NOVEL USES

FIELD OF THE INVENTION

[0001] The field relates to the administration of inhibitors of phosphodiesterase 1 (PDE1) inhibitors for promoting the resolution of inflammation, for example through the polarization of Ml macrophages to M2 macrophages, and the treatment and prophylaxis of diseases or disorders related to inflammation.

BACKGROUND OF THE INVENTION

[0002] Eleven families of phosphodiesterases (PDEs) have been identified but only PDEs in Family I, the Ca ²⁺ -calmodulin-dependent phosphodiesterases (CaM-PDEs), are activated by the Ca ²⁺ -calmodulin and have been shown to mediate the calcium and cyclic nucleotide (e.g. cAMP and cGMP) signaling pathways. These PDEs are therefore active in stimulated conditions when intra-cellular calcium levels rise, leading to increased hydrolysis of cyclic nucleotides. The three known CaM-PDE genes, PDE1A, PDE1B, and PDE1C, are all expressed in central nervous system tissue. In the brain, the predominant expression of PDE1A is in the cortex and neostriatum, PDE1B is expressed in the neostriatum, prefrontal cortex, hippocampus, and olfactory tubercle, and PDE1C is more ubiquitously expressed.

[0003] PDE4 is the major cAMP-metabolizing enzyme found in inflammatory and immune cells, and PDE4 inhibitors are of interest as anti-inflammatory drugs. PDE1, however, has not been thought to play a major role in the inflammatory response, although PDE-1 is induced in monocyte-to-macrophage differentiation mediated by the cytokine granulocyte-macrophage colony- stimulating factor (GM-CSF). The PDE1 inhibitor vinpocetine has been shown to be anti inflammatory, but the anti-inflammatory action of vinpocetine is believed to be caused by a direct inhibition of the IKB kinase complex (IKK) rather than PDE blockade.

[0004] Macrophages have a central role in maintaining homeostasis and mediating inflammation in the body. Macrophages are capable of polarization by which a macrophage expresses different functional programs in response to microenvironmental signals. There are several activated forms of macrophages, but the two main groups are designated as Ml and M2. Ml macrophages, also referred to as“classically activated macrophages,” are activated by LPS and IFN-gamma, and secrete high levels of IL-12 and low levels of IL-10 for a pro-inflammatory effect. In contrast, the M2 designation, also referred to as“alternatively activated macrophages,” broadly refers to macrophages that function in constructive processes like wound healing and tissue repair, and those that turn off damaging immune system activation by producing anti inflammatory cytokines like IL-10. M2 macrophages produce high levels of IL-10, TGF-beta and low levels of IL-12. Prolonged Ml type of macrophages is harmful for the organism and that is why tissue repair and restoration is necessary.

[0005] When tissues are challenged by pathogens, inflammatory monocytes in circulation are recruited and differentiated into macrophages. Generally, macrophages are polarized toward an Ml phenotype in the early stages of bacterial infection. When the bacteria are recognized by pathogen recognition receptors, macrophages are activated and produce a large amount of pro- inflammatory mediators including TNF-a, IL-l, and nitric oxide (NO), which kill the invading organisms and activate the adaptive immunity. For example, this mechanism has been considered to be involved in infection with Salmonella typhi, Salmonella typhimurium, Listeria

monocytogenes, and the early phases of infection with Mycobacterium tuberculosis,

Mycobacterium ulcerans, and Mycobacterium avium. If macrophage-mediated inflammatory response cannot be quickly controlled, a cytokine storm is formed, thereby contributing to the pathogenesis of severe sepsis. In order to counteract the excessive inflammatory response, macrophages undergo apoptosis or polarize to an M2 phenotype to protect the host from excessive injury and facilitate wound healing.

[0006] Macrophage polarization is also involved in virus infection, in which M2 phenotype macrophages can also suppress inflammation and promote tissue healing. Influenza virus augments the phagocytic function of human macrophages, which is a major feature of M2 phenotype, to clear apoptotic cells and accelerate the resolution of inflammation. In severe acute respiratory syndrome (SARS)-Cov infection, M2 phenotype macrophages are critical to regulate immune response and protect host from the long-term progression to fibrotic lung disease by a STAT dependent pathway. In addition, severe respiratory syncytial virus (RSV) induced bronchiolitis is closely associated with mixed Ml and M2 macrophages.

[0007] Many viruses elicit mechanisms to adapt and modulate macrophage polarization. In human monocyte-derived macrophages, HIV-l infection has been observed to skew cells toward a Ml-like status, which correlates with downregulation of M2-status markers (i.e., CD163, CD206, CCL18, and IL-10) and increased secretion of Ml -associated chemokines including CCL3, CCL4, and CCL5.

[0008] Macrophage polarization has also been shown to play a significant role in various inflammatory diseases and disorders, such as nonalcoholic steatohepatitis (NASH),

atherosclerosis, metabolic disease, systemic lupus erythematosus, among many others.

[0009] It has not been previously shown that PDE1 has a significant role in mediating resolution of inflammation, or that it would have a significant effect on inflammatory diseases. Inflammatory processes in general, and diseases and disorders related to inflammation, are numerous, and the mechanisms and actions are still not well understood. Currently, there is a largely unmet need for an effective way of treating inflammation and inflammatory related diseases and disorders, especially with regard to inflammation occurring in the brain.

BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 depicts the number of leukocytes detected at the site of inflammation following sterile insult when treated with Compound 1.

[0011] Figure 2A depicts the number of macrophages detected at the site of inflammation following sterile insult when treated with Compound 1.

[0012] Figure 2B depicts the number of macrophages expressed as percent of total leukocytes detected at the site of inflammation following sterile insult when treated with Compound 1.

[0013] Figure 3A depicts the number of neutrophils detected at the site of inflammation following sterile insult when treated with Compound 1.

[0014] Figure 3B depicts the amount of neutrophils expressed as percent of total leukocytes detected at the site of inflammation following sterile insult when treated with Compound 1.

[0015] Figure 4A depicts the amount of Ml macrophages expressed as a percentage of total macrophages detected at the site of inflammation following sterile insult when treated with Compound 1.

[0016] Figure 4B depicts the amount of M2 macrophages expressed as a percentage of total macrophages detected at the site of inflammation following sterile insult when treated with Compound 1. [0017] Figure 5A depicts the number of Ml macrophages detected at the site of inflammation in the M2 activation state following sterile insult when treated with Compound 1.

[0018] Figure 5B depicts the number of M2 macrophages detected at the site of

inflammation following sterile insult when treated with Compound 1.

[0019] Figure 6A depicts the mean fluorescent intensity (MFI) of CD38 expression on macrophage populations detected at the site of inflammation following sterile insult when treated with Compound 1.

[0020] Figure 6B depicts the mean fluorescent intensity (MFI) of CD38 expression on macrophage populations detected at the site of inflammation following sterile insult when treated with Compound 1.

[0021] Figure 7 depicts cytokine production in plasma in test subjects following sterile insult when treated with Compound 1.

