878 A1, U.S. Patent Application Publication No. 2010-0273754 A1, U.S. Patent Application Publication No. 2010-0273753 A1, WO 2010 / 065153, WO 2010 / 065151, WO 2010 / 065152, WO 2010 / 065153, WO 2010 / 065154, WO 2010 / 065155, WO 2010 / 065156, WO 2010 / 065157, WO 2010 / 065159, WO 2010 / 065158, WO 2010 / 065159 ... The compounds may be prepared using the methods described in WO 2010 / 065151, WO 2010 / 065149, WO 2010 / 065147, WO 2010 / 065152, WO 2011 / 153129, WO 2011 / 133224, WO 2011 / 153135, WO 2011 / 153136, and WO 2011 / 153138. All documents cited herein are incorporated by reference in their entirety.

[0163] The compounds (PDE1 inhibitors) of the present disclosure include their enantiomers, diastereomers and racemates, as well as their polymorphs, hydrates, solvates and complexes.Some individual compounds within the scope of the present disclosure contain double bonds.The expression of double bonds in the present disclosure is meant to include both the E and Z isomers of the double bond.In addition, some compounds within the scope of the present disclosure contain one or more asymmetric centers.The present disclosure includes the use of any of the optically pure stereoisomers and combinations of stereoisomers.

[0164] The compounds (PDE1 inhibitors) of the present disclosure are also intended to include their stable and unstable isotopes. Stable isotopes are non-radioactive isotopes that contain one additional neutron compared to the abundant nuclide of the same species (i.e., element). The activity of compounds containing such isotopes is expected to be retained, and such compounds are also useful for measuring the pharmacokinetics of non-isotopic analogs. For example, hydrogen atoms at certain positions of the compounds of the present disclosure can be replaced with deuterium (non-radioactive stable isotope). Examples of known stable isotopes include, but are not limited to, deuterium, 13C, 15N, 18O. Alternatively, unstable isotopes that are radioactive isotopes that contain an additional neutron compared to the abundant nuclide of the same species (i.e., element), such as 123I, 131I, 125I, 11C, 18F, can be replaced with the corresponding abundant species of I, C, and F. Another example of a useful isotope of the compounds of the present disclosure is the 11C isotope. These radioisotopes are useful for radioimaging and / or pharmacokinetic studies of the compounds of the present disclosure.

[0165] The terms "treatment" and "treating" should be understood as encompassing treatment of the cause of the disease as well as treatment or amelioration of symptoms of disease, as appropriate.

[0166] For the treatment method, the term "effective amount" is intended to include a therapeutically effective amount for treating breast cancer, for example, a therapeutically effective amount for inhibiting the growth (volume or weight) of breast cancer, when a PDE-1 inhibitor and an immune checkpoint inhibitor are administered in combination. The effective amount of the PDE-1 inhibitor or immune checkpoint may be lower than when the PDE-1 inhibitor or immune checkpoint is administered as a monotherapy.

[0167] The terms "patient" and "subject" include human or non-human (i.e., animal) patients and are understood to be interchangeable within the context of this disclosure. In certain embodiments, the disclosure encompasses both humans and non-humans. In other embodiments, the disclosure encompasses non-humans. In other embodiments, the terms encompass humans.

[0168] As used in this disclosure, the term "comprising" is intended to be open-ended and does not exclude additional, unrecited elements or method steps.

[0169] The dosages used in carrying out the present disclosure will of course vary depending on, for example, the type of breast cancer to be treated, the specific PDE-1 inhibitor or immune checkpoint inhibitor, the mode of administration, and the desired treatment.The PDE-1 inhibitor can be administered by any suitable route, including orally, parenterally (intravenously, intramuscularly or subcutaneously), transdermally, or by inhalation, and preferably orally.In certain embodiments, the PDE-1 inhibitor, for example the PDE-1 inhibitor in a depot formulation, is preferably administered parenterally, for example by injection.The immune checkpoint inhibitor can be administered by any suitable route, including orally, parenterally (intravenously, intramuscularly or subcutaneously), transdermally, or by inhalation, and preferably intravenously.

[0170] In general, for example, for the treatment of breast cancer, oral administration of PDE-1 inhibitors at doses of the order of about 0.01-2.0 mg / kg has been shown to provide satisfactory results.Accordingly, in larger mammals, such as humans, the recommended daily dose for oral administration of PDE-1 inhibitors ranges from about 0.50-300 mg, conveniently administered once a day or 2-4 times a day in divided doses, or in sustained release form.Thus, for example, a unit dosage form for oral administration can contain about 0.2-150 or 300 mg of PDE-1 inhibitor, for example about 0.2 or 2.0-10, 25, 50, 75, 100, 150 or 200 mg, together with a pharma- ceutically acceptable diluent or carrier therefor.

[0171] PDE1 inhibitors and immune checkpoint inhibitors can be used in combination with one or more additional therapeutic agents, particularly at lower doses than when each individual agent is used as monotherapy, to enhance the therapeutic activity of the combined agents without causing the undesirable side effects that commonly occur in conventional monotherapy.Therefore, PDE1 inhibitors and immune checkpoint inhibitors can be administered simultaneously, separately, sequentially, or contemporaneously with other agents useful for treating disease.In another example, side effects can be reduced or minimized by administering PDE1 inhibitors and immune checkpoint inhibitors in combination with one or more additional therapeutic agents in free or salt form, where the dose of (i) the additional therapeutic agent or (ii) PDE1 inhibitors and immune checkpoint inhibitors is lower than when the agent / inhibitor is administered as monotherapy.

[0172] The term "concurrently" when referring to therapeutic applications means that two or more active ingredients are administered at the same time or at about the same time and by the same route of administration.

[0173] The term "separately" when referring to therapeutic applications means that two or more active ingredients are administered simultaneously or at about the same time and by different routes of administration.

