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Definitions

• the present invention relates to compounds of Formula I or Formula II, which are activators of long form cyclic nucleotide phosphodiesterase-4 (PDE4) enzymes (isoforms) and to therapies using these activators.
• PDE4 cyclic nucleotide phosphodiesterase-4
• the invention relates to these activator compounds for use in a method for the treatment or prevention of disorders requiring a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP).
• cAMP cyclic 3',5'-adenosine monophosphate
• Cyclic 3',5'-adenosine monophosphate - "cAMP" - is a critical intracellular biochemical messenger that is involved in the transduction of the cellular effects of a variety of hormones, neurotransmitters, and other extracellular biological factors in most animal and human cells.
• the intracellular concentration of cAMP is controlled by the relative balance between its rate of production and degradation.
• cAMP is generated by biosynthetic enzymes of the adenylyl cyclase superfamily and degraded by members of the cyclic nucleotide phosphodiesterase (PDE) superfamily.
• PDE4 Certain members of the PDE superfamily, such as PDE4, specifically degrade cAMP, while others either specifically degrade cyclic guanosine monophosphate (cGMP) or degrade both cAMP and cGMP. PDE4 enzymes inactivate cAMP, thereby terminating its signalling, by hydrolysing cAMP to 5'-AMP (Lugnier, C. Pharmacol Ther. 109: 366-398, 2006).
• PDE4A, PDE4B, PDE4C and PDE4D encodes a number of different enzyme isoforms through the use of alternative promoters and mRNA splicing.
• the catalytically active PDE4 splice variants can be classified as “long”, “short” or “super-short” forms (Houslay, M.D. Prog Nucleic Acid Res Mol Biol. 69: 249-315, 2001).
• a "dead short” form also exists, which is not catalytically active (Houslay, M.D., Baillie, G.S. and Maurice, D.H. Circ Res. 100: 950-66, 2007).
• UCR1 and UCR2 upstream conserved regions 1 and 2
• the UCR1 domain is absent in short forms, whereas the super-short forms not only lack UCR1 , but also have a truncated UCR2 domain (Houslay, M.D., Schafer, P. and Zhang, K. Drug Discovery Today 10: 1503-1519, 2005).
• PDE4 long forms, but not short forms, associate into dimers within cells (Richter, W and Conti, M. J. Biol. Chem. 277: 40212-40221 , 2002; Bolger, G. B. et al., Cell. Signal. 27: 756- 769, 2015).
• a proposed negative allosteric modulation of PDE4 long forms by small molecules has been reported (Burgin A. B. et al., Nat. Biotechnol. 28: 63-70, 2010; Gurney M. E. et al., Handb. Exp. Pharmacol. 204: 167-192, 2011).
• PDE4 long forms may be activated by endogenous cellular mechanisms, such as phosphorylation (MacKenzie, S. J. et al., Br. J. Pharmacol. 136: 421- 433, 2002) and phosphatidic acid (Grange et al., J. Biol. Chem. 275: 33379-33387, 2000).
• Activation of PDE4 long forms by ectopic expression of a 57 amino acid protein (called 'UCR1 C') whose precise sequence reflects part of that of the upstream conserved region 1 of PDE4D ('UCR1 C' sequence reflects that of amino acids 80-136 while UCR is amino acids 17-136: numbering based on the PDE4D3 long isoform) has recently been reported (Wang, L. et al., Cell. Signal. 27: 908-922, 2015: "UCR1 C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy"). The authors hypothesised that PDE4 activation might be used as a potential therapeutic strategy for preventing cardiac hypertrophy.
• PDE4 activation might be used as a potential therapeutic strategy for preventing cardiac hypertrophy.
• Small molecules that act as activators of PDE4 long forms have not previously been disclosed. Small molecule activators would be desirable for a number of reasons, including ease of manufacture and formulation and improved pharmacokinetic properties. It is amongst the objects of the present invention to provide small molecule activators of at least one of the long forms of PDE4 of Formula I or Formula II for use in a method of therapy, as well as specific disease treatment or prevention.
• R 1 is H, (C1-4) alkyl or (C1-4)alkyloxy, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 2 and R 6 are independently selected from H, (C1-4)alkyl, (C1-4)alkyloxy, -CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 3 , R 4 and R 5 are independently selected from H, (C1-4)alkyl, (C1-4)alkyloxy, (C1- 4)alkylsulfonyl, C(0)-NR 16 R 17 , C(0)-OR 16 , S(0) 2 -NR 16 R 17 , -CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 7 , R 8 , R 0 and R 1 are independently selected from H and F;
• R 9 is selected from H, (C1 -4)alkyl, (C1-4)alkyloxy, (C1 -4)alkylsulfonyl, C(0)-NR 16 R 17 , C(O)- OR 16 , S(0) 2 -NR 16 R 17 , CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 12 , R 13 , R 14 and R 15 are independently selected from H and (C1-4)alkyl; each R 16 and R 17 , when present, is independently selected from H and (C1 -4)alkyl; or a pharmaceutically acceptable salt thereof.
• the triazole derivative compounds of Formula 1 are shown in the Examples to activate PDE4 long form enzymes, and to provide therapeutically useful effects on cells.
• the present invention provides a compound of Formula 1 for use in therapy.
• the therapy is the treatment or prevention of a disease or disorder mediated by excessive intracellular cAMP signalling.
• a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) should provide a therapeutic benefit.
• a method of treating or preventing a disease or disorder mediated by excessive intracellular cAMP signalling comprising the step of administering an effective amount of a compound of Formula 1 to a patient in need thereof.
• the use of a compound of Formula 1 in the manufacture of a medicament for treating or preventing a disease or disorder mediated by excessive intracellular cAMP signalling.
• the invention also provides a compound of Formula 2, for use in therapy:
• R 1 is H, (C1-6)alkyl or (C3-7)cycloalkyl, the (C1-6)alkyl and (C3-7)cycloalkyl groups being optionally substituted with 1 to 3 substituents selected from OH, (C1-4)alkyloxy, (C1-4)alkyl- sulfonyl, C(0)-NR 1S R 17 , C(0)-OR 16 , S(0) 2 -NR 16 R 17 , CN and halogen;
• R 2 and R 6 are independently selected from H, (C1-4)alkyl, (C1-4)alkyloxy, -CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 3 , R 4 and R 5 are independently selected from H, (C1-4)alkyl, (C1-4)alkyloxy, (Cl4)alkylsulfonyl, C(0)-NR 16 R 17 , C(0)-OR 16 , S(0) 2 -NR 16 R 17 , -CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 7 , R 8 , R 10 and R 11 are independently selected from H and F;
• R 9 is selected from H, (C1-4)alkyl, (C1-4)alkyloxy, (C1-4)alkylsulfonyl, C(0)-NR 16 R 17 , C(O)- OR 16 , S(0) 2 -NR 16 R 17 , -CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
• R 12 , R 13 , R 14 and R 15 are independently selected from H and (C1-4)alkyl; each R 16 and R 17 , when present, is independently selected from H and (C1-4)alkyl; or a pharmaceutically acceptable salt thereof.
• Compounds of Formula 2 are shown in the Examples to activate a long form cyclic phosphodiesterase-4 (PDE4) enzyme and to provide therapeutically useful effects on cells.
• a compound of Formula 1 or Formula 2 can be provided in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
• the compounds of the invention are provided for the treatment or prevention of a condition selected from hyperthyroidism, Jansens's metaphyseal chondrodysplasia, hyperparathyroidism, familial male-limited precocious puberty, pituitary adenomas, Cushing's disease, polycystic kidney disease, polycystic liver disease, McCune-Albright syndrome, cholera, whooping cough, anthrax, tuberculosis, HIV, AIDS, Common Variable Immunodeficiency (CVID), melanoma, pancreatic cancer, leukaemia, prostate cancer, adrenocortical tumours, testicular cancer, primary pigmented nodular adrenocortical diseases (PPNAD), Carney Complex, autosomal dominant polycystic kidney disease (ADPKD)
• PNAD primary pigment
• the invention provides a method of preparing a compound of Formula 1 or a pharmaceutically acceptable salt thereof, comprising the step of reacting a compound of Formula 1A
• the invention also provides a method of preparing a compound of Formula 1 or a pharmaceutically acceptable salt thereof, comprising the step of reacting a compound of Formula 1C
• the invention provides a method of identifying a compound able to activate a long form PDE4 enzyme, comprising the steps of: a. contacting a long form PDE4 enzyme with a candidate compound;
• the method may be performed according to one of the methods in the Examples.
• the method further comprises the prior or subsequent step of determining whether the candidate compound activates a short or super-short PDE4 enzyme.
• a compound that activates a long form PDE4 and does not activate a short or super-short form PDE4 is selective for long form PDE4.
