WO2002070469A2 - Selective pde3b inhibitors and use of the same in therapy - Google Patents

Selective pde3b inhibitors and use of the same in therapy Download PDF

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WO2002070469A2
WO2002070469A2 PCT/US2002/005624 US0205624W WO02070469A2 WO 2002070469 A2 WO2002070469 A2 WO 2002070469A2 US 0205624 W US0205624 W US 0205624W WO 02070469 A2 WO02070469 A2 WO 02070469A2
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mmol
group
pde3b
alkyl
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WO2002070469A3 (en
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Peter B. Snyder
Graham Beaton
Jaimie K. Rueter
Dewey L. Fanning
Stephen D. Warren
Sara S. Hadida-Ruah
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Icos Corporation
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Definitions

  • the present invention relates to a series of compounds, to methods of preparing the compounds, to pharmaceutical compositions containing the compounds, and to their use as therapeutic agents.
  • the present invention relates to compounds that are potent and selective inhibitors of cGMP- inhibited phosphodiesterase (cGI-PDE) , and particularly type 3B phosphodiesterase (PDE3B) , and have utility in a variety of therapeutic areas whei'ein such inhibition is considered beneficial, including the treatment of obesity and diabetes.
  • cGI-PDE cGMP- inhibited phosphodiesterase
  • PDE3B type 3B phosphodiesterase
  • Cyclic nucleotide phosphodiesterases are enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) to their respective 5 ' -monophosphate forms. Therefore, PDEs regulate intracellular levels of cyclic nucleotides, and play an important role in modulating cellular signaling.
  • Known mammalian PDEs are classified into eleven families, based on their structure, regulation, and kinetic proper- ties. See S.H. Francis et al . , Prog. Nucleic Acid Res . , 65 , pp. 1-52 (2001) .
  • the present invention is directed to inhibitors of the type-3 phosphodiesterase (PDE3) , also referred to as the cGMP- inhibited phosphodiesterase (cGI-PDE) , and in particular to inhibitors of PDE3B.
  • the PDE3 family contains two members, PDE3A and PDE3B. See Y. Shakur et al . , Prog. Nucleic Acid Res . Molec . Bi ol . , 66, pp. 241-277 (2001) .
  • the two different PDE3 isoforms, encoded by different genes, resemble other mammalian PDEs in having a conserved catalytic domain in the carboxy- terminal portion of the protein and regulatory/- localization motifs located in the amino-terminal portion.
  • the catalytic domains of PDE3A and PDE3B are approximately 85% identical to one another. E. Meacci et al . , Proc . Natl . Acad . Sci .
  • PDE3A is the predominant isoform in cardiac myocytes, vascular and nonvascular smooth muscle cells, and platelets, leading to its designation as the "cardiovascular" isoform.
  • PDE3B is prominent in adipocytes, hepatocytes, endocrine pancreas, kidney epithelium, spermatocytes, embryonic neuroepithelium, and immune cells. See, R.R. Reinhardt et al . , J “ . Clin . Invest . 95, pp.
  • PDE3 inhibitors have been used to treat various diseases and conditions, for example, acute cardiac support in heart failure and for the treatment of intermittent claudication.
  • various diseases and conditions for example, acute cardiac support in heart failure and for the treatment of intermittent claudication.
  • the therapeutic use of PDE3 inhibitors has disadvantages, particularly adverse cardiac effects .
  • Cilostazol also was found to be effective in preclinical models of cerebral infarct and pulmonary thromboembolism, again attributed to the vasodilatory and antiplate- let activities of PDE3 inhibition. See Y. Ikeda, Thromb . Hae ost . , 82, pp. 435-438 (1999).
  • PDE3 inhibitors also inhibit proliferation of VSMC. See M.T. Osinski, Biochem . Pharmacol . , 60 , pp. 381-387 (2000) , suggesting a potential utility of PDE3 inhibitors in the treatment and prevention of atherosclerosis.
  • PDE3 inhibitors reversed in- timal thickening in mouse and rat models of athero- sclerosis, while cilostazol was found to reduce restenosis in humans following coronary angioplasty.
  • K. Kondo et al . Atherosclerosis, 142, pp. 133 -• 138 (1999); Y. Inoue et al . , Br. J. Pharmacol , 130 , pp. 231-241 (2000), and T. Kunishima et al . , Clin . Ther. , 19, pp. 1058-1066 (1997).
  • PDE3 inhibitors also have an effect in the kidney. For example, proliferation of kidney mesangial cells is an indication of several types of glomerular nephritis. PDE3 inhibitors have been found to suppresses proliferation of cultured rat mesangial cells (K. Matousovic et al . , J. Clin . Invest . , 96, pp. 401-410 (1995)). Furthermore, the PDE3 inhibitor lixazinone, when administered in conjunction with the PDE4 inhibitor rolipram, sup- pressed the development of experimentally induced glomerular nephritis in rats. See Y. Tsuboi et al . , J. Clin . Invest .
  • PDE3 inhibitors Human lymphocytes express PDE3B, and PDE3 inhibition has been found to inhibit T-cell proliferation in some, but not all, studies (D. Ekholm et al . , J " .
  • PDE3 inhibitors can potentiate the antiproliferative effects of the PDE4 inhibitor rolipram (M. Giembycz et al . , Br. J. Pharmacol . , 118 , pp. 1945-1958 (1996) and P. Marcoz et al., Molec .
  • PDE3 inhibitors alone have a comparative - ly low ability to inhibit release of inflammatory cytokines, but can act synergistically with PDE4 inhibitors in this process.
  • PDE3 inhibitors also are bronchodilators, and are capable of potentiating the bronchodilatory effects of PDE4 inhibitors. See T.J. Torphy, Am . J Consumer Respir. Cri t . Care Med . , 151, pp. 351-370 (1998).
  • PDE3 inhibitors may be useful in the treatment of asthma and chronic obstructive pulmonary disease (COPD) .
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • cilostazol reverses meth- acholine-induced bronchoconstriction in normal and asthmatic patients (M. Fuj imura et al . , Am . J. Respir. Cri t . Care Med . , 151 , pp. 222-225 (1995)), while enoximone was found to reduce airway resistance in patients suffering from COPD (M. Leeman et al., Chest, 91 , pp. 662
  • PDE3A is present in the smooth muscle of the corpus cavernosum where it plays a role in regulating cavernosal blood pressure and penile erection.
  • Local injection of milrinone into the cavernosum of anesthetized cats increased intra- cavernosal pressure and penile length (P.C. Doherty et al. U.S. Patent No. 6,156,753).
  • PDE3A also is expressed in the oocyte, and
  • PDE3 inhibitors block maturation of mouse oocytes both in vi tro and in vivo, suggesting effectiveness as contraceptives. See A. Tsafriri et al . , Dev. Biol . , 178 , pp. 393-402 (1996) and A. Wiersma et. al., J " . Clin . Invest . , 102 , pp. 532-537 (1998).
  • PDE3 inhibitors induce lipolysis (i.e., hydrolysis of stored triglycerides to free fatty acids and glycerol) in cultured adipocytes (M.L. Elks et al . , Endocrinology, 115, pp. 1262-1268 (1984)).
  • lipolysis i.e., hydrolysis of stored triglycerides to free fatty acids and glycerol
  • amrinone stimulated lipolysis and increased resting energy expenditure (Y. Ruttimann et al., Crit. Care Med. , 22, pp. 1235-1240 (1994)). Therefore, by mobilizing fat reserves, PDE3 inhibitors have been suggested as an effective treatment for obesity (P.B. Snyder, Emerging Therapeutic Targets, 3 , pp.
  • cilostazol lowered serum triglyceride levels and increased serum HDL cholesterol levels in individuals being treated for intermittent claudication. See M.B. Elam et al . , Arterioscler. Thromb. Vase . Biol . , 18 , pp. 1942-1947 (1998).
  • PDE3B isoform. Therefore, it would be desirable to provide a selective PDE3B inhibitor to realize the benefits of PDE3B inhibition, e.g., lipolysis, while avoiding the adverse effects of PDE3A inhibition, e.g., adverse cardiac effects.
  • PDE3 inhibitors also potentiate glucose- stimulated insulin release from pancreatic ⁇ -cells both in vi tro and in vivo . See R. Shafiee-Nick et al., Br. J. Pharmacol , 115, pp. 1486-1492 (1995); M. El-Metwally et al . , Eur. J. Pharmacol , 324 , pp. 227- 232 (1997); and J.C. Parker et al . , Biochem . Biophys, Res . Commun . , 236, pp. 665-669 (1997). Addi- tionally, long-term treatment of diabetic rats with PDE3 inhibitors leads to improved insulin sensitiv- ity of peripheral tissues (Y.
  • PDE3 inhibitors can provide beneficial effects in type 2 diabetes.
  • PDE3 inhibitors can be used in a therapy for a cluster of obesity- associated pathologies, e.g., insulin resistance, hyperinsulinemia, hyperlipidemia, atherosclerosis, and high blood pressure, collectively termed "metabolic syndrome.”
  • a PDE3 in- hibitor has been hampered by adverse side effects associated with present-day PDE3 inhibitors.
  • obesity is a major risk factor for conditions such as diabetes, hyperlipi- demia, hypertension, and coronary artery disease.
  • diabetes hyperlipi- demia
  • hypertension hypertension
  • coronary artery disease the proportion of the population that is obese has been increasing in recent decades (e.g., reaching one-third of the adult pop- ulation of the United States in 1991) . See,
  • weight loss usually occurs only during the first three to six months of treatment, with no further loss observed even when drug treatment is continued for a year or more. This phenomenon is attributed to an increase in energy efficiency following weight loss. According to this model, as body mass decreases, fewer consumed calories are required to sustain resting metabolic activity. As a result, energy expenditure eventually drops to match the reduced level of energy intake, and subsequently no further change in body weight occurs. Thus, the development of pharmacological agents that increase resting metabolic rate and do not exhibit adverse side effects is an important, but elusive, goal in antiobesity research.
  • an antiobesity pharmacological agent increases resting metabolic rate by stimulation of two processes: (1) hydrolysis of triglyc- erides stored in adipose tissue to glycerol and free fatty acids (lipolysis) ; and (2) oxidation of excess free fatty acids by a pathway coupled to the production of heat (thermogenesis) rather than to generation of ATP.
  • lipolytic/thermogenic agents reduce metabolic efficiency by causing a greater fraction of the total caloric intake to be dissi- pated as heat rather than harnessed for useful cellular work. These agents thereby increase metabolic rate because more calories are utilized to sustain basic cellular processes.
  • Elevation of cAMP can be achieved either by stimulation of cAMP synthesis (catalyzed by adenylyl cyclases) or by inhibition of cAMP degradation (catalyzed by cyclic nucleotide PDEs) .
  • PDE3 which is primarily associated with the par- ticulate fraction
  • PDE4 which is the principal cytosolic cAMP-PDE in this cell type.
  • PDE3B is present in adipocytes, as opposed to PDE3A. Accordingly, PDE3B inhibition is an attrac- tive target in providing an antiobesity agent.
  • PDE3B also is the predominant isoform in immune cells
  • selective PDE3B inhibitors also should have utility for the treatment of inflammatory and autoimmune diseases, and for the prevention of graft rejection during organ transplant . It also has been theorized that, in most cell types, PDE3A is predominantly a cytosolic enzyme, while PDE3B is associated with a membrane fraction (H. Liu et al . , Br. J. Pharmacol . , 125, pp. 1501-1510 (1998)). These different intracellular localizations suggest that, within a single cell type ' , the two isoforms serve distinct cellular functions, and that in some cases it is desirable to inhibit one PDE3 isoform selectively. Thus, isoform-selective inhibitors represent a novel mechanism for selectively inhibiting PDE3 activity in a specific intracellular compartment.
  • PDE3 inhibitor has been shown to posses significant selectivity between the two PDE3 isoforms (M-J. Leroy et al . , Biochemistry, 35, pp. 10194-10202 (1996); Snyder, (1999)).
  • the most selective agent reported in the literature is cGMP, which is capable of inhibiting hydrolysis of cAMP with a three-fold higher potency against PDE3A than PDE3B (Leroy et al . , 1996).
  • the present invention is directed to a series of compounds that exhibit a selective inhibition of PDE3B over PDE3A.
  • the present invention also is directed to the use of such compounds in the treatment of diseases and conditions wherein inhibition of PDE3B provides a benefit, while minimizing or eliminating adverse effects associated with PDE3A inhibition.
  • the present invention is directed to selective PDE3B inhibitors, and to use of the selective PDE3B inhibitors in therapy. More particular- ly, the present invention is directed to compounds disclosed herein, to compositions containing a compound disclosed herein, and to methods of treating a condition or disease wherein selective inhibition of PDE3B provides a therapeutic benefit.
  • PDE3 type-3 phosphodiesterase
  • PDE3 inhibitors potentially are useful in the treatment of obesity, diabetes, hyperlipidemia, atherosclerosis, coronary restenosis, thrombocythemia, glomer- ular nephritis, asthma, inflammatory diseases, autoimmune disorders, organ transplant rejection, and erectile dysfunction.
  • present-day PDE3 inhibitors are nonselective with respect to PDE3A inhibition versus PDE3B inhibition, and the simultaneous inhibition of both the PDE3A and PDE3B isoforms leads to adverse side effects. These side effects can be minimized or eliminated by a selective inhibition of PDE3B over PDE3A. Accordingly, the present invention is directed to a selective PDE3B inhibitor having a structural formula (I) :
  • R° groups are taken together to form a 5- or 6-membered nonaromatic ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur;
  • R 1 is Ogalkyl or halo;
  • R 2 is selected from the group consisting of hydrogen, C h alky!, aryl, heteroaryl, C- ⁇ alkylene- aryl, C 1 . 3 alkyleneheteroaryl , C 3 - 8 cycloalkyl , C 3 _ 8 - heterocycloalkyl , and
  • Y is selected from the group consisting of
  • R 3 and R 4 independently, are selected from the group consisting of hydrogen, aryl, heteroaryl, and halo;
  • A is aryl
  • B is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring and. optionally substituted fused bicyclic and polycyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
  • R a and R are selected from the group consisting of hydrogen, C ⁇ alkyl, aryl, arylC j ⁇ alkyl , C ⁇ alkylenearyl , heteroaryl, hetero- arylC 1 . 3 alkyl , , C 1 _ 3 alkoxyC 1 . 6 alkyl, C 3 _ 8 cycloalkyl , C 1 . 3 alkyleneC 3 . 8 - cycloalkyl, C 3 . 8 heterocycloalkyl , C 1 . 3 alkyleneOC 1 _ 3 - alkylenearyl , C 1 . 4 alkyleneOC 1 . 3 alkyl , C 1 .
  • R c is selected from the group consisting of hydrogen, aryl, heteroaryl, C x _ 6 alkyl, C 3 _ 8 cycloalkyl , C 3 _ 8 heterocycloalkyl , C 1 _ 3 alkylenearyl , C 1 .
  • Another aspect of the present invention is to provide a selective PDE3B inhibitor having a structural formula (II) or (III) :
  • Still another aspect of the present invention is to provide a selective PDE3B inhibitor having a structural formula (IV) :
  • the present invention also is directed to selective PDE3B inhibitors having the following structures : (a)
  • a compound of the present invention is defined as a compound encompassed by structural formula (I), (II), (III), or (IV), compounds (a) - (i) listed above, Examples 1- 41, and compounds identified at page 1 through page 39 in Appendix A, which constitutes a porti.on of this disclosure.
  • Another aspect of the present invention is to provide a PDE3B inhibitor having an IC 50 value vs. human recombinant PDE3B of about 10 ⁇ M or less, preferably about 1 ⁇ M or less, more preferably about 500 nM or less, and most preferably about 100 nM or less.
  • a PDE5 inhibitor of the present invention has an IC 50 value versus human recombinant PDE3B of about 700 picomolar to about 1,000 ⁇ m.
  • Yet another aspect of the present inven- 5 tion is to provide a preferred PDE3B inhibitor having a ratio of an IC S0 vs. human recombinant PDE3A to an IC 50 vs. human recombinant PDE3B of at least 5, preferably at least 10, more preferably at least 20, and most preferably at least 30.
  • compositions comprising a compound of the present invention and a physiologically acceptable diluent or carrier.
  • Another aspect of the present invention is
  • the method minimizes or eliminates adverse side effects attributed to PDE3A inhibition.
  • Still another aspect of the present invention is to provide a method of treating an individ-
  • ' 25 suffering from obesity, diabetes, hyperlipidemia, asthma, an inflammatory disease, an autoimmune disorder, or organ transplant rejection, comprising administering a therapeutically effective amount of a compound of the present invention, or a composi-
  • Another aspect of the present invention is to provide therapeutic compounds and methods for an efficacious treatment of obesity that minimize or eliminate adverse side effects associated with prior compounds and methods used to treat obesity.
  • the present invention provides a method of inducing, promoting, or causing lipolysis in mammalian cells comprising the step of contacting mammalian cells with a compound of the present invention at a concentration effective to stimulate lipolysis in the cells.
  • the mammalian cells are mammalian adipo- cytes, and preferably human adipocytes, for example, human brown adipose cells or white adipose cells.
  • Another aspect of the present invention is to provide a combination therapy comprising administration of therapeutically effective amounts of (a) a compound of the present invention, or pharmaceutically acceptable salts and solvates thereof, and (b) a second therapeutically active agent, to an individual for simultaneous, separate, or sequent i ⁇ .1 use in the treatment of a disease or condition wherein inhibition of PDE3B provides a benefit, such as obesity or diabetes.