[0174] The pharmaceutical compositions may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus, oral dosage forms may include tablets, capsules, solutions, suspensions, and the like. EXAMPLES

[0175] Example – Effect of PDE1 inhibitor treatment alone or in combination with anti-PD-1 treatment in a mouse model of breast cancer

[0176] Example 1 material and method In vivo tumor implantation and treatment E0771 mouse breast cancer cells are obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium containing 10% fetal bovine serum, 4 mM glutamine, 20 mM HEPES, and 1% penicillin / streptomycin. The cells are cultured in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. E0771 tumor cells used for implantation are harvested and diluted to 2.5 x 10 6 The cells were resuspended at a concentration of 0.5 × 10 cells / mL. Nine-week-old female C57Bl / 6 mice (C57Bl / 6J, Jackson Laboratory) were inoculated into the right flank with 0.5 × 10 6 0.2 mL of cold PBS containing 100 x 100 cells is injected subcutaneously. After 10 days, the mice have palpable tumors of the designated size (approximately 100 mm) at which treatment can begin. 3 Tumors are measured bidimensionally with calipers to monitor size, and mice are then randomly assigned to groups. Tumor size is calculated using the following formula: tumor volume (mm 3 )=(w 2 × l) / 2 (where w is width and l is length in mm). Tumors were measured twice weekly for the duration of the study. Each mouse had a 1500 mm 3 Mice are euthanized when the tumor volume endpoint is reached or when they show any type of discomfort, whichever occurs first.

[0177] Mice are treated with Compound A alone, anti-PD-1 antibody alone, or a combination of Compound A and anti-PD-1 antibody. Compound A is synthesized at Intra-Cellular Therapies Inc., and Compound A diet is used to treat mice. Compound A diet 900 ppm (Picolab Rodent Diet 5053 supplemented with 900 mg / kg Compound A) is manufactured by Envigo (Madison WI). Mice are administered vehicle or Compound A 5 days per week.

[0178] Anti-PD-1 RMP1-14 (Lot No. 800121F12A) and isotype (Lot No. 749620N1) antibodies were purchased from BioXCell. Stock solutions were diluted with PBS to obtain 0.1 or 1 mg / mL dosing solutions, with a dose of 0.2 mL (10 mL / kg for 20 mg mice) to deliver 1 or 10 mg / kg, respectively. Mice were intraperitoneally administered isotype or anti-PD-1 RMP1-14 at 0.2 mL (10 mL / kg for 20 mg mice) twice weekly.

[0179] Animals are weighed twice weekly until the end of the study. Animals are observed frequently for signs of toxicity and any notable clinical findings are recorded. Animal weights are monitored and animals that experience greater than 15% weight loss at any one time are euthanized.

[0180] Flow cytometry and cell sorting Tumors are harvested and minced in digestion solution containing 2 mg / mL collagenase D (Sigma-Aldrich) and 1 mg / mL DNase I (Sigma-Aldrich). Samples are incubated at 37 °C for 30-45 min and passed through a 70 μm nylon cell strainer (Corning). The suspension is centrifuged at 1200 rpm for 3 min at 4 °C. Pelleted cells are harvested, resuspended in red blood cell lysis buffer (Sigma-Aldrich), incubated at room temperature for 5 min, washed with PBS, and centrifuged at 1200 rpm for 3 min at 4 °C. Cells are resuspended in 2% Fc Block (BD Bioscience) and blocked for 30 min. Fluorophore-conjugated primary antibodies are then incubated in FACS buffer (1X HBSS (Thermo Fisher Scientific), 2% BSA and 0.5 mM EDTA) for 30 min at room temperature. Antibodies used for the macrophage panel are PerCp-Cy5.5 anti-mouse CD45 (BD Bioscience), APC-Cy7 anti-mouse F4 / 80 (Invitrogen), PE anti-mouse CD11b (Biolegend), FITC anti-mouse INOS (BD Bioscience), BV510 anti-mouse CD80 (BD Bioscience), APC anti-mouse CD206 (Biolegend), and PE-Cy7 anti-mouse Arg1 (Invitrogen). Antibodies used for the T cell panel are PerCp-Cy5.5 anti-mouse CD45 (BD Bioscience), APC-Cy7 anti-mouse CD3 (Biolegend), PE-Cy7 anti-mouse CD8 (BD Bioscience), BV610 anti-mouse CD4 (Biolegend), and BV786 anti-mouse NK1.1 (Biolegend). The antibodies used to select macrophages are PerCp-Cy5.5 anti-mouse CD45 (BD Bioscience), APC anti-mouse F4 / 80 (Invitrogen), PE anti-mouse CD11b (Biolegend), and BV785 anti-mouse CD11c (Invitrogen). All antibodies are used at 1:200.After washing once with FACS buffer, cells are resuspended in FACS buffer and analyzed using an in-house CytoFLEX (Beckman Coulter) or FACSAria II cell sorter (BD Biosciences) for sorting and recovery of macrophages (M1 and M2).

[0181] RNAseq Tumors are extracted with PureLINK RNA Mini Kit (Invitrogen) and sorted macrophages are extracted with PicoPure RNA Isolation Kit (Thermo Fisher scientific), and the quality and quantity are measured using Agilent 2100 Bioanalyzer (Agilent Technologies). RNAseq libraries are prepared using TrueSeq Stranded mRNA Kit for tumors and Clontech SMARTer Stranded Total RNA Seq Kit - Pico Input Mammalian for sorted macrophages, according to the manufacturer's protocol. Libraries are purified using AMPure beads, equimolar pooled and paired-end reads are run on a HiSeq 6000. FASTQ files are obtained. Read data are mapped to the mouse mm10 genome and gene-level and differential expression analysis is performed using Rosalind tools. Genes with p-value < 0.05 and FC (fold change) < -1.5 or > 1.5 between conditions are determined to be differentially expressed. Heatmaps and pathway analyses are generated using Rosalind (https: / / www.rosalind.bio / ).

[0182] LC / MS / MS quantification of compound A Tumor, liver, and fecal samples are diluted in phosphate buffer and homogenized using an ultrasonicator. Plasma and homogenized tissue samples along with a set of standards and quality control samples are extracted by protein precipitation on Ostro plates (Waters) using 1% formic acid in acetonitrile spiked with internal standards. 2 μL of filtered sample is injected by autosampler and chromatographically separated using a Phenomenex, Synergi TM, 2.5 μm, 50 × 3 mm, Polar-RP column, an isocratic mobile phase combination of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40 / 60) at a flow rate of 0.8 mL / min. A Sciex Qtrap 6500 mass spectrometer is used as the detector with positive electrospray in MRM ionization mode and an ion source temperature of 650 °C. Quantification of compound A is performed using a calibration curve established using standards of corresponding analyte concentration, retention time, and mass profile.

[0183] 4. Data Analysis Statistical analysis was performed using GraphPad Prism 9.1.0. Data are reported as mean ± SD. In general, two-tailed unpaired Student's t-test was used when comparing two groups, and a p-value <0.05 was considered significant, with the level of significance being 1. * Results are presented as p<0.05. For in vivo experiments, n is the number of animals. Samples were randomly assigned to experimental groups and no data were excluded in the analysis.