• One or more of the compounds exemplified herein may be used as the reference or control. Brief description of the Figures
• Figure 1 shows activation of PDE4D5, a long form of PDE4, by Examples A, B, D and L;
• FIG. 1 shows activation of PDE4A4, another long form of PDE4, by Example A;
• Figure 3 shows activation of PDE4B1 , another long form of PDE4, by Example A;
• Figure 4 shows a lack of activation of PDE4B2, a short form of PDE4, by Examples A, B, D and L using the method of Experiment 1 , demonstrating selectivity for activation of PDE4 long forms over this PDE4 short form;
• Figure 5 shows a reduction in intracellular cAMP levels in HEK293 cells treated with a PDE4 long form activator (10 ⁇ ) - Example A - for 10 min prior to forskolin (F) (10 ⁇ ) for 2 min;
• Figure 6 shows a reduction in intracellular cAMP levels in MDCK cells treated with PDE4 long form activators (10 ⁇ ) - Examples A, C and D - for 10 min prior to forskolin (F) (10 ⁇ ) for 2 min;
• Figure 7 shows inhibition of in vitro cyst formation in MDCK cells treated with a PDE4 long form activator - Example A - using the method described in Experiment 3, with addition of the specified concentrations of the test compounds every 2 days from day 0 to day 20;
• Figure 8 shows a reversal of in vitro cyst formation in MDCK cells treated with a PDE4 long form activator - Example A - using the method described in Experiment 4, with addition of the specified concentrations of the test compounds every 2 days from day 10 to day 20;
• Figure 9 shows (normalised cell index at 72 hours) that a PDE4 long form activator - Example A - inhibits the proliferation of androgen-sensitive (AS) LNCaP human prostate cancer cells in a concentration dependent manner, using the method described in Experiment 5;
• Figure 10 shows (normalised cell index at 72 hours) that, using the method described in Experiment 5, a PDE4 long form activator - Example A - inhibits the proliferation of androgen-insensitive (Al) LNCaP human prostate cancer cells in a concentration dependent manner;
• Figure 11 shows the mouse pharmacokinetic profile of Example A, determined by whole blood analysis at defined time points after i.v. and p.o. administration to male C57 mice;
• Figure 12 shows the rat pharmacokinetic profile of Example A, determined by blood plasma analysis at defined time points after i.v. and p.o. administration to male SD rats.
• Figure 13 shows (A) concentration dependent inhibition and (B) concentration dependent reversal of in vitro cyst formation in a human patient-derived conditionally immortalised OX161 cell line by Example A.
• the invention is based on the surprising identification of new compounds that are able to activate long isoforms of PDE4 enzymes.
• the compounds are small molecules and so are expected to be easier and cheaper to make and formulate into pharmaceuticals than large biological molecules such as polypeptides, proteins or antibodies.
• the compounds can be chemically synthesized, as demonstrated in the Examples.
• the Examples demonstrate that a number of compounds of Formula 1 and Formula 2 are able to activate long isoforms of PDE4.
• the Examples go on to demonstrate that certain tested compounds of the invention: do not activate a short form of PDE4, (thereby demonstrating selectivity for activation of PDE4 long forms over PDE4 short forms); reduce intracellular cAMP levels in human and dog cells; inhibit and even reverse in vitro cyst formation in human and dog cells; inhibit the proliferation of human prostate cancer cells; and have favourable pharmacokinetic properties.
• the compounds of the invention are therefore surprisingly advantageous.
• Compounds - Formula 1 A first aspect provides a compound of Formula 1 , as set out above.
• R 7 and R 11 are H.
• R 11 , R 12 , R 13 , R 14 and R 15 are H.
• R 9 is selected from (C1-4)alkyl, (C1- 4)alkyloxy, CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 7 , R 11 , R 12 , R 13 , R 14 and R 15 are H and R 9 is selected from (C1-4)alkyl, (C1-4)alkyloxy, CN and halogen, the (C1-4)alkyl and (C1- 4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 1 is H, methyl, or methoxy. In an embodiment of the compound of Formula 1 , R 1 is methyl.
• R 2 and R 5 are each independently selected from H and halogen.
• R 2 and R s are each independently selected from H and fluoro.
• R 2 and R 6 are each H.
• R 3 , R 4 and R 5 are each independently selected from H, halogen, CN, (C1-4)alkyl and (C1-4)alkyloxy, the (C1-4)alkyl and (C1- 4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 3 and R 5 are each independently selected from H, fluoro, chloro, methoxy, CN, thfluoromethyl, methoxy and trifluoromethoxy.
• R 4 is selected from H, fluoro and methoxy.
• R 9 is selected from methyl, chloro and trifluoromethoxy. In an embodiment of the compound of Formula 1 , R 9 is chloro.
• one of R 8 and R 10 is H and the other is fluoro.
• R 8 and R 10 are both H.
• R 9 is chloro
• one of R 8 and R 10 is H and the other of R 8 and R 10 is fluoro.
• the compound is selected from:
• a further aspect provides a compound of Formula 2, as set out above.
• R 7 and R 11 are H.
• R 11 , R 12 , R 13 , R 14 and R 15 are H.
• R 9 is selected from (C1-4)alkyl, (C1- 4)alkyloxy, CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 7 , R 11 , R 12 , R 13 , R 14 and R 15 are H and R 9 is selected from (C1-4)alkyl, (C1-4)alkyloxy, CN and halogen, the (C1-4)alkyl and (C1- 4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 1 is H, methyl, or methoxy. In an embodiment of the compound of Formula 2, R 1 is methyl.
• R 2 and R s are each independently selected from H and halogen.
• R 2 and R 5 are each independently selected from H and fluoro. In an embodiment of the compound of Formula 2, R 2 and R 6 are each H.
• R 3 , R 4 and R 5 are each independently selected from H, halogen, CN, (C1-4)alkyl and (C1-4)alkyloxy, the (C1-4)alkyl and (C1- 4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 3 and R 5 each independently selected from H, fluoro, chloro, methoxy, CN, trifluoromethyl, methoxy and trifluoromethoxy.
• R 4 is selected from H, fluoro and methoxy.
• R 9 is selected from methyl, chloro and trifluoromethoxy.
• R 9 is chloro. In an embodiment of the compound of Formula 2, one of R 8 and R 10 is H and the other is fluoro.
• R 8 and R 10 are both H.
• R 9 is chloro
• one of R 8 and R 10 is H and the other of R 8 and R 10 is fluoro.
• R 7 and R 1 1 are H.
• R 9 is selected from (C1 -4)alkyl, (C1- 4)alkyloxy, CN and halogen, the (C1 -4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 7 and R 1 1 are H and R 9 is selected from (C1 -4)alkyl, (C1 -4)alkyloxy, CN and halogen, the (C1 -4)alkyl and (C1 -4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 1 is H, methyl, or methoxy. In an embodiment of the compound of Formula 1A, R 1 is methyl.
• R 9 is selected from methyl, chloro and trifluoromethoxy.
• R 9 is chloro
• one of R 8 and R 10 is H and the other is fluoro.
• R 8 and R 10 are both H.
• R 9 is chloro
• one of R 8 and R 10 is H and the other of R 8 and R 10 is fluoro.
• R 7 and R 1 1 are H.
• R , R 12 and R 13 are H.
• R 9 is selected from (C1-4)alkyl, (C1- 4)alkyloxy, CN and halogen, the (C1-4)alkyl and (C1-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 7 , R 11 , R 12 and R 13 are H and R 9 is selected from (C1-4)alkyl, (C1-4)alkyloxy, CN and halogen, the (C1-4)alkyl and (C1- 4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
• R 1 is H, methyl, or methoxy.
• R 1 is methyl.
• R 9 is selected from methyl, chloro and trifluoromethoxy.
• R 9 is chloro
• one of R 8 and R 10 is H and the other is fluoro. In an embodiment of the compound of Formula 1C, R 8 and R 10 are both H.
• R 9 is chloro
• one of R 8 and R 10 is H and the other of R 8 and R 10 is fluoro.
• (C1-4)alkyl as used herein means a branched or unbranched alkyl group having 1-4 carbon atoms, optionally containing a ring.
• Examples of (C1-4)alkyl include butyl, isobutyl, cyclobutyl, tertiary butyl, propyl, isopropyl, cyclopropyl, ethyl and methyl.
• (C1-4)alkyl may be substituted, for example with 1 to 3 fluoros.
• a particularly preferred example of a substituted (C1-4)alkyl is trifluoromethyl.
• (C1-4)alkyl may be unsubstituted.
• (C1-4)alkyloxy means -0-(C1-4)alkyl wherein (C1-4)alkyl has the meaning as defined above.
• Examples of (C1-4)alkyloxy include methoxy, ethoxy, propoxy, isopropoxy, butyoxy, isobutoxy and tertiary butoxy.
• (C1- 4)alkyloxy may be substituted, for example with 1 to 3 fluoros.
• a particularly preferred example of a substituted (C1-4)alkyloxy is trifluoromethoxy.
• (C1-4)alkyloxy may be unsubstituted.
• alkyloxy is attached to the rest of the molecule by the "oxy" moiety.
• halogen means F, CI, Br or I. F and CI are particularly preferred.
• the 1 ,2,4-triazole derivatives of Formula 1 or 2 may be prepared by methods known in the art of organic chemistry in general. Suitable methods for constructing 1 ,2,4-triazole rings are, for example, described in the general reference Katritzky, A.R.: Comprehensive heterocyclic chemistry (First Edition, Pergamon Press, 1984, see especially Volume 5, Part 4A, Five- membered rings with two or more nitrogen atoms). Suitable protecting groups for functional groups which are to be temporarily protected during syntheses are known in the art, for example from Wuts, P.G.M. and Greene, T.W.: Protective Groups in Organic Synthesis, Fourth Edition, Wiley, New York, 2006.
• PDE4 long isoforms have two regulatory regions, upstream conserved region 1 (UCR1) and upstream conserved region 2 (UCR2). These are between the isoform-specific N-terminal portion and the catalytic domain.
• the UCR1 domain is missing in the short forms, whereas the super-short forms not only lack UCR1 , but also have a N-terminal truncated UCR2 domain (Houslay, M. D., Schafer, P. and Zhang, K. Drug Discovery Today 10: 1503-1519, 2005).
• PDE4 families PDE4A, PDE4B, PDE4C and PDE4D.
• the present invention concerns compounds that are capable of activating one or more of the long isoforms from one or more of these four families.
• the long isoform PDE4 may therefore be long isoform PDE4A, long isoform PDE4B, long isoform PDE4C or long isoform PDE4D.
• a long isoform PDE4 comprises a UCR1 region.