  • the second therapeutically active agent can be a PDE4 inhibitor, for example.
  • the present invention provides a kit for the treatment of obesity compris- ing a compound of the present invention, or a composition containing the same, packaged with instructions for administration of the compound, or composition, to a mammal, including a human, to promote weight loss.
  • the compound of the present invention and a second therapeutically active ingredient for the treatment of obesity are packaged together in separate vials, separate tablets, or the like.
  • Still another aspect of the present invention is to provide a method of preparing a compound of the present invention.
  • a compound is considered to be a PDE inhibitor if the compound effectively inhibits phosphodiesterase activity of a PDE at a physiologically compatible concentration. • To be- useful as a therapeutic compound, the compound also must be practi- cally nontoxic to a cell at such a concentration.
  • Effective inhibition typically is defined as a compound that inhibits PDE activity by at least 50%, preferably at least 80%, and more preferably at least 90%, at a physiologically compatible concen- ration.
  • a PDE inhibitor is referred to as being selective if the compound effectively inhibits a particular PDE of interest, and only marginally inhibits, or fails to inhibit, other PDEs. In this case, all the PDEs, except the particular PDE of interest, perform their normal functions, and the activity of the PDE of interest is reduced sufficiently to provide a benefit.
  • enzyme inhibition typically is measured using a dose-response assay in which a sensitive assay system is contacted with a compound of interest over a range of concentrations at which no or minimal effect is observed, through higher concentrations at which partial effect is observed, to saturating concentrations at which a maximum effect is observed.
  • assays of the dose-response effect of inhibitor compounds can be described as a sigmoidal curve, expressing a degree of inhibition as a function of concentration. The curve also theoretically passes through a point at which the concentration is sufficient to reduce activity of the PDE enzyme to a level that is 50% that of the difference between minimal and maximal enzyme activity in the assay.
  • This concentration is defined as the Inhibitory Concentration (50%) or IC S0 .
  • the potency of inhibitor compounds can be related in terms of the Effective Concentration (50%) or EC 50 , which is a measure of dose-respcnse activity in a cell -based or animal- based model .
  • EC 5 ⁇ measurements are useful to relate properties of the compound that can influence its clinical utility, such as compound solubility, ability to penetrate cell membranes, partition coefficient, bioavailability, and the like.
  • Two compounds can exhibit a divergence in comparative IC 50 and EC 50 values, i.e., one compound can be more potent in a biochemical assay and the second compound more potent in a cell-based assay simply due to different properties of the compounds.
  • a measure of comparative selectivity is a ratio of IC 5C values for a compound with respect to two different enzymes.
  • the selectivity of a compound is sufficiently high such that, at an effective concen- tration to inhibit the PDE inhibitor of interest, the compound has minimal to no effect on other enzymes .
  • the present, invention is directed, to potent inhibitors of PDE3 , and that further exhibit: a selective inhibition of PDE3B over PDE3A.
  • a selective PDE3B inhibitor is a compound that inhibits PDE3B at least 5-fold, and preferably at least 10 -fold, more effectively than the compound inhibits PDE3A.
  • a selective PDE3B inhibitor inhibits PDE3B at least 25-fold, and even more preferably at least 30-fold, more effectively than the compound inhibits PDE3A.
  • Selective PDE3B inhibitors are expected to promote lipolysis m adipocytes, for example, with fewer adverse side effects (e.g., adverse cardiac effects) than a nonselective PDE3A and PDE3B inhibitor.
  • a PDE3 inhibitor that is selective, or specific, for PDE3B is highly preferred in the treatment of obesity and conditions related to or resulting from obesity, e.g., diabetes .
  • R° groups are taken together to form a 5- or 6-membered nonaromatic ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur;
  • R 1 is Ci.galkyl or ha o
  • R 2 is selected from the group consisting of hydrogen, C h alky!, aryl, heteroaryl, C ⁇ alkylene- aryl , C 1 . 3 alkyleneheteroaryl , C 3 8 cycloalkyl , C 3 frustrating 8 het- erocycloalkyl , and
  • R 3 and R 4 are selected from the group consisting of hydrogen, C 1 . 6 alkyl , aryl, heteroaryl, and halo;
  • A is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring, either carbo- cyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, or A is null ;
  • B is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring and optionally substituted fused bicyclic ana polycyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
  • R a and R b are selected from the group consisting of hydrogen, C t t alkyl , aryl, arylC 1 . 3 alkyl , C-- 6 alkylenearyl , heteroaryl, hetero- arylCj_ 3 alkyl , C 1 _ 3 alkyleneheteroaryl , naloOgalkyl , C 1 _ 3 alkoxyC 1 . 6 alkyl, C 3 . 8 cycloalkyl , C 1 - 3 alkyleneC 3 _ £ - cycloalkyl, C 3 . 8 heterocy ⁇ loalkyl, C ⁇ alkyleneOC ⁇ - alkylenearyi , C 1 .
  • R c is selected from the group consisting of hydrogen, aryl, heteroaryl, Ogalkyl, C 3 . 8 cycloalkyl , C 3 . 8 heterocycloalkyl, C ⁇ alkylenearyl , C ⁇ 3 al ⁇ yleneC 3 . 8 - heterocycloalkyl, C 1 . 3 alkyleneheteroaryl , arylC ⁇ - alkyl, and heteroarylC- ⁇ alkyl ; n is 0, 1, 2, 3, or 4; p is 0 or 1 ; and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
  • the selective PDE3B has the structural formula (II) or (III) . !R°> HX
  • a selective PDE3B inhibitor has a structural formula (TV) :
  • alkyl includes straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups .
  • the hydrocarbon group can contain up to 16 carbon atoms.
  • alkyl includes "bridged alkyl," i.e., a C 6 -C 16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl , adamantyl , bicyclo [2.2.2] octyl , bicyclo [2.2.1] heptyl , bicyclo [3.2.1] octyi , or deca- hydronaphthyl .
  • Alkyl groups can be substituted, for example, with hydroxy (OH) or amino (NH 2 ) groups.
  • cycloalkyl is defined as a cyclic C-C 8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl .
  • Hetero- cycloalkyl is defined similarly as cycloalkyl except the ring concains one to three heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Cycloalkyl and heterocycloalkyl groups can be substituted for example, with one to three groups, independently selected from groups OH.
  • alkenyl is defined identically as “alkyl,” except for containing a carbon-carbon double bond. "Cycloalkenyl " is defined similarly to, and is encompassed by, the term cycloalkyl, except a carbon -carbon double bond is present in the ring .
  • alkylene refers to an alkyl group having a substituent.
  • C- ⁇ alkylenearyl refers to an alkyl group containing one to three carbon atoms, and substituted with an aryl group .
  • halo or halogen is defined herein to include fluorine, bromine, chlorine, and iodine .
  • haloalkyl is defined herein as an alkyl group substituted with one or more halo substituents, either fluoro, chloro, bromo, iodo, or combinations thereof.
  • Halocycloalkyl is encompassed by the term “haloalkyl,” and is defined as a cycloalkyl group having one or more halo substitu- ents.
  • aryl alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g.. phenyl or naphthyl.
  • an "aryl” group can be unsubstituted or substituted, for example, with one or more, and in particular one to three, halo, alkyl, alkenyl, heterocycloalkyl, trifluoromethyl , C ⁇ - alkyleneOCi.galkyl, trif uoromethoxy, S0 2 NR a R , NR a R, benzyloxy, thiobenzyl , C h alk leneObenzyl , OC 3 ._ 5 al.kyl- enearyl , OC j 3 alkyleneheter ⁇ aryl , OC_ ( 8 heterocyclo- aikyl , OCi.
  • Exemplary aryl groups include phenyl, naphthyl, tetrahydronaphthyl , 2-chlorophenyl , 3 -chlorophenyl , 4-chloropnenyl , 2- methylphenyl , 4-methoxyphenyl , 3-trifluoromethyl- phenyl, 4-nitrophenyl , and the like.
  • arylC 1 . 3 alkyl and “heteroarylC- ⁇ alkyl” are defined as an aryl or heteroaryl group having a C 1 _ 3 alkyl substituent .
  • heteroaryl is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, for example, the substituents listed above for aryl groups.
  • heteroaryl groups include thienyl, furyl , pyridyl, oxazolyl, quinolyl , isoquinolyl, indolyl , triazolyl, isothiazolyl , isoxazolyl, imidizolyl, benzothiazol- yl , pyrazinyl, pyrirnidinyl , thiazolyl, and thiadi- azolyl .
  • hydroxy is defined as -OH.
  • alkoxy is defined as -OR, wherein R is alkyl, including cycloalkyl.
  • alkoxyalkyl is defined as an alkyl group wherein a hydrogen has been replaced by an alkoxy group.
  • the alkoxyalkyl group can be sub- stituted with an aryl or heteroaryl group.
  • (alkylthio) alkyl is defined similarly as alkoxyalkyl, except a' sulfur atom, rather than an oxygen atom, is present .
  • hydroxyalkyl is defined as a hydroxy group appended to an alkyl group.
  • amino is defined as -NH 2
  • alkylamino is defined as -NR 2 , wherein at least one R is alkyl and the second R is alkyl or hydrogen .
  • 5- or 6-membered aryl or heteroaryl group refers to carbocyclic and heterocyclic aromatic groups, including, but not limited to, phenyl, thiophenyl, furyl , pyrrolyl, imidazolyl, pyrirnidinyl, and pyridinyl .
  • acylamino is defined as RC-
  • alkylthio and arylthio are defined as -SR, wherein R is alkyl or aryl, respectively.
  • alkylsulfinyl is defined as R-S0 2 , wherein R is alkyl.
  • alkylsulfonyl is defined as R-S0 3 , wherein R is alkyl.
  • nitro is defined as -N0 2 .
  • cyano is defined as -CN.
  • the carbon atom content of hydrocarbon- containing moieties is indicated by a subscript designating the minimum and maximum number of carbon atoms in the moiety-, e.g., refers to an alkyl group having one to six carbon atoms, inclusive .
  • the substituent is methyl, for example,
  • R-C is R-C-H and R-N is R-NH ? .
  • S (O) 0 _ 2 is S0 2
  • Y 15. is CH(R 4 ) or MR C
  • p is 0.
  • q is 1 or 2
  • G independently, is C(R a 2 / 0, S, or NR a .
  • q is 1 or 2
  • G independently, are C(R a ) 2 or O.
  • B is phenyl
  • R 2 is hydrogen
  • R* and R 4 are hydrogen
  • R c is hydrogen or alkyl.
  • a and B independently, are selected from the group consisting of
  • B also can be selected from the group consisting of:
  • R is hydrogen; R 2 is selected from the group consisting of hydrogen, C ⁇ alkyl, and aryl; S (O) 0 _ 2 is SO-, Y is CH(R 4 ), R 3 and R 4 , independently, are hydrogen, C 1 . 3 alkyl, phenyl, fluoro, or chloro; X is CH 2 CH 2 OH or CH 2 CH 2 NH 2 ; A is selected from the group consisting of
  • Compounds of the present invention can contain one or more asymmetric center, and, there- fore, can exist as stereoisomers.
  • the present invention includes both mixtures and separate individual stereoisomers of the compounds of the present invention.
  • Compounds of the present invention also can exist in tautomeric forms, and the invention includes both mixtures and separate individual tautomers thereof .
  • Pharmaceutically acceptable salts of compounds of the present invention can be acid a.ddition salts formed with pharmaceutically acceptable acids.
  • suitable salts include, but are not limited to, the hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lac- tate, citrate, tartrate, gluconate, methanesulfon- ate, benzenesulfonate, and p-toluenesulfonate salts.
  • the compounds of the present invention also can provide pharmaceutically acceptable metal salts, in particular alkali metal salts and alkaline earth metal salts, with bases.
  • Example ' s include the sodium, potassium, magnesium, and calcium salts.
  • Compounds of the present invention are potent and selective inhibitors of PDE3B.
  • the compounds are of interest for use in therapy, spe- cifically in the treatment of a variety of conditions where selective inhibition of PDE3B is considered beneficial.
  • PDE3B is a particularly attractive target for inhibition because a potent and selective inhibitor of PDE3B provides effects that are beneficial in the treatment of various disease states.
  • the biochemical, physiological, and clinical effects of PDE3B inhibitors therefore suggest their utility m a variety of disorders and disease states, including obesity, diabetes, hyperlipidemia, inflammatory diseases, autoimmune disorders, and organ transplant rejection.
  • compounds of the present invention are useful in treating the above and other diseases and conditions, while minimizing or eliminating adverse side effects associated with PDE3A inhibition.
  • the present invention is directed to the use of the disclosed compounds, a pharmaceutically acceptable salt t.Viereof, or a pharmaceutical composition containing either entity, for the manufacture of a medicament for the curative or prophylactic treatment of obesity and diabetes in a mammal, including humans.
  • the present invention also is directed the use of a compound of the present invention for the manufacture of a medicament for the treatment of the other above-noted conditions and disorders.
  • treatment includes preventing, lowering, stopping, or reversing the progression or severity of the condition or symptoms being treated.
  • treatment includes both medical therapeutic and/or prophylactic administration, as appropriate, including, but not i.imiced to, the diseases and con- ditions discussed above.
  • compounds of the present invention are envisioned primarily for the treatment of obesity and diabetes in humans, they also can be used for the treatment of other disease states, including, but not limited to, the diseases and conditions discussed above.
  • a compound of the present invention or . a physiologically acceptable salt or solvate thereof, can be administered as the neat compound, or as a pharmaceutical composi- tion containing either entity.
  • the present invention also is directed to a method of treating the conditions and disorders wherein inhibition of PDE3B provides a benefit, in a human or nonhuman animal body, comprising adminis- tering to said body a therapeutically effective amount of a compound of the present invention.
  • the present invention includes a method of inducing weight loss in a mammal comprising the steps of administering to the mammal (a) a compound that selectively inhibits PDE3B and (b) an optional second active compound for effecting weight loss, wherein the compound or com- pounds are administered in amounts effective to promote weight loss in the mammal .
  • Administration to humans is specifically contemplated, but administration to other animals, including pets, live- stock, zoo specimens, wildlife, and the like, also is contemplated. Treatment of humans who are clinically diagnosed as obese is specifically contemplated.
  • Compounds of the present invention can be administered by any suitable route, for example by oral, buccal, inhalation, sublingual, rectal, vaginal, transurethral , nasal, topical, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, and mfcra- coronary) administration.
  • Parenteral administration can be accompl shed using a needle and syringe, or using a high pressure technique, like POWDERJECTTM.
  • Oral administration of a compound of the invention is a preferred route. Oral administration is the most convenient and avoids the disadvantages associated with other routes of administration.
  • the drug can be administered parenter- ally, e.g., sublingually or buccally.
  • Compounds and pharmaceutical compositions suitable for use in the present invention include those wherein the active ingredient is administered in an effective amount to achieve its intended pur- pose. More specifically, a “therapeutically effective amount” means an amount effective to prevent development of, or to alleviate the existing symptoms of, the subject being treated. Therefore, a “therapeutically effective dose” refers to that amount of a compound that results m achieving the desired effect. Determination of an effectOe amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the toxicity and therapeutic efficacy of compounds of the present invention can be determined by standard pharmaceutical procedures in cell cul- tures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from such data can be used in formulating a dosage range for use in humans .
  • the dosage of such compounds prefe - ably lies within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. The exact formulation, route of administration, and dosage is selected by the individual physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide plasma levels of a compound of the pre ⁇ - ent invention which are sufficient to maintain therapeutic effects. Therefore, the amount of a compound of the present invention administered is related to the subject being treated, including the subject's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
  • dosages of a compound of the present invention generally are about 0.1 to about 1000 mg daily fox an average adult patient ('70 kg) .
  • individual doses for example, tablets or capsules, contain 0.1 to 500 mg of active compound, in a suitable pharmaceutically acceptable vehicle or carrier, for administration in single or multiple doses, once or several times per- day.
  • Dcs- ages for intravenous, buccal, or sublingual administration typically also are 0.1 to 500 mg per single dose as required.
  • the physician determines the actual dosing regimen which is most suitable for an individual patient , and the dosage varies with the age, weight, and response of the particular patient.
  • the above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention.
  • a compound of the present invention can be administered alone, but generally is administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration a d standard pharmaceutical practice.
  • Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in a conventional manner usinct one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of compounds of the present invention into compositions that can be used pharmaceutically.
  • Such pharmaceutical compositions can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is related to the route of administration chosen.
  • the composition typically is in the form of a tablet, capsule, powder, solution, oielixir.
  • the composition can additionally contain a solid carrier, such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain about 5% 'to about 95% compound of the present invention, and preferably from about 25% to about 90% compound of the present invention.
  • a liquid ' carrier such as water, petroleum, or oils of animal or plant origin can be added.
  • the liquid form of the composition can further contain physiological saline solution, dextrose or other saccharide solutions, or glycols.
  • the composition contains about 0.5% to about 90% by weight of a compound of the present invention, and preferably about 1% to about 50% of a com- pound of the present invention.
  • compositions When a therapeutically effective amount of a compound of the present invention is administered by intravenous, cutaneous, or subcutaneous injec- tion, the composition is in the form of a pyrogen- free, parenterally acceptable aqueous solution.