[0184] result The effect of PDE1 inhibitors alone or in combination with sub-effective doses of programmed cell death-1 (PD-1) immune checkpoint inhibitors on tumor growth inhibition is examined in a syngeneic mouse model of triple-negative breast cancer. The effect of dietary delivered Compound A (900 ppm) on tumor growth is evaluated as monotherapy or in combination with an anti-PD1 antibody (RMP1-14) at a dose of 10 mg / kg. [ka]

[0185] Neither compound A (900 ppm) treatment alone nor anti-PD-1 antibody (10 mg / kg) treatment alone shows a significant effect on tumor volume at any given measurement time point (days 3, 7, 10, 14 and 17) (Figure 1 and Figure 2) or tumor weight at terminal sacrifice (day 17 of treatment) (Figure 3). However, the combination of compound A (900 ppm) treatment + anti-PD1 antibody (10 mg / kg) treatment results in a significant reduction in tumor volume and tumor weight compared to the isotype control (Figures 1-3). No significant differences in food consumption or mouse body weight were observed between groups. Furthermore, drug exposure to compound A in plasma, tissues and feces is also comparable in all groups administered compound A. These results demonstrate the synergistic ability of compound A and anti-PD-1 antibody to inhibit tumor growth in a mouse model of breast cancer.

[0186] Next, tumors from the isotype (control), compound A (900 ppm) alone, anti-PD1 antibody (10 mg / kg) and compound A (900 ppm) + anti-PD1 antibody (10 mg / kg) combination groups are analyzed for drug-associated changes in immune cells (macrophages, T cells and NK cells) in the tumor microenvironment by flow cytometry (Figures 4 and 5). The results show that compound A + anti-PD1 antibody combination treatment does not change the total number of macrophages in the tumor microenvironment (Figure 4A), but the combination treatment affects the M1 / M2 ratio (Figure 4B). M1 macrophages are inflammatory, while M2 macrophages are anti-inflammatory. Compound A + anti-PD1 antibody combination treatment significantly increases the M1 / M2 macrophage ratio compared to the isotype group (Figure 4B). The combination treatment did not significantly affect T cell populations (CD8+, CD4+) and NK cells as measured by flow cytometry (Figure 5).

[0187] RNAseq analysis is performed to examine drug-related gene expression changes in Compound A + anti-PD1 (10 mg / kg) tumors compared to the isotype (control) group. Comparative volcano plots are shown in Figure 6. The volcano plots show that 48 genes are downregulated and 136 genes are upregulated in Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors (fold change <-1.5 or >1.5; p<0.05). To characterize the role of these differentially expressed genes in Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors, pathway analysis is performed. Pathways enriched for differentially expressed genes in Compound A (900 ppm) + anti-PD1 (10 mg / kg) tumors are shown in Figure 7. Transcriptional regulators associated with upregulated or downregulated genes in Compound A + anti-PD1 (10 mg / kg) tumors are shown in Figure 7. RNAseq analysis shows that the combination therapy significantly downregulated genes involved in cell proliferation, survival and migration pathways, while upregulating genes involved in inflammatory pathways. These results suggest that PDE-1 inhibitors combined with anti-PD-1 antibodies promote antitumor immunity, leading to tumor growth inhibition.

[0188] Example 2 material and method In vivo tumor implantation and treatment 4T1 mouse breast cancer cells are obtained from the American Type Culture Collection (ATCC) and maintained in RPMI medium containing 10% fetal bovine serum, 4 mM glutamine, and 1% penicillin / streptomycin. The cells are cultured in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. 4T1 tumor cells used for implantation are harvested and diluted to 50 x 10 3 The cells were resuspended at a concentration of 10 × 10 cells / mL. 30.2 mL of cold PBS containing 100 x 100 cells is injected subcutaneously. After 10 days, the mice have palpable tumors of the designated size (approximately 100 mm) at which treatment can begin. 3 Tumors are measured bidimensionally with calipers to monitor size, and mice are then randomly assigned to groups. Tumor size is calculated using the following formula: tumor volume (mm 3 )=(w 2 × l) / 2 (where w is width and l is length in mm). Tumors were measured twice weekly for the duration of the study. Each mouse had a 1500 mm 3 Mice are euthanized when the tumor volume endpoint is reached or when they show any type of discomfort, whichever occurs first.

[0189] Mice are treated with Compound A alone, anti-PD-1 antibody alone, or a combination of Compound A and anti-PD-1 antibody. Compound A is synthesized at Intra-Cellular Therapies Inc., and Compound A diet is used to treat mice. Compound A diet 300 ppm or 900 ppm (Picolab Rodent Diet 5053 supplemented with 300 mg or 900 mg / kg Compound A diet) is manufactured by Envigo (Madison WI). Mice are administered vehicle or Compound A 5 days per week.

[0190] Anti-PD-1 RMP1-14 (Lot No. 800121F12A) and isotype (Lot No. 749620N1) antibodies were purchased from BioXCell. Stock solutions were diluted with PBS to obtain 0.1 or 1 mg / mL dosing solutions, with a dose of 0.2 mL (10 mL / kg for 20 mg mice) to deliver 1 or 10 mg / kg, respectively. Mice were intraperitoneally administered isotype or anti-PD-1 RMP1-14 at 0.2 mL (10 mL / kg for 20 mg mice) twice weekly.

[0191] Animals are weighed twice weekly until the end of the study. Animals are observed frequently for signs of toxicity and any notable clinical findings are recorded. Animal weights are monitored and animals that experience greater than 15% weight loss at any one time are euthanized.

[0192] LC / MS / MS quantification of compound A Tumor, liver, and fecal samples are diluted in phosphate buffer and homogenized using an ultrasonicator. Plasma and homogenized tissue samples along with a set of standards and quality control samples are extracted by protein precipitation on Ostro plates (Waters) using 1% formic acid in acetonitrile spiked with internal standards. 2 μL of filtered sample is injected by autosampler and chromatographically separated using a Phenomenex, Synergi TM, 2.5 μm, 50 × 3 mm, Polar-RP column, an isocratic mobile phase combination of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40 / 60) at a flow rate of 0.8 mL / min. A Sciex Qtrap 6500 mass spectrometer is used as the detector with positive electrospray in MRM ionization mode and an ion source temperature of 650 °C. Quantification of compound A is performed using a calibration curve established using standards of corresponding analyte concentration, retention time, and mass profile.