• the long isoform PDE4 is human.
• UCR1 is conserved within mammalian species (Houslay, MD, Sullivan, M and Bolger GB Adv Pharmacol. 1998;44:225-34), so in other embodiments, the long isoform PDE4 can be from a non-human mammal.
• PDE4 long form activators of Formula I or Formula II of the present invention are small molecules that are believed to bind directly to PDE4 long forms and induce structural changes that increase, stabilise, uncover and/or maintain the catalytic activity of these enzymes.
• a small molecule is defined as a low molecular weight organic compound that may serve as a regulator of biological processes.
• a small molecule activator according to the present invention has a molecular weight of less than or equal to 700 Daltons. This allows for the possibility to rapidly diffuse across cell membranes and reach intracellular sites of action (Veber, D. F. et a ⁇ ., J. Med. Chem. 45: 2615-2623, 2002).
• the preferred molecular weight for a small molecule activator according to the present invention is greater than or equal to 200 Daltons and less than or equal to 600 Daltons.
• Especially preferred small molecule activators according to the present invention have molecular weights of greater than or equal to 250 Daltons and less than or equal to 500 Daltons (Lipinski, C. A. Drug Discovery Today: Technologies 1 : 337-341 , 2004).
• One suitable method of detecting whether or not a compound is capable of serving as an activator of a PDE4 long form is using a two-step radio-assay procedure described in Experiment 1.
• the method involves incubating a PDE4 long form with a test small molecule activator, together with [ 3 H]-labelled cAMP to assess alterations in the breakdown of cAMP to the 5'- adenosine monophosphate (5'-AMP) product.
• a sample of the reaction mixture from such an incubation is subsequently treated with snake venom 5'- nucleotidase to allow conversion of the nucleotide [ 3 H]-labelled 5'-AM P to the uncharged nucleoside [ 3 H]-labelled adenosine, which can be separated and quantified to assess PDE4 activity and the effect of the test compound (Thompson, W. J. and Appleman, M. M. Biochemistry 10: 31 1 -316, 1971 , with some modifications as described in: Marchmont, R. J. and Houslay, M. D. Biochem J. 187: 381-92, 1980).
• preferred small molecule activators according to the present invention produce an increase in the background activity of one or more PDE4 long forms of more than 50% at a test compound concentration of 100 micromolar or less.
• Especially preferred small molecule activators according to the present invention produce an increase in the background activity of one or more PDE4 long forms of more than 50% at a test compound concentration of 10 micromolar, or less, for example 3 micromolar.
• the compounds of the present invention may be selective for the long form of the PDE4 enzyme and, as such, do not act or act to a lesser extent as activators of the short or super- short isoforms of the PDE4 enzyme.
• the short or super-short isoform PDE4 may therefore be short or super-short isoform PDE4A, short or super-short isoform PDE4B, short or super- short isoform PDE4C, or short or super-short isoform PDE4D.
• short and super-short isoforms of PDE4 lack a UCR1 domain.
• Super-short isoforms comprise a truncated UCR2 domain.
• the short or super-short isoform PDE4 is human, but may also be from other mammalian species (where UCR2 is conserved, see Houslay, MD, Sullivan, M and Bolger GB Adv Pharmacol. 1998;44:225-34).
• the small molecule activators according to the present invention typically produce a less than 50% increase in the background activity of the short or super-short forms of the PDE4A, PDE4B, PDE4C or PDE4D enzymes at a test compound concentration of 100 micromolar, or less.
• Typical compounds may therefore provide a positive result in an assay for activation of a long form PDE4 and a negative result in an assay for activation of a short form (or super- short form) of PDE4.
• the compound activates long isoform PDE4A and does not activate either of short and super-short isoform PDE4A.
• the compound activates long isoform PDE4B and does not activate either of short and super-short isoform PDE4B. In a further embodiment, the compound activates long isoform PDE4C and does not activate either of short and super-short isoform PDE4C. In another embodiment, the compound activates long isoform PDE4D and does not activate either of short and super-short isoform PDE4D.
• PDE4 long isoforms include those now known as PDE4A4, PDE4A4/5, PDE4A5, PDE4A8, PDE4A10 , PDE4A1 1 , PDE4B1 , PDE4B3, PDE4B4, PDE4C1 , PDE4C2, PDE4C3, PDE4D3, PDE4D4, PDE4D5, PDE4D7, PDE4D8, PDE4D9 and PDE4D1 1.
• Further long isoforms may be or have already been identified or called by different nomenclature from any of the four PDE4 sub-families.
• PDE4 short isoforms include PDE4A1 , PDE4B2, PDE4D1 and PDE4D2. Further short isoforms may be or have already been identified or called by different nomenclature from any of the four PDE4 sub-families.
• PDE4 super-short isoforms include PDE4B5, PDE4D6 and PDE4D10. Further super-short isoforms may be or have already been identified or called by different nomenclature from any of the four PDE4 sub-families.
• PDE4A4B clone is correct while PDE4A4A has a cloning artefact and PDE4A4C is a truncation artefact.
• the compounds of the present invention may function by reducing cAMP levels in one or more intracellular compartments.
• the PDE4 long form activators of the present invention may thus provide a means to regulate certain cellular processes that are dependent upon cAMP. Excessive intracellular cAMP signalling mediates a number of diseases and disorders. Therefore, the compounds of the invention are expected to be of utility for the treatment of diseases associated with abnormally elevated cAMP levels, increased cAMP-mediated signalling and/or reduced cAMP elimination, enzymatic or otherwise (e.g. via efflux).
• the treatment is typically of a human, but may also be of a non-human animal, such as a non-human mammal (e.g. veterinary treatment).
• the present invention provides a small molecule activator of a PDE4 long form of Formula 1 or Formula 2, for use in a method for the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required.
• cAMP cyclic 3',5'-adenosine monophosphate
• gain-of-function gene mutations in proteins involved in driving cAMP signalling upstream of adenylyl cyclase can lead to abnormal excessive cAMP activity with pathological consequences (Lania A, Mantovani G, Spada A. Ann Endocrinol (Paris). 73: 73-75, 2012.; Thompson, M. D. et a/., Methods Mol. Biol. 448: 109- 137, 2008; Weinstein LS, Liu J, Sakamoto A, Xie T, Chen M. Endocrinology. 145: 5459- 5464, 2004; Lania A, Mantovani G, Spada A. Eur J Endocnnol.
• PDE4 long form activators of the present invention possessing the ability to accelerate the termination of cAMP action, would therefore be expected to be effective in the treatment, prevention or partial control of diseases characterised by undesirably high cAMP levels, or activity, as detailed below. Diseases characterised by elevated cAMP levels
• TSH thyroid-stimulating hormone
• TSHR thyroid-stimulating hormone receptor
• the most common cause of hyperthyroidism is Graves' disease, an autoimmune disorder in which antibodies mimic TSH action at the TSHR, leading to excessive cAMP activity in thyroid follicle cells and consequently a state of hyperthyroidism.
• PDE4 long form activators of the present invention are therefore expected to be effective in the treatment, prevention or partial control of hyperthyroidism.
• the hyperthyroidism is associated with Graves' disease.
• JMC Jansens's Metaphyseal Chondrodysplasia
• PTH parathyroid hormone receptor 1
• PTHR1 parathyroid hormone receptor 1
• the constitutive activation of the PTHR1 which couples to adenylyl cyclase as effector is associated with excessive cAMP signalling primarily in bone and kidney, leading to dysregulation of ion homeostasis characterised by hypercalcemia and hypophosphatemia (Calvi, L.M. and Schipani, E. J. Endocrinol. Invest. 23: 545-554, 2000) and developmental (e.g. short stature) and physical (e.g. protruding eyes) abnormalities.
• PDE4 long form activators of the present invention are therefore expected to be effective in the treatment, prevention or partial control of JMC and hyperparathyroidism.
• FMPP Familial male-limited precocious puberty
• familial sexual precocity or gonadotropin-independent testotoxicosis is a disorder in which boys generally develop signs of precocious puberty in early childhood.
• FMPP is an autosomal dominant condition with constitutively activating mutations in the luteinizing hormone (LH) receptor, which leads to increased cAMP production, associated with Leydig cell hyperplasia and low sperm cell count (Latronico, A.C. et a/., J Clin. Endocrinol. Metab. 80: 2490-2494, 1995; Kosugi, S. et a/., Hum. Mol. Genet. 4: 183-188, 1995).
• LH luteinizing hormone
• Non-cancerous tumours of the pituitary gland are collectively referred to as pituitary adenomas and can lead to hypersecretion of adenohypophyseal hormones (e.g. growth hormone, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone and adrenocorticotrophic hormone), which exert their action through GPCRs coupled to Gs and cAMP generation.
• adenohypophyseal hormones e.g. growth hormone, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone and adrenocorticotrophic hormone
• pituitary adenomas can lead to a state of enhanced cAMP mediated signalling in a variety of endocrine tissues which can precipitate a number of hormonal disorders such as acromegly (mainly due to excess growth hormone secretion), Cushing's disease (due to overproduction of adrenocorticotrophic hormone (ACTH) and the subsequent hypercortisolemia) and/or general hyperpituitarism (associated with excess release of multiple anterior pituitary hormones).
• Current treatment options for pituitary adenomas include treatment with dopamine receptor agonists, which reduce tumour size and lower pituitary hormonal output through a mechanism involving lowering of intracellular cAMP levels.
• PDE4 long form activators of the present invention may also be expected to attenuate the pathological effects of pituitary hormones in their target tissues, such as the adrenal glands.
• PTD Polycystic kidney disease
• ADPKD autosomal dominant polycystic kidney disease
• ARPKD autosomal recessive polycystic kidney disease
• ARPKD affects around 1 :20,000 live births and is typically identified in the first few weeks after birth. Pulmonary hypoplasia results in a 30-50% death rate in neonates with ARPKD.