  • parenterally acceptable solutions having due regard to pH, isotonicity, stabil- ity, and the like, is within the skill in the art.
  • a preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains, in addition to a compound of the present invention, an isotonic vehicle.
  • the compounds can be formulated readily by combining a compound of the present invention with pharmaceutically acceptable carriers well known in the art .
  • Such carriers enable the present compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by adding a compound of the present invention with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, for example, fillers and cellulose prepara- tions. If desired, disintegrating agents can be added .
  • compounds of the present invention are conveniently delivered in the form of an aerosol spray presen- tation from pressurized packs or a nebulizer, with the use of a suitable propellant .
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. ⁇ Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • a present composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Compounds of the present invention also can be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases.
  • the compounds also can be formulated as a depot preparation.
  • Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a compound of the present invention or a nontoxic salt thereof is administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian readily can determine the dosing regimen and route of administration that is most appropriate for a particular animal .
  • the present invention provides a pharmaceutical composition comprising a compound of the present invention, together with a pharmaceutically acceptable diluent or carrier therefor.
  • the present invention also provides a process of preparing a pharmaceutical composition comprising a com- pound of the present invention, which process comprises mixing a compound of the present invention, together with a pharmaceutically acceptable diluent or carrier therefor.
  • the invention includes a pharmaceutical composition for the curative or prophylactic treatment of obesity, diabetes, and other diseases and conditions wherein selective inhibition of PDE3B provides a benefit in a mammal, including humans, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable diluent or carrier.
  • a composition of the present invention can be administered to an individual in concert with a second therapeutically active agent.
  • the second therapeutically active agent is a compound useful in treating the disease or condition afflicting the individual, and for which the individual is receiving treatment with a compound of the present invention.
  • the individual can be administered a therapeutically effective amount of a com- pound of the present invention and a second therapeutically active agent useful in the treatment of obesity, for example, a PDE4 inhibitor, like roli- pram and CDP840.
  • a PDE4 inhibitor like roli- pram and CDP840.
  • the selective PDE3B inhibitor of the present invention and second therapeutically active agent can be administered either simultaneously, separately, or sequentially. If administered sequentially, either the PDE3B inhibitor or second therapeutically active agent can be administered first .
  • PDE4 inhibitors useful in conjunction with a compound of the present invention in the treatment of obesity and other diseases and conditions can be categorized into various classes, such as xanthine derivatives, rolipram analogs, and quinazolinedi- ones, and include benafentrine, tolafentrine, zardaverine, Org 20241, nitraquazone, RS 5344, BRL 1063, SB 207499, SCZ MKS 492, CDP 840, CP 80,633, D- 22888, AWD-12-281, D-4418, RP 73401, WAY-PDA- 641, LAS 31025, tibenelast, benbufylline, and substituted pyrrolidines (U.S. Patent No. 5,665,754, incorporated herein by reference, for example) .
  • PDE4 inhibitors have been described in the litera- ture, e.g., Dent et al . , in Phosphodiesterase
  • Compounds of the present invention can be prepared by any suitable method known in the art, or by the following processes which form part of the present invention. In the methods below, R°, R 1 , R 2 , R 3 , and R 4 , as well as A, B, X, and Y are defined as in the present invention above.
  • compounds of the present invention can be prepared according to the following synthetic scheme, which comprises reacting compounds of formulae (V) and (VI) in a Suzuki coupling reaction.
  • a compound of formula (V) can be prepared by the scheme outlined below.
  • protecting groups can be utilized in accordance with general principles of synthetic organic chemistry to provide compounds of the present invention.
  • Protecting group-forming reagents like benzyl chloroformate and trichloroethyl chloroformate, are well known to persons skilled in the art, for example, see T.W. Greene et al . , "Protective Groups in Organic Synthesis, Third Edition," John Wiley and Sons, Inc., NY, NY (1999) .
  • These protecting groups are removed when necessary by appropriate basic, acidic, or hydro- genolytic conditions known to persons skilled in the art. Accordingly, compounds of the present invention not specifically exemplified herein can be prepared by persons skilled in the art.
  • a compound of the present invention can be converted to another compound of the present invention.
  • a particular R substituent can be interconverted to prepare another suitably substituted compound of the present invention.
  • appropriate inter- conversions include, but are not limited to, OR to hydroxy by suitable means (e.g., using an agent such as BBr 3 or a palladium catalyst, like palladium-on- carbon, with hydrogen) , or amino to substituted amino, such as acylamino or sulphonylamino, using standard acylating or sulfonylating conditions.
  • Compounds of the present invention can be prepared by the method above as individual stereoisomers or as a racemic mixture.
  • Individual stereo- isomers of the compounds of the invention can be prepared from racemates by resolution using methods known in the art for the separation of racemic mixtures into their constituent stereoisomers, for example, using HPLC on a chiral column, such as Hypersil naphthyl urea, or using separation of salts of stereoisomers.
  • Compounds of the invention can be isolated in association with solvent molecules by crystallization from, or evaporation of, an appropriate solvent .
  • the pharmaceutically acceptable acid addition salts of the compounds of the present invention that contain a basic center can be prepared in a conventional manner.
  • a solution of the free base can be treated with a suitable acid, either neat or in a suitable solution, and the resulting salt isolated either by filtration or by evaporation under vacuum of the reaction solvent.
  • Pharmaceutically acceptable base addition salts can be obtained in an analogous manner by treating a solution of a compound of the present invention with a suitable base. Both types of salt can be formed or interconverted using ion-exchange resin techniques.
  • a method for preparing a compound of the present invention or a salt or solvate e.g., hydrate
  • salt formation e.g., hydrate
  • Amides were prepared by saponification of an appropriate ester with sodium hydroxide followed by reaction of the resulting carboxylic acid with a suitable amine.
  • the sulfone methyl ester (3 g, 9.5 mmol) was dissolved in 150 mL dioxane and 10 mL water and cooled to 0°C.
  • Sodium hydroxide (14.3 mL, IN, 1.5 eq) was added dropwise.
  • the resulting solution was stirred at 0°C for 2.5h, at which time the reaction was judged complete by TLC.
  • the solution was neutralized and concentrated to 50 mL, added to 350 mL EtOAc, and acidified with 10% NaHS0 4 . Then the organic layer was extracted with 300mL EtOAc . The combined organic layers were washed with brine, dried over MgS0 4 , and evaporated to give the pure acid as a slightly yellow solid (2.63 g, 92%) .
  • reaction mixture was concentrated under reduced pressure, and reconstituted in AcOH until the pH was acidic.
  • the reaction then was heated to 110 °C and stirred for 4h.
  • the reaction mixture was allowed to cool, and the acetic acid was removed under reduced pressure.
  • the concentrated product was partitioned between methylene chloride and water, extracted four times, dried over magnesium sulfate, filtered, and concen- trated in vacuo to an off-white solid. The solid was triturated with a small amount of ethyl acetate and filtered. Four grams of pure product was recovered as a white solid.
  • nitrile ester (10 g, 56.5 mmol) was dissolved in 20 mL MeOH and cooled to -10°C. Hydro- gen sulfide gas was bubbled through the solution for 45 min. DMAP (0.07 eq) was added and the reaction mixture was sealed and heated overnight at 50 °C. The solvents were removed and the crude product was used without further purification.
  • Methyl thioglycolate (95%, Aldrich) (200 g, 1.88 mol) was placed in a 500 mL roundbottom flask. Ethanolamine (99.5%, Aldrich) (109 g, 1.79 mol) was added. Magnetic stirring was initiated with a vent in place because the reaction is exo- thermic. A vacuum was slowly applied to distill off the methanol. The reaction was maintained under a high vacuum with magnetic stirring for 6 hours to overnight to yield N- (hydroxyethyl) -2 -mercapto acetamide as a clear, highly viscous material that was used without further purification.
  • Step 2
  • Example 6 4 -Bromo-2 - fluorophenylmethanesulfonyl-N- hydroxyethyl acetamide (50 mg, 0.141 mmol), palladium acetate (1 mg, 0.0046 mmol), triphenylphosphine attached to solid support (3 mmol/g) (4.5 mg, 0.0135 mmol), and trimethoxyphenyl boronic acid (29.9 mg, 0.141 mmol) were added to a mixture of DMF (1.5 ml) and 2M potassium carbonate (0.5 ml) . The mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 6 (16 mg, 97% purity) .
  • Phosphorus tribromide (1.25 ml, 13.1 mmol) was added dropwise to a solution of 2-bromo-5-hy- droxymethylthiophene from Step 1 in dichloromethane (52.4 ml) below 10°C. The solution was stirred for 2h at 20°C, then washed with ice-water, dried, and evaporated to give a white solid that was used without further purification.
  • N- (hydroxyethyl) -2 -mercapto acetamide (5.3 g, 39.3 mmol) and 2-bromo-5-bromomethylthiophene (26.2 mmol) from Step 2 were combined in DMF (30 ml) with K 2 C0 3 (10.8 g, 78.6 mmol), and stirred for 12h at room temperature under N 2 .
  • the DMF was removed in vacuo, and the compound was partitioned between EtOAc and H 2 0.
  • the EtOAc layer was washed with H 2 0 and brine.
  • the organics were dried over MgS0 4 , filtered, and concentrated to a yellow oil that was used without further purification.
  • Step 4
  • N- (hydroxyethyl) -2 -mercapto acetamide (134 mg, 0.99mmol) and chloromethyl oxadiazole prepared in Step 1 (0.66 mmol) were combined with DMF (2 ml) and K 2 C0 3 (274 mg, 1.98 mmol), and stirred for 12h at room temperature under N 2 .
  • the DMF was removed in vacuo, and the compound was partitioned between EtOAc and H 2 0.
  • the EtOAc layer was washed with H 2 0 and brine.
  • the organics were dried over MgS0 4 , filtered, and concentrated to a residue that was carried to next step without purification.
  • Example 12 The sulfide prepared in Step 2 (0.66 mmol) was dissolved in acetic acid (1.3 ml) . Water peroxide (30%, 255 ⁇ l, 1.98 mmol) was added, and the mixture was heated at 80°C for 2 hours. The solvent was evaporated, the sample dissolved in DMSO (2 ml), and purified by reverse phase chromatography to yield Example 12 (4.7 g, 100% purity).
  • Step 2
  • Step 3
  • N- (hydroxyethyl) -2-mercaptoacetamide (187 mg, 1.38 mmol) and chloromethyl oxadiazole prepared in step 2 (0.92 mmol) were combined with DMF (2 ml) and K 2 C0 3 (381 mg, 2.76 mmol) , and stirred for 12h at room temperature .
  • the DMF was removed in vacuo and the compound was partitioned between EtOAc and H 2 0.
  • the EtOAc layer was washed with H 2 0 and brine.
  • the organics were dried over MgS0 4 , filtered, and concentrated to a residue that was used without further purification.
  • Step 4
  • Example 14 was prepared by this method from 8-hydroxy-2-bromonaphthalene :
  • 5-Bromo-2 , 3-dimethoxybenzaldehyde (100 mg, 0.408 mmol), ammonium acetate (314 mg, 4.08 mmol), and 2 , 3-butanedione (52 mg, 0.608 mmol) were added to a tube, dissolved in 3 ml of acetic acid, then heated to 100°C for 2h. The solvent was removed under reduced pressure, and the residue was coevap- orated three times with water to remove excess acetic acid. The residue was dried under reduced pressure and was used without any further purification.
  • Example 22 was prepared using a similar synthetic sequence.
  • Step 3 To a solution of phenol prepared in Step 1 (50 mg, 0.21 mmol) in acetone (1 ml) were added anhydrous K 2 C0 3 (59 mg, 0.43 mmol) and benzyl bromide (76.3 ml, 0.64 mmol) . The reaction mixture was stirred at 60°C for 12 hours. The suspension was filtered using a disposable filter device (0.45 mM PTFE membrane) . The solvent was evaporated, the crude residue dissolved in DMSO, and purified by reverse phase HPLC to give l-benzyloxy-5-bromo-2 , 3- dimethoxybenzene (39 mg, 56% yield) as a white solid. Step 3
  • Step 1 Preparation of Sulfonyl Chlorides (Soon- Kyoung et al . , Arzneim Forsch, 46 (10) , pp. 966-971 (1986) )
  • Step 3
  • Sulfonamide prepared in Step 2 was dis- solved in DMF (1.5 ml) .
  • Boronic acid, pinacol ester (67 mg, 0.176 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenylphosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added.
  • the mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield product example 25 (36.5 mg, 92% purity).
  • the sulfide prepared in Step 1 (42 mg, 0.166 mmol) was dissolved in DMF (1.5 ml) .
  • Boronic acid, pinacol ester (63 mg, 0.166 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenylphosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added.
  • the mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 26 (9.2 mg, 100% purity) .
  • the thiaxolidinone prepared in Step 1 (48.5 mg, 0.155 mmol) was dissolved in DMF (1.5 ml) , Boronic acid, pinacol ester (60 mg, 0.155 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenyl- phosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added. The mixture was heated at 80°C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 27 (9.4 mg, 97.1% purity) .
  • Step 1
  • the thiazolidinone prepared in Step 1 (0.72 mmol) was dissolved in acetic acid (1.5 ml) .
  • Water peroxide (30%, 247 ⁇ l , 2.16 mmol) was added and the mixture was stirred at 25°C for 12 hours.
  • the solvent was evaporated and the thiazolidinone sulfoxide was used without further purification.
  • Step 2 (48.5 mg, 0.155 mmol) was dissolved in DMF (1.5 ml) .
  • Boronic acid, pinacol ester 60 mg, 0.155 mmol
  • palladium acetate 1.2 mg, 0.0053 mmol
  • tri- phenylphosphine attached to solid support 3 mmol/g
  • 2M potassium carbonate 0.5 ml
  • Step 1
  • Step 1 J. Org. Chem . , 63 , pp. 9594-9596 (1998).
  • Step 1 Mol . Cryst . Liq. Cryst . , 172 , pp. 165-189 (1989)
  • 4-Bromo-2 , 6-difluorophenylamine (15 g, 72 mmol) was mechanically stirred with 70 L of concentrated 1:1 sulfuric acid/water. The suspension was heated until a solution was obtained, followed by cooling to -10°C with a salt/ice bath. A solution of sodium nitrite (8.28 g, 120 mmol) in water (20 mL) was added dropwise to the stirring aniline such that the temperature was maintained below -5°C. The mixture was stirred for 2.5h after the addition was complete .
  • the aqueous phase was extracted twice with ether, and the combined organics were washed sequentially with water, 1 M sodium hydroxide, and water. The organics were dried with anhydrous magnesium sulfate and removed under reduced pressure. The resulting solid was crystallized twice from ethanol to yield 6 g of product .
  • the crude material was chromatographed on silica using a four-step gradient of hexanes/ethyl acetate (0% ethyl acetate, 5%, 10%, and 15%) to yield 1.1 g of 4-bromo-2-fluoro-6-methoxybenzo- nitrile .
  • Step 3
  • Example 32 was prepared according to procedure set forth for the preparation of a 3-alkoxy- 4-cyanobiphenyl using 4-fluoro-3-nitrobromobenzene as the starting aryl bromide.
  • Example 34 (0.4 mg, 100% purity) .
  • Compounds of the present invention were tested for an ability to inhibit PDE3B.
  • the ability of a compound to inhibit PDE3B activity is related to the IC 50 value for the compound, i.e., the concentration of inhibitor required for 50% inhibition of enzyme activity.
  • the IC 50 value for compounds of the present invention were determined using recombinant human PDE3B.
  • the in vi tro phosphodiesterase activity inhibitory IC S0 values were determined by measuring the inhibition as a function of the concentration of the test compound over the range of 0 to 1 mM.
  • the IC 50 values of the compounds tested in the aforementioned assay ranged from about 0.01 ⁇ M to about 10 ⁇ M.
  • the compounds of the present invention typically exhibit an IC 50 value against recombinant human PDE3B of less than about 10 ⁇ M, and preferably less than about 5 ⁇ M, and more preferably less than about 1 M.
  • the compounds of the present invention typically exhibit an IC 50 value against recombinant human PDE3B of less than about 1000 nM, and often less than about 100 nM.
  • a present PDE3B inhibitor has an IC 50 of about 700 pM (picomolar) to about 10 ⁇ M .
  • the IC 50 values for the compounds were determined from concentration-response curves typically using concentrations ranging from 0.1 pM to 500 ⁇ M .
  • Plasmid pBB-PDElA3/6 contained the complete open reading frame of PDE1A3 (Loughney et al . , J. Biol . Chem . , 271 , pp. 796-806 (1996)) in pBlueBacIII.
  • Plasmid Hcam3aBB contained the complete open reading frame of PDE1C3 (Loughney et al .
  • Plasmid pBB-PDE3A contained the complete open reading frame of PDE3A (Meacci et al., Proc . Na tl . Acad . Sci . , USA, 89 , pp. 3721-3725
  • Plasmid pFB-PDE3B contains the complete open reading frame of PDE3B (Miki et al., Genomics, 36, pp. 476-485 (1996)).
  • Recombinant virus stocks were produced using either the MaxBac system (Invitrogen) or the FastBac ® system (Gibco-BRL) according to the manu- facturer's protocols. In both cases, expression of recombinant human PDEs in the resultant viruses was driven off the viral polyhedron promoter. When using the MaxBac ® system, virus was plaque purified twice in order to insure that no wild type (occ+) virus contaminated the preparation. Protein expression was carried out as follows.