[0193] 4. Data Analysis Statistical analysis was performed using GraphPad Prism 9.1.0. Data are reported as mean ± SD. In general, two-tailed unpaired Student's t-test was used when comparing two groups, and a p-value <0.05 was considered significant, with the level of significance being 1. * p < 0.05; ** p<0.01; and *** Shown as p<0.001. For in vivo experiments, n is the number of animals. Samples were randomly assigned to experimental groups and no data were excluded in the analysis.

[0194] result The effect of compound A (300 ppm or 900 ppm) delivered in the diet on 4T1 tumor growth is evaluated as monotherapy or in combination with anti-PD1 antibody (RMP1-14) at a dose of 10 mg / kg. Tumor volumes (Figures 8 and 9) and tumor weights (Figure 10) for the anti-PD1 (10 mg / kg) group are not different from the isotype (control) at any given measurement time point or at the time of terminal sacrifice (day 14 of treatment). However, as shown in Figures 8-10, treatment with different compound A monotherapy doses or different combinations with anti-PD1 significantly reduces tumor volume or tumor weight compared to the isotype (control). Animal survival is also improved in the monotherapy and combination groups (Figure 11). Compound A exposure in plasma and tissues is comparable in groups administered the drug in the diet in the absence or presence of anti-PD1.

[0195] Example 3 material and method In vivo tumor implantation and treatment E0771 mouse breast cancer cells are obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium containing 10% fetal bovine serum, 4 mM glutamine, 20 mM HEPES, and 1% penicillin / streptomycin. The cells are cultured in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. E0771 tumor cells used for implantation are harvested and diluted to 2.5 x 10 6 The cells were resuspended at a concentration of 0.5 × 10 cells / mL. Nine-week-old female C57Bl / 6 mice (C57Bl / 6J, Jackson Laboratory) were inoculated into the right flank with 0.5 × 10 6 0.2 mL of cold PBS containing the cells is injected subcutaneously. After 10 days, the mice have palpable tumors of the designated size (approximately 100 mm) to start treatment. 3 Tumors are measured bidimensionally with calipers to monitor size, and mice are then randomly assigned to groups. Tumor size is calculated using the following formula: tumor volume (mm 3 )=(w 2× l) / 2 (where w is width and l is length in mm). Tumors were measured twice weekly for the duration of the study. Each mouse had a 1500 mm 3 Mice are euthanized when the tumor volume endpoint is reached or when they show any type of discomfort, whichever occurs first.

[0196] Mice are treated with Compound B alone, anti-PD-1 antibody alone, or a combination of Compound B and anti-PD-1 antibody. Compound B is synthesized at Intra-Cellular Therapies Inc., and Compound B diet is used to treat mice. Compound B diet 100 ppm, 300 ppm, or 900 ppm (Picolab Rodent Diet 5053 supplemented with 100, 300, or 900 mg / kg Compound B diet) is manufactured by Envigo (Madison WI). Mice are administered vehicle or Compound B 5 days per week.

[0197] Anti-PD-1 RMP1-14 (Lot No. 800121F12A) and isotype (Lot No. 749620N1) antibodies were purchased from BioXCell. Stock solutions were diluted with PBS to obtain 0.1 or 1 mg / mL dosing solutions, with a dose of 0.2 mL (10 mL / kg for 20 mg mice) to deliver 1 or 10 mg / kg, respectively. Mice were intraperitoneally administered isotype or anti-PD-1 RMP1-14 at 0.2 mL (10 mL / kg for 20 mg mice) twice weekly.

[0198] Animals are weighed twice weekly until the end of the study. Animals are observed frequently for signs of toxicity and any notable clinical findings are recorded. Animal weights are monitored and animals that experience greater than 15% weight loss at any one time are euthanized.

[0199] LC / MS / MS quantification of compound B Tumor, liver, and fecal samples are diluted with phosphate buffer and homogenized using an ultrasonicator. Plasma and homogenized tissue samples along with a set of standards and quality control samples are extracted by protein precipitation on Ostro plates (Waters) using 1% formic acid in acetonitrile spiked with internal standards. 2 μL of filtered sample is injected by autosampler and chromatographically separated using a Phenomenex, Synergi TM, 2.5 μm, 50 × 3 mm, Polar-RP column, an isocratic mobile phase combination of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40 / 60) at a flow rate of 0.8 mL / min. A Sciex Qtrap 6500 mass spectrometer is used as the detector with positive electrospray in MRM ionization mode and an ion source temperature of 650 °C. Quantification of compound B is performed using a calibration curve established using standards of corresponding analyte concentration, retention time, and mass profile.

[0200] 4. Data Analysis Statistical analysis was performed using GraphPad Prism 9.1.0 for PC. Data are reported as mean ± SD. In general, two-tailed unpaired Student's t-test was used when comparing two groups, and a p-value <0.05 was considered significant, with the significance level being 0.01. * p < 0.05; ** p<0.01; and *** Shown as p<0.001. For in vivo experiments, n is the number of animals. Samples were randomly assigned to experimental groups and no data were excluded in the analysis.

[0201] result The effect of dietary delivered Compound B (100 ppm, 300 ppm or 900 ppm) on E0771 tumor growth is evaluated as monotherapy or in combination with an anti-PD1 antibody (RMP1-14) at a dose of 1 mg / kg. [ka]

[0202] Tumor volumes (Figures 12 and 13) and tumor weights (Figure 14) in mice treated with anti-PD1 (1 mg / kg) alone are not different from isotype (control) at any given measurement time point or at the time of terminal sacrifice (day 17 of treatment). However, as shown in Figures 12-14, treatment with different doses of Compound B as monotherapy or in combination with anti-PD1 significantly reduces tumor volume or tumor weight compared to isotype (control) and / or anti-PD1 alone. Compound B exposure in plasma and tissues is comparable in groups that received the drug in the diet in the absence or presence of anti-PD1.

[0203] Example 4 material and method In vivo tumor implantation and treatment 4T1 mouse breast cancer cells are obtained from the American Type Culture Collection (ATCC) and maintained in RPMI medium containing 10% fetal bovine serum, 4 mM glutamine, and 1% penicillin / streptomycin. The cells are cultured in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. 4T1 tumor cells used for implantation are harvested and diluted to 50 x 10 3 The cells were resuspended at a concentration of 10 × 10 cells / mL. 3 0.2 mL of cold PBS containing 100 x 100 cells is injected subcutaneously. After 10 days, the mice have palpable tumors of the designated size (approximately 100 mm) at which treatment can begin. 3 Tumors are measured bidimensionally with calipers to monitor size, and mice are then randomly assigned to groups. Tumor size is calculated using the following formula: tumor volume (mm 3 )=(w 2 × l) / 2 (where w is width and l is length in mm). Tumors were measured twice weekly for the duration of the study. Each mouse had a 1500 mm 3 Mice are euthanized when the tumor volume endpoint is reached or when they show any type of discomfort, whichever occurs first.