• ADPKD Alzheimer's disease .
• PC-1 polycystin-1
• PC-2 polycystin-2
• Cyclic AMP has been identified as an important stimulus for proliferation and cyst expansion in polycystic kidney cells but not in normal human kidney cells (Yamaguchi, T. et a/., Kidney Int. 57: 1460-1471 , 2000).
• a considerable body of evidence has now developed to implicate cAMP as an important facilitator of renal cystogenesis (Masoumi, A.
• Phosphodiesterase activation has been suggested as a therapeutic strategy for PKD treatment (Sun, Y., Zhou, H. and Yang, B-X. Acta Pharmacologica Sinica 32: 805-816, 2011).
• PDE4 long form activators of the present invention are therefore expected to be effective in the treatment, prevention or partial control of polycystic kidney disease.
• the Examples, in particular Figure 13 show the inhibition and reversal of in vitro cyst formation in human OX161 cells treated with a PDE4 long form activator - Example A - using the methods described in Experiment 9, with addition of the specified concentrations of the test compound every 2 days from day 0 to day 10, and Experiment 10, with addition of the specified concentrations of the test compound every 2 days from day 10 to day 20.
• PLD Polycystic liver disease
• Increased cholangiocyte proliferation, neovascularisation and elevated fluid secretion act to drive liver cyst formation through dysregulation of multiple signal transduction pathways, including cAMP-mediated signalling. Elevation of hepatic cAMP levels stimulates cAMP-dependent chloride and fluid secretion in biliary epithelial cells and increases cholangiocyte proliferation (Janssen, M. J. et a/., J. Hepatol. 52: 432-440, 2010). Somatostatin, which acts through a Gi-coupled mechanism to lower cAMP levels, reduced cholangiocyte proliferation and fluid secretion (Gong, A.Y. et a/., Am. J. Physiol. Cell. Physiol.
• PDE4 long form activators of the present invention may therefore be effective in the treatment, prevention or partial control of polycystic liver disease due at least in part to cAMP.
• MODY5 is a form of non-insulin-dependent diabetes mellitus associated with renal cysts. It is an autosomal dominant disorder caused by mutations in the gene encoding hepatocyte nuclear factor- ⁇ ⁇ (HNF- ⁇ ⁇ ). The predominant clinical feature of patients affected by MODY5 is renal dysfunction, frequently diagnosed before the onset of diabetes. In some patients, HNF- ⁇ ⁇ mutations can result in additional phenotypic features, such as pancreatic atrophy, abnormal liver function and genital tract abnormalities. Studies in mice suggest that the mechanism responsible for renal cyst formation, associated with mutations of HNF- ⁇ ⁇ , involves a severe defect of the transcriptional activation of PKD2, in addition to effects on uromodulin (UMOD) and PKD1 genes.
• UMOD uromodulin
• HNF- ⁇ ⁇ also binds to the PDE4C promoter and regulates the expression of PDE4C (Ma et a/., PNAS 104: 20386, 2007).
• PDE4 long form activators of the present invention are therefore expected to be effective in the treatment, prevention or partial control of the symptoms of MODY5.
• Cardiomyocyte hypertrophy is induced via elevated cAMP and activation of its downstream effectors, including PKA and Epac (Wang, L. et al., Cell. Signal. 27: 908-922, 2015 and references therein). Cardiomyocyte hypertrophy increases the risk of heart failure and arrhythmia.
• PDE4 long form activators of the present invention may therefore be effective in the treatment, prevention or partial control of cardiac hypertrophy, heart failure and/or arrhythmia.
• the G-protein Gs acts as a transducer for GPCRs that stimulate adenylyl cyclase activity and exert their biological effects by increasing intracellular cAMP levels.
• Gs is a heterotrimeric protein composed of ⁇ , ⁇ and ⁇ subunits. Activating mutations in the gene, GNAS1 , for the a-subunit have been identified which lead to exaggerated abnormal cAMP signalling in a variety of tissues and give rise to a range of disorders.
• McCune-Albright syndrome is a rare genetic disorder typically characterised by three dominating features of precocious puberty, fibrous dysplasia of bone and cafe au lait lesions.
• the underlying molecular pathology for MAS involves an activating mutation of the GNAS1 gene (Diaz, A. Danon, M. and Crawford, J. J. Pediatr. Endocrinol. Metab. 20: 853-880, 2007).
• PDE4 long form activators of the present invention would therefore be expected to be effective in the treatment, prevention or partial control of disorders associated with activating mutations of GNASI , including McCune-Albright syndrome.
• Adenylyl cyclase the enzyme responsible for production of cAMP, is a key biological target thought to be involved in mediating the effects of many bacterial toxins (Ahuja et al., Critical Reviews in Microbiology, 30: 187-196, 2004). These toxins produce their effects by raising cAMP levels through enhancement of host immune cell and/or pathogen related adenylyl cyclase activity. PDE4 long form activators of the present invention, by reducing cAMP levels, would therefore be expected to be of utility in the treatment or partial control of symptoms of infectious diseases that are associated with elevated cAMP activity. The following are some examples of such infectious diseases:
• Vibrio choierae produces cholera toxin, which through adenosine disphosphate ribosylation of the a subunit of Gs leads to host cell adenylyl cyclase activation and cAMP production. Diarrhoea caused by cholera toxin is believed to be a result of excessive cAMP accumulation in the cells of the gastrointestinal tract.
• Bordetella pertussis is the pathogen responsible for the childhood disease whooping cough. Bordetella pertussis toxin stimulates adenosine disphosphate ribosylation of the a subunit of Gi and indirectly augments cAMP levels in target cells. The bacterium also secretes an invasive adenylyl cyclase, which produces toxic cAMP levels and impairs host immune defence.
• Anthrax is caused by Bacillus anthracis and whilst it is primarily an animal disease it can be transmitted to humans through contact.
• Anthrax infections are associated with widespread oedema, the development of which is thought to be driven by oedema toxin.
• the latter is an adenylyl cyclase and is activated by host calmodulin to produce abnormally high levels of cAMP that have a toxic effect on host immune cells.
• Tuberculosis Mycobactrium tuberculosis expresses a large and diverse range of adenylyl cyclases, which may play a role in virulence and generation of disease pathology.
• adenylyl cyclase subtype RV0386
• PDE4 long form activators of the present invention may therefore be effective in the treatment, prevention or partial control of infectious diseases such as cholera, whooping cough, anthrax and tuberculosis.
• cAMP activates protein kinase A (PKA), which is also known as cAMP-dependent protein kinase.
• PKA protein kinase A
• PKA is normally inactive as a tetrameric holoenzyme, consisting of two catalytic and two regulatory units, with the regulatory units blocking the catalytic centres of the catalytic units.
• cAMP binds to specific locations on the regulatory units of PKA and causes dissociation between the regulatory and catalytic units, thus activating the catalytic units.
• the active catalytic units catalyse the transfer of phosphate from ATP to specific residues of protein substrates, which may modulate the function of those protein substrates.
• PDE4 long form activation reduces cAMP levels and cAMP mediated activation of PKA.
• PDE4 long form activators of the present invention would therefore be expected to be of utility in the treatment or partial control of disorders where inhibitors of PKA show evidence of therapeutic effects.
• Rp-8-Br-cAMPS is an analogue of cAMP that occupies the cAMP binding sites of PKA, preventing its dissociation and activation.
• HIV infection and AIDS T cells from H IV-infected patients have increased levels of cAM P and are more sensitive to inhibition by Rp-8-Br-cAMPS than are normal T cells.
• Excessive activation of PKA by cAM P has been associated with the progressive T cell dysfunction in HIV infection (Aandahl, E. M. et al., FASEB J. 12: 855-862, 1998).
• PDE4 long form activators of the present invention are therefore expected to be of utility in the treatment, prevention or partial control of HIV infection and AIDS.
• CVID Common Variable Immunodeficiency
• CVI D Common Variable Immunodeficiency
• Epac exchange protein directly activated by cAMP
• Epac proteins exert their functions through interactions with a number of other cellular partners at specific cellular loci. Pathophysiological changes in Epac signalling have been associated with a wide range of diseases (Breckler, M. et al., Cell. Signal. 23: 1257-1266, 2011 ).
• Epac inhibitors such as ESI-09, a novel non-cyclic nucleotide Epad and Epac2 antagonist that is capable of specifically blocking intracellular Epac- mediated Rap1 activation and Akt phosphorylation, as well as Epac-mediated insulin secretion in pancreatic beta cells (Almahariq, M. et al., Mol. Pharmacol. 83: 122-128, 2013).
• Melanoma Epad has been implicated in promoting migration and metastasis in melanoma (Baljinnyam, E. et al., Pigment Cell Melanoma Res. 24: 680-687, 2011 and references cited therein).
• PDE4 long form activators of the present invention are therefore expected to be of utility in the treatment, prevention or partial control of melanoma.
• Pancreatic cancer It has recently been shown that Epad is markedly elevated in human pancreatic cancer cells as compared with normal pancreas or surrounding tissue (Lorenz, R. et al., Pancreas 37: 102-103, 2008).
• Pancreatic cancer is often resistant to treatments that are usually effective for other types of cancer.
• Epac inhibitor ESI-09 a functional role of Epad overexpression in pancreatic cancer cell migration and invasion was demonstrated (Almahariq, M. et al., Mol. Pharmacol. 83: 122-128, 2013).
• PDE4 long form activators of the present invention would therefore be expected to be of utility in the treatment, prevention or partial control of pancreatic cancer.