  • Sf9 cells were grown at 27°C in Grace's Insect culture medium (Gibco-BRL) supplemented with 10% fetal bovine serum, 0.33% TC yeastolate, 0.33% lactalbumin hydro- lysate, 4.2 mM NaHC0 3 , 10 ⁇ g/ml gentamycin, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • Exponentially growing cells were infected at a multiplicity of approximately 2 to 3 virus particles per cell and incubated for 48 hours. Cells were collected by centrifugation, washed with nonsupple- ented Grace's medium, and quick-frozen for storage.
  • yeast ADH2 promoter and termi- nator sequences and the Saccharomyces cerevisiae host was the protease-deficient strain BJ2-54 deposited on August 31, 1998 with the American Type Culture Collection, Manassas, Virginia, under accession number ATCC 74465. Transformed host cells were grown in 2X SC-leu medium, pH 6.2, with trace metals, and vitamins. After 24 hours, YEP medium- containing glycerol was added to a final concentration of 2X YET/3% glycerol. Approximately 24 hr later, cells were harvested, washed, and stored at -70°C.
  • Calmodulin used for activation of the PDE1 enzymes was purified from bovine testes essentially as described by Dedman et al . , Methods in Enzymology, 102, pp. 1-8 (1983) using the Pharmacia Phenyl -Sepharose ® procedure.
  • Calmodulin was immobilized on BioRad Affi- Gel ® 15 per manufacturer's instructions.
  • Phosphodiesterase activity of the preparations was determined as follows. PDE assays utilizing a charcoal separation technique were performed essentially as described in Loughney et al . (1996). In this assay, PDE activity converts [32P] cAMP or [32P]cGMP to the corresponding [32P] 5 ' -AMP or [32P]5'-GMP in proportion to the amount of PDE activity present. The [32P]5'-AMP or [32P]5'-GMP then was quantitatively converted to free [32P] phosphate and unlabeled adenosine or guanosine by the action of snake venom 5 ' -nucleotidase .
  • the amount of [32P] phosphate liberated is proportional to en- zyme activity.
  • the assay was performed at 30°C in a 100 ⁇ L reaction mixture containing (final concentrations) 40 mM Tris HCl (pH 8.0) , 1 ⁇ M ZnS0 4 , 5 mM MgCl 2 , and 0.1 mg/ml bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • incubation mixtures further incorporated the use of 0.1 mM CaCl 2 and 10 ⁇ g/ml calmodulin.
  • PDE enzyme was present in quantities that yield ⁇ 30% total hydrolysis of substrate (linear assay conditions) .
  • the assay was initiated by addition of sub- strate (1 mM [32P] cAMP or cGMP) , and the mixture was incubated for 12 minutes. Seventy- five (75) ⁇ g of Crotalus atrox venom then was added, and the incubation was continued for 3 minutes (15 minutes total) . The reaction was stopped by addition of 200 ⁇ L of activated charcoal (25 mg/ml suspension in 0.1 M
  • Lysis Buffer 50 mM MOPS pH 7.5 , 2 mM dithio- threitol (DTT) , 2 mM benzamidine HCl, 5 ⁇ M ZnS0 4 ,
  • the column was washed with 5 volumes of Column Buffer B (Column Buffer A without NaCl) and followed by 5 volumes of Column Buffer C (Column Buffer A containing 250 mM NaCl) .
  • the column was eluted with Column Buffer D (50 mM MOPS pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benz- amidine HCl, 100 mM NaCl, 20 ⁇ g/ml calpain inhibitors I and II, and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin) by applying one column volume at 0.1 mL/min, stopping flow for 1 hour, and then continuing elution at the same flow rate.
  • Column Buffer D 50 mM MOPS pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benz- amidine HCl,
  • Fractions of 0.5 ml were collected. Fractions displaying activity were pooled, and first dialyzed against dialysis buffer containing 25 mM MOPS pH 7.5, 100 mM NaCl, 10 ⁇ M ZnS0 4 , 1 mM CaCl 2 , 1 M DTT, and 1 mM benzamidine HCl. A subsequent dialysis against dialysis buffer containing 50% glycerol was performed prior to quick-freezing the sample with dry ice and storage at -70°C. The resultant prepara- tions were about 10 to 15% pure by SDS-PAGE. These preparations had specific activities of about 5 to 20 ⁇ mol cAMP hydrolyzed per minute per milligram protein.
  • Yeast cells 50 g were thawed by mixing with 100 ml glass beads (0.5 mM, acid washed) and 200 ml Buffer A at room temperature. Buffer A con- sisted of 50 mM MOPS pH 7.5, 1 mM DTT, 2 mM benzamidine HCl, 0.01 mM ZnS0 4 , 5 mM MgCl 2 , 20 ⁇ g/ml calpain inhibitors I and II, and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin.
  • the mixture was cooled to 4°C, transferred to a Bead-Beater ® , and the cells lysed by rapid mixing for 6 cycles of 30 seconds each.
  • the homogenate was centrifuged for 15 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at 9,000 rpm and 4°C.
  • the supernatant was recovered and centrifuged in a Beckman XL-80 ultracentrifuge using a TI45 rotor at 36,000 rpm for 45 minutes at 4°C.
  • the supernatant was recovered and PDE1B was precipitated by the addition of solid ammonium sulfate (0.33 g/ml supernatant) while stirring in an ice bath and maintaining the pH between 7.0 and 7.5. This mixture then was centrifuged for 22 minutes in a Beckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000 X g) .
  • the supernatant was discarded and the pellet was dissolved in 100 ml of buffer B (50 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mM ZnS0 4 , 2 mM MgCl 2 , 2 mM CaCl 2 , and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin) .
  • the pH and conductivity were corrected to 7.5 and 15-20 milli- Siemens (mS) , respectively.
  • This solution was loaded onto a 20 ml column of calmodulin-Agarose that had been equilibrated with 10 column volumes of Buffer B at a rate of 1 mL/min.
  • the flow-through was reapplied to the column at least 5 times.
  • the column was washed with 5 volumes of Buffer B, 5 volumes of buffer B containing 250 mM NaCl, and 2 volumes of Buffer B without NaCl again.
  • Elution was accomplished by applying one volume of Buffer C (50 mM MOPS pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benzamidine HCl) at 0.33 mL/min, then stopping flow for 1 hour before continuing the elution.
  • Fractions of about 4 ml were collected and assayed for PDE activity. Active fractions were pooled and concentrated to a volume of 5 mL, using an Amicon ultra- filtration system.
  • the concentrate was then applied to a 320 ml Sephacryl ® S-300 column (1.6 X 150 cm) that had been equilibrated with at least 2 volumes of Buffer D (25 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mM ZnS0 4 , 2 mM CaCl 2 , and 100 mM NaCl) .
  • Buffer D 25 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mM ZnS0 4 , 2 mM CaCl 2 , and 100 mM NaCl
  • the column was developed at a flow rate of 1 mL/min (11 cm/hr) , and 5 ml fractions were collected.
  • the activity peak was pooled and dialyzed overnight against Buffer D containing 50% glycerol.
  • the purified enzyme was frozen on dry ice and stored at
  • Frozen cell pellets (2 x 10 10 ) cells were resuspended in Lysis Buffer (50 mM Na MOPS (pH 7.2), 1 mM dithiothreitol, 2 mM benzamidine HCl, 5 ⁇ M ZnS0 4 , 20 ⁇ g/ml calpain inhibitors I and II, and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin) .
  • Lysis Buffer 50 mM Na MOPS (pH 7.2), 1 mM dithiothreitol, 2 mM benzamidine HCl, 5 ⁇ M ZnS0 4 , 20 ⁇ g/ml calpain inhibitors I and II, and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin
  • This material was loaded onto a Cibacron blue Seph- arose column that had been equilibrated with at least 10 volumes of Buffer A.
  • the column was washed with 10 volumes of Buffer A and eluted with Buffer B (50 mM Na MOPS (pH 7.2) , 1 mM dithiothreitol, 2 mM benzamidine HCL, 5 ⁇ M ZnS0 4 , 100 mM NaCl and 1-5 mM cAMP) .
  • Buffer B 50 mM Na MOPS (pH 7.2) , 1 mM dithiothreitol, 2 mM benzamidine HCL, 5 ⁇ M ZnS0 4 , 100 mM NaCl and 1-5 mM cAMP
  • the resultant material was exchanged into a Buffer C (50 mM Na MOPS (pH 7.2), 2 mM dithiothreitol, 2 mM benzamidine HCl, 10 ⁇ M ZnS0 4 , 2 mM CaCl 2 , and 200 mM NaCl) using a Sephadex G25 column and an equal volume of glycerol was added. Aliquots were quick frozen in dry ice and stored at -70°C.
  • Buffer C 50 mM Na MOPS (pH 7.2), 2 mM dithiothreitol, 2 mM benzamidine HCl, 10 ⁇ M ZnS0 4 , 2 mM CaCl 2 , and 200 mM NaCl
  • Frozen yeast cell pellets from strain YI34 (lOg, stored at -70°C) were allowed to thaw on ice in 25 ml of Lysis Buffer (50 mM MOPS, pH 7.2 , 1 mM EDTA, 1 mM EGTA, 0.1 mM DTT, 0.1 mM 4-(2-amino- ethyl) benzenesulfonyl fluoride (AEBSF) , 1 ⁇ g/ml of pepstatin, leupeptin, aprotinin, calpain inhibitors I and II, and 2 mM benzamidine) .
  • Lysis Buffer 50 mM MOPS, pH 7.2 , 1 mM EDTA, 1 mM EGTA, 0.1 mM DTT, 0.1 mM 4-(2-amino- ethyl) benzenesulfonyl fluoride (AEBSF) , 1 ⁇ g
  • Cells (2 x 1010) were suspended in Lysis Buffer containing 50 mM MPOS pH 7.5, 2 mM DTT-, 2 mM benzamidine HCl, 5 ⁇ M ZnS0 4 , 0.1 mM CaCl 2 , 20 ⁇ g/ml calpain inhibitors I and II, and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin.
  • the mixture was sonicated twice for 30 seconds and the cells were lysed in a French ® pressure cell (SLM-Aminco ® , Spectronic Instruments) at 4°C. The lysate was centrifuged 100,000 x g for 45 minutes.
  • SLM-Aminco ® French ® pressure cell
  • Yeast cells 150 g of yeast strain YI23 harboring HDTJN2.32 were thawed by mixing with 100 ml glass beads (0.5 mM, acid washed) and 150 ml Lysis Buffer (50 mM MOPS pH 7.2 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, 5 ⁇ g/ml each of pepstatin, leupeptin, aprotinin, calpain inhibitors I and II) at room temperature. The mixture was cooled to 4°C, transferred to a Bead-Beater ® , and the cells lysed by rapid mixing for 6 cycles of 30 seconds each.
  • Lysis Buffer 50 mM MOPS pH 7.2 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, 5 ⁇ g/ml each of pepstatin,
  • the homogenate was centrifuged for 22 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at 9,000 rpm and 4°C.
  • the supernatant was recovered and centrifuged in a Beckman XL- 80 ultra- centrifuge using a TI45 rotor at 36,000 rpm for 45 minutes at 4°C.
  • the supernatant was recovered and PDE4B was precipitated by the addition of solid ammonium sulfate (0.26 g/ml supernatant) while stir- ring in an ice bath and maintaining the pH between 7.0 and 7.5.
  • the resuspended sample was loaded onto a 1.6 X 200 cm column (25 mL) of Sigma Cibacron Blue Agarose-type 300 equilibrated in Buffer A. The sample was cycled through the column 4 to 6 times over the course of 12 hours . The column was washed in succession with 125 to 250 ml of Buffer A, 125 to 250 ml of Buffer A containing 1.5 M NaCl, and 25 to 50 ml of Buffer A.
  • the enzyme was eluted with 50 to 75 ml of Buffer E (50 mM Tris HCl pH 8 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and 20 mM cAMP) and 50 to 75 ml of Buffer E containing 1 M NaCl.
  • Buffer E 50 mM Tris HCl pH 8 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and 20 mM cAMP
  • Buffer E 50 mM Tris HCl pH 8 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and 20 mM cAMP
  • the PDE activity peak was pooled, and precipitated with ammonium sulfate (0.4 g/ml enzyme pool) to remove excess
  • the enzyme pool was dialyzed over- night against Buffer X containing 50% glycerol.
  • This enzyme was quick- frozen in a dry ice/ethanol bath and stored at -70°C.
  • the resultant preparations were about >90% pure by SDS-PAGE. These preparations had specific activities of about 10 to 50 ⁇ mol cAMP hydrolyzed per minute per milligram protein.
  • Lysis Buffer 25 mM Tris HCl, pH 8, 5 mM MgCl 2 , 0.25 mM DTT, 1 mM benzamidine, and 10 ⁇ M ZnS0 4 .
  • Cells were lysed in a Microfluid- izer ® (Microfluidics Corp.) using nitrogen at 20,000 psi. The lysate was centrifuged and filtered through 0.45 ⁇ m disposable filters. The filtrate was applied to a 150 ml column of Q SEPHAROSE ® Fast- Flow (Pharmacia) .
  • the column was washed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl,, 0.25 mM DTT, 10 ⁇ M ZnS0 4 ) and eluted with a step gradient of 125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mM NaCl in Buffer A. Active fractions from the linear gradient were applied to a 180 ml hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane (pH 6.8), 1 mM MgCl 2 , 0.25 mM DTT, 10 ⁇ M ZnS0 4 , and 250 mM KCI) .
  • Buffer A 20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl,, 0.25 mM DTT, 10 ⁇ M ZnS0 4 , and 250 mM KCI
  • the column was washed with 2 volumes of Buffer B and eluted with a linear gradient of 0-125 mM potassium phosphate in Buffer B. Active fractions were pooled, precipitated with 60% ammonium sulfate, and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mM NaCl, 0.5 mM DTT, and 10 ⁇ M ZnS0 4 ) . The pool was applied to a 140 ml column of SEPHA- CRYL ® S-300 HR and eluted with Buffer C. Active fractions were diluted to 50% glycerol and stored at -20°C.
  • the resultant preparations were about 85% pure by SDS-PAGE. These preparations had specific activities of about 3 ⁇ mol cGMP hydrolyzed per minute per milligram protein.
  • Cell pellets (126 g) were thawed and resuspended at room temperature for about 30 minutes with an equal volume of Lysis Buffer (50 mM Tris HCl, pH 8, 1 mM EDTA, 1 mM DTT, 50 mM NaCl, 2 mM benzamidine HCl, and 5 ⁇ g/ml each of pepstatin, leupeptin, and aprotinin) .
  • Lysis Buffer 50 mM Tris HCl, pH 8, 1 mM EDTA, 1 mM DTT, 50 mM NaCl, 2 mM benzamidine HCl, and 5 ⁇ g/ml each of pepstatin, leupeptin, and aprotinin.
  • the cells were lysed at 0-4°C with the aid of glass beads (125 mL) in a Bead-Beater ® for 6 X 30 second cycles.
  • the lysate was centrif
  • the filtered extract (178 mL) was distributed into 4 ml aliquots, quick-frozen with dry ice, and stored in a freezer at -70°C. These preparations were stable to several cycles of freezing and thawing and had specific activities of about 50 to 100 pmol cAMP hydrolyzed per minute per milligram protein.
  • Frozen cell pellets (2.2 g) were mixed with 2.2 mL of Lysis Buffer (50 mM Tris-CI (pH 8.5),
  • Frozen cell pellets (7.8 g) were resus- pended in 8 L of Lysis Buffer (25 mM Tris (pH 8.0), 5 mM EDTA, 0.1 mM DTT, and 10 ⁇ g/ml each of pepstatin, leupeptin, and aprotinin) .
  • the mixture was chilled on ice and lysed by passing through a French ® pressure cell (SLM-Amino ® , Speetonic Instruments) at 20,000 psi.
  • the lysate was centrifuged at 25,000 x g for 20 minutes at 4°C. The supernatant was removed, and an equal volume 80% glycerol was added prior to storage at -20°C.
  • Frozen cell pellets (5 x 10 11 cells) were resuspended in Lysis Buffer (50 mM MOPS (pH 7.2), 1 mM dithiothreitol, 2 mM benzamidine HCl, 5 ⁇ M ZnS0 4 , 20 ⁇ g/ml calpain inhibitors I and II, and 5 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin) . The mixture was sonicated twice for 30 seconds and the cells were lysed in a French ® pressure cell (SLM- Aminco ® , Spectronic Instruments) at 20,000 psi (4°C) .
  • SLM- Aminco ® French ® pressure cell
  • Table 1 contains the IC S0 value vs. PDE3B for Examples 1-34 (left column) , and the ratio of IC 50 (vs. PDE3A) to IC 50 (vs. PDE3B) (right column) .
  • the potency of the inhibitors is illustrated in the IC 50 value vs. PDE3B, which is about 8.5 ⁇ M or less for each example.
  • the IC 50 value vs. PDE3B is about 0.01 ⁇ M (i.e., 10 nM) .
  • Table 1 further shows the PDE3B inhibition selectivity of compounds of the present invention.
  • the selectivity of the inhibitors for PDE3B is illustrated in the IC S0 value ratio, which shows a strong selectivity for PDE3B over PDE3A, i.e., up to a factor of about 30, and typically a factor of at least about 5 to about 20.