[0022] Figure 8 depicts the number of macrophages in the Ml activation state detected at the site of inflammation following sterile insult when treated with Compound 2.

[0023] Figure 9 depicts the number of macrophages in the M2 activation state detected at the site of inflammation following sterile insult when treated with Compound 2.

[0024] Figure 10 depicts the results of Compound 1 on the motility of BV2 cells in a microglia chemotaxis assay.

[0025] Figure 11 A depicts the amount of CD80+ macrophages expressed as a percentage of total macrophages detected at the site of inflammation.

[0026] Figure 11B depicts the amount of iNOS+ macrophages expressed as a percentage of total macrophages detected at the site of inflammation.

[0027] Figure 12A depicts the amount of Argl+ macrophages expressed as a percentage of total macrophages detected at the site of inflammation.

[0028] Figure 12A depicts the amount of CD206+ macrophages expressed as a percentage of total macrophages detected at the site of inflammation.

SUMMARY OF THE INVENTION

[0029] Surprisingly, we have discovered that PDE1 mediates the expression of certain pro- inflammatory cytokines and chemokines and that PDE1 inhibitors have specific anti

inflammatory effects. In one aspect, inhibition of PDE1 regulates inflammatory activity in macrophages, reducing expression of pro-inflammatory genes, thereby providing novel treatments for a variety of disorders and conditions characterized by macrophage mediation.

[0030] Positive regulation of inflammatory resolution responses in macrophages by elevated intracellular cyclic nucleotide levels provides a promising area for therapeutic intervention. It is known that PDE1B is present in monocytes and involved in the differentiation into macrophage via growth factor activation signals such as GM-CSF. Bender AT, et al., Selective up-regulation of PDE1B2 upon monocyte-to-macrophage differentiation, Proc Natl Acad Sci U S A. 2005 Jan 11; 102(2): 497-502. Cyclic guanosine monophosphate (cGMP) has been demonstrated to be a key modulator of the differentiation pathways in macrophages. cGMP also plays a role in modulation of inflammatory processes, such as inducible NO synthase induction and TNF-a release. Therefore, the marked up-regulation of PDE1B may be critical in the regulation of these processes in differentiated macrophages. This suggests that PDE1 inhibitors, such as those disclosed herein, may prove beneficial in diseases associated with, for example, inflammation disorders relating to macrophage activation.

[0031] In one embodiment, therefore, the invention provides using various PDE1 inhibitory compounds to treat inflammation, and/or diseases or disorders related to inflammation. Without being bound by theory, one possible mechanism for this activity is that inhibition of PDE1B may affect macrophage activation in the blood and/or microglial activation in the CNS, so as to reduce Ml activation and the release of pro-inflammatory cytokines, and to promote the polarization of macrophages to M2 type through the up-regulation of anti-inflammatory cytokines such as IL-10. Discussion of the treatment of and prophylaxis of inflammation and/or diseases or disorders related to inflammation as they relate to the microglia e.g.,

neuroinflammation, is discussed in International Publication WO 2018/049417, which is hereby incorporated by reference in its entirety.

[0032] The regulation of Ml to M2 type activation in macrophages is central to

inflammatory pathways in a number of disorders. The role of Ml to M2 polarization in macrophages is important in a number of inflammatory-related disorders including bacterial infections (e.g., Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes,

Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium avium infections); viral infections (e.g., African Swine Fever Virus, Classical Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human Immunodeficiency Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus-2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome

Coronavirus, West Nile Virus, Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C)); parasitic infestations (e.g., Taenia crassiceps, Toxoplasma gondii, Leishmania infantum, Schistosoma mansoni infestations); atopic dermatitis; pneumonia; cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance; asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD); neuropathic pain; stroke; diabetes; sepsis; nonalcoholic

steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

[0033] Targeted inhibition of PDE1 with a compound of the present invention is believed to affect macrophage activation and promote production of anti-inflammatory cytokines and factors involved in resolution of macrophage mediated inflammation.

[0034] Accordingly, in one embodiment, the invention provides a method of promoting resolution of inflammation for the treatment or prophylaxis of inflammation or disease associated with inflammation, the method comprising administering a specific inhibitor of

phosphodiesterase type I (e.g., PDE1 inhibitor, e.g., a PDE1B inhibitor) (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described).

[0035] In one embodiment, the invention provides a method of promoting macrophage activation to the M2 activation state, the method comprising administering a specific inhibitor of phosphodiesterase type I (e.g., PDE1 inhibitor, e.g., a PDE1B inhibitor) (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described).

[0036] In one embodiment, the invention provides a method of treating inflammation and/or diseases or disorders associated with inflammation and/or microglial function, e.g., including bacterial infections (e.g., Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium avium infections); viral infections (e.g., African Swine Fever Virus, Classical Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human Immunodeficiency Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus-2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome

Coronavirus, West Nile Virus, Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C)); parasitic infestations (e.g., Taenia crassiceps, Toxoplasma gondii, Leishmania infantum, Schistosoma mansoni infestations); atopic dermatitis; pneumonia; cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance; asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD); neuropathic pain; stroke; diabetes; sepsis; nonalcoholic

steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis, the method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described), e.g., an amount effective to promote macrophage activation from the Ml activation state to the M2 activation state in a patient in need thereof.

[0037] Further embodiments of the invention are set forth or evident from the detailed description below and the examples herein.

DETAILED DESCRIPTION OF THE INVENTION

Compounds for use in the methods of the invention

[0038] In one embodiment, the PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are optionally substituted 7,8-dihydro-imidazo[l,2-a]pyrazolo[4,3- e]pyrimidin-4-one compounds and 7,8,9-trihydro-[lH or 2H]-pyrimido [l,2-a]pyrazolo[4,3- e]pyrimidin-4(5H)-one compounds, in free or pharmaceutically acceptable salt form.

[0039] In still yet another embodiment, the invention provides that the PDE1 inhibitors for use in the methods of treatment and prophylaxis which are described herein are selected from any of the Applicant’s own publications: US 2008-0188492 Al, US 2010-0173878 Al, US 2010-0273754 Al, US 2010-0273753 Al, WO 2010/065153, WO 2010/065151, WO

2010/065151, WO 2010/065149, WO 2010/065147, WO 2010/065152, WO 2011/153129, WO 2011/133224, WO 2011/153135, WO 2011/153136, and WO 2011/153138, the entire contents of each of which are incorporated herein by reference in their entireties.

[0044] Further examples of PDE1 inhibitors suitable for use in the methods and treatments discussed herein can be found in International Publication WO2006133261A2; U.S. Patent 8,273,750; U.S. Patent 9,000,001; U.S. Patent 9,624,230; International Publication

W02009075784A1; U.S. Patent 8,273,751; U.S. Patent 8,829,008; U.S. Patent 9,403,836; International Publication W02014151409A1, U.S. Patent 9,073,936; U.S. Patent 9,598,426; U.S. Patent 9,556,186; U.S. Publication 2017/0231994A1, International Publication

WO2016022893A1, and U.S. Publication 2017/0226117A1, each of which are incorporated by reference in their entirety.