• PDE4 long form activators of the present invention would therefore be expected to be of utility in the treatment of disorders where inhibitors of cAMP-gated ion channels show evidence of therapeutic effects.
• cAMP response element binding protein is an important transcription factor involved in the regulation of a variety of cellular functions such as cell proliferation, differentiation, survival, and apoptosis (Cho et al., Crit Rev Oncog, 16: 37-46, 2011).
• CREB activity is regulated by kinase dependant phosphorylation through a range of extracellular signals, such as stress, growth factors and neurotransmitters. Phosphorylation leads to dimerisation of CREB, and together with other co-activator partner proteins, enables it to bind to promoter regions of target genes containing the cAMP response element (CRE sites) and initiate transcriptional activity.
• the cAMP pathway e.g.
• PDE4 long form activators of the present invention are therefore expected to be of utility in the treatment, prevention or partial control of disorders associated with elevated CREB activity.
• Bone marrow cells from acute lymphoid and myeloid leukaemia patients have been reported to overexpress CREB protein and mRNA (Crans-Vargas et al., Blood, 99: 2617-9, 2002; Cho et al., Crit Rev Oncog, 16: 37-46, 2011). Furthermore, the increased CREB level correlates with poor clinical response in subjects with acute myeloid leukaemia (Crans-Vargas et al., Blood, 99: 2617-9, 2002; Shankar et al., Cancer Cell, 7:351-62, 2005). Upregulation of CREB is associated with stimulation of human leukaemia cell growth whilst downregulation inhibits myeloid cell proliferation and survival.
• PDE4 long form activators of the present invention would be expected to reduce CREB activity and function through attenuation of cAMP mediated stimulation of CREB and therefore expected to have utility in the treatment, prevention or partial control of acute lymphoid and myeloid leukaemia.
• Cyclic AMP elevating agents such as forskolin can enhance androgen receptor activity through multiple intracellular mechanisms including androgen receptor activation through phosphorylation and/or interaction with CREB. Epad activation has also been implicated in promoting cellular proliferation in prostate cancer (Misra, U. K. and Pizzo, S. V. J. Cell. Biochem.
• PDE4 long form activators of the present invention are therefore expected to have utility in the treatment, prevention or partial control of prostate cancer.
• Loss-of-function mutations in gene(s) for cAMP-hydrolysing PDE isoforms other than PDE4, such as PDE8 and PDE1 1 have been detected in a number of diseases (Vezzosi, D. and Bertherat, J., Eur. J. Endocrinol. 165: 177-188, 2011 ; Levy, I. et al., Curr. Opin. Pharmacol. 11 : 689-697, 2011 ; Azevedo, M. F. and Stratakis, C. A. Endocr. Pract. 17 Suppl 3: 2-7, 2011).
• PDE4 long form activators of the present invention are therefore expected to be of utility in the treatment, prevention or partial control of these diseases, such as adrenocortical tumours, testicular cancer, PPNAD and Carney Complex.
• Adrenocortical tumours such as adrenocortical tumours, testicular cancer, PPNAD and Carney Complex.
• Adrenocortical tumours associated with an inactivating point mutation in the gene encoding PDE11A4 have decreased expression of PDE11A4 and increased cAMP levels (Horvath, A. et al., Nat Genet. 38: 794-800, 2006; Horvath, A. et al., Cancer Res. 66: 11571-11575, 2006; Libe, R., et al., Clin. Cancer Res. 14: 4016-4024, 2008).
• PNAD Primary pigmented nodular adrenocortical diseases
• Carney Complex In Carney Complex (CNC) caused by PRKAR1A mutations some patients also have defects in PDE1 1A that may exert a synergistic effect to enhance abnormal activation of the cAMP signal transduction pathway, leading to adrenal and testicular cancer (Libe, R. et a/., J. Clin. Endocrinol. Metab. 96: E208-214, 2011).
• treatment herein is meant the treatment by therapy, whether of a human or a non- human animal (e.g., in veterinary applications) typically a non-human mammal, in which some desired therapeutic effect on the condition is achieved; for example, the inhibition of the progress of the disorder, including a reduction in the rate of progress, a halt in the rate of progress, amelioration of the disorder or cure of the condition.
• Treatment as a prophylactic measure is also included.
• References herein to prevention or prophylaxis herein do not indicate or require complete prevention of a condition; its manifestation may instead be reduced or delayed via prophylaxis or prevention according to the present invention.
• a “therapeutically effective amount” herein is meant an amount of the one or more compounds of the invention or a pharmaceutical formulation comprising such one or more compounds, which is effective for producing such a therapeutic effect, commensurate with a reasonable benefit/risk ratio.
• appropriate dosages of the compounds of the invention may vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
• the selected dosage level will depend on a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination and the age, sex, weight, condition, general health and prior medical history of the patient.
• the amount of compound(s) and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action so as to achieve the desired effect.
• Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to a person skilled in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
• a suitable dose of the one or more compounds of the invention may be in the range of about 0.001 to 50 mg/kg body weight of the subject per day, preferably in a dosage of 0.01-25 mg per kg body weight per day, e.g., 0.01 , 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 10 or 25 mg/kg per day.
• the compound(s) is a salt, solvate, prodrug or the like
• the amount administered may be calculated on the basis of the parent compound and so the actual weight to be used may be increased proportionately.
• the compounds of the invention may also find application in mimicking or enhancing the effects of drugs known to produce their therapeutic effect through lowering of intracellular cAMP levels.
• a number of therapeutically beneficial drugs have a primary mode of action involving lowering intracellular cAMP levels and/or cAMP-mediated activity, as summarised below. Since PDE4 long form activators of the present invention will also act to lower cAMP levels it is expected that these agents will mimic and / or augment the pharmacological properties and therapeutic utility of drugs operating through a down-regulation of cAMP-mediated signalling.
• a compound of the invention is therefore provided as part of a combination therapy with another agent that lowers intracellular cAMP levels and/or cAMP-mediated activity.
• the combination therapy may be administered simultaneously, contemporaneously, sequentially or separately.
• the compound of the invention and the separate cAMP lowering agent are provided in a single composition, as described in more detail below.
• the combination therapy may comprise a compound of the invention and one or more of:
• a presynaptic a-2 adrenergic receptor agonist optionally clonidine, dexmedetomidine, or guanfacine
• a ⁇ -1 Adrenergic receptor antagonist (“beta-blocker"), optionally Atenolol, Metoprolol, Bisoprolol, Acebutolol, or Betaxolol.
• a-2 Adrenergic receptor stimulation is known to reduce cAMP levels through a G, protein- mediated inhibition of adenylyl cyclase activity in a broad range of tissues.
• presynaptic a-2 adrenergic receptor activation inhibits noradrenaline release and noradrenergic activity.
• Drugs e.g. clonidine, dexmedetomidine, guanfacine
• Clonidine the prototypic agent, has shown therapeutic utility in the treatment of hypertension, neuropathic pain, opioid detoxification, insomnia, ADHD, Tourette syndrome, sleep hyperhidrosis, addiction (narcotic, alcohol and nicotine withdrawal symptoms), migraine, hyperarousal, anxiety and also as a veterinary anaesthetic.
• Lowering of cAMP levels by PDE4 long form activation may be expected to yield similar effects to drugs acting through a-2 adrenergic receptor stimulation.
• PDE4 long form activators of the present invention may be expected to potentiate the pharmacodynamic effects of a-2 adrenergic receptor agonists when used in combination.
• ⁇ -1 Adrenergic receptor antagonists are used in the treatment a range of cardiovascular indications including hypertension, cardiac arrhythmias and cardioprotection following myocardial infarction. Their primary mechanism of action involves reducing the effects of excessive circulating adrenaline and sympathetic activity, mediated by noradrenaline, particularly at cardiac ⁇ -1 adrenergic receptors. Endogenous and synthetic ⁇ -1 adrenergic receptor agonists stimulate adenylyl cyclase activity through G s activation and raise intracellular cAMP levels in a variety of tissues such as heart and kidney.
• PDE4 long form activators of the present invention may be expected to find utility in the treatment or partial control of hypertension, cardiac arrhythmias, congestive heart failure and cardioprotection. Additional non-cardiovascular therapeutic utility may be expected in disorders such as post-traumatic stress related disorder, anxiety, essential tremor and glaucoma, which also respond to ⁇ -1 adrenergic antagonist treatment. Furthermore, PDE4 long form activators of the present invention may be expected to potentiate the pharmacodynamic effects of ⁇ -1 adrenergic receptor antagonists when used in combination. Methods of treatment
• the present invention provides a small molecule activator of a PDE4 long form of Formula 1 or Formula 2 for use in a method for the treatment or prevention of a disease or disorder in a patient in need of such therapy.
• the disease or disorder may be any disease of disorder described herein, including: a disease associated with increased cAMP production and signalling (such as hyperthyroidism, Jansens's metaphyseal chondrodysplasia, hyperparathyroidism, familial male-limited precocious puberty, pituitary adenomas, Cushing's disease, polycystic kidney disease, polycystic liver disease, MODY5 and cardiac hypertrophy); diseases known to be associated with increased cAMP-mediated signalling, including disorders associated with activating mutations of the alpha subunit of the G protein (GNAS1) (such as McCune-Albright syndrome); amelioration of toxin-induced increases in adenylyl cyclase activity in infectious diseases (such as chol
• compound of Formula 2 etc. include pharmaceutically acceptable derivatives thereof and polymorphs, isomers and isotopically labelled variants thereof. Furthermore, these terms include all the sub-embodiments of those compounds disclosed herein.
• compositions comprising a compound of the invention, including a pharmaceutically acceptable salt, solvate, ester, hydrate or amide thereof, in admixture with a pharmaceutically acceptable excipient(s), and optionally other therapeutic agents.