  • Table 1 allows the selection of a very potent or a very selective PDE3B inhibitor, or a PDE3B inhibitor that exhibits both potency and selectivity.
  • the choice of a particular compound for use in therapy is related to several factors, including the toxicity, physical properties, and pharmacological properties of the compound, the condition or disease to be treated, and the intended route of administration.
  • the selectivity of a compound of the present invention to inhibit PDE3B over PDE3A and other PDE enzymes is illustrated in the following Table 2.
  • Table 2 shows that the compounds of Examples 4, 35, and 36 are potent inhibitors of PDE3B, i.e., have an IC 50 value vs. PDE3B of 0.085, 0.59, and 1.05 ⁇ M, respectively.
  • Table 2 also shows that the compounds are selective inhibitors of PDE3B over PDE3A, i.e., have a ratio of IC 50 (vs. PDE3A) to IC 50 (vs. PDE3B) of 14.1, 23.7, and 12.6, respectively.
  • Table 2 further shows a selective inhibition of PDE3B over other listed PDE isoforms by a factor of 141 to 8129 for Example 4, 23.7 to 347.5 for Example 35, and 28.6 to 952.4 for Example 36.
  • Adipocytes store excess energy in the form of triglyceride. Synthesis of adipocyte triglycer- ide is regulated hormonally, and is a crucial aspect of body weight regulation. The breakdown of triglyceride to glycerol and free fatty acids is known as lipolysis. Agents that induce lipolysis are expected to have utility in the treatment of obesity. PDE3 inhibitors have been known to induce lipolysis both in vi tro and in vivo (P.B. Snyder, Emerging Therapeutic Targets, 3 , pp. 587-599 (1999)). Because adipocytes express the PDE3B isoform (M. Taira et al . , J " . Biol . Chem . , 268, pp. 18573-18579 (1993)), the effect of selective PDE3B inhibitors on lipolysis in cultured human adipocytes was tested.
  • Human adipocyte were purchased from Zen-Bio, Inc. (Research Triangle Park, NC) . The cells were derived from surgical specimens of subcutaneous adipose tissue by collagenase digestion. The cells were grown to con- fluence in 96-well tissue culture plates, and induced to differentiate into adipocytes by addition of a medium containing adipogenic and lipogenic hormones (see Hauner et al . , J " . Clin . Invest . , 84 , pp. 1663-1670 (1989) for a representative protocol) .
  • the cells were maintained in a medium consisting of DMEM/Ham's F-10 Medium (1:1), 15 mM HEPES (pH 7.4) supplemented with 3% FCS, 33 ⁇ M Biotin, 17 ⁇ M pantothenate, 100 nM human insulin, 1 ⁇ M dexamethasone , 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 0.25 ⁇ g/ml amphotericin B (Maintenance Medium) .
  • the cells were maintained at 37°C in a 5% C0 2 atmosphere and were fed every 2 to 3 days by removing 100 ⁇ l of culture supernatant and replacing it with 100 ⁇ l of fresh, prewarmed Maintenance Medium.
  • Lipolysis assay Adipocytes were treated with inhibitors that were diluted to the appropriate concentration in a diluent consisting of DMEM/Ham's F-12 (1:1 v/v), 15 mM HEPES supplemented with 33 ⁇ M biotin, 17 ⁇ M pantothenate, and 4% BSA. Because the inhibitor stocks were dissolved in 100% DMSO, a vehicle control containing the same final concentra- tion of DMSO as in the drug dilutions also was tested. Each test condition was performed in triplicate. Cells were incubated with inhibitors or vehicle for 6 hours at 37°C in an atmosphere containing 5% C0 2 , after which supernatants from the treated cells were collected and stored at -20°C until ready for analysis.
  • Lipolysis was measured by determining the concentration of glycerol (a breakdown product of triglyceride) in the culture supernatants.
  • the assay was performed in a 96-well format as follows. Culture supernatant (100 ⁇ l) was mixed with an equal volume of GPO Trinder Reagent A (Sigma Diagnostics, Inc., St. Louis, MO). The reaction was allowed to proceed for 15 minutes at room temperature and the absorbance of the samples was determined at 540 nm. The value of a blank (containing only diluent) was subtracted from each sample. The mean and the standard error of the mean were determined for each set of three replicates and these values were expressed as a percentage of the value for the vehicle control. EC 50 values were determined by fitting the data to a four-parameter logistic dose-response model using Table Curve 2D (version 4) software (SSPS, Inc., Birmingham, UK).
  • the following nine selective PDE3B inhibitors of the present invention were tested for induction of lipolysis in cultured human adipocytes. Each of these nine compounds had an IC 50 value vs. recombinant human PDE3B of less than 100 nM (see Table 3) . The selectivity for inhibition of PDE3B over PDE3A of these nine compounds ranged from about three-fold to about 14-fold.
  • the EC 50 of each compound for induction of lipolysis was determined by incubating cultured human adipocytes in the presence of various concentrations of drug and measuring the amount of glycerol released into the culture medium during a six-hour incubation. The results were expressed as a percentage of the response of cells treated with vehicle alone (0.33% DMSO). Each data point corresponded to the mean of three replicate samples . Test compounds :

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Abstract

Selective PDE3B inhibitors, including compounds of the general structural formula (I), and use of the selective PDE3B inhibitors and salts and solvates thereof as therapeutic agents, are disclosed.

Description

SELECTIVE PDE3B INHIBITORS AND USE OF THE SAME IN THERAPY
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. provisional application Serial No. 60/273,497, filed March 5, 2001.
FIELD OF THE INVENTION
The present invention relates to a series of compounds, to methods of preparing the compounds, to pharmaceutical compositions containing the compounds, and to their use as therapeutic agents. In particular, the present invention relates to compounds that are potent and selective inhibitors of cGMP- inhibited phosphodiesterase (cGI-PDE) , and particularly type 3B phosphodiesterase (PDE3B) , and have utility in a variety of therapeutic areas whei'ein such inhibition is considered beneficial, including the treatment of obesity and diabetes.
BACKGROUND OF THE INVENTION
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) to their respective 5 ' -monophosphate forms. Therefore, PDEs regulate intracellular levels of cyclic nucleotides, and play an important role in modulating cellular signaling. Known mammalian PDEs are classified into eleven families, based on their structure, regulation, and kinetic proper- ties. See S.H. Francis et al . , Prog. Nucleic Acid Res . , 65 , pp. 1-52 (2001) . The present invention is directed to inhibitors of the type-3 phosphodiesterase (PDE3) , also referred to as the cGMP- inhibited phosphodiesterase (cGI-PDE) , and in particular to inhibitors of PDE3B.
The PDE3 family contains two members, PDE3A and PDE3B. See Y. Shakur et al . , Prog. Nucleic Acid Res . Molec . Bi ol . , 66, pp. 241-277 (2001) . The two different PDE3 isoforms, encoded by different genes, resemble other mammalian PDEs in having a conserved catalytic domain in the carboxy- terminal portion of the protein and regulatory/- localization motifs located in the amino-terminal portion. The catalytic domains of PDE3A and PDE3B are approximately 85% identical to one another. E. Meacci et al . , Proc . Natl . Acad . Sci . USA, 89 , pp. 3721-3725 (1992) and M. Taira et al . , J". Biol . Chem . , 268 , pp. 18573-18579 (1993). PDE3A is the predominant isoform in cardiac myocytes, vascular and nonvascular smooth muscle cells, and platelets, leading to its designation as the "cardiovascular" isoform. PDE3B is prominent in adipocytes, hepatocytes, endocrine pancreas, kidney epithelium, spermatocytes, embryonic neuroepithelium, and immune cells. See, R.R. Reinhardt et al . , J". Clin . Invest . 95, pp. 1528-1538 (1995) and C. Schudt et al . , Pulmonary Pharmacol . Ther. , 12 , pp. 123-129 (1999). PDE3 inhibitors have been used to treat various diseases and conditions, for example, acute cardiac support in heart failure and for the treatment of intermittent claudication. However, as dis- cussed below, the therapeutic use of PDE3 inhibitors has disadvantages, particularly adverse cardiac effects .
With respect to cardiovascular effects, inhibition of PDE3 in cardiac myocytes leads to increased force of cardiac contraction (positive inotropy) . In addition, PDE3 inhibition of vascular smooth muscle cells (VSMC) leads to vasorelaxation. These hemodynamic effects are expected to benefit individuals suffering from congestive heart failure (CHF) , and this has been demonstrated in studies wherein PDE3 inhibitors were administered acutely.
However, for unknown reasons, chronic treatment of severe CHF patients with PDE3 inhibi- tors led to increased mortality. See J.M. Cruick- shank, Cardiovasc . Drugs Ther. , 7, pp. 655-660 (1993) . Consequently, PDE3 inhibitors currently are used only for acute cardiac support in heart failure (e.g., as a bridge to heart transplant) . However, recent studies suggest that relatively long-term
(e.g., 12 -week) administration of the PDE3 inhibitor enoximone, at low doses, may be safe and effective in the treatment of chronic heart failure. See B.D. Lowes et al . , J". Am . College Cardiol . , 26, pp. 501-508 (2000) .
In addition, inhibition of platelet PDE3 prevents platelet aggregation. See T. Tani et al . , Adv. Second Messenger Phosphoprotein Res . , 25, pp. 215-229 (1992) . A combination of antiaggregation and vasorelaxant properties has been proposed to explain the efficacy of the PDE3 inhibitor cilosta- zol in the treatment of intermittent claudication. Claudication is a partial restriction of blood flow to the extremities, which is a common condition in diabetics and the elderly. See D.L. Dawson et al . , Circulation, 98 , pp. 678-686 (1998) . Cilostazol also was found to be effective in preclinical models of cerebral infarct and pulmonary thromboembolism, again attributed to the vasodilatory and antiplate- let activities of PDE3 inhibition. See Y. Ikeda, Thromb . Hae ost . , 82, pp. 435-438 (1999).
PDE3 inhibitors also inhibit proliferation of VSMC. See M.T. Osinski, Biochem . Pharmacol . , 60 , pp. 381-387 (2000) , suggesting a potential utility of PDE3 inhibitors in the treatment and prevention of atherosclerosis. PDE3 inhibitors reversed in- timal thickening in mouse and rat models of athero- sclerosis, while cilostazol was found to reduce restenosis in humans following coronary angioplasty. See K. Kondo et al . , Atherosclerosis, 142, pp. 133 -• 138 (1999); Y. Inoue et al . , Br. J. Pharmacol , 130 , pp. 231-241 (2000), and T. Kunishima et al . , Clin . Ther. , 19, pp. 1058-1066 (1997).
PDE3 inhibitors also have an effect in the kidney. For example, proliferation of kidney mesangial cells is an indication of several types of glomerular nephritis. PDE3 inhibitors have been found to suppresses proliferation of cultured rat mesangial cells (K. Matousovic et al . , J. Clin . Invest . , 96, pp. 401-410 (1995)). Furthermore, the PDE3 inhibitor lixazinone, when administered in conjunction with the PDE4 inhibitor rolipram, sup- pressed the development of experimentally induced glomerular nephritis in rats. See Y. Tsuboi et al . , J. Clin . Invest . , 98 , pp. 262-270 (1996). Antiinflammatory and bronchodilatory effects also are demonstrated by PDE3 inhibitors. Human lymphocytes express PDE3B, and PDE3 inhibition has been found to inhibit T-cell proliferation in some, but not all, studies (D. Ekholm et al . , J".
Immunol . , 159, pp. 1520-1529 (1997); S.B. Sheth et al . , Br. J. Haematol , 99, pp. 784-789 (1997); and D.M. Essayan et al . , J". Pharmacol . Exp . Ther . , 282 , pp. 505-512 (1997). However, PDE3 inhibitors can potentiate the antiproliferative effects of the PDE4 inhibitor rolipram (M. Giembycz et al . , Br. J. Pharmacol . , 118 , pp. 1945-1958 (1996) and P. Marcoz et al., Molec . Pharmacol . , 44 , pp. 1027-1035 (1993). Similarly, PDE3 inhibitors alone have a comparative - ly low ability to inhibit release of inflammatory cytokines, but can act synergistically with PDE4 inhibitors in this process.
PDE3 inhibitors also are bronchodilators, and are capable of potentiating the bronchodilatory effects of PDE4 inhibitors. See T.J. Torphy, Am . J„ Respir. Cri t . Care Med . , 151, pp. 351-370 (1998). Hence, PDE3 inhibitors may be useful in the treatment of asthma and chronic obstructive pulmonary disease (COPD) . Indeed, cilostazol reverses meth- acholine-induced bronchoconstriction in normal and asthmatic patients (M. Fuj imura et al . , Am . J. Respir. Cri t . Care Med . , 151 , pp. 222-225 (1995)), while enoximone was found to reduce airway resistance in patients suffering from COPD (M. Leeman et al., Chest, 91 , pp. 662-666 (1987)).
PDE3A is present in the smooth muscle of the corpus cavernosum where it plays a role in regulating cavernosal blood pressure and penile erection. Local injection of milrinone into the cavernosum of anesthetized cats increased intra- cavernosal pressure and penile length (P.C. Doherty et al. U.S. Patent No. 6,156,753). PDE3A also is expressed in the oocyte, and
PDE3 inhibitors block maturation of mouse oocytes both in vi tro and in vivo, suggesting effectiveness as contraceptives. See A. Tsafriri et al . , Dev. Biol . , 178 , pp. 393-402 (1996) and A. Wiersma et. al., J". Clin . Invest . , 102 , pp. 532-537 (1998).
With respect to metabolic effects, PDE3 inhibitors induce lipolysis (i.e., hydrolysis of stored triglycerides to free fatty acids and glycerol) in cultured adipocytes (M.L. Elks et al . , Endocrinology, 115, pp. 1262-1268 (1984)). In addition, when administered to humans, the PDE3 inhibitor amrinone stimulated lipolysis and increased resting energy expenditure (Y. Ruttimann et al., Crit. Care Med. , 22, pp. 1235-1240 (1994)). Therefore, by mobilizing fat reserves, PDE3 inhibitors have been suggested as an effective treatment for obesity (P.B. Snyder, Emerging Therapeutic Targets, 3 , pp. 587-599 (1999)). Furthermore, cilostazol lowered serum triglyceride levels and increased serum HDL cholesterol levels in individuals being treated for intermittent claudication. See M.B. Elam et al . , Arterioscler. Thromb. Vase . Biol . , 18 , pp. 1942-1947 (1998).
As previously stated, two isoforms of PDE3 have been identified, i.e., PDE3A and PDE3B. Although a number of potent inhibitors that are selective for PDE3 are available (i.e., are selective for PDE3 over PDE2 , PDES, PDE5, etc.), none of these inhibitors have been shown to possess significant inhibitory selectivity for PDE3B over PDE3A. However, because only PDE3B has been detected in adipo- cytes, the effects of PDE3 inhibitors on adipocytes reasonably can be attributed to inhibition of the
PDE3B isoform. Therefore, it would be desirable to provide a selective PDE3B inhibitor to realize the benefits of PDE3B inhibition, e.g., lipolysis, while avoiding the adverse effects of PDE3A inhibition, e.g., adverse cardiac effects.
PDE3 inhibitors also potentiate glucose- stimulated insulin release from pancreatic β-cells both in vi tro and in vivo . See R. Shafiee-Nick et al., Br. J. Pharmacol , 115, pp. 1486-1492 (1995); M. El-Metwally et al . , Eur. J. Pharmacol , 324 , pp. 227- 232 (1997); and J.C. Parker et al . , Biochem . Biophys, Res . Commun . , 236, pp. 665-669 (1997). Addi- tionally, long-term treatment of diabetic rats with PDE3 inhibitors leads to improved insulin sensitiv- ity of peripheral tissues (Y. Nakaya et al . , Diabetes Obesi ty Metabol . , 1 , pp. 37-41 (1999)). These observations also suggest that PDE3 inhibitors can provide beneficial effects in type 2 diabetes. These observations indicate that PDE3 inhibitors can be used in a therapy for a cluster of obesity- associated pathologies, e.g., insulin resistance, hyperinsulinemia, hyperlipidemia, atherosclerosis, and high blood pressure, collectively termed "metabolic syndrome." However, such use of a PDE3 in- hibitor has been hampered by adverse side effects associated with present-day PDE3 inhibitors.
In particular, obesity is a major risk factor for conditions such as diabetes, hyperlipi- demia, hypertension, and coronary artery disease. In developed nations, the proportion of the population that is obese has been increasing in recent decades (e.g., reaching one-third of the adult pop- ulation of the United States in 1991) . See,
Kuczmarski et al . , J". Amer. Med. Assoc . , 272 , pp. 205-211 (1995); and Seidell et al . , Handbook of Obesi ty, pp. 79-91 (1998). Obesity also has been identified as an important cause of the unfolding epidemic of diabetes in the U.S.
In the U.S. alone, the cost of treating conditions linked to obesity has been estimated at more than $20 billion annually. The effectiveness of behavioral strategies (i.e., diet and exercise) in reducing weight is limited due to poor patient compliance. As a result, there is a need for pharm- acotherapy as an adjunct for treatment of obese individuals with associated pathologies.
Current antiobesity drugs primarily are appetite suppressants that work by potentiating the effect of satiety- inducing neurotransmitters in the central nervous system. These antiobesity drugs produce a modest weight reduction in obese patients, but also can produce both cardiovascular and central nervous system side effects (Bray, Handbook of Obesi ty, pp. 953-975 (1998)).