[0045] Still further examples of PDE1 inhibitors suitable for use in the methods and treatments discussed herein can be found in International Publication W02018007249A1; U.S. Publication 2018/0000786; International Publication W02015118097A1; U.S. Patent 9,718,832;

International Publication W02015091805A1; U.S. Patent 9,701,665; U.S. Publication

2015/0175584A1; U.S. Publication 2017/0267664A1; International Publication

WO2016055618A1; U.S. Publication 2017/0298072A1; International Publication

W02016170064A1; U.S. Publication 2016/0311831A1; International Publication

WO2015150254A1; U.S. Publication 2017/0022186A1; International Publication

WO2016174188A1; U.S. Publication 2016/0318939A1; U.S. Publication 2017/0291903 Al; International Publication WO2018073251A1; International Publication WO2017178350A1; and U.S. Publication 2017/0291901A1; each of which are incorporated by reference in their entirety. In any situation in which the statements of any documents incorporated by reference contradict

or are incompatible with any statements made in the present disclosure, the statements of the present disclosure shall be understood as controlling.

[0042] In yet another embodiment the invention provides that the PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are compounds of Formula I:

Formula I

wherein

(i) Ri is H or C _M alkyl (e.g., methyl);

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

(e.g., R ₂ and R ₃ are both methyl, or R ₂ is H and R ₃ is isopropyl), aryl, heteroaryl,

(optionally hetero)arylalkoxy, or (optionally hetero)arylalkyl; or

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

(pref. wherein the R ₃ and R ₄ together have the cis configuration, e.g., where the carbons carrying R ₃ and R ₄ have the R and S configurations, respectively);

(iii) R5 is a substituted heteroarylalkyl, e.g., substituted with haloalkyl;

or R ₅ is attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A

Formula A wherein X, Y and Z are, independently, N or C, and Rs, R ₉ , R ₁₁ and R ₁₂ are

independently H or halogen (e.g., Cl or F), and Rio is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoro methyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl (for example pyrid-2-yl) optionally substituted with halogen, or thiadiazolyl (e.g., l,2,3-thiadiazol-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, Rs, R ₉ , or Rio, respectively, is not present; and

(iv) R ₆ is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g., phenylamino), heterarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N- (arylakyl) amino (e.g., N-phenyl-N-(l,l’-biphen-4-ylmethyl)amino); and

(v) n=0 or 1 ;

(vi) when n=l, A is -C(Ri ₃ Ri4)- wherein R ₁₃ and R _14, are, independently, H or C _1-4 alkyl, aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl;

in free, salt or prodrug form, including its enantiomers, diastereoisomers and racemates. [0043] In another embodiment the invention provides that the PDE1 inhibitors for use in the methods as described herein are Formula la:

Formula la

wherein

(i) R ₂ and R5 are independently H or hydroxy and R ₃ and R ₄ together form a tri- or tetra- methylene bridge [pref. with the carbons carrying R ₃ and R ₄ having the R and S configuration respectively]; or R ₂ and R ₃ are each methyl and R ₄ and R5 are each H; or R ₂ , R ₄ and R5 are H and R ₃ is isopropyl [pref. the carbon carrying R ₃ having the R configuration];

(ii) R ₆ is (optionally halo- or hydroxy-substituted) phenylamino, (optionally halo- or hydroxy-substituted) benzylamino, Ci- ₄ alkyl, or Ci- ₄ alkyl sulfide; for example, phenylamino or 4-fluorophenylamino ;

(iii) Rio is Ci- ₄ alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, (optionally halo- or hydroxy-substituted) phenyl, (optionally halo- or hydroxy-substituted) pyridyl (for example 6-fluoropyrid-2-yl), or thiadiazolyl (e.g., l,2,3-thiadiazol-4-yl); and

X and Y are independently C or N,

in free, pharmaceutically acceptable salt or prodrug form, including its enantiomers,

diastereoisomers and racemates. [0044] In another embodiment the invention provides that the PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are compounds of Formula II:

Formula II

(i) X is Ci- ₆ alkylene (e.g., methylene, ethylene or prop-2-yn-l-ylene);

(ii) Y is a single bond, alkynylene (e.g.,— CºC— ), 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), haloCi- ₆ alkyl (e.g., trifluoromethyl),— C(O)— R ¹ ,— N(R ² )(R ³ ), or C _3- 7cycloalkyl optionally containing at least one atom selected from a group consisting of N or O (e.g., cyclopentyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, or morpholinyl);

(iv) R ¹ is Ci- ₆ alkyl, haloCi- ₆ alkyl,— OH or— OCi- ₆ alkyl (e.g.,— OCH ₃ );

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

(vi) R ⁴ and R ⁵ are independently H, Ci- ₆ alky or aryl (e.g., phenyl) optionally 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 Ci- ₆ alkoxy;

(vii) wherein X, Y and Z are independently and optionally substituted with one or more halo (e.g., F, Cl or Br), C 1 ₆ al ky 1 (e.g., methyl), haloCi- ₆ alkyl (e.g., trifluoromethyl), for example, Z is heteroaryl, e.g., pyridyl 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), haloCi- ₆ alkyl (e.g., 5-trifluoromethylpyrid-2-yl) or Ci- ₆ -alkyl (e.g., 5-methylpyrid-2-yl), or Z is aryl, e.g., phenyl, substituted with one or more halo (e.g., 4-fluorophenyl),

in free, salt or prodrug form.

[0045] In yet another embodiment the invention provides that the PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are Formula III:

Formula III wherein

(i) Rl is H or C _1-4 alkyl (e.g., methyl or ethyl);

(ii) R ₂ and R ₃ are independently H or Ci- ₆ alkyl (e.g., methyl or ethyl);

(iii) R ₄ is H or C _M alkyl (e.g., methyl or ethyl);

(iv) R5 is aryl (e.g., phenyl) optionally substituted with one or more groups independently selected from -C(=0)-Ci- ₆ alkyl (e.g., -C(=0)-CH ₃ ) and Ci _-6 -hydroxyalkyl (e.g., 1- hydroxyethyl);

(v) R ₆ and R ₇ are independently H or aryl (e.g., phenyl) optionally substituted with one or more groups independently selected from Ci _-6 alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), for example unsubstituted phenyl or phenyl substituted with one or more halogen (e.g., F) or phenyl substituted with one or more Ci _-6 alkyl and one or more halogen or phenyl substituted with one Ci _-6 alkyl and one halogen, for example 4- fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3-methylphenyl; and

(vi) n is 1, 2, 3, or 4, in free or salt form.