• accepted means being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
• Compositions include e.g. those suitable for oral, sublingual, subcutaneous, intravenous, epidural, intrathecal, intramuscular, transdermal, intranasal, pulmonary, topical, local, or rectal administration, and the like, typically in unit dosage forms for administration.
• pharmaceutically acceptable salt includes a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids and bases.
• Compounds of the invention which contain basic, e.g. amino, groups are capable of forming pharmaceutically acceptable salts with acids.
• Examples of pharmaceutically acceptable acid addition salts of the compounds according to the invention include acid addition salts formed with organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
• organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids
• organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzen
• Pharmaceutically acceptable basic salts of the compounds of the invention include, but are not limited to, metal salts such as alkali metal or alkaline earth metal salts (e.g. sodium, potassium, magnesium or calcium salts) and zinc or aluminium salts and salts formed with ammonia or pharmaceutically acceptable organic amines or heterocyclic bases such as ethanolamines (e.g. diethanolamine), benzylamines, N-methyl-glucamine, amino acids (e.g. lysine) or pyridine. Hemisalts of acids and bases may also be formed, e.g. hemisulphate salts.
• metal salts such as alkali metal or alkaline earth metal salts (e.g. sodium, potassium, magnesium or calcium salts) and zinc or aluminium salts and salts formed with ammonia or pharmaceutically acceptable organic amines or heterocyclic bases such as ethanolamines (e.g. diethanolamine), benzylamines, N-methyl-glucamine, amino acids (e.g. ly
• compositions of the compounds of the invention may be prepared by methods well-known in the art.
• pharmaceutically acceptable salts see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002).
• the invention includes prodrugs of the compounds of Formulae 1 and 2.
• Prodrugs are derivatives of compounds of Formula 1 or 2 (which may have little or no pharmacological activity themselves), which can, when administered in vivo, be converted into compounds of Formula 1 or 2.
• Prodrugs can, for example, be produced by replacing functionalities present in the compounds of Formula 1 or 2 with appropriate moieties which are metabolized in vivo to form a compound of Formula 1 or 2.
• the design of prodrugs is well-known in the art, as discussed in Bundgaard, Design of Prodrugs 1985 (Elsevier), The Practice of Medicinal Chemistry 2003, 2 nd Ed, 561-585 and Leinweber, Drug Metab. Res. 1987, 18: 379.
• prodrugs of compounds of Formula 1 or 2 are amides and esters of those compounds.
• the compound of Formula 1 or 2 contains a carboxylic acid group (-COOH)
• the hydrogen atom of the carboxylic acid group may be replaced in order to form an ester (e.g. the hydrogen atom may be replaced by Ci-6alkyl).
• the hydrogen atom of the alcohol group may be replaced in order to form an ester ⁇ e.g. the hydrogen atom may be replaced by -C(0)Ci-6alkyl.
• solvate is used herein to refer to a complex of solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a mono-hydrate, di- hydrate, tri-hydrate etc, depending on the number of water molecules present per molecule of substrate.
• Stereoisomers It will be appreciated that the compounds of the present invention may exist in various stereoisomeric forms and the compounds of the present invention as hereinbefore defined include all stereoisomeric forms and mixtures thereof, including enantiomers and racemic mixtures.
• the present invention includes within its scope the use of any such stereoisomeric form or mixture of stereoisomers, including the individual enantiomers of the compounds of Formula 1 or 2 as well as wholly or partially racemic mixtures of such enantiomers. Where appropriate isomers can be separated from their mixtures by the application or adaptation of known methods (e.g. chromatographic techniques and recrystallisation techniques). Where appropriate isomers can be prepared by the application or adaptation of known methods (e.g. asymmetric synthesis).
• Isotopes The invention includes pharmaceutically acceptable isotopically-labelled compounds of Formula 1 or 2 wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2 H and 3 H, carbon, such as 11 C, 13 C and 14 C, chlorine, such as 3S CI, fluorine, such as 18 F, iodine, such as 123 l and 125 l, nitrogen, such as 13 N and 15 N, oxygen, such as 15 0, 17 0 and 18 0, and sulphur, such as 35 S.
• isotopically-labelled compounds of Formula 1 or 2 are useful in drug and/or substrate tissue distribution studies.
• the radioactive isotopes 3 H and 14 C are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
• Substitution with positron emitting isotopes, such as 11 C, 18 F, 15 0 and 13 N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
• Isotopically-labelled compounds of Formula 1 or 2 can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.
• the active ingredient may be presented as discrete units, such as tablets, capsules, powders, granulates, solutions, suspensions, and the like.
• Formulations suitable for oral administration may also be designed to deliver the compounds of the invention in an immediate release manner or in a rate-sustaining manner, wherein the release profile can be delayed, pulsed, controlled, sustained, or delayed and sustained or modified in such a manner which optimises the therapeutic efficacy of the said compounds.
• Means to deliver compounds in a rate-sustaining manner are known in the art and include slow release polymers that can be formulated with the said compounds to control their release.
• rate-sustaining polymers include degradable and non-degradable polymers that can be used to release the said compounds by diffusion or a combination of diffusion and polymer erosion.
• rate-sustaining polymers include hydroxypropyl methylcellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, xanthum gum, polymethacrylates, polyethylene oxide and polyethylene glycol.
• Liquid (including multiple phases and dispersed systems) formulations include emulsions, suspensions, solutions, syrups and elixirs. Such formulations may be presented as fillers in soft or hard capsules (made, for example, from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
• the compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Liang and Chen, Expert Opinion in Therapeutic Patents 2001 , 1 1 (6): 981-986.
• the active ingredient may be presented in the form of a dry powder from a dry powder inhaler or in the form of an aerosol spray of a solution or suspension from a pressurised container, pump, spray, atomiser or nebuliser.
• the pharmaceutical composition of the invention may be presented in unit-dose or multi-dose containers, e.g. injection liquids in predetermined amounts, for example in sealed vials and ampoules, and may also be stored in a freeze dried (lyophilized) condition requiring only the addition of sterile liquid carrier, e.g. water, prior to use.
• sterile liquid carrier e.g. water
• the compounds of the invention may be administered directly into the blood stream, into subcutaneous tissue, into muscle, or into an internal organ.
• Suitable means for administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous.
• Suitable devices for administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
• Parenteral formulations are typically aqueous or oily solutions.
• excipients such as sugars (including but restricted to glucose, mannitol, sorbitol, etc.) salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen- free water (WFI).
• WFI sterile, pyrogen- free water
• Parenteral formulations may include implants derived from degradable polymers such as polyesters (i.e. polylactic acid, polylactide, polylactide-co-glycolide, polycapro-lactone, polyhydroxybutyrate), polyorthoesters and polyanhydrides. These formulations may be administered via surgical incision into the subcutaneous tissue, muscular tissue or directly into specific organs.
• degradable polymers such as polyesters (i.e. polylactic acid, polylactide, polylactide-co-glycolide, polycapro-lactone, polyhydroxybutyrate), polyorthoesters and polyanhydrides.
• parenteral formulations under sterile conditions may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
• solubility of compounds of the invention used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of co-solvents and/or solubility-enhancing agents such as surfactants, micelle structures and cyclodextrins.
• the active agent may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules, suppositories or patches.
• the active agent can be applied as a fluid composition, e.g. as an injection preparation or as an aerosol spray, in the form of a solution, suspension, or emulsion.
• Suitable carriers with which the active agent of the invention can be administered as solid compositions include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.
• aqueous suspensions, isotonic saline solutions and sterile injectable solutions may be used, containing pharmaceutically acceptable dispersing agents and/or wetting agents, such as propylene glycol or butylene glycol.
• the invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material suitable for said composition, said packaging material including instructions for the use of the composition for the use as hereinbefore described.
• the one or more compounds of the present invention may be used in combination therapies for the treatment of the described conditions i.e. , in conjunction with other therapeutic agents.
• the two or more treatments may be given in individually varying dose schedules and via different routes.
• a compound of the invention is administered in combination therapy with one, two, three, four or more, preferably one or two, preferably one other therapeutic agents
• the compounds can be administered simultaneously or sequentially.
• they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1 , 2, 3, 4 or more hours apart, or even longer period apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
• the invention provides a product comprising a compound of the invention and another therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy.
• the therapy is the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required.
• Products provided as a combined preparation include a composition comprising a compound of the invention and the other therapeutic agent together in the same pharmaceutical composition, or the compound of the invention and the other therapeutic agent in separate form, e.g. in the form of a kit.
• the invention provides a pharmaceutical composition comprising a compound of the invention and another therapeutic agent.
• the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, as described above.
• the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention.
• the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
• An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like.
• the kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another.
• the kit of the invention typically comprises directions for administration.
• the compound of the invention and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of the invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the compound of the invention and the other therapeutic agent.
• the invention also provides the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the medicament is prepared for administration with another therapeutic agent.
• the invention also provides the use of another therapeutic agent in the manufacture of medicament for treating a disease or condition mediated by cAMP for the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the medicament is prepared for administration with a compound of the invention.
• the invention also provides a compound of the invention for use in the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the compound of the invention is prepared for administration with another therapeutic agent.
• the invention also provides another therapeutic agent for use in the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the other therapeutic agent is prepared for administration with a compound of the invention.
• the invention also provides a compound of the invention for use in for the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the compound of the invention is administered with another therapeutic agent.
• cAMP cyclic 3',5'-adenosine monophosphate
• the invention also provides another therapeutic agent for use in the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAM P) is required, wherein the other therapeutic agent is administered with a compound of the invention.
• cAM P cyclic 3',5'-adenosine monophosphate
• the invention also provides the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the patient has previously (e.g. within 24 hours) been treated with another therapeutic agent.