In addition, weight loss usually occurs only during the first three to six months of treatment, with no further loss observed even when drug treatment is continued for a year or more. This phenomenon is attributed to an increase in energy efficiency following weight loss. According to this model, as body mass decreases, fewer consumed calories are required to sustain resting metabolic activity. As a result, energy expenditure eventually drops to match the reduced level of energy intake, and subsequently no further change in body weight occurs. Thus, the development of pharmacological agents that increase resting metabolic rate and do not exhibit adverse side effects is an important, but elusive, goal in antiobesity research. Ideally, an antiobesity pharmacological agent increases resting metabolic rate by stimulation of two processes: (1) hydrolysis of triglyc- erides stored in adipose tissue to glycerol and free fatty acids (lipolysis) ; and (2) oxidation of excess free fatty acids by a pathway coupled to the production of heat (thermogenesis) rather than to generation of ATP. Such lipolytic/thermogenic agents reduce metabolic efficiency by causing a greater fraction of the total caloric intake to be dissi- pated as heat rather than harnessed for useful cellular work. These agents thereby increase metabolic rate because more calories are utilized to sustain basic cellular processes.
In the adipocyte, elevation of intracellu- lar cAMP leads to stimulation of lipolysis and fatty acid oxidation. Hence, agents that cause an elevation of cAMP are candidates for antiobesity therapeutics. Elevation of cAMP can be achieved either by stimulation of cAMP synthesis (catalyzed by adenylyl cyclases) or by inhibition of cAMP degradation (catalyzed by cyclic nucleotide PDEs) . There are two primary cAMP-hydrolyzing PDEs in adipocytes : PDE3 , which is primarily associated with the par- ticulate fraction, and PDE4 , which is the principal cytosolic cAMP-PDE in this cell type. More particularly, PDE3B is present in adipocytes, as opposed to PDE3A. Accordingly, PDE3B inhibition is an attrac- tive target in providing an antiobesity agent.
The use of current PDE3 inhibitors as therapeutic agents is significantly limited because of their cardiotonic activity. For example, amrinone acutely lowers the diastolic blood pressure and increases the heart rate in normal subjects
(Ruttimann et al . , Crit. Care Med . , 22, pp. 1235- 1240 (1994)). Because the two isoforms of PDE3 display distinct anatomical patterns of expression, selective inhibition of individual isoforms may provide a means of obtaining pharmacological agents with more favorable side -effect profiles. For example, cardiovascular tissues contain predominantly PDE3A, while PDE3B predominates in adipocytes and endocrine pancreas. Thus, selective inhibition of PDE3B would be beneficial for treating obesity and diabetes (e.g., by stimulating lipolysis and resting energy expenditure, improving insulin sensitivity, and lowering serum lipid levels) without causing adverse hemodynamic side effects. Because PDΞ3B also is the predominant isoform in immune cells, selective PDE3B inhibitors also should have utility for the treatment of inflammatory and autoimmune diseases, and for the prevention of graft rejection during organ transplant . It also has been theorized that, in most cell types, PDE3A is predominantly a cytosolic enzyme, while PDE3B is associated with a membrane fraction (H. Liu et al . , Br. J. Pharmacol . , 125, pp. 1501-1510 (1998)). These different intracellular localizations suggest that, within a single cell type', the two isoforms serve distinct cellular functions, and that in some cases it is desirable to inhibit one PDE3 isoform selectively. Thus, isoform-selective inhibitors represent a novel mechanism for selectively inhibiting PDE3 activity in a specific intracellular compartment.
However, no known PDE3 inhibitor has been shown to posses significant selectivity between the two PDE3 isoforms (M-J. Leroy et al . , Biochemistry, 35, pp. 10194-10202 (1996); Snyder, (1999)). The most selective agent reported in the literature is cGMP, which is capable of inhibiting hydrolysis of cAMP with a three-fold higher potency against PDE3A than PDE3B (Leroy et al . , 1996). The present invention is directed to a series of compounds that exhibit a selective inhibition of PDE3B over PDE3A. The present invention also is directed to the use of such compounds in the treatment of diseases and conditions wherein inhibition of PDE3B provides a benefit, while minimizing or eliminating adverse effects associated with PDE3A inhibition.
SUMMARY OF THE INVENTION
The present invention is directed to selective PDE3B inhibitors, and to use of the selective PDE3B inhibitors in therapy. More particular- ly, the present invention is directed to compounds disclosed herein, to compositions containing a compound disclosed herein, and to methods of treating a condition or disease wherein selective inhibition of PDE3B provides a therapeutic benefit.
Present-day selective inhibitors of the type-3 phosphodiesterase (PDE3) currently are used for acute cardiac support in heart failure and for the treatment of intermittent claudication. PDE3 inhibitors potentially are useful in the treatment of obesity, diabetes, hyperlipidemia, atherosclerosis, coronary restenosis, thrombocythemia, glomer- ular nephritis, asthma, inflammatory diseases, autoimmune disorders, organ transplant rejection, and erectile dysfunction.
However, present-day PDE3 inhibitors are nonselective with respect to PDE3A inhibition versus PDE3B inhibition, and the simultaneous inhibition of both the PDE3A and PDE3B isoforms leads to adverse side effects. These side effects can be minimized or eliminated by a selective inhibition of PDE3B over PDE3A. Accordingly, the present invention is directed to a selective PDE3B inhibitor having a structural formula (I) :
( ° ) -NRΛ -X
Figure imgf000013_0001
wherein R° , independently, is selected from the group consisting of halo , Chalky! , C2.6alkenyl , C-L-ghaloalkyl , C3_8cycloalkyl , C3_8heterocycloalkyl , aryl , heteroaryl , C ( =0) Ra , 0C ( =0) Ra , C ( =0) ORa , C^- alkyleneNRaRb, C^alkyleneOR9, C(=0)NRaRb, C(=0)OCj.3- alkyleneC(=0)Ra, ORa, NRaRb, NRaC1.4alkyleneNRaRb, NRaC(=0)Rb, NRaC(=0)NRaRb, N (S02Cα_4alkyl) 2, NRa(S02C1.4- alkyl) , nitro, tri luoromethyl , trif luoromethoxy, cyano, C1_3alkyleneCN, SRa, S02Ra, S02NRaRb, N02, and
Figure imgf000014_0001
or two R° groups are taken together to form a 5- or 6-membered nonaromatic ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur; R1 is Ogalkyl or halo;
R2 is selected from the group consisting of hydrogen, Chalky!, aryl, heteroaryl, C-^alkylene- aryl, C1.3alkyleneheteroaryl , C3-8cycloalkyl , C3_8- heterocycloalkyl , and
CHC(=0)OCH3
CH90H
Y is selected from the group consisting of
CH(R4) , CH2CH(R4) , CH(R4)CH2, RC |7 C (=0) (CH2) ^S (CH2) 0_2 , O(CH2)0.4, NRcC(=0) (CH2)0-2/ and S02NRa (CH2) 0.2 , or Y i s nui 1 ; R3 and R4, independently, are selected from the group consisting of hydrogen,
Figure imgf000015_0001
aryl, heteroaryl, and halo;
X is selected from the group consisting of hydrogen, OH, OC^alkyl, cycloalkyl, CH(Rc)CH2OH, CH2CH(Rc)OH, NRaRb, NHCH (Rc) CH2OH, NHCH2CH (R°) OH, CH(RC)CH2NH2, CH2CH(RC)NH2, NHC(=0)Ra, NHC(=S)NHRa, NHC(=0)ORa, NHC(=0)C(=0)NRaRb, and NHC(=0)NHRa, or X is a bond between NR2 and an atom of ring A or ring B, or R2 and X are taken together to form an optionally substituted 5- or 6-membered nonaromatic ring containing one to three heteroatoms selected from oxygen, sulfur, and nitrogen; A is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, or A is null ;
B is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring and. optionally substituted fused bicyclic and polycyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
Ra and R , independently, are selected from the group consisting of hydrogen, C^alkyl, aryl, arylCj^alkyl , C^alkylenearyl , heteroaryl, hetero- arylC1.3alkyl ,
Figure imgf000015_0002
, C1_3alkoxyC1.6alkyl, C3_8cycloalkyl , C1.3alkyleneC3.8- cycloalkyl, C3.8heterocycloalkyl , C1.3alkyleneOC1_3- alkylenearyl , C1.4alkyleneOC1.3alkyl , C1.4alkyleneSi- (C1.3alkyl) 3, and C1.3alkyleneheterocycloalkyl , or Ra and Rb are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and optionally substituted with
Figure imgf000016_0001
C1.3alkylenearyl , and C(=0)NRaRb; Rc is selected from the group consisting of hydrogen, aryl, heteroaryl, Cx_6alkyl, C3_8cycloalkyl , C3_8heterocycloalkyl , C1_3alkylenearyl , C1.3alkyleneC3.8- heterocycloalkyl , C1.3alkyleneheteroaryl , arylC-^- alkyl, and heteroarylC1.3alkyl ; n is 0, 1, 2, 3, or 4; p is 0 or 1 ; and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
Another aspect of the present invention is to provide a selective PDE3B inhibitor having a structural formula (II) or (III) :
Figure imgf000016_0002
(ID
Figure imgf000017_0001
(III)
and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
Still another aspect of the present invention is to provide a selective PDE3B inhibitor having a structural formula (IV) :
Figure imgf000017_0002
(IV)
and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
The present invention also is directed to selective PDE3B inhibitors having the following structures : (a)
Figure imgf000018_0001
(b)
Figure imgf000018_0002
Figure imgf000019_0001
(d)
Figure imgf000019_0002
(e)
Figure imgf000020_0001
(f:
Figure imgf000020_0002
(g)
Figure imgf000020_0003
( h)
Figure imgf000021_0001
( i )
Figure imgf000021_0002
As used herein, the term "a compound of the present invention" is defined as a compound encompassed by structural formula (I), (II), (III), or (IV), compounds (a) - (i) listed above, Examples 1- 41, and compounds identified at page 1 through page 39 in Appendix A, which constitutes a porti.on of this disclosure.
Another aspect of the present invention is to provide a PDE3B inhibitor having an IC50 value vs. human recombinant PDE3B of about 10 μM or less, preferably about 1 μM or less, more preferably about 500 nM or less, and most preferably about 100 nM or less. For example, a PDE5 inhibitor of the present invention has an IC50 value versus human recombinant PDE3B of about 700 picomolar to about 1,000 μm.
Yet another aspect of the present inven- 5 tion is to provide a preferred PDE3B inhibitor having a ratio of an ICS0 vs. human recombinant PDE3A to an IC50 vs. human recombinant PDE3B of at least 5, preferably at least 10, more preferably at least 20, and most preferably at least 30.
10 Another aspect of the present inventi.on is to provide a. composition comprising a compound of the present invention and a physiologically acceptable diluent or carrier.
Another aspect of the present invention is
15 to provide a method of treating an individual suffering from a disease or condition wherein inhibition of PDE3B provides a benefit comprising administering a therapeutically effective amount of a compound of the present invention, or a composition
20 containing the same, to the individual. The method minimizes or eliminates adverse side effects attributed to PDE3A inhibition.
Still another aspect of the present invention is to provide a method of treating an individ-
'25 ual suffering from obesity, diabetes, hyperlipidemia, asthma, an inflammatory disease, an autoimmune disorder, or organ transplant rejection, comprising administering a therapeutically effective amount of a compound of the present invention, or a composi-
.30 tion containing the same, to the individual.
Another aspect of the present invention is to provide therapeutic compounds and methods for an efficacious treatment of obesity that minimize or eliminate adverse side effects associated with prior compounds and methods used to treat obesity.
In another aspect, the present invention, provides a method of inducing, promoting, or causing lipolysis in mammalian cells comprising the step of contacting mammalian cells with a compound of the present invention at a concentration effective to stimulate lipolysis in the cells. In a preferred embodiment, the mammalian cells are mammalian adipo- cytes, and preferably human adipocytes, for example, human brown adipose cells or white adipose cells.
Another aspect of the present invention is to provide a combination therapy comprising administration of therapeutically effective amounts of (a) a compound of the present invention, or pharmaceutically acceptable salts and solvates thereof, and (b) a second therapeutically active agent, to an individual for simultaneous, separate, or sequent iε.1 use in the treatment of a disease or condition wherein inhibition of PDE3B provides a benefit, such as obesity or diabetes. The second therapeutically active agent can be a PDE4 inhibitor, for example. In another aspect, the present invention provides a kit for the treatment of obesity compris- ing a compound of the present invention, or a composition containing the same, packaged with instructions for administration of the compound, or composition, to a mammal, including a human, to promote weight loss. In one variation, the compound of the present invention and a second therapeutically active ingredient for the treatment of obesity are packaged together in separate vials, separate tablets, or the like. Still another aspect of the present invention is to provide a method of preparing a compound of the present invention.
These and other aspects of the present invention will become apparent from the following detailed description of the preferred embodiment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A compound is considered to be a PDE inhibitor if the compound effectively inhibits phosphodiesterase activity of a PDE at a physiologically compatible concentration. • To be- useful as a therapeutic compound, the compound also must be practi- cally nontoxic to a cell at such a concentration.
Effective inhibition typically is defined as a compound that inhibits PDE activity by at least 50%, preferably at least 80%, and more preferably at least 90%, at a physiologically compatible concen- ration.
A PDE inhibitor is referred to as being selective if the compound effectively inhibits a particular PDE of interest, and only marginally inhibits, or fails to inhibit, other PDEs. In this case, all the PDEs, except the particular PDE of interest, perform their normal functions, and the activity of the PDE of interest is reduced sufficiently to provide a benefit.
As discussed in more detail hereafter, enzyme inhibition typically is measured using a dose-response assay in which a sensitive assay system is contacted with a compound of interest over a range of concentrations at which no or minimal effect is observed, through higher concentrations at which partial effect is observed, to saturating concentrations at which a maximum effect is observed. Theoretically, such assays of the dose-response effect of inhibitor compounds can be described as a sigmoidal curve, expressing a degree of inhibition as a function of concentration. The curve also theoretically passes through a point at which the concentration is sufficient to reduce activity of the PDE enzyme to a level that is 50% that of the difference between minimal and maximal enzyme activity in the assay. This concentration is defined as the Inhibitory Concentration (50%) or ICS0. Comparisons between the efficacy of inhibitors often are provided with reference to comparative 1C50 concentrations, wherein a higher ICb0 indicates that the test compound is less potent, and a lower ICS0 indicates that the compound is more potent, than a reference compound. Similarly, the potency of inhibitor compounds can be related in terms of the Effective Concentration (50%) or EC50, which is a measure of dose-respcnse activity in a cell -based or animal- based model . EC5ϋ measurements are useful to relate properties of the compound that can influence its clinical utility, such as compound solubility, ability to penetrate cell membranes, partition coefficient, bioavailability, and the like. Two compounds can exhibit a divergence in comparative IC50 and EC50 values, i.e., one compound can be more potent in a biochemical assay and the second compound more potent in a cell-based assay simply due to different properties of the compounds. A measure of comparative selectivity is a ratio of IC5C values for a compound with respect to two different enzymes. To illustrate, if a compound has an IC50 for enzyme A of 1 μM, and an IC50 for enzyme B of 10 μM, then the compound is said to have a 10-fold selectivity for enzyme A over enzyme B: IC50 B/IC50 A=10 μM/1 μM=10. In most cases, it is desirable that the selectivity of a compound is sufficiently high such that, at an effective concen- tration to inhibit the PDE inhibitor of interest, the compound has minimal to no effect on other enzymes .
The present, invention is directed, to potent inhibitors of PDE3 , and that further exhibit: a selective inhibition of PDE3B over PDE3A. As used herein, a "selective PDE3B inhibitor" is a compound that inhibits PDE3B at least 5-fold, and preferably at least 10 -fold, more effectively than the compound inhibits PDE3A. To achieve the full advantage of the present invention, a selective PDE3B inhibitor inhibits PDE3B at least 25-fold, and even more preferably at least 30-fold, more effectively than the compound inhibits PDE3A. Selective PDE3B inhibitors are expected to promote lipolysis m adipocytes, for example, with fewer adverse side effects (e.g., adverse cardiac effects) than a nonselective PDE3A and PDE3B inhibitor.
In particular, because the PDE3 enzyme exists in at least two isoforms, PDE3A and PDE3B, and because adipocytes apparently only express PDE3B to any significant extent, a PDE3 inhibitor that is selective, or specific, for PDE3B is highly preferred in the treatment of obesity and conditions related to or resulting from obesity, e.g., diabetes .