[0204] Mice are treated with Compound B alone, anti-PD-1 antibody alone, or a combination of Compound B and anti-PD-1 antibody. Compound B is synthesized at Intra-Cellular Therapies Inc., and Compound B diet is used to treat mice. Compound B diet 100 ppm, 300 ppm, or 900 ppm (Picolab Rodent Diet 5053 supplemented with 100, 300, or 900 mg / kg Compound B diet) is manufactured by Envigo (Madison WI). Mice are administered vehicle or Compound B 5 days per week.

[0205] Anti-PD-1 RMP1-14 (Lot No. 800121F12A) and isotype (Lot No. 749620N1) antibodies were purchased from BioXCell. Stock solutions were diluted with PBS to obtain 0.1 or 1 mg / mL dosing solutions, with a dose of 0.2 mL (10 mL / kg for 20 mg mice) to deliver 1 or 10 mg / kg, respectively. Mice were intraperitoneally administered isotype or anti-PD-1 RMP1-14 at 0.2 mL (10 mL / kg for 20 mg mice) twice weekly.

[0206] Animals are weighed twice weekly until the end of the study. Animals are observed frequently for signs of toxicity and any notable clinical findings are recorded. Animal weights are monitored and animals that experience greater than 15% weight loss at any one time are euthanized.

[0207] Flow cytometry and cell sorting Flow cytometry and cell sorting are performed essentially as described in Example 1.

[0208] RNAseq RNAseq is performed essentially as described in Example 1.

[0209] LC / MS / MS quantification of compound B Tumor, liver, and fecal samples are diluted in phosphate buffer and homogenized using an ultrasonicator. Plasma and homogenized tissue samples along with a set of standards and quality control samples are extracted by protein precipitation on Ostro plates (Waters) using 1% formic acid in acetonitrile spiked with internal standards. 2 μL of filtered sample is injected by autosampler and chromatographically separated using a Phenomenex, Synergi TM, 2.5 μm, 50 × 3 mm, Polar-RP column, an isocratic mobile phase combination of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40 / 60) at a flow rate of 0.8 mL / min. A Sciex Qtrap 6500 mass spectrometer is used as the detector with positive electrospray in MRM ionization mode and an ion source temperature of 650 °C. Quantification of compound A is performed using a calibration curve established using standards of corresponding analyte concentration, retention time, and mass profile.

[0210] 4. Data Analysis Statistical analysis was performed using GraphPad Prism 9.1.0. Data are reported as mean ± SD. In general, two-tailed unpaired Student's t-test was used when comparing two groups, and a p-value <0.05 was considered significant, with the level of significance being 1. * p < 0.05; ** p<0.01; and *** Shown as p<0.001. For in vivo experiments, n is the number of animals. Samples were randomly assigned to experimental groups and no data were excluded in the analysis.

[0211] result The effect of compound B (100 ppm, 300 ppm or 900 ppm) delivered in the diet on 4T1 tumor growth is evaluated, either as monotherapy or in combination with anti-PD1 antibody (RMP1-14) at a dose of 10 mg / kg. Tumor volumes (Figures 15 and 16) and tumor weights (Figure 17) in mice treated with anti-PD1 (10 mg / kg) alone are not different from isotype (control) in the period after experimental day 5 or at the time of terminal sacrifice (day 14 of treatment). However, as shown in Figures 15-17, treatment with different doses of compound B as monotherapy or treatment with different doses of compound B in combination with anti-PD1 significantly reduces tumor volume or tumor weight compared to isotype (control) and / or anti-PD1 alone. Compound B exposure in plasma and tissues is comparable in groups receiving the drug in the diet in the absence or presence of anti-PD1.

[0212] Next, tumors from the isotype (control), compound B (900 ppm) alone, anti-PD1 antibody (10 mg / kg) and compound B (900 ppm) + anti-PD1 antibody (10 mg / kg) combination groups are analyzed for drug-associated changes in immune cells (macrophages, T cells and NK cells) in the tumor microenvironment by flow cytometry (Figures 18 and 19). The results show that compound B + anti-PD1 antibody combination treatment does not change the total number of macrophages in the tumor microenvironment (Figure 18A), but the combination treatment affects the M1 / M2 ratio (Figure 18B). M1 macrophages are inflammatory, while M2 macrophages are anti-inflammatory. Compound B + anti-PD1 antibody combination treatment significantly increases the M1 / M2 macrophage ratio compared to the isotype group (Figure 18B). The combination treatment does not significantly affect the T cell population as measured by flow cytometry (Figure 19).

[0213] RNAseq analysis is performed to examine drug-related gene expression changes in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors compared to the isotype (control) group. A comparative volcano plot is shown in Figure 20. The volcano plot shows that 708 genes are upregulated and 281 genes are downregulated in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors (fold change <-1.5 or >1.5; p<0.05). Pathway analysis is performed to characterize the role of these differentially expressed genes in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors. Pathways enriched in differentially expressed genes (including genes regulating inflammatory processes such as type II interferon signaling, or chemokine signaling pathways including cytokine and inflammatory responses) and downregulated pathways (including those involved in cell proliferation, survival and migration pathways) in Compound B + anti-PD1 (10 mg / kg) tumors are shown in Figure 21A. Transcriptional regulators associated with upregulated or downregulated genes in Compound B (900 ppm) + anti-PD1 (10 mg / kg) tumors are shown in Figure 21B. RNAseq analysis shows that the combination therapy significantly downregulated genes involved in cell proliferation, survival and migration pathways (TFAP2A, SP1, TEAD2 and FOS), while upregulated genes involved in inflammatory pathways (PRDM1, IRF8, NFKβ1, HIF1A, STAT1 and NR3C1). These results suggest that Compound B in combination with anti-PD1 antibody promotes anti-tumor immunity, leading to tumor growth inhibition.