• the invention also provides the use of another therapeutic agent in the manufacture of a medicament for the treatment or prevention of disorders where a reduction of second messenger responses mediated by cyclic 3',5'-adenosine monophosphate (cAMP) is required, wherein the patient has previously (e.g. within 24 hours) been treated with a compound of the invention.
• the other therapeutic agent is:
• a presynaptic a-2 adrenergic receptor agonist optionally clonidine, dexmedetomidine, or guanfacine
• a ⁇ -1 Adrenergic receptor antagonist (“beta-blocker"), optionally Atenolol, Metoprolol, Bisoprolol, Acebutolol, or Betaxolol.
• Table 1 shows examples of novel small molecule PDE4 long form activators of Formula 1 and Formula 2 (Examples A to L), according to the present invention
• Table 2 shows activation of PDE4D5, a long form of PDE4, by Examples A to L.
• Table 3 shows activation of PDE4A4, another long form of PDE4, by Example A.
• Table 4 shows activation of PDE4B1 , another long form of PDE4, by Example A.
• Figure 1 shows activation of PDE4D5, a long form of PDE4, by Examples A, B, D and L.
• FIG. 1 shows activation of PDE4A4, another long form of PDE4, by Example A.
• Figure 3 shows activation of PDE4B1 , another long form of PDE4, by Example A.
• Figure 4 shows a lack of effect on PDE4B2, a short form of PDE4, Examples A, B, D and L.
• Figure 5 shows a reduction in intracellular cAMP levels in HEK 293 cells treated with a PDE4 long form activator of the present invention (10 ⁇ ) for 10 min prior to forskolin (F) (10 ⁇ ) for 2 min.
• Figure 6 shows a reduction in intracellular cAMP levels in Madin-Darby canine kidney (MDCK) cells treated with PDE4 long form activators of the present invention (10 ⁇ ) for 10 min prior to forskolin (F) (10 ⁇ ) for 2 min.
• Figure 7 shows inhibition of in vitro cyst formation in MDCK cells treated with a PDE4 long form activator of the present invention.
• Figure 8 shows reversal of in vitro cyst formation in MDCK cells treated with a PDE4 long form activator of the present invention.
• Figure 9 shows inhibition of proliferation of androgen-sensitive (AS) LNCaP human prostate cancer cells treated with a PDE4 long form activator of the present invention.
• Figure 10 shows inhibition of proliferation of androgen-insensitive (Al) LNCaP human prostate cancer cells treated with a PDE4 long form activator of the present invention.
• Figure 11 shows the in vivo mouse pharmacokinetic profile of Example A, determined by whole blood analysis at defined time points after i.v. and p.o. administration to male C57 mice.
• Figure 12 shows the in vivo rat pharmacokinetic profile of Example A, determined by blood plasma analysis at defined time points after i.v. and p.o. administration to male SD rats.
• Figure 13 shows (A) inhibition and (B) reversal of in vitro cyst formation in OX161 cells treated with Example A, a PDE4 long form activator of the present invention.
• NMR spectra were recorded using a Bruker AV300 spectrometer at 25 °C. The following abbreviations are used in the assignment of NMR signals: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet), dd (doublet of doublet), dt (doublet of triplet).
• Example A /V-(3-Fluorobenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 y-1 ,2,4-triazol-1- yllacetamide Step 1 : A/-(3-Fluorobenzyl)-2-chloroacetamide
• Step 2 /V-(3-Fluorobenzyl)-2-iodoacetamide
• Step 4 3-(4-Chloro-3-fluorophenyl)-5-ethyl-1H-1 ,2,4-triazole
• a mixture of 4-chloro-3-fluorobenzene carboximidic acid methyl ester, hydrochloride salt (187 mg, 0.83 mmol), hydrazine hydrate (0.88 ml_, 18.0 mmol) and aluminium chloride (120 mg, 0.90 mmol) in toluene (30 ml_) was heated to reflux for 3h. The mixture was concentrated under reduced pressure, taken up in toluene and concentrated under reduced pressure twice more.
• Step 5 /V-(3-Fluorobenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 W-1 ,2,4-triazol-1- yl]acetamide
• Example B A/-Benzyl-2-[3-(4-chlorophenyl)-5-methoxymethyl-1 - -1 ,2,4-triazol-1-yllacetamide Step 1 : A/-Benzyl-2-chloroacetamide
• Step 3 3-(4-Chlorophenyl)-5-methoxymethyl-1 -/-1 ,2,4-triazole
• 4- chlorobenzonitrile (1 .38 g, 10 mmol).
• the resulting suspension was stirred at room temperature under argon for 2.5h.
• a solution of methoxyacetic acid hydrazide (1 .04 g, 10 mmol) in dry methanol (10 ml.) was added to the mixture, resulting in a clear solution, which was heated to reflux for 3h and then stirred at room temperature overnight.
• Step 4 /V-Benzyl-2-[3-(4-chlorophenyl)-5-methoxymethyl-1 H-1 ,2,4-triazol-1-yl]acetamide
• Example C A/-Benzyl-2-[3-(4-chlorophenylV5-rTiethyl-1 -/-1 ,2,4-triazol-1-yl1acetamide
• the title compound was prepared according to the method of Example B, using acethydrazide instead of methoxyacetic acid hydrazide.
• Example D /V-(3-Fluorobenzyl)-2- ⁇ 3-[4-(trifluoromethoxy)phenyll-5-methoxymethyl-1 HA 2A- tri azo I- 1 -yl ⁇ acetam i d e
• the title compound was prepared according to the method of Example B, steps 3 and 4, using 4-(trifluoromethoxy)benzonitrile instead of 4-chlorobenzonitrile and /V-(3-fluorobenzyl)- 2-iodoacetamide (as prepared in Example A) instead of /V-benzyl-2-iodoacetamide.
• Step 1 3-(4-Chloro-3-fluorophenyl)-5-ethyl-1H-1 ,2,4-triazole
• the triazole was prepared according to the method of Example B, step 3, using 4-chloro-3- fluorobenzonitrile instead of 4-chlorobenzonitrile and propionic acid hydrazide instead of methoxyacetic acid hydrazide.
• Step 2 0-tert-butyl-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 H-1 ,2,4-triazol-1-yl]acetate
• Step 3 2-[3-(4-Chloro-3-fluorophenyl)-5-ethyl-1H-1 ,2,4-triazol-1-yl]acetic acid
• Step 4 2-[3-(4-Chloro-3-fluorophenyl)-5-ethyl-1H-1 ,2,4-triazol-1-yl]acetyl chloride
• 2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 H-1 ,2,4-triazol-1-yl]acetic acid (crude material from step 3, ca. 1.94 mmol) in dichloromethane (30 mL) was added thionyl chloride (2.32 mL, 32.0 mmol). The mixture was heated under reflux for 6h.
• Step 5 /V-(3-Chlorobenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 HA ,2,4-triazol-1- yl]acetamide
• Example G A/-r3-(Trifluoromethyl)benzyll-2-r3-(4-chloro-3-fluorophenyl)-5-ethyl-1 /-/-1 ,2,4- triazol-1-yllacetamide
• Example H A/-(3-Methoxybenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 y-1 ,2,4-triazol-1- yllacetamide
• the title compound was prepared according to the method of Example E, using 3- methoxybenzylamine instead of 3-chlorobenzylamine in Step 5.
• Example I /V-r3-(Trifluoromethoxy)benzyll-2-r3-(4-chloro-3-fluorophenyl)-5-ethyl-1 /-/-1 ,2,4- triazol-1-yllacetamide
• Example J A/-(2-Fluorobenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 y-1 ,2,4-triazol-1- yllacetamide
• the title compound was prepared according to the method of Example E, using 2- fluorobenzylamine instead of 3-chlorobenzylamine in Step 5.
• Example K /V-(4-Fluorobenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1 y-1 ,2,4-triazol-1- yllacetamide
• Example L A/-(3,4-Dimethoxybenzyl)-2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1H-1 ,2,4-triazol- 1-yllacetamide
• HEK 293 and MDCK cells Public Health England, Cell Culture Collections
• DMEM fetal bovine serum
• 2 mM L-glutamine 1 ,000 U penicillin and 1 ,000 ⁇ g streptomycin (Life Technologies); termed complete media.
• Clonal HEK 293 cell lines were maintained in complete media supplemented with 0.6 mg/mL G418 (Enzo Life Sciences).
• HEK 293 cells were transfected with pDESTTM PDE4 expression vectors using Lipofectamine LTX/Plus reagent (Invitrogen) as outlined by the manufacturer and clonal isolates expanded to obtain cell lines that stably expressed the full-length human PDE4D5, PDE4A4 and PDE4B1 long isoforms and the full length human PDE4B2 short isoform. These were called the HEK-PDE4D5, HEK-PDE4A4, HEK-PDE4B1 and HEK-PDE4B2 cell lines, respectively.
• HEK-PDE4D5 cells were seeded out in 100mm plates and incubated at 37°C in an atmosphere of 5% CO ⁇ , 95% air.
• Cell lysates were prepared using KHEM buffer [50mM KCI, 10mM EGTA, 50mM HEPES (pH 7.2), 1.92mM MgCI 2 ].
• the 100mm plates containing the cells were placed on ice and washed with ice-cold PBS (phosphate buffered saline, pH 7.4). KHEM buffer (500 ⁇ ) was added to the cells. Cells were then scraped off the plate and triturated using a needle (BD MicrolanceTM 0.8, 40mm). The lysed cells were then centrifuged at 2000rpm for 10 minutes to remove cell debris and the supernatant (cell lysate containing recombinant PDE4D5) was transferred to a fresh tube and kept on ice.