Such inhibitors are exemplified by the compounds of the present invention:
-NR2-X
Figure imgf000027_0001
wherein R°, independently, is selected from the group consisting of halo, C1_6alkyl, C2.6alkenyl,
Figure imgf000027_0002
, C3.8cycloalkyl , C3.8heterocycloalkyl , aryl, heteroaryl, C(=0)Ra, 0C(-=0)Ra, C(=0)ORa, Cj.,- alkyleneNRaR , C1.4alkyleneOR\ C(=0)NRaRb, C(=0)OCj_3- alkyleneC(=0)Ra, ORa, NRaRD, NRaC1-4aikyleneNRaRb, NRaC(=0)Rb, NRaC(=0)NRaRb, N (S02C1.4alkyl ) 2, NRa(S02C1„4- alkyl) , nitro, trif luoromethyl , trif luoromethoxy, cyano, C-^alkyleneCN, SRa, S02Ra, S02NRaRb, N02, and
Figure imgf000027_0003
or two R° groups are taken together to form a 5- or 6-membered nonaromatic ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur;
R1 is Ci.galkyl or ha o;
R2 is selected from the group consisting of hydrogen, Chalky!, aryl, heteroaryl, C^alkylene- aryl , C1.3alkyleneheteroaryl , C3 8cycloalkyl , C3„8het- erocycloalkyl , and
Figure imgf000028_0001
Y is selected from the group consisting of CH(R4), CH2CH(R4), CH(R4 CH2, NRC, C (=0) (CH2) X.2S (CH2) 0.2 , O(CH2)0.4, NRcC(=0) (CH2)0_2, and SO2NRa(CH2)0_,, or Y is null;
R3 and R4 , independently, are selected from the group consisting of hydrogen, C1.6alkyl , aryl, heteroaryl, and halo;
X is selected from the group consisting of hydrogen, OH, 0C1.3alkyl, cycloalkyl, CH(Rc)CH2OH, CH2CH(Rc)0H, NRaRb, NHCH (Rc) CH2OH, NHCH2CH (Rc) OH, CH(RC)CH2NH2, CH2CH(RC)NH2, NHC(=0)Ra, NHC(=S)NHRa, NHC(=0)0Ra, NHC(=0)C(=0)NRaRb, and NHC(=0)NHRa, or X is a bond between NR2 and an atom of ring A or ring B, or R2 and X are taken together to form an optionally substituted 5- or 6-membered nonaromatic ring containing one to three heteroatoms selected from oxygen, sulfur, and nitrogen;
A is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring, either carbo- cyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, or A is null ; B is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring and optionally substituted fused bicyclic ana polycyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
Ra and Rb, independently, are selected from the group consisting of hydrogen, Ct talkyl , aryl, arylC1.3alkyl , C--6alkylenearyl , heteroaryl, hetero- arylCj_3alkyl , C1_3alkyleneheteroaryl , naloOgalkyl , C1_3alkoxyC1.6alkyl, C3.8cycloalkyl , C1-3alkyleneC3_£- cycloalkyl, C3.8heterocyσloalkyl, C^alkyleneOC^- alkylenearyi , C1.4alkyleneOC1.3alkyl , C].4aikyleneSi- (C1.3alkyl) 3, and C1_3alkyleneheterocycloalkyl , or Ra and R are taken together co form a 5- or 6-membered ring, optionally containing at east one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and op- tionally substituted with C, ealkyl , Ci.3alkylenearyl , and C(=0)NRaRb;
Rc is selected from the group consisting of hydrogen, aryl, heteroaryl, Ogalkyl, C3.8cycloalkyl , C3.8heterocycloalkyl, C^alkylenearyl , Cα 3alκyleneC3.8- heterocycloalkyl, C1.3alkyleneheteroaryl , arylC^- alkyl, and heteroarylC-^alkyl ; n is 0, 1, 2, 3, or 4; p is 0 or 1 ; and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
In some preferred embodiments, the selective PDE3B has the structural formula (II) or (III) . !R°> HX
Figure imgf000030_0001
(ID
Figure imgf000030_0002
(III)
and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
In another preferred embodiment of the present invention, a selective PDE3B inhibitor has a structural formula (TV) :
Figure imgf000030_0003
(IV)
and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof.
As used herein, the term "alkyl" includes straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups . The hydrocarbon group can contain up to 16 carbon atoms. The term "alkyl" includes "bridged alkyl," i.e., a C6-C16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl , adamantyl , bicyclo [2.2.2] octyl , bicyclo [2.2.1] heptyl , bicyclo [3.2.1] octyi , or deca- hydronaphthyl . Alkyl groups can be substituted, for example, with hydroxy (OH) or amino (NH2) groups.
The term "cycloalkyl" is defined as a cyclic C-C8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl . "Hetero- cycloalkyl" is defined similarly as cycloalkyl except the ring concains one to three heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Cycloalkyl and heterocycloalkyl groups can be substituted for example, with one to three groups, independently selected from groups
Figure imgf000031_0001
OH.
The term "alkenyl" is defined identically as "alkyl," except for containing a carbon-carbon double bond. "Cycloalkenyl " is defined similarly to, and is encompassed by, the term cycloalkyl, except a carbon -carbon double bond is present in the ring .
The term "alkylene" refers to an alkyl group having a substituent. For example, the term "C-^alkylenearyl" refers to an alkyl group containing one to three carbon atoms, and substituted with an aryl group .
The term "halo" or "halogen" is defined herein to include fluorine, bromine, chlorine, and iodine .
The term "haloalkyl" is defined herein as an alkyl group substituted with one or more halo substituents, either fluoro, chloro, bromo, iodo, or combinations thereof. "Halocycloalkyl " is encompassed by the term "haloalkyl," and is defined as a cycloalkyl group having one or more halo substitu- ents.
The term "aryl," alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g.. phenyl or naphthyl. Unless otherwise indicated, an "aryl" group can be unsubstituted or substituted, for example, with one or more, and in particular one to three, halo, alkyl, alkenyl, heterocycloalkyl, trifluoromethyl , C^- alkyleneOCi.galkyl, trif uoromethoxy, S02NRaR , NRaR, benzyloxy, thiobenzyl , Chalk leneObenzyl , OC3._5al.kyl- enearyl , OCj 3alkyleneheterυaryl , OC_( 8heterocyclo- aikyl , OCi.jalkyleneC^.gheterocycloalkyi , hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, cyano, amino, alkylamino, acylamino, alkylthio, arylthio, C(=0)- NRaRb, C(=0)Ra, NHC(=--0)C1_3alkyl, OC1_3alkyleneNRaRb, alkylsulfinyl , and alkylsulfonyl . Exemplary aryl groups include phenyl, naphthyl, tetrahydronaphthyl , 2-chlorophenyl , 3 -chlorophenyl , 4-chloropnenyl , 2- methylphenyl , 4-methoxyphenyl , 3-trifluoromethyl- phenyl, 4-nitrophenyl , and the like. The terms
"arylC1.3alkyl " and "heteroarylC-^alkyl " are defined as an aryl or heteroaryl group having a C1_3alkyl substituent .
The term "heteroaryl" is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, for example, the substituents listed above for aryl groups. Examples of heteroaryl groups include thienyl, furyl , pyridyl, oxazolyl, quinolyl , isoquinolyl, indolyl , triazolyl, isothiazolyl , isoxazolyl, imidizolyl, benzothiazol- yl , pyrazinyl, pyrirnidinyl , thiazolyl, and thiadi- azolyl .
The term "hydroxy" is defined as -OH. The term "alkoxy" is defined as -OR, wherein R is alkyl, including cycloalkyl.
The term "alkoxyalkyl " is defined as an alkyl group wherein a hydrogen has been replaced by an alkoxy group. The alkoxyalkyl group can be sub- stituted with an aryl or heteroaryl group. The term " (alkylthio) alkyl" is defined similarly as alkoxyalkyl, except a' sulfur atom, rather than an oxygen atom, is present .
The term "hydroxyalkyl" is defined as a hydroxy group appended to an alkyl group.
The term "amino" is defined as -NH2, and the term "alkylamino" is defined as -NR2, wherein at least one R is alkyl and the second R is alkyl or hydrogen . The term "5- or 6-membered aryl or heteroaryl group" as used herein refers to carbocyclic and heterocyclic aromatic groups, including, but not limited to, phenyl, thiophenyl, furyl , pyrrolyl, imidazolyl, pyrirnidinyl, and pyridinyl . The term "acylamino" is defined as RC-
(=0)N, wherein R is alkyl or aryl. The term "alkylthio" and "arylthio" are defined as -SR, wherein R is alkyl or aryl, respectively.
The term "alkylsulfinyl " is defined as R-S02, wherein R is alkyl.
The term "alkylsulfonyl " is defined as R-S03, wherein R is alkyl.
The term "nitro" is defined as -N02.
The term "trifluoromethvl " is defined as
■CF,
The term " trifluoromethoxy" is defined as
■OCF,
The term "cyano" is defined as -CN.
The carbon atom content of hydrocarbon- containing moieties is indicated by a subscript designating the minimum and maximum number of carbon atoms in the moiety-, e.g.,
Figure imgf000034_0001
refers to an alkyl group having one to six carbon atoms, inclusive .
In the structures herein, for a bond lacking a substituent, the substituent is methyl, for example,
Figure imgf000034_0002
Figure imgf000034_0003
When no substituent is indicated as attached to a carbon atom on a ring, it is understood that the carbon atom contains the appropriate number of hydrogen atoms. In addition, when no substituent is indicated as attached to a carbonyl group or a nitrogen atom, for example, the substituent is understood to be hydrogen, e.g.,
0 0
10
R-C is R-C-H and R-N is R-NH? .
In a preferred embodiment, S (O) 0_2 is S02, Y 15. is CH(R4) or MRC, and p is 0. In other preferred embodiments, n is greater than 0, and R°, independently, are selected from 'the group consisting of Ci.gal yl, aryl, heteroaryl, CN, ORa, Ojalkyle eCR3, C(=0)ORa,
Figure imgf000035_0001
C(=0)Ra, SO,NRaRb, 20 NRaRb, C3_8cycloalkyl, C(=0)NRaRb, and
Figure imgf000035_0002
25
or two R° groups are taken together to form with the B ring,
30
Figure imgf000036_0001
wherein q is 1 or 2, and G, independently, is C(Ra 2 / 0, S, or NRa. In a more preferred group of compounds of the present invention, q is 1 or 2, and G, independently, are C(Ra)2 or O. Examples wherein B is phenyl, include
Figure imgf000036_0002
Figure imgf000036_0003
, and
Figure imgf000036_0004
In another preferred group of compounds of the present invention, R2 is hydrogen, R* and R4 are hydrogen, and Rc is hydrogen or alkyl. In preferred embodiments, A and B, independently, are selected from the group consisting of
Figure imgf000037_0001
phenyl
Figure imgf000037_0002
furanyl
Figure imgf000037_0003
t l
H
py Orrolyl
Figure imgf000037_0004
oxazolyl
Figure imgf000037_0005
thiazolyl H
Figure imgf000038_0001
imidazolyl
H
V
N
10 yy pyrazolyl
Figure imgf000038_0002
15 isoxazolyl
Figure imgf000038_0003
20 isothiazolvl
Figure imgf000038_0004
25 1, 2 , 3-oxadiazolyl
Figure imgf000038_0005
1,2, 3-triazolyl
Figure imgf000039_0001
1,3, 4-thiadiazolyl
Figure imgf000039_0002
10 1, 2 , 4-oxadiazolyl
,0
N *N
15 1, 2, 5-oxadiazolyl
Figure imgf000039_0003
20 1,3, 4-oxadiazolyl
0 .
{ N
N N
25 1,2,3, -oxatriazolyl
Figure imgf000039_0004
30 1,2,3, 5-oxatriazolyl
Figure imgf000040_0001
pyridinyl
Figure imgf000040_0002
pyridazinyl
10
Figure imgf000040_0003
pyrazinyl
20
Figure imgf000040_0004
1,3, 5-triazinyl
25
Figure imgf000040_0005
1,2, 4-triazinyl
Figure imgf000041_0001
1,2, 3-triazinyl
In addition to the above aromatic ring systems, B also can be selected from the group consisting of:
Figure imgf000041_0002
indolizi.nyl
Figure imgf000041_0003
indolyl
Figure imgf000041_0004
isoindolyl
Figure imgf000042_0001
benzo (b) f uranyl
Figure imgf000042_0002
benzothienyl
H
Figure imgf000042_0003
IH-indazolyl
H
Figure imgf000042_0004
benzimidazolyl
Figure imgf000042_0005
benzthiazonyl H
Figure imgf000043_0001
purinyl
Figure imgf000043_0002
10
4H-quinolizinyl
Figure imgf000043_0003
qυinolinyl
Figure imgf000043_0004
isoquinolinyl
Figure imgf000043_0005
indenyl
30
Figure imgf000044_0001
naphthyl
In especially preferred embodiments, Rf is selected from the group consisting of chloro, methyl, methoxy, benzyloxy, C (=0) NHCH2C6H5, CH2OCH2C6H5, CN, S02N(CH3)2,
OCH,
Figure imgf000044_0002
and..
Figure imgf000044_0003
R: is hydrogen; R2 is selected from the group consisting of hydrogen, C^alkyl, and aryl; S (O) 0_2 is SO-, Y is CH(R4), R3 and R4, independently, are hydrogen, C1.3alkyl, phenyl, fluoro, or chloro; X is CH2CH2OH or CH2CH2NH2; A is selected from the group consisting of
Figure imgf000045_0001
Figure imgf000045_0002
Figure imgf000045_0003
Figure imgf000045_0004
, and
Figure imgf000045_0005
and B is selected from the group consisting of
Figure imgf000046_0001
Figure imgf000046_0002
Figure imgf000046_0003
20
Figure imgf000046_0004
Figure imgf000047_0001
Figure imgf000047_0002
Figure imgf000047_0003
20
Figure imgf000047_0004
25
Figure imgf000047_0005
, and
Figure imgf000048_0001
Compounds of the present invention can contain one or more asymmetric center, and, there- fore, can exist as stereoisomers. The present invention includes both mixtures and separate individual stereoisomers of the compounds of the present invention. Compounds of the present invention also can exist in tautomeric forms, and the invention includes both mixtures and separate individual tautomers thereof .
Pharmaceutically acceptable salts of compounds of the present invention can be acid a.ddition salts formed with pharmaceutically acceptable acids. Examples of suitable salts include, but are not limited to, the hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lac- tate, citrate, tartrate, gluconate, methanesulfon- ate, benzenesulfonate, and p-toluenesulfonate salts. The compounds of the present invention also can provide pharmaceutically acceptable metal salts, in particular alkali metal salts and alkaline earth metal salts, with bases. Example's include the sodium, potassium, magnesium, and calcium salts.
Compounds of the present invention are potent and selective inhibitors of PDE3B. Thus, the compounds are of interest for use in therapy, spe- cifically in the treatment of a variety of conditions where selective inhibition of PDE3B is considered beneficial.
PDE3B is a particularly attractive target for inhibition because a potent and selective inhibitor of PDE3B provides effects that are beneficial in the treatment of various disease states. The biochemical, physiological, and clinical effects of PDE3B inhibitors therefore suggest their utility m a variety of disorders and disease states, including obesity, diabetes, hyperlipidemia, inflammatory diseases, autoimmune disorders, and organ transplant rejection. As selective PDE3B inhibitors, compounds of the present invention are useful in treating the above and other diseases and conditions, while minimizing or eliminating adverse side effects associated with PDE3A inhibition.
An especially important use of the compounds of the present invention is the treatment of obesity, which is a common medical problem and a major contributor to the onset of diabetes. Therefore, a further important use of the present PDE3B inhibitors is the prophylactic and therapeutic treatment of diabetes. Thus, the present invention is directed to the use of the disclosed compounds, a pharmaceutically acceptable salt t.Viereof, or a pharmaceutical composition containing either entity, for the manufacture of a medicament for the curative or prophylactic treatment of obesity and diabetes in a mammal, including humans. The present invention also is directed the use of a compound of the present invention for the manufacture of a medicament for the treatment of the other above-noted conditions and disorders.
As used above and hereafter, the term "treatment" includes preventing, lowering, stopping, or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" includes both medical therapeutic and/or prophylactic administration, as appropriate, including, but not i.imiced to, the diseases and con- ditions discussed above. Although compounds of the present invention are envisioned primarily for the treatment of obesity and diabetes in humans, they also can be used for the treatment of other disease states, including, but not limited to, the diseases and conditions discussed above.
It also is understood that "a compound of the present invention," or . a physiologically acceptable salt or solvate thereof, can be administered as the neat compound, or as a pharmaceutical composi- tion containing either entity.
The present invention, also is directed to a method of treating the conditions and disorders wherein inhibition of PDE3B provides a benefit, in a human or nonhuman animal body, comprising adminis- tering to said body a therapeutically effective amount of a compound of the present invention.
In vivo methods of treatment are specifically contemplated. Thus, for example, the present invention includes a method of inducing weight loss in a mammal comprising the steps of administering to the mammal (a) a compound that selectively inhibits PDE3B and (b) an optional second active compound for effecting weight loss, wherein the compound or com- pounds are administered in amounts effective to promote weight loss in the mammal . Administration to humans is specifically contemplated, but administration to other animals, including pets, live- stock, zoo specimens, wildlife, and the like, also is contemplated. Treatment of humans who are clinically diagnosed as obese is specifically contemplated.
Compounds of the present invention can be administered by any suitable route, for example by oral, buccal, inhalation, sublingual, rectal, vaginal, transurethral , nasal, topical, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, and mfcra- coronary) administration. Parenteral administration can be accompl shed using a needle and syringe, or using a high pressure technique, like POWDERJECT™. Oral administration of a compound of the invention is a preferred route. Oral administration is the most convenient and avoids the disadvantages associated with other routes of administration. For patients suffering from a swallowing disorder or from impairment of drug absorption after oral administration, the drug can be administered parenter- ally, e.g., sublingually or buccally.
Compounds and pharmaceutical compositions suitable for use in the present invention include those wherein the active ingredient is administered in an effective amount to achieve its intended pur- pose. More specifically, a "therapeutically effective amount" means an amount effective to prevent development of, or to alleviate the existing symptoms of, the subject being treated. Therefore, a "therapeutically effective dose" refers to that amount of a compound that results m achieving the desired effect. Determination of an effectOe amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
The toxicity and therapeutic efficacy of compounds of the present invention can be determined by standard pharmaceutical procedures in cell cul- tures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from such data can be used in formulating a dosage range for use in humans . The dosage of such compounds prefe - ably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. The exact formulation, route of administration, and dosage is selected by the individual physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide plasma levels of a compound of the preε- ent invention which are sufficient to maintain therapeutic effects. Therefore, the amount of a compound of the present invention administered is related to the subject being treated, including the subject's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
Specifically, for administration to a human in the curative or prophylactic treatment of the conditions and disorders identified above, dosages of a compound of the present invention generally are about 0.1 to about 1000 mg daily fox an average adult patient ('70 kg) . Thus, for a typical adult patient, individual doses, for example, tablets or capsules, contain 0.1 to 500 mg of active compound, in a suitable pharmaceutically acceptable vehicle or carrier, for administration in single or multiple doses, once or several times per- day. Dcs- ages for intravenous, buccal, or sublingual administration typically also are 0.1 to 500 mg per single dose as required. In practice, the physician determines the actual dosing regimen which is most suitable for an individual patient , and the dosage varies with the age, weight, and response of the particular patient. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention.
For human use, a compound of the present invention can be administered alone, but generally is administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration a d standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in a conventional manner usinct one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of compounds of the present invention into compositions that can be used pharmaceutically. Such pharmaceutical compositions can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is related to the route of administration chosen. When a therapeutically effective amount of a compound of the present invention is administered orally, the composition typically is in the form of a tablet, capsule, powder, solution, oielixir. When administered -in tablet form, the composition can additionally contain a solid carrier, such as a gelatin or an adjuvant. The tablet, capsule, and powder contain about 5% 'to about 95% compound of the present invention, and preferably from about 25% to about 90% compound of the present invention. When administered in liquid form, a liquid ' carrier such as water, petroleum, or oils of animal or plant origin can be added. The liquid form of the composition can further contain physiological saline solution, dextrose or other saccharide solutions, or glycols. When administered in liquid form, the composition contains about 0.5% to about 90% by weight of a compound of the present invention, and preferably about 1% to about 50% of a com- pound of the present invention.
When a therapeutically effective amount of a compound of the present invention is administered by intravenous, cutaneous, or subcutaneous injec- tion, the composition is in the form of a pyrogen- free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stabil- ity, and the like, is within the skill in the art. A preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains, in addition to a compound of the present invention, an isotonic vehicle. For oral administration, the compounds can be formulated readily by combining a compound of the present invention with pharmaceutically acceptable carriers well known in the art . Such carriers enable the present compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a compound of the present invention with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose prepara- tions. If desired, disintegrating agents can be added .
For administration by inhalation, compounds of the present invention are conveniently delivered in the form of an aerosol spray presen- tation from pressurized packs or a nebulizer, with the use of a suitable propellant . In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. ■ Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, a present composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Compounds of the present invention also can be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases. In addition to the formulations described previously, the compounds also can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. For veterinary use, a compound of the present invention or a nontoxic salt thereof, is administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian readily can determine the dosing regimen and route of administration that is most appropriate for a particular animal .
Thus, the present invention provides a pharmaceutical composition comprising a compound of the present invention, together with a pharmaceutically acceptable diluent or carrier therefor. The present invention also provides a process of preparing a pharmaceutical composition comprising a com- pound of the present invention, which process comprises mixing a compound of the present invention, together with a pharmaceutically acceptable diluent or carrier therefor.
In a particular embodiment, the invention includes a pharmaceutical composition for the curative or prophylactic treatment of obesity, diabetes, and other diseases and conditions wherein selective inhibition of PDE3B provides a benefit in a mammal, including humans, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable diluent or carrier. A composition of the present invention can be administered to an individual in concert with a second therapeutically active agent. The second therapeutically active agent is a compound useful in treating the disease or condition afflicting the individual, and for which the individual is receiving treatment with a compound of the present invention. For example, if an individual is being treated for obesity, the individual can be administered a therapeutically effective amount of a com- pound of the present invention and a second therapeutically active agent useful in the treatment of obesity, for example, a PDE4 inhibitor, like roli- pram and CDP840. The selective PDE3B inhibitor of the present invention and second therapeutically active agent can be administered either simultaneously, separately, or sequentially. If administered sequentially, either the PDE3B inhibitor or second therapeutically active agent can be administered first . PDE4 inhibitors useful in conjunction with a compound of the present invention in the treatment of obesity and other diseases and conditions can be categorized into various classes, such as xanthine derivatives, rolipram analogs, and quinazolinedi- ones, and include benafentrine, tolafentrine, zardaverine, Org 20241, nitraquazone, RS 5344, BRL 1063, SB 207499, SCZ MKS 492, CDP 840, CP 80,633, D- 22888, AWD-12-281, D-4418, RP 73401, WAY-PDA- 641, LAS 31025, tibenelast, benbufylline, and substituted pyrrolidines (U.S. Patent No. 5,665,754, incorporated herein by reference, for example) . Several PDE4 inhibitors have been described in the litera- ture, e.g., Dent et al . , in Phosphodiesterase
Inhibi tors, Schudt et al . , eds., Academic Press, San Diego, pp. 111-126 (1996); and Crocker et al . , Drugs of Today, 35 (7) , pp. 519-535 (1999), all incorporated herein by reference in their entirety. Addi- tional PDE4 inhibitors are known in the art and are contemplated for use in conjunction with a present selective PDE3B inhibitor.
Compounds of the present invention can be prepared by any suitable method known in the art, or by the following processes which form part of the present invention. In the methods below, R°, R1, R2, R3, and R4, as well as A, B, X, and Y are defined as in the present invention above. For example, compounds of the present invention can be prepared according to the following synthetic scheme, which comprises reacting compounds of formulae (V) and (VI) in a Suzuki coupling reaction.
Figure imgf000059_0001
(V)
Figure imgf000060_0001
(VI )
A compound of formula (V) can be prepared by the scheme outlined below.
It should be understood that protecting groups can be utilized in accordance with general principles of synthetic organic chemistry to provide compounds of the present invention. Protecting group- forming reagents, like benzyl chloroformate and trichloroethyl chloroformate, are well known to persons skilled in the art, for example, see T.W. Greene et al . , "Protective Groups in Organic Synthesis, Third Edition," John Wiley and Sons, Inc., NY, NY (1999) . These protecting groups are removed when necessary by appropriate basic, acidic, or hydro- genolytic conditions known to persons skilled in the art. Accordingly, compounds of the present invention not specifically exemplified herein can be prepared by persons skilled in the art.
In addition, a compound of the present invention can be converted to another compound of the present invention. Thus, for example, a particular R substituent can be interconverted to prepare another suitably substituted compound of the present invention. Examples of appropriate inter- conversions include, but are not limited to, OR to hydroxy by suitable means (e.g., using an agent such as BBr3 or a palladium catalyst, like palladium-on- carbon, with hydrogen) , or amino to substituted amino, such as acylamino or sulphonylamino, using standard acylating or sulfonylating conditions.
Compounds of the present invention can be prepared by the method above as individual stereoisomers or as a racemic mixture. Individual stereo- isomers of the compounds of the invention can be prepared from racemates by resolution using methods known in the art for the separation of racemic mixtures into their constituent stereoisomers, for example, using HPLC on a chiral column, such as Hypersil naphthyl urea, or using separation of salts of stereoisomers. Compounds of the invention can be isolated in association with solvent molecules by crystallization from, or evaporation of, an appropriate solvent . The pharmaceutically acceptable acid addition salts of the compounds of the present invention that contain a basic center can be prepared in a conventional manner. For example, a solution of the free base can be treated with a suitable acid, either neat or in a suitable solution, and the resulting salt isolated either by filtration or by evaporation under vacuum of the reaction solvent. Pharmaceutically acceptable base addition salts can be obtained in an analogous manner by treating a solution of a compound of the present invention with a suitable base. Both types of salt can be formed or interconverted using ion-exchange resin techniques. Thus, according to a further aspect of the invention, a method for preparing a compound of the present invention or a salt or solvate (e.g., hydrate) is provided, followed by (i) salt formation, or (ii) solvate (e.g., hydrate) formation. The following additional abbreviations are used hereafter in the accompanying examples : HPLC (high-performance liquid chromatography) , TLC (thin layer chromatography) , rt (room temperature) , min (minute), h (hour), g (gram), mmol (millimole) , m.p. (melting point) , eq (equivalents) , L (liter) , mL
(milliliter) , μL (microliter) , saturated (sat.), mol (mole) , amu (atomic mass unit) , nm (nanometer) , UV (ultraviolet), cone, (concentrated), H20 (water), DMSO (dimethyl sulfoxide) , CH2C12 (dichloromethane) , KHMDS (potassium bis-trimethylsilyl amide) , IPA (isopropyl alcohol) , TFA (trifluoroacetic acid) , EtOH (ethanol) , MeOH (methanol) , Me (methyl) , DMF (dimethylformamide) , CHC13 (chloroform) , NaOH (sodium hydroxide) , EtOAc (ethyl acetate) , MgS04 (magnesium sulfate) , NaHC03 (sodium bicarbonate) , Et3N (triethylamine) , CuBr (cuprous bromide), Cu(0Ac)2 (cupric acetate) , K2C03 (potassium carbonate) , NaHS203 (sodium hydrogen thiosulfate) , KMn04 (potassium permanag- anate) , N2 (nitrogen gas) , HC1 (hydrochloric acid) , KOAc (potassium acetate) , HBr (hydrobromic acid) , Br2 (bromine) , H2S04 (sulfuric acid) , SOCl, (thionyl chloride) , TEA (triethanolamine) , NaHS04 (sodium hydrogen sulfate), NaN02 (sodium nitrite), Pd(OAc)2 (palladium acetate) , CC14 (carbon tetrachloride) , AcOH (acetic acid) , and THF (tetrahydrofuran) .
All following compounds, unless otherwise stated, were purified using standard chromatographic techniques and analyzed by HPLC utilizing a YMC (ODS-A column) . The flow rate through the column was 3.5 ml/minute. About 100 μl/min were introduced into the mass spectrometer. The mass scan ranged from 200 to 700 amu. The purity of the samples was determined through a UV detector (wavelengths: 220 and 254 nm) . The gradient commenced with 90% of A (0.05% TFA in water) and 10% of B (0.035% of TFA in acetonitrile) and linearly ramped to 90% of mobile phase B over a 5-minute period (Analytical Method 2) or a 3.5-minute period (Rapid Method 2) .
GENERAL SYNTHESES AND PREPARATION OF EXAMPLES
Preparation of Sulfide/Sulfone Amides
Amides were prepared by saponification of an appropriate ester with sodium hydroxide followed by reaction of the resulting carboxylic acid with a suitable amine.
Figure imgf000063_0001
Preparation of Example 1
Figure imgf000063_0002
The sulfone methyl ester (3 g, 9.5 mmol) was dissolved in 150 mL dioxane and 10 mL water and cooled to 0°C. Sodium hydroxide (14.3 mL, IN, 1.5 eq) was added dropwise. The resulting solution was stirred at 0°C for 2.5h, at which time the reaction was judged complete by TLC. The solution was neutralized and concentrated to 50 mL, added to 350 mL EtOAc, and acidified with 10% NaHS04. Then the organic layer was extracted with 300mL EtOAc . The combined organic layers were washed with brine, dried over MgS04, and evaporated to give the pure acid as a slightly yellow solid (2.63 g, 92%) .
Figure imgf000064_0001
Example 1
The acid (0.045 mmol) was dissolved in DMF
(250 mL) with pyridine (5.5 mL, 1.5 eq.) and penta- fluorophenyl trifluoroacetate (9.3 mL, 1.2 eq) , then stirred for 45 minutes at room temperature. To this solution was added the thiosemicarbazide dissolved in 750 μL of DMF, and the resulting mixture was stirred overnight at room temperature. The product was purified by reverse phase HPLC. (Yield 4 mg, 86% purity) . Preparation of Example 2 (Dioxobenzothiazinones)
Figure imgf000065_0001
7 -Bromo-2 -methyl-4H-benzo [1,4] thiazin-3-one
In a 250 ml flask charged with a stir bar was added 2-amino-6-bromobenzothiazole (5.0 g, 21.8 mmol) , 75 ml NaOH (20% w/w) solution, and 10 ml of 2 -methoxyethanol . The flask was fitted with a condenser and heated at 110 °C for 16 hours under a nitrogen atmosphere. The reaction mixture then was cooled to 80°C, upon which 2-bromopropionic acid (5.15 g, 26.26 mmol) in 20 ml (10% aqueous NaOH solution) was added. The resulting mixture then was stirred at 80°C. After one hour, the reaction mixture was concentrated under reduced pressure, and reconstituted in AcOH until the pH was acidic. The reaction then was heated to 110 °C and stirred for 4h. The reaction mixture was allowed to cool, and the acetic acid was removed under reduced pressure. The concentrated product was partitioned between methylene chloride and water, extracted four times, dried over magnesium sulfate, filtered, and concen- trated in vacuo to an off-white solid. The solid was triturated with a small amount of ethyl acetate and filtered. Four grams of pure product was recovered as a white solid.
Figure imgf000066_0001
7-Bromo-2-methyl-l, 1-dioxo-l, 4-dihydro-2H-lλs- benzo [1,4] thiazin-3 -one
7-Bromo-2-methyl-4H-benzo [1,4] thiazin-3 - one (1.2 g, 4.14 mmol) was suspended in 250 ml of acetic acid and cooled to 0°C. Twenty-seven milli- liters of a KMn04 (1.0 g, 5.8 mmol) solution (0.42 mmol/2 ml H20) then was added dropwise and the reaction was allowed to warm to room temperature over two hours. Sodium hydrogen sulfite then was added to the reaction mixture until reaction turned transparent. The mixture was concentrated in vacuo, partitioned between methyl chloride and water, extracted four times, dried over magnesium sulfate, filtered, and concentrated down to an off-white solid. The product was purified by trituration in ethyl acetate/hexanes to provide 550 mg of desired product as a white solid.
Figure imgf000066_0002
2-Methyl-7-napthalen-l-yl-l, 1-dioxo-l, 4-dihydro-2H- lλ6-benzo [1,4] thiazin-3-one
To a tube charged with of 7-bromo-2- methyl-1, 1-dioxo-l, 4-dihydro-2H-lλs-benzo [1,4] - thiazin-3-one (25.0 mg, 0.0862 mmol), Pd(0Ac)2 (1.1 mg, 0.0051 mmol), triphenylphosphine polymer supported (2.6 mg, 0.0077 mmol), and K2C03 (23.8 mg, 0.172 mmol) in 500 ml of H20 was added 1-napthyl boronic acid in 2 ml of DMF. The tube was sealed with a cap, then heated at 85°C for 3 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac to give Example 2.
Preparation of Example 3 (Thiazoles)
Figure imgf000067_0001
The nitrile ester (10 g, 56.5 mmol) was dissolved in 20 mL MeOH and cooled to -10°C. Hydro- gen sulfide gas was bubbled through the solution for 45 min. DMAP (0.07 eq) was added and the reaction mixture was sealed and heated overnight at 50 °C. The solvents were removed and the crude product was used without further purification.
Figure imgf000067_0002
The resulting thioamide (1 mmol) was combined with the appropriate bromoketone (1 mmol) in 1 mL 50% DMF/MeOH and heated at 50°C for lh.
Figure imgf000068_0001
Example 3
Sodium hydroxide (1.5 mL, IN) was added, then the reaction mixture was stirred for lh at room temperature. The mixture was concentrated, acidi- fied with 10% NaHS04, and extracted with EtOAc.
After drying the organic layer, the crude product was used without further purification. The thiazcle acid (0.1 mmol) was dissolved in DMF (200 mL) with pyridine (1.5 eq) and pentafluorophenyl trifluoro- acetate (1.2 eq) , then stirred for 45 min. at room temperature. The amine was dissolved in 300 mL DMF and added to the acid solution. The mixture was stirred overnight at room temperature. The product was purified by reverse phase HPLC (yield 4.3 mg, 94% purity) .
Preparation of 4-bromophenylmethanesulfonyl-N- hydroxyethyl acetamide
1st step :
»o0^ H.N
OH HS of OH
Methyl thioglycolate (95%, Aldrich) (200 g, 1.88 mol) was placed in a 500 mL roundbottom flask. Ethanolamine (99.5%, Aldrich) (109 g, 1.79 mol) was added. Magnetic stirring was initiated with a vent in place because the reaction is exo- thermic. A vacuum was slowly applied to distill off the methanol. The reaction was maintained under a high vacuum with magnetic stirring for 6 hours to overnight to yield N- (hydroxyethyl) -2 -mercapto acetamide as a clear, highly viscous material that was used without further purification.
2nd step :
JX " ♦
Figure imgf000069_0001
Γ" 0H
N- (hydroxyethyl) -2 -mercapto acetamide
(35.1 g, 260 mmol) and 4-bromobenzyl bromide (50.0 g, 200 mmol) were combined in DMF (500 ml) with K2C03 (55 g, 400 