Claims

1. A pharmaceutical product for the treatment of breast cancer comprising a pharmaceutically acceptable amount of a PDE1 inhibitor alone or in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor, wherein the PDE1 inhibitor is (A) Formula I in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Chemistry 1】 [In the formula, (i) R1 is H or C1-4 alkyl (e.g., methyl); (ii) R4 is H or C1-4 alkyl, and R2 and R3 are independently H or C1-4 alkyl (e.g., both R2 and R3 are methyl, or R2 is H and R3 is isopropyl), aryl, heteroaryl, (may be hetero) arylalkoxy, or (may be hetero) arylalkyl; or R2 is H, and R3 and R4 together form a dimethylene, trimethylene, or tetramethylene bridge (preferably R3 and R4 together have a cis configuration, for example, the carbon supporting R3 and R4 has an R configuration and an S configuration, respectively); (iii) R 5 is a substituted heteroarylalkyl, for example, substituted with a haloalkyl; or R5 is bonded to one of the nitrogen atoms in the pyrazoro portion of formula I, and formula A: 【Chemistry 2】 (wherein X, Y, and Z are independently N or C; R8, R9, R11, and R12 are independently H or halogen (e.g., Cl or F); and R10 is a halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), possibly halogen-substituted heteroaryl (e.g., pyridyl (e.g., pyrida-2-yl), or thiadiazolyl (e.g., 1,2,3-thiazole-4-yl)), diazolyl, triazolyl, tetrazolyl, arylcarbonyl (e.g., benzoyl), alkylsulfonyl (e.g., methylsulfonyl), heteroarylcarbonyl, or alkoxycarbonyl; however, if X, Y, or Z is nitrogen, R8, R9, or R10 are absent, respectively.) This is the part indicated by; (iv) R 6 is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g., phenylamino), heteroarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-(arylalkyl)amino (e.g., N-phenyl-N-(1,1'-bifen-4-ylmethyl)amino); (v) n is 0 or 1; (vi) When n is 1, A is -C(R13 R14)- (where R13 and R14 are independently H, or C1-4 alkyl, aryl, heteroaryl, (may be hetero)arylalkoxy or (may be hetero)arylalkyl); (B) Formula II in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Transformation 3】 (i) X is a C1-6 alkylene (e.g., methylene, ethylene, or prop-2-in-1-ylene); (ii) Y is a single bond, an alkynylene (e.g., -C≡C-), an arylene (e.g., phenylene), or a heteroarylene (e.g., pyridylene); (iii) Z is a C3-7 cycloalkyl (e.g., cyclopentyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, or morpholinyl) which may contain at least one atom selected from the group consisting of H, aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrida-2-yl), halo (e.g., F, Br, Cl), halo C1-6 alkyl (e.g., trifluoromethyl), -C(O)-R1, -N(R2)(R3), or N or O; (iv) R1 is a C1-6 alkyl, a halo C1-6 alkyl, -OH, or -OC1-6 alkyl (e.g., -OCH3); (v) R2 and R3 are independently H or C1-6 alkyl; (vi) R4 and R5 are independently H, C1-6 alkyl, or aryl (e.g., phenyl) which may be substituted with one or more halo (e.g., fluorophenyl, e.g., 4-fluorophenyl), hydroxy (e.g., hydroxyphenyl, e.g., 4-hydroxyphenyl or 2-hydroxyphenyl), or C1-6 alkoxy; (vii) X, Y, and Z may be independently substituted with one or more halos (e.g., F, Cl, or Br), C1-6 alkyls (e.g., methyl), or halo-C1-6 alkyls (e.g., trifluoromethyl), for example, Z may be a heteroaryl, e.g., pyridyl, substituted with one or more halos (e.g., 6-fluoropyrida-2-yl, 5-fluoropyrida-2-yl, 6-fluoropyrida-2-yl, 3-fluoropyrida-2-yl, 4-fluoropyrida-2-yl, 4,6-dichloropyrida-2-yl), halo-C1-6 alkyls (e.g., 5-trifluoromethylpyrida-2-yl), or C1-6 alkyls (e.g., 5-methylpyrida-2-yl), or Z may be an aryl, e.g., phenyl, substituted with one or more halos (e.g., 4-fluorophenyl); (C) Formula III in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Chemistry 4】 [In the formula, (i) R1 is H or C1-4 alkyl (e.g., methyl or ethyl); (ii) R2 and R3 are independently H or C1-6 alkyl (e.g., methyl or ethyl); (iii) R 4 is H or C 1-4 alkyl (e.g., methyl or ethyl); (iv) R5 is an aryl (e.g., phenyl) which may be independently substituted with one or more groups selected from -C(=O)-C1-6 alkyl (e.g., -C(=O)-CH3) and C1-6-hydroxyalkyl (e.g., 1-hydroxyethyl); (v) R6 and R7 are independently H, or aryl (e.g., phenyl) which may be independently substituted with one or more groups selected from C1-6 alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), e.g., unsubstituted phenyl or phenyl substituted with one or more halogens (e.g., F), or phenyl substituted with one or more C1-6 alkyl and one or more halogens, or phenyl substituted with one C1-6 alkyl and one halogen, e.g., 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3-methylphenyl; (vi)n is 1, 2, 3 or 4]; (D) Formula IV in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates) 【Transformation 5】 [In the formula, (i) R1 is a C1-4 alkyl (e.g., methyl or ethyl), or -NH(R2), where R2 is a phenyl which may be substituted with a halo (e.g., fluoro), e.g., 4-fluorophenyl; (ii) X, Y, and Z are independently N or C; (iii) R3, R4 and R5 are independently H or C1-4 alkyl (e.g., methyl); or R3 is H and R4 and R5 together form a trimethylene bridge (preferably R4 and R5 together have a cis configuration, for example the carbon supporting R4 and R5 each having an R configuration and an S configuration), (iv) R6, R7 and R8 are independent of each other. H, C1-4 alkyl (e.g., methyl), Pyrida-2-yl substituted with hydroxyl, or -S(O) 2 -NH 2 And; However, if X, Y, and / or Z are N, then R6, R7, and / or R8 do not exist, respectively; and if X, Y, and Z are all C, then at least one of R6, R7, or R8 is -S(O)2-NH2 or pyrida-2-yl substituted with hydroxyl. (E) Formula 1a in free form, pharmaceutically acceptable salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Transformation 6】 [In the formula, (i) R2 and R5 are independently H or hydroxyl, and R3 and R4 together form a trimethylene crosslink or a tetramethylene crosslink [preferably, the carbon supporting R3 and R4 has an R configuration and an S configuration, respectively]; or R2 and R3 are each methyl, and R4 and R5 are each H; or R2, R4 and R5 are H, and R3 is isopropyl [preferably, the carbon supporting R3 has an R configuration]; (ii) R 6 is a (halosubstituted) phenylamino, a (halosubstituted) benzylamino, a C1-4 alkyl, or a C1-4 alkyl sulfide; for example, phenylamino or 4-fluorophenylamino; (iii) R 10 is C1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, (halosubstituted or hydroxysubstituted) phenyl, (halosubstituted or hydroxysubstituted) pyridyl (e.g., 6-fluoropyrida-2-yl), or thiadiazolyl (e.g., 1,2,3-thiazole-4-yl); (iv) X and Y are independently C or N]; (F) In free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates), formula V 【Transformation 7】 [In the formula, (i) R1 is -NH(R4), where R4 is a phenyl which may be substituted with a halo (e.g., fluoro), such as 4-fluorophenyl; (ii) R2 is H or C1-6 alkyl (e.g., methyl, isobutyl, or neopentyl); (iii) R3 is -SO₂NH₂ or -COOH; and / or (G) In free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates), formula V 【Transformation 8】 [In the formula, (i) R1 is -NH(R4), where R4 is a phenyl which may be substituted with a halo (e.g., fluoro), such as 4-fluorophenyl; (ii) R2 is H or C1-6 alkyl (e.g., methyl or ethyl); (iii) R3 is H, halogen (e.g., bromo), C1-6 alkyl (e.g., methyl), halogen-substituted aryl (e.g., 4-fluorophenyl), halogen-substituted heteroaryl (e.g., 6-fluoropyrida-2-yl or pyrida-2-yl), or acyl (e.g., acetyl); A pharmaceutical compound selected from among these.