[0046] In yet another embodiment the invention provides that the PDE1 inhibitors for use in the methods of treatment and prophylaxis described herein are Formula IV

Formula IV

in free or salt form, wherein

(i) Ri is Ci- ₄ alkyl (e.g., methyl or ethyl), or -NH(R ₂ ), wherein R ₂ is phenyl optionally

substituted with halo (e.g., fluoro), for example, 4-fluorophenyl;

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

(iii) R ₃ , R ₄ and R5 are independently H or Ci- ₄ alkyl (e.g., methyl); or R ₃ is H and R ₄ and R5 together form a tri-methylene bridge (pref. wherein the R ₄ and R5 together have the cis configuration, e.g., where the carbons carrying R ₄ and Rshave the R and S

configurations, respectively),

(iv) R ₆ , R7 and Rs are independently:

H,

Ci- ₄ alkyl (e.g., methyl),

pyrid-2-yl substituted with hydroxy, or

-S(0) ₂ -NH ₂ ;

(v) Provided that when X, Y and/or Z are N, then R ₆ , R ₇ and/or Rs, respectively, are not present; and when X, Y and Z are all C, then at least one of R ₆ , R ₇ or Rs is -S(0) ₂ -NH ₂ or pyrid-2-yl substituted with hydroxy. [0047] In another embodiment the invention provides that the PDE1 inhibitors for use in the methods as described herein are Formula V :

Formula V

wherein

(i) Ri is -NH(R ₄ ), wherein R ₄ is phenyl optionally substituted with halo (e.g., fluoro), for example, 4-fluorophenyl;

(ii) R ₂ is H or Ci-r,alkyl (e.g., methyl, isobutyl or neopentyl);

(iii) R ₃ is -SO2NH2 or -COOH;

in free or salt form.

[0048] In another embodiment the invention provides that the PDE1 inhibitors for use in the methods as described herein are Formula VI:

Formula VI

wherein

(i) Ri is -NH(R ₄ ), wherein R ₄ is phenyl optionally substituted with halo (e.g., fluoro), for example, 4-fluorophenyl; (ii) R ₂ is H or Ci-r,alkyl (e.g., methyl or ethyl);

(iii) R ₃ is H, halogen (e.g., bromo), C i ₆ al ky 1 (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),

in free or salt form.

[0049] In one embodiment, the present disclosure provides for administration of a PDE1 inhibitor for use in the methods described herein (e.g., a compound according to Formulas I, la, II, III, IV, V, and/or VI), wherein the inhibitor is a compound according to the following:



[0050] In one embodiment the invention provides administration of a PDE1 inhibitor for treatment or prophylaxis of inflammation or an inflammatory related disease or disorder, wherein the inhibitor is a compound according to the following:

in free or pharmaceutically acceptable salt form.

[0051] In still another embodiment, the invention provides administration of a PDE1 inhibitor for treatment or prophylaxis of inflammation or an inflammatory related disease or disorder, wherein the inhibitor is a compound according to the following:

in free or pharmaceutically acceptable salt form. [0052] In still another embodiment, the invention provides administration of a PDE1 inhibitor for treatment or prophylaxis of inflammation or an inflammatory related disease or disorder, wherein the inhibitor is a compound according to the following:

in free or pharmaceutically acceptable salt form.

[0053] In still another embodiment, the invention provides administration of a PDE1 inhibitor for treatment or prophylaxis of inflammation or an inflammatory related disease or disorder, wherein the inhibitor is a compound according to the following:

in free or pharmaceutically acceptable salt form.

[0054] In one embodiment, selective PDE1 inhibitors of the any of the preceding formulae (e.g., Formulas I, la, II, III, IV, V, and/or VI) are compounds that inhibit phosphodiesterase- mediated (e.g., PDE1 -mediated, especially PDE1B -mediated) hydrolysis of cGMP, e.g., the preferred compounds have an IC50 of less than ImM, preferably less than 500 nM, preferably less than 50 nM, and preferably less than 5nM in an immohili zed-metal affinity particle reagent PDE assay, in free or salt form.

[0055] If not otherwise specified or clear from context, the following terms herein have the following meanings:

“Alkyl” as used herein is a saturated or unsaturated hydrocarbon moiety, preferably saturated, preferably having one to six carbon atoms, which may be linear or branched, and may be optionally mono-, di- or tri- substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or carboxy.

“Cycloalkyl” as used herein is a saturated or unsaturated nonaromatic hydrocarbon moiety, preferably saturated, preferably comprising three to nine carbon atoms, at least some of which form a nonaromatic mono- or bicyclic, or bridged cyclic structure, and which may be optionally substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or carboxy. Wherein the cycloalkyl optionally contains one or more atoms selected from N and O and/or S, said cycloalkyl may also be a heterocycloalkyl.

“Heterocycloalkyl” is, unless otherwise indicated, saturated or unsaturated nonaromatic hydrocarbon moiety, preferably saturated, preferably comprising three to nine carbon atoms, at least some of which form a nonaromatic mono- or bicyclic, or bridged cyclic structure, wherein at least one carbon atom is replaced with N, O or S, which

heterocycloalkyl may be optionally substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or carboxy.

“Aryl” as used herein is a mono or bicyclic aromatic hydrocarbon, preferably phenyl, optionally substituted, e.g., with alkyl (e.g., methyl), halogen (e.g., chloro or fluoro), haloalkyl (e.g., trifluoro methyl), hydroxy, carboxy, or an additional aryl or heteroaryl (e.g., biphenyl or pyridylphenyl).

“Heteroaryl” as used herein is an aromatic moiety wherein one or more of the atoms making up 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.

[0056] Compounds of the Invention, e.g., optionally substituted 7,8-dihydro-imidazo[l,2- a]pyrazolo[4,3-e]pyrimidin-4-one compounds and 7,8,9-trihydro-[lH or 2H]-pyrimido [1,2- a]pyrazolo[4,3-e]pyrimidin-4(5H)-one compounds, in free or pharmaceutically acceptable salt form, e.g., Compounds of Formulas I, la, II, III, IV, V, and/or VI, may exist in free or salt form, e.g., as acid addition salts. In this specification unless otherwise indicated, language such as “Compounds of the Invention” is to be understood as embracing the compounds in any form, for example free or acid addition salt form, or where the compounds contain acidic substituents, in base addition salt form. The Compounds of the Invention are intended for use as

pharmaceuticals, therefore pharmaceutically acceptable salts are preferred. Salts which are unsuitable for pharmaceutical uses may be useful, for example, for the isolation or purification of free Compounds of the Invention or their pharmaceutically acceptable salts, are therefore also included.

[0057] Compounds of the Invention may in some cases also exist in prodrug form. A prodrug form is compound which converts in the body to a Compound of the Invention. For example when the Compounds of the Invention contain hydroxy or carboxy substituents, these substituents may form physiologically hydrolysable and acceptable esters. As used herein, “physiologically hydrolysable and acceptable ester” means esters of Compounds of the Invention which are hydrolysable under physiological conditions to yield acids (in the case of Compounds of the Invention which have hydroxy substituents) or alcohols (in the case of Compounds of the Invention which have carboxy substituents) which are themselves physiologically tolerable at doses to be administered. Therefore, wherein the Compound of the Invention contains a hydroxy group, for example, Compound-OH, the acyl ester prodrug of such compound, i.e., Compound- 0-C(0)-Ci- ₄ alkyl, can hydrolyze in the body to form physiologically hydrolysable alcohol (Compound-OH) on the one hand and acid on the other (e.g., HOC(0)-Ci- ₄ alkyl). Alternatively, wherein the Compound of the Invention contains a carboxylic acid, for example, Compound- C(0)OH, the acid ester prodrug of such compound, Compound-C(0)0-Ci- ₄ alkyl can hydrolyze

to form Compound-C(0)OH and HO-Ci- ₄ alkyl. As will be appreciated the term thus embraces conventional pharmaceutical prodrug forms.

[0058] In another embodiment, the invention further provides a pharmaceutical composition comprising a Compound of the Invention, in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable carrier, for use as an anti-inflammatory agent.

[0059] Compounds of the Invention may in some cases also exist in prodrug form. A prodrug form is compound which converts in the body to a Compound of the Invention. For example when the Compounds of the Invention contain hydroxy or carboxy substituents, these substituents may form physiologically hydrolysable and acceptable esters. As used herein, “physiologically hydrolysable and acceptable ester” means esters of Compounds of the Invention which are hydrolysable under physiological conditions to yield acids (in the case of Compounds of the Invention which have hydroxy substituents) or alcohols (in the case of Compounds of the Invention which have carboxy substituents) which are themselves physiologically tolerable at doses to be administered. Therefore, wherein the Compound of the Invention contains a hydroxy group, for example, Compound-OH, the acyl ester prodrug of such compound, i.e., Compound- 0-C(0)-Ci- ₄ alkyl, can hydrolyze in the body to form physiologically hydrolysable alcohol (Compound-OH) on the one hand and acid on the other (e.g., HOC(0)-Ci- ₄ alkyl). Alternatively, wherein the Compound of the Invention contains a carboxylic acid, for example, Compound- C(0)OH, the acid ester prodrug of such compound, Compound-C(0)0-Ci- ₄ alkyl can hydrolyze to form Compound-C(0)OH and HO-Ci- ₄ alkyl. As will be appreciated the term thus embraces conventional pharmaceutical prodrug forms.

[0060] In another embodiment, the invention further provides a pharmaceutical composition comprising a Compound of the Invention, in free, pharmaceutically acceptable salt or prodrug form, in admixture with a pharmaceutically acceptable carrier, for use as an anti-inflammatory agent.

Methods of Making Compounds of the Invention

[0061] The compounds of the Invention and their pharmaceutically acceptable salts may be made using the methods as described and exemplified herein and by methods similar thereto and by methods known in the chemical art. Such methods include, but not limited to, those described below. If not commercially available, starting materials for these processes may be made by procedures, which are selected from the chemical art using techniques which are similar or analogous to the synthesis of known compounds.

[0062] Various starting materials and/or Compounds of the Invention may be prepared using methods described in US 2008-0188492 Al, US 2010-0173878 Al, US 2010-0273754 Al, US 2010-0273753 Al, WO 2010/065153, WO 2010/065151, WO 2010/065151, WO 2010/065149, WO 2010/065147, WO 2010/065152, WO 2011/153129, WO 2011/133224, WO 2011/153135, WO 2011/153136, WO 2011/153138, and US Pat. 9,073,936, the contents of each of which herein are hereby incorporated by reference in their entireties.

[0063] The Compounds of the Invention include their enantiomers, diastereoisomers and racemates, as well as their polymorphs, hydrates, solvates and complexes. Some individual compounds within the scope of this invention may contain double bonds. Representations of double bonds in this invention are meant to include both the E and the Z isomer of the double bond. In addition, some compounds within the scope of this invention may contain one or more asymmetric centers. This invention includes the use of any of the optically pure stereoisomers as well as any combination of stereoisomers.

[0064] It is also intended that the Compounds of the Invention encompass their stable and unstable isotopes. Stable isotopes are nonradioactive isotopes which contain one additional neutron compared to the abundant nuclides of the same species (i.e., element). It is expected that the activity of compounds comprising such isotopes would be retained, and such compound would also have utility for measuring pharmacokinetics of the non-isotopic analogs. For example, the hydrogen atom at a certain position on the Compounds of the Invention may be replaced with deuterium (a stable isotope which is non-radioactive). Examples of known stable isotopes include, but not limited to, deuterium, ¹³ C, ¹⁵ N, ¹⁸ O. Alternatively, unstable isotopes, which are radioactive isotopes which contain additional neutrons compared to the abundant nuclides of the same species (i.e., element), e.g., ¹²³ 1, ¹³¹ I, ¹²⁵ I, ^U C, ¹⁸ F, may replace the corresponding abundant species of I, C and F. Another example of useful isotope of the compound of the invention is the ^U C isotope. These radio isotopes are useful for radio-imaging and/or pharmacokinetic studies of the compounds of the invention.

[0065] Melting points are uncorrected and (dec) indicates decomposition. Temperature are given in degrees Celsius (°C); unless otherwise stated, operations are carried out at room or ambient temperature, that is, at a temperature in the range of 18-25 °C. Chromatography means flash chromatography on silica gel; thin layer chromatography (TLC) is carried out on silica gel plates. NMR data is in the delta values of major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Conventional abbreviations for signal shape are used. Coupling constants (J) are given in Hz. For mass spectra (MS), the lowest mass major ion is reported for molecules where isotope splitting results in multiple mass spectral peaks. Solvent mixture compositions are given as volume percentages or volume ratios. In cases where the NMR spectra are complex, only diagnostic signals are reported.

Methods of using Compounds of the Invention

[0066] The Compounds of the Invention are useful in the treatment of inflammatory diseases or conditions, particularly inflammatory diseases or conditions. Therefore, administration or use of a preferred PDE1 inhibitor as described herein, e.g., a PDE1 inhibitor as hereinbefore described, e.g., a Compound of Formulas I, la, II, III, IV, V, and/or VI provides a means to regulate inflammation (e.g., prevent, reduce, and/or reverse inflammation, and diseases or disorders related to inflammation), and in certain embodiments provide a treatment for various inflammatory diseases and disorders.

[0067] In one embodiment, the invention provides a method (Method 1) of promoting resolution of inflammation comprising administering an effective amount of a specific inhibitor of phosphodiesterase type I (PDE1), to a patient in need thereof, for example:

[0068] For example, in one embodiment the invention provides a method (Method 1) of promoting resolution of inflammation for the treatment or prophylaxis of inflammation or disease associated with inflammation comprising administering an effective amount of a specific inhibitor of phosphodiesterase type I (PDE1), to a patient in need thereof, for example: Method 1 wherein the patient is suffering from inflammation and/or a disease or disorder mediated by macrophage activation.

Method 1 or 1.1, wherein promoting resolution of inflammation comprises promoting activation of M2 macrophages.

Method 1 or 1.1 wherein the disease or condition to be treated is selected from bacterial infections (e.g., Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium ulcerans, and

Mycobacterium avium infections); viral infections (e.g., African Swine Fever Virus, Classical Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human Immunodeficiency Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus- 2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus, West Nile Virus, Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C));

parasitic infestations (e.g., Taenia crassiceps, Toxoplasma gondii, Leishmania infantum, Schistosoma mansoni infestations); atopic dermatitis; pneumonia;

cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance;

asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD);

neuropathic pain; stroke; diabetes; sepsis; nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

Any foregoing method wherein the disease or condition to be treated is a bacterial infection.

Any foregoing method wherein the disease or condition to be treated is a Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes, Mycobacterium

tuberculosis, Mycobacterium ulcerans, or Mycobacterium avium infection.

Method 1-1.3, wherein the disease or condition to be treated is a viral infection.

Method 1.6, wherein the viral infection is African Swine Fever Virus, Classical Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human

Immunodeficiency Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus- 2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus, West Nile Virus, or Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C).

Method 1-1.3, wherein the disease or condition to be treated is a parasitic infestation. Method 1.8, wherein the parasitic infestation is a Taenia crassiceps, Toxoplasma gondii, Leishmania infantum, or Schistosoma mansoni infestation.

Method 1-1.3, wherein the disease or condition to be treated is atopic dermatitis; pneumonia; cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance; asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD); neuropathic pain; stroke; diabetes; sepsis; nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

Method 1-1.3 or 1.10, wherein the disease or condition to be treated is nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

Any foregoing method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described) in an amount effective to (i) reduce or inhibit activation of Ml macrophages, and/or (ii) an amount effective to reduce levels of one or more pro-inflammatory cytokines (e.g., IL1 b, TNF- a, IL6 and Ccl2, or

combination thereof); to a patient in need thereof.

Any foregoing method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described) to a patient in need thereof, in an amount effective to (i) promote activation of M2 macrophages, and/or (ii) an amount effective to promote anti-inflammatory cytokines (e.g., IL-10).

Any foregoing method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described) to a patient in need thereof, in an amount effective to reduce levels of macrophages of the Ml phenotype and/or enhance levels of macrophages of the M2 phenotype. Any foregoing method wherein the PDE1 inhibitor is a Compound of Formulas I, la, II, III, IV, V, and/or VI.

Any foregoing method wherein the inflammation is associated with increased expression and/or activation of macrophages (e.g., Ml macrophages).

Any foregoing method wherein the PDE1 inhibitor blunts or inhibits the expression and/or activity of pro-inflammatory cytokines, e.g., selected from the group consisting of: IL1B, IL-6, TNF- a, Ccl2, Nitric Oxide (NO), and Reactive Oxygen Species (ROS).

Any foregoing method wherein the PDE1 inhibitor in administered in combination with a PDE4 inhibitor (e.g., rolipram).

Any foregoing method wherein the patient exhibits increased levels of pro- inflammatory cytokines (e.g., IL1B, IL6, TNF-alpha, Ccl2).

Any foregoing method wherein“PDE1 inhibitor” describes a compound(s) which selectively inhibit phosphodiesterase-mediated (e.g., PDE1 -mediated, especially PDE IB -mediated) hydrolysis of cGMP, e.g., with an IC50 of less than ImM, preferably less than 750 nM, more preferably less than 500 nM, more preferably less than 50 nM in an immohili zed-metal affinity particle reagent PDE assay.

Any foregoing method wherein the PDE1 inhibitor inhibits the activity of PDE1 (e.g., bovine PDE1 in the assay described in Example 1) with an IC50 of less than 10 nM, e.g., wherein the PDE1 inhibitor does not inhibit the activity of PDE types other than PDE1, e.g., has an IC50 at least 1000 times greater for PDE types other than PDE1. Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable form.

Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable form.

Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable form.

1.25. Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable form.

1.26. Any of the foregoing method wherein the patient has elevated levels of one or more pro-inflammatory cytokines (e.g., selected from IL1 b, TNFa, Ccl2, IL-6, and combinations thereof).

1.27. Any of the foregoing method wherein the patient has reduced levels of one or more anti-inflammatory cytokines (e.g., IL-10).

1.28. Any of the foregoing method wherein the patient has elevated levels of macrophages of the Ml phenotype compared to macrophages of the M2 phenotype.

1.29. Any of the foregoing methods wherein the patient is also administered one or more of an antibiotic agent, antiviral agent, corticosteroids or NSAIDs.

[0069] For example, in one embodiment the invention provides a method (Method 2) of promoting macrophage activation to the M2 activation state, the method comprising

administering an effective amount of a specific inhibitor of phosphodiesterase type I (PDE1), to a patient suffering from inflammation or a disease or condition associated with inflammation

(e.g., macrophage-mediated inflammation), for example:

2.1 Method 2, wherein the disease or condition to be treated is selected from bacterial infections (e.g., Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium avium infections); viral infections (e.g., African Swine Fever Virus, Classical Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human Immunodeficiency Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus-2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus, West Nile Virus, Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C)); parasitic infestations (e.g., Taenia crassiceps, Toxoplasma gondii, Leishmania infantum, Schistosoma mansoni infestations); atopic dermatitis; pneumonia; cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance; asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD); neuropathic pain; stroke; diabetes; sepsis; nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis;

systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

2.2 Any foregoing method wherein the disease or condition to be treated is a bacterial infection.

2.3 Any foregoing method wherein the disease or condition to be treated is a Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes, Mycobacterium

tuberculosis, Mycobacterium ulcerans, or Mycobacterium avium infection.

2.4 Method 2 or 2.1, wherein the disease or condition to be treated is a viral infection.

2.5 Method 2 or 2.4, wherein the viral infection is African Swine Fever Virus, Classical

Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human Immunodeficiency Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus- 2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus, West Nile Virus, or Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C). Method 2 or 2.1, wherein the disease or condition to be treated is a parasitic infestation.

Method 2.7, wherein the parasitic infestation is a Taenia crassiceps, Toxoplasma gondii, Leishmania infantum, or Schistosoma mansoni infestation.

Method 2 or 2.1, wherein the disease or condition to be treated is atopic dermatitis; pneumonia; cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance; asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD); neuropathic pain; stroke; diabetes; sepsis; nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

Method 2 or 2.8, wherein the disease or condition to be treated is nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

Any foregoing method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described) in an amount effective to (i) reduce or inhibit activation of Ml macrophages, and/or (ii) an amount effective to reduce levels of one or more pro-inflammatory cytokines (e.g., IL1 b, TNF- a, IL6 and Ccl2, or

combination thereof); to a patient in need thereof.

Any foregoing method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described) to a patient in need thereof, in an amount effective to (i) promote activation of M2 macrophages, and/or (ii) an amount effective to promote anti-inflammatory cytokines (e.g., IL-10).

Any foregoing method comprising administering an effective amount of a PDE1 inhibitor of the current invention (e.g., a PDE1 inhibitor of Formulas I, la, II, III, IV, V, and/or VI as herein described) to a patient in need thereof, in an amount effective to reduce levels of macrophages of the Ml phenotype and/or enhance levels of macrophages of the M2 phenotype.

Any foregoing method wherein the PDE1 inhibitor is a Compound of Formulas I, la, II, III, IV, V, and/or VI. Any foregoing method wherein the inflammation is associated with increased expression and/or activation of macrophages (e.g., Ml macrophages).

Any foregoing method wherein the PDE1 inhibitor blunts or inhibits the expression and/or activity of pro-inflammatory cytokines, e.g., selected from the group consisting of: IL1B, IL-6, TNF- a, Ccl2, Nitric Oxide (NO), and Reactive Oxygen Species (ROS).

Any foregoing method wherein the PDE1 inhibitor in administered in combination with a PDE4 inhibitor (e.g., rolipram).

Any foregoing method wherein the patient exhibits increased levels of pro- inflammatory cytokines (e.g., IL1B, IL6, TNF-alpha, Ccl2).

Any foregoing method wherein“PDE1 inhibitor” describes a compound(s) which selectively inhibit phosphodiesterase-mediated (e.g., PDE1 -mediated, especially PDE IB -mediated) hydrolysis of cGMP, e.g., with an IC50 of less than ImM, preferably less than 750 nM, more preferably less than 500 nM, more preferably less than 50 nM in an immohili zed-metal affinity particle reagent PDE assay.

Any foregoing method wherein the PDE1 inhibitor inhibits the activity of PDE1 (e.g., bovine PDE1 in the assay described in Example 1) with an IC50 of less than 10 nM, e.g., wherein the PDE1 inhibitor does not inhibit the activity of PDE types other than PDE1, e.g., has an IC50 at least 1000 times greater for PDE types other than PDE1. Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable salt form.

Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable salt form.

Any foregoing method, wherein the PDE1 inhibitor is the following: in free or pharmaceutically acceptable form.

2.23 Any foregoing method, wherein the PDE1 inhibitor is the following:

in free or pharmaceutically acceptable form.

2.24 Any of the foregoing method wherein the patient has elevated levels of one or more pro-inflammatory cytokines (e.g., selected from IL1 b, TNFa, Ccl2, IL-6, and combinations thereof).

2.25 Any of the foregoing method wherein the patient has reduced levels of one or more anti-inflammatory cytokines (e.g., IL-10).

2.26 Any of the foregoing method wherein the patient has elevated levels of macrophages of the Ml phenotype compared to macrophages of the M2 phenotype.

2.27 Any of the foregoing methods wherein the patient is also administered one or more of an antibiotic agent, antiviral agent, corticosteroids or NSAIDs.

[0070] The invention further provides the use of a PDE1 inhibitor, e.g., any of a Compound of Formulas I, la, II, III, IV, V, and/or VI in the manufacture of a medicament for use in any of Methods 1, et seq. [0071] The invention further provides a PDE1 inhibitor, e.g., any of a Compound of

Formulas I, la, II, III, IV, V, and/or VI for use in any of Methods 1, et seq.

[0072] The invention further provides a pharmaceutical composition comprising a PDE1 inhibitor, e.g., any of a Formulas I, la, II, III, IV, V, and/or VI for use in any of Methods 1 et seq.

[0073] The phrase“Compounds of the Invention” or“PDE 1 inhibitors of the Invention”, or like terms, encompasses any and all of the compounds disclosed herewith, e.g., a Compound of Formulas I, la, II, III, IV, V, and/or VI.

[0074] The words "treatment" and "treating" are to be understood accordingly as embracing prophylaxis and treatment or amelioration of symptoms of disease as well as treatment of the cause of the disease.

[0075] For methods of treatment, the word“effective amount” is intended to encompass a therapeutically effective amount to treat or mitigate a specific disease or disorder, and/or a symptom thereof, and/or to reduce inflammatory cytokines, e.g., as produced by macrophages, and/or to reduce Ml macrophage activation, and/or to increase anti-inflammatory cytokines, e.g., as produced by macrophages, and/or to enhance M2 macrophage activation.

[0076] The term“patient” includes a human or non-human (i.e., animal) patient. In a particular embodiment, the invention encompasses both humans and nonhuman animals. In another embodiment, the invention encompasses nonhuman animals. In other embodiments, the term encompasses humans.

[0077] The term“comprising” as used in this disclosure is intended to be open-ended and does not exclude additional, unrecited elements or method steps.

[0078] Compounds of the Invention, e.g., Formulas I, la, II, III, IV, V, and/or VI as hereinbefore described, in free or pharmaceutically acceptable salt form, may be used as a sole therapeutic agent, but may also be used in combination or for co-administration with other active agents.

[0079] For example, in certain embodiments, the Compounds of the Invention, e.g.,

Formulas I, la, II, III, IV, V, and/or VI as hereinbefore described, in free or pharmaceutically acceptable salt form, may be administered in combination (e.g. administered sequentially or simultaneously or within a 24 hour period) with other active agents, e.g., with one or more antidepressant agents, e.g., with one or more compounds in free or pharmaceutically acceptable salt form, selected from selective serotonin reuptake inhibitors (SSRIs), ) serotonin- norepinephrine reuptake inhibitors (SNRIs), c) tricyclic antidepressants (TCAs), and atypical antipsychotics.

[0080] Dosages employed in practicing the present invention will of course vary depending, e.g. on the particular disease or condition to be treated, the particular Compound of the Invention used, the mode of administration, and the therapy desired. Compounds of the Invention may be administered by any suitable route, including orally, parenterally, transdermally, or by inhalation, but are preferably administered orally. In general, satisfactory results, e.g. for the treatment of diseases as hereinbefore set forth are indicated to be obtained on oral administration at dosages of the order from about 0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicated daily dosage for oral administration will accordingly be in the range of from about 0.75 to 150 mg (depending on the drug to be administered and the condition to be treated, for example in the case of Compound 214, 0.5 to 25 mg, e.g., 1 to 10 mg, per diem, e.g., in monophosphate salt form, for treatment of inflammatory conditions), conveniently administered once, or in divided doses 2 to 4 times, daily or in sustained release form. Unit dosage forms for oral administration thus for example may comprise from about 0.2 to 75 or 150 mg, e.g. from about 0.2 or 2.0 to 50, 75 or 100 mg (e.g., 1, 2.5, 5, 10, or 20 mg) of a Compound of the Invention, e.g., together with a pharmaceutically acceptable diluent or carrier therefor.

[0081] Pharmaceutical compositions comprising Compounds of the Invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets, capsules, solutions, suspensions and the like.

EXAMPLES

Example 1: Peripheral Inflammation Assessment Using Mouse Zymosan Pleurisy Model

[0082] Zymosan is injected into the pleural cavities of mice in order to induce sterile inflammation. Infiltration of leukocytes, neutrophils, and macrophages are monitored at days 3 and 7 following injection. Detection of various cell types are identified according to the gating strategy outlined in Table 1 below.

Table 1: Cell types and identifiable markers for flow cytometry

[0083] In this model, injection of zymosan causes the recruitment of various waves of leukocytes, which are observed and recorded. Exudate volume increases to a maximum over a period of 24 hours, and neutrophils increase within 4 hours and reach a maximum by 48 hours. Lymphocytes of the adaptive immune system enter at a later stage, after three days, which is signaled by macrophages presenting antigens. A resolution phase is well documented in this model and is accompanied by decreased total macrophage number and transition into M2 phenotype.

[0084] In the studies, Compounds 1 and 2 were administered to the test subjects, and the effect the compounds had on infiltration of leukocytes, neutrophils, and macrophages was observed.

Compound 1 Compound 2

[0085] As shown in the accompanying figures 1-9, it was observed that the subjects treated with Compound 1 or 2 showed enhanced inflammatory resolution by promoting shift from Ml to M2. The data show that in the treated specimens, inflammation due to Ml macrophages was consistently decreased, while M2 activation was promoted. As shown in Figure 1, 1 mg of Zymosan i.p. injection into the peritoneal cavity resulted in significant total CD45+ leukocyte infiltration. This increased total number of leukocytes resulted in a general increase in total macrophage numbers on day 3 and 7 following Zymosan injection in disease only animals compared to naive (Figure 2A). The percentage of the macrophages based on the total number of leukocytes (Figure 2B) slightly decreased between days 3 and 7.

[0086] The number of neutrophils dropped significantly on day 7 in the disease only and vehicle tested animals, while the animals administered Compound 1 showed a less dramatic decrease (Figure 3A). These results are reflected in Figure 3B, which showed that the overall percentage of neutrophils relative to CD45+ leukocytes dropped significantly for all subjects.

[0087] To further assess the CD38 and Egr2 expression on macrophages, total numbers of CD38+ macrophages and Egr2+ macrophages were analyzed. Total numbers of CD38+ macrophages were increased in disease and vehicle day 3 animal groups, but decreased on day 3 for animals treated with Compound 1 (Figure 5A). The number of Egr2+ macrophages was decreased for all animal groups at day 3 (Figure 5B). The mean fluorescence intensity (MFI) of both CD38 and Egr2 was also analyzed on macrophages in Figures 6 A and 6B. MFI provides a number that relates to the relative expression of a given marker on a cellular population. MFI for CD38+ showed an increase on day 3 for all animal groups, with the lowest value for the group treated with Compound 1, and decreased on day 7 for all groups. On the other hand, the MFI for Egr2+ was decreased on all animal groups on days 3 and 7, when compared to naive.

[0088] Overall, the results indicate that the number of CD38+ cells tended to decrease and the number of Egr2+ cell number and percent tended to increase indicating a trend to increase the resolution phase of the inflammatory insult on day 7. As shown in Figure 7, animals treated with Compound 1 also tended to show less inflammatory biomarkers (MCP-1/CCL2) at 3 and 7 days in comparison with control groups.

[0089] Similar tests were conducted with Compound 2, the results of which are illustrated in Figures 8 and 9. Treatment with 2 mg/kg of Dexamethasone had no significant effect on the number of CD38+ or Erg2+ macrophage populations on day 3 or 7. Treatment with 3 mg/kg of Compound 2, however, resulted in a significant drop of CD38+ macrophages, which

corresponded with a sharp and significant increase in Erg2+ macrophages on day 7. An additional test was carried out according to the same method. Mice were injected intraperitoneally with lmg Zymosan followed by lOmg/kg Compound 1. Macrophage levels were recorded at injection, then at 4, 8, 16, 24, 48 and 72 hours post-injection. The results are summarized in Figures 11A, 11B, 12A and 12B. As shown in Figures 11A and 11B, treatment with Compound 1 resulted in lower Ml macrophage levels at all observed times, with a significant different observed at day 7 in CD80+ macrophages. Correspondingly, Argl-i- M2 macrophages increased at 4 hours, and CD206+ M2 macrophages significantly increased at relative to control at 2 and 3 days.

Example 2: Effect of PDE1 Inhibitor on Microglia Chemotaxis Assay

[0090] BV2 cells were added to upper chamber of a 5 pm pore Transwell 96-well plate over a reservoir containing 100 pM ADP and incubated at 37°C with 5% C02 for 4 hours. After the incubation cells were harvested with pre-warmed cell detachment solution for 30 minutes in the same incubation conditions. 75 pl of this cell detachment solution was combined with 75 pl of culture medium in a new 96 well plate compatible with a fluorescence reader. Cell number in bottom chamber was determined by adding CyQuant® GR dye and reading in the Envision fluorescence reader at 480 nm EX /520 nm EM. CyQuant® GR dye exhibits strong fluorescence when bound to nucleic acid and is accurate enough to measure differences down to single cells. As shown in Figure 10, the presence of the PDE1 inhibitor Compound 1 showed a marked dampening effect on the motility of the BV2 cells across the membrane, providing additional evidence that Compound 1 dampens the release of pro-inflammatory markers.

Example 3: Detection of Inflammatory Biomarkers Using Mouse Zymosan Pleurisy Model

[0091] Zymosan was injected into the pleural cavities of mice in order to induce sterile inflammation by the methods discussed in Example 1. Compound 1 was administered to test subjects to observe the effects on a variety of inflammatory biomarkers. Results were recorded after 4 hours. The subjects showed a clear decrease in cytokine markers following

administration of Compound 1. IFNy, IL-Ib, MCPl-b and TNF-a decreased following administration of Compound 1 in all serum and plasma samples. IL10 showed a decrease in serum.

[0092] Lipids are known to be involved in regulation of a multitude of cellular responses including cell growth and death, and inflammation/infection, via receptor- mediated pathways. Various lipids are involved in both the initiation and resolution of inflammation. Pro-resolving lipid mediators are produced naturally in the body from unsaturated fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA). Further studies were carried out to identify metabolites of AA and DHA, which are summarized below in Tables 2 and 3.

Table 2: Detection of Arachidonic Acid Metabolites

Table 3: Detection of Docosahexaenoic Acid Metabolites

[0093] As shown above in relation to AA metabolism, 12-HETE and 15-HETE, both intermediate mediators leading to resolution of inflammation, show increased occurrence compared with controls, while pro-inflammatory mediators TXB2, PGE2 and LTB4 all decrease. For the metabolism of DHA, each of 17-HDOHE, RVD5 and 14-HDOHE increase, all of which are related to resolution of inflammation. This profile of lipid biomarkers suggests that the tested compound induces metabolites of 15-LOX and 12-LOX pathways, indicating a mobilization of pro-resolution pathways. It also shows that the tested compound does not induce metabolites of 5-LOX, which is a pro-inflammatory pathway.