• PBS phosphate buffered saline, pH 7.4
• KHEM buffer 500 ⁇
• Cells were then scraped off the plate and triturated using a needle (BD MicrolanceTM 0.8, 40mm).
• the lysed cells were then centrifuged at 2000rpm for 10 minutes to remove cell debris and the supernatant (cell lysate containing recombinant PDE4D5) was transferred to a
• Cytosol fraction preparation (using PDE4D5 as a typical example) The cell lysate containing recombinant PDE4D5 was transferred into a centrifuge tube and placed into an ultracentrifuge (BECKMAN COULTER) and spun at high speed (100,000g) for 30 minutes at 4°C. The cytosol fraction was then collected and its protein amount determined using a BCA protein assay.
• PDE Assay (using PDE4D5 as a typical example) PDE assays were performed using a final concentration of 10 mM Tris/5 mM MgC and 1 ⁇ [3H]-cAMP (Perkin Elmer) plus PDE4D5 cell lysate cytosol fraction, containing over- expressed PDE4D5, with and without test compound. Incubations were performed at 30°C for 5 minutes. The samples were then placed in a boiling water bath for 2 minutes to denature the PDE enzyme, and returned to ice. Samples were allowed to cool for 10 min after which snake venom 5'-nucleotidase (25 ⁇ , 1 mg/ml; Sigma) was added.
• snake venom 5'-nucleotidase 25 ⁇ , 1 mg/ml; Sigma
• the tubes were vortexed and incubated in a water bath at 30°C for 10 minutes, to attain conversion of [3H]- 5'-AMP to [3H]-adenosine, and then placed on ice.
• Dowex ion exchange resin (Sigma) prepared as a 1 :1 Dowex: water stock was thoroughly re-suspended and diluted 2: 1 with ethanol. The resulting Dowex suspension (0.4 ml) was added to each tube. Tubes were vortexed to mix and then incubated on ice for at least 15 minutes before a final vortex.
• HEK 293 or MDCK cells were seeded at 100,000 cells per well, and left to adhere overnight. The cells were then treated with the compound indicated (10 ⁇ ) for 10 minutes, prior to stimulation with forskolin (10 ⁇ , Sigma) for 2 minutes. Media was aspirated, and hydrochloric acid (0.1M) was added to lyse the cells. The cAMP assay (Enzo Life Sciences) was performed according to the manufacturer's instructions.
• Compounds of the present invention reduced intracellular cAMP levels in forskolin stimulated HEK 293 or MDCK cells.
• MDCK cells (50,000 cells/well) were seeded into collagen (Life Technologies; final concentration 1 img/mL), containing 17 mM NaOH in DMEM, supplemented with 2% (v/v) FBS, 2 mM L- glutamine and 2 mM L-glutamine, 1 ,000 U penicillin and 1 ,000 pg streptomycin (DMEM- 2%FBS), on ice.
• DMEM- 2%FBS Upon gelling at 37°C, 1 mL of DMEM-2%FBS was added along with the test compound indicated in the presence of 10 ⁇ forskolin (Sigma) and 1 ⁇ g/mL [Arg 8 ]- vasopressin acetate salt (Sigma). Media was replenished every 2 days for 20 days; at every feed, test compound, forskolin and vasopressin were added.
• Phase-contrast images were obtained on the Motic microscope (x200 magnification) every 2 days for 20 days. Per condition, 10 images were taken (in duplicate) and the average cyst radius measured.
• LNCaP ceil culture Androgen-sensitive (AS) LNCaP cells were maintained in RPMI1640 supplemented with 10% FBS (Seralabs), 2mM L-glutamine and 1 ,000U penicillin-streptomycin.
• LNCaP androgen-insensitive (AI) cells were generated in-house by culturing the LNCaP-AS cells in RPMI1640 supplemented with 10% charcoal stripped FBS, 2mM L-glutamine and 1 ,00011 penicillin-streptomycin for a minimum of four weeks. All tissue culture reagents were from Life Technologies.
• Cell proliferation is measured as a function of changing electrical impedance. Values are represented by cell index number, a dimensionless unit of measurement representing the cell status, which increases as cells adhere to 96-well electrode plates and divide.
• LNCaP AI/AS cells were plated at a density of 25,000 cells per well in a 96-well electrode plate (in triplicate), in the presence/absence of various concentrations of test compound.
• Example A The effects of Example A on proliferation of androgen-insensitive (Al) LNCaP human prostate cancer cells are shown in Figure 10.
• Example 6 Measurement of in vitro clearance of Example A using human hepatocytes Human hepatocyte stability is considered the gold standard method for evaluating the hepatic metabolism of drugs in vitro. The in vitro clearance of Example A was evaluated using cryopreserved human hepatocytes according to the method of Lau et al. (Lau, Y. Y. et al. Drug Metab. Dispos. 30: 1446-1454, 2002) with minor modifications.
• Cryopreserved hepatocytes were thawed in a water bath at 37°C and transferred to a tube containing 50 ml of hepatocyte thaw medium.
• the hepatocytes were centrifuged at 500 rpm for 3 min.
• the supernatant was removed and the hepatocytes were resuspended in medium, mixed gently and centrifuged again at 500 rpm for 3 min.
• the supernatant was discarded and the hepatocyte pellet was gently resuspended in medium to a final density of 2 million cells/ml. Incubations were carried out at a final test compound concentration of 1 ⁇ .
• Example A is moderately stable in human hepatocytes.
• Example A was dissolved in 5%DMA+10%Solutol+85%(10%HPBCD in water) to afford a clear dosing solution of 1 mg/ml_.
• the dosing solution was administered to three mice at 2 mg/kg i.v. via the tail vein and to three further mice at 10 mg/kg p.o. via oral gavage.
• Blood samples (ca. 20 ⁇ _) were collected at 2, 5, 20 min, 1 , 2, 4, 8 and 12 h after i.v. dosing and at 15, 30 min, 1 , 2, 4, 8, 12 and 24 h after oral dosing.
• the blood samples were diluted with 3 volumes of distilled water and stored at -80°C until analysis. The samples were analysed by UPLC-MS/MS (API 5500).
• Example A exhibited 66% oral bioavailability in male C57 mice, with a terminal half life of 4.9 hours after oral dosing and 4.2 hours after i.v. dosing. No abnormal effects were observed in the mice.
• the mean whole blood concentration-time profiles of Example A after i.v. and p.o. dosing are shown in Figure 1 1 .
• Example A Pharmacokinetic profile of Example A in male SD rats
• Example A was dissolved in 5%DMA+10%Solutol+85%(10%HPBCD in saline) to afford a clear dosing solution of 1 mg/mL.
• the dosing solution was administered to three rats at 2 mg/kg i.v. via the foot dorsal vein and to three further rats at 10 mg/kg p.o. via oral gavage.
• Blood samples (ca. 150 ⁇ _) were collected at 2, 5, 20 min, 1 , 2, 4, 8, 12 and 24 h after i.v. dosing and at 5, 15, 30 min, 1 , 2, 4, 8, 12 and 24 h after oral dosing.
• the blood samples were centrifuged at 4°C (2000 g, 5 min) within 15 min of sample collection to obtain plasma samples. Plasma samples were stored at -80°C until analysis. The samples were analysed by UPLC-MS/MS (API 4000).
• Example A exhibited 100% oral bioavailability in male SD rats, with a terminal half life of 9.1 hours after oral dosing and 2.2 hours after i.v. dosing. No abnormal effects were observed in the rats.
• the mean plasma concentration-time profiles of Example A after i.v. and p.o. dosing are shown in Figure 12.
• a human ADPKD patient-derived (OX161) cell line was used to investigate the effects of PDE4 long form activators on the formation of kidney cysts in vitro and evaluate their potential in the treatment of polycystic kidney diseases.
• Conditionally immortalised OX161 cystic tubular epithelial cells were generated from human kidneys removed for clinical indications from ADPKD patients with characterised PKD1 (PC1) mutations (Parker, E., Newby, L. J., Sharpe, C. C, Rossetti, S., Streets, A. J., Harris, P. C, O'Hare, M. J., Ong, A. C. M. Kidney Int. 72(2): 157-165, 2007).
• OX161 cells (50,000 cells/well) were seeded into collagen (Life Technologies; final concentration 1 mg/mL), containing 17 mM NaOH in DMEM, supplemented with 2% (v/v) FBS, 2 mM L-glutamine and 2 mM L-glutamine, 1 ,000 U penicillin and 1 ,000 pg streptomycin (DMEM-2%FBS), on ice.
• DMEM-2%FBS penicillin and 1 ,000 pg streptomycin
• Phase-contrast images were obtained every 2 days for 10 days. Per condition, 10 images were taken (in duplicate) and the average cyst area calculated.
• Example A inhibited in vitro cyst formation in the OX161 cells in a concentration dependent manner.
• the day 10 phase-contrast images and cyst area graphs for Example A are shown in Figure 13 (Panel A).
• Experiment 10 Reversal of in vitro cyst formation in OX161 cells treated with PDE4 long form activators of the present invention
• the potential of PDE4 long form activators to reverse the formation of preformed cysts was evaluated in a human ADPKD patient-derived OX161 cell line.
• the experiment was carried out according to the method of Experiment 9. For the first 10 days, forskolin was added at every feed (every 2 days) but no test compound was added. For the next 10 days, the test compound and forskolin were added at every feed (every 2 days). Phase-contrast images were obtained every 2 days for 20 days. Per condition, 10 images were taken (in duplicate) and the average cyst area calculated.
• Table 2 Activation of PDE4D5, a long form of PDE4, by Examples A to L Using the method described in Experiment 1 , the following concentration / PDE4D5 activity data were obtained for Examples A to L. Data are shown in graphical form in Figure 1 .

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