2. The pharmaceutical product according to claim 1, wherein the breast cancer is triple-negative breast cancer (TNBC), and the TNBC may be high-risk early-stage TNBC.

3. The pharmaceutical product according to claim 2, wherein the treatment is adjunctive therapy after surgical removal of TNBC.

4. The pharmacopoeia according to claim 2, wherein the subject is a locally recurrent, unresectable or metastatic TNBC (compound positive score (CPS) ≥ 1) tumor expressing PD-L1.

5. The pharmaceutical product according to claim 1, wherein the immune checkpoint inhibitor is selected from one or more inhibitors of CTLA-4, PD-1, and / or PD-L1, and the immune checkpoint inhibitor may be a PD-1 inhibitor, or the immune checkpoint inhibitor may be an anti-PD-1 antibody.

6. The pharmacopoeia according to claim 1, wherein the immune checkpoint inhibitor comprises one or more members selected from nivolumab, pembrolizumab, semiprimab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

7. The pharmaceutical product according to claim 1, wherein the subject is suffering from a systemic inflammatory response, gastrointestinal inflammation-related disorder, endocrine inflammation-related disorder, skin inflammation-related disorder, ocular inflammation-related disorder, neurological inflammation-related disorder, hematological inflammation-related disorder, genitourinary inflammation-related disorder, respiratory inflammation-related disorder, musculoskeletal inflammation-related disorder, cardiac inflammation-related disorder, or a defined systemic inflammation-related disorder.

8. The pharmacopoeia according to any one of claims 1 to 7, wherein administration to a subject in a pharmaceutically acceptable amount of a PDE1 inhibitor alone or in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor increases the M1 / M2 ratio of macrophages in the tumor microenvironment.

9. A pharmaceutical product comprising a pharmaceutically acceptable amount of a PDE1 inhibitor for the prevention or reduction of disease, impairment or adverse effects resulting from the administration of immune checkpoint inhibitor therapy to a subject with breast cancer, comprising administering a reduced amount of the checkpoint inhibitor to the subject, wherein the PDE1 inhibitor is (A) Formula I in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Chemistry 9】 [In the formula, (i) R1 is H or C1-4 alkyl (e.g., methyl); (ii) R4 is H or C1-4 alkyl, and R2 and R3 are independently H or C1-4 alkyl (e.g., both R2 and R3 are methyl, or R2 is H and R3 is isopropyl), aryl, heteroaryl, (may be hetero) arylalkoxy, or (may be hetero) arylalkyl; or R2 is H, and R3 and R4 together form a dimethylene, trimethylene, or tetramethylene bridge (preferably R3 and R4 together have a cis configuration, for example, the carbon supporting R3 and R4 has an R configuration and an S configuration, respectively); (iii) R 5 is a substituted heteroarylalkyl, for example, substituted with a haloalkyl; or R5 is bonded to one of the nitrogen atoms in the pyrazoro portion of formula I, and formula A: 【Chemistry 10】 (wherein X, Y, and Z are independently N or C; R8, R9, R11, and R12 are independently H or halogen (e.g., Cl or F); and R10 is a halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), possibly halogen-substituted heteroaryl (e.g., pyridyl (e.g., pyrida-2-yl), or thiadiazolyl (e.g., 1,2,3-thiazole-4-yl)), diazolyl, triazolyl, tetrazolyl, arylcarbonyl (e.g., benzoyl), alkylsulfonyl (e.g., methylsulfonyl), heteroarylcarbonyl, or alkoxycarbonyl; however, if X, Y, or Z is nitrogen, R8, R9, or R10 are absent, respectively.) This is the part indicated by; (iv) R 6 is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g., phenylamino), heteroarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-(arylalkyl)amino (e.g., N-phenyl-N-(1,1'-bifen-4-ylmethyl)amino); (v) n is 0 or 1; (vi) When n is 1, A is -C(R13 R14)- (where R13 and R14 are independently H, or C1-4 alkyl, aryl, heteroaryl, (may be hetero)arylalkoxy or (may be hetero)arylalkyl); (B) Formula II in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Chemistry 11】 (i) X is a C1-6 alkylene (e.g., methylene, ethylene, or prop-2-in-1-ylene); (ii) Y is a single bond, an alkynylene (e.g., -C≡C-), an arylene (e.g., phenylene), or a heteroarylene (e.g., pyridylene); (iii) Z is a C3-7 cycloalkyl (e.g., cyclopentyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, or morpholinyl) which may contain at least one atom selected from the group consisting of H, aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrida-2-yl), halo (e.g., F, Br, Cl), halo C1-6 alkyl (e.g., trifluoromethyl), -C(O)-R1, -N(R2)(R3), or N or O; (iv) R1 is a C1-6 alkyl, a halo C1-6 alkyl, -OH, or -OC1-6 alkyl (e.g., -OCH3); (v) R2 and R3 are independently H or C1-6 alkyl; (vi) R4 and R5 are independently H, C1-6 alkyl, or aryl (e.g., phenyl) which may be substituted with one or more halo (e.g., fluorophenyl, e.g., 4-fluorophenyl), hydroxy (e.g., hydroxyphenyl, e.g., 4-hydroxyphenyl or 2-hydroxyphenyl), or C1-6 alkoxy; (vii) X, Y, and Z may be independently substituted with one or more halos (e.g., F, Cl, or Br), C1-6 alkyls (e.g., methyl), or halo-C1-6 alkyls (e.g., trifluoromethyl), for example, Z may be a heteroaryl, e.g., pyridyl, substituted with one or more halos (e.g., 6-fluoropyrida-2-yl, 5-fluoropyrida-2-yl, 6-fluoropyrida-2-yl, 3-fluoropyrida-2-yl, 4-fluoropyrida-2-yl, 4,6-dichloropyrida-2-yl), halo-C1-6 alkyls (e.g., 5-trifluoromethylpyrida-2-yl), or C1-6 alkyls (e.g., 5-methylpyrida-2-yl), or Z may be an aryl, e.g., phenyl, substituted with one or more halos (e.g., 4-fluorophenyl); (C) Formula III in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Chemistry 12】 [In the formula, (i) R1 is H or C1-4 alkyl (e.g., methyl or ethyl); (ii) R2 and R3 are independently H or C1-6 alkyl (e.g., methyl or ethyl); (iii) R 4 is H or C 1-4 alkyl (e.g., methyl or ethyl); (iv) R5 is an aryl (e.g., phenyl) which may be independently substituted with one or more groups selected from -C(=O)-C1-6 alkyl (e.g., -C(=O)-CH3) and C1-6-hydroxyalkyl (e.g., 1-hydroxyethyl); (v) R6 and R7 are independently H, or aryl (e.g., phenyl) which may be independently substituted with one or more groups selected from C1-6 alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), e.g., unsubstituted phenyl or phenyl substituted with one or more halogens (e.g., F), or phenyl substituted with one or more C1-6 alkyl and one or more halogens, or phenyl substituted with one C1-6 alkyl and one halogen, e.g., 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3-methylphenyl; (vi)n is 1, 2, 3 or 4]; (D) Formula IV in free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates) 【Chemistry 13】 [In the formula, (i) R1 is a C1-4 alkyl (e.g., methyl or ethyl), or -NH(R2), where R2 is a phenyl which may be substituted with a halo (e.g., fluoro), e.g., 4-fluorophenyl; (ii) X, Y, and Z are independently N or C; (iii) R3, R4 and R5 are independently H or C1-4 alkyl (e.g., methyl); or R3 is H and R4 and R5 together form a trimethylene bridge (preferably R4 and R5 together have a cis configuration, for example the carbon supporting R4 and R5 each having an R configuration and an S configuration), (iv) R6, R7 and R8 are independent of each other. H, C1-4 alkyl (e.g., methyl), Pyrida-2-yl substituted with hydroxyl, or -S(O) 2 -NH 2 And; However, if X, Y, and / or Z are N, then R6, R7, and / or R8 do not exist, respectively; and if X, Y, and Z are all C, then at least one of R6, R7, or R8 is -S(O)2-NH2 or pyrida-2-yl substituted with hydroxyl. (E) Formula 1a in free form, pharmaceutically acceptable salt form or prodrug form (including its enantiomers, diastereoisomers and racemates): 【Chemistry 14】 [In the formula, (i) R2 and R5 are independently H or hydroxyl, and R3 and R4 together form a trimethylene crosslink or a tetramethylene crosslink [preferably, the carbon supporting R3 and R4 has an R configuration and an S configuration, respectively]; or R2 and R3 are each methyl, and R4 and R5 are each H; or R2, R4 and R5 are H, and R3 is isopropyl [preferably, the carbon supporting R3 has an R configuration]; (ii) R 6 is a (halosubstituted) phenylamino, a (halosubstituted) benzylamino, a C1-4 alkyl, or a C1-4 alkyl sulfide; for example, phenylamino or 4-fluorophenylamino; (iii) R 10 is C1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, (halosubstituted or hydroxysubstituted) phenyl, (halosubstituted or hydroxysubstituted) pyridyl (e.g., 6-fluoropyrida-2-yl), or thiadiazolyl (e.g., 1,2,3-thiazole-4-yl); (iv) X and Y are independently C or N]; (F) In free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates), formula V 【Chemistry 15】 [In the formula, (i) R1 is -NH(R4), where R4 is a phenyl which may be substituted with a halo (e.g., fluoro), such as 4-fluorophenyl; (ii) R2 is H or C1-6 alkyl (e.g., methyl, isobutyl, or neopentyl); (iii) R3 is -SO₂NH₂ or -COOH; and / or (G) In free form, salt form or prodrug form (including its enantiomers, diastereoisomers and racemates), formula V 【Chemistry 16】 [In the formula, (i) R1 is -NH(R4), where R4 is a phenyl which may be substituted with a halo (e.g., fluoro), such as 4-fluorophenyl; (ii) R2 is H or C1-6 alkyl (e.g., methyl or ethyl); (iii) R3 is H, halogen (e.g., bromo), C1-6 alkyl (e.g., methyl), halogen-substituted aryl (e.g., 4-fluorophenyl), halogen-substituted heteroaryl (e.g., 6-fluoropyrida-2-yl or pyrida-2-yl), or acyl (e.g., acetyl); A pharmaceutical compound selected from among these.

10. The pharmaceutical product according to claim 9, wherein the breast cancer is triple-negative breast cancer (TNBC), the checkpoint inhibitor may be selected from one or more inhibitors of CTLA-4, PD-1, and / or PD-L1, and the immune checkpoint inhibitor may be an anti-PD-1 antibody.

11. PDE1 inhibitors, in free form or pharmaceutically acceptable salt form, 【Chemistry 17】 A pharmaceutical product according to claim 1 or 9, selected from any of the following.

12. PDE1 inhibitors are available in free form or pharmaceutically acceptable salt form, for example, monophosphate form. [Chemistry 18] The pharmaceutical product according to claim 11.

13. PDE1 inhibitors, in free form or pharmaceutically acceptable salt form, 【Chemistry 19】 The pharmaceutical product according to claim 11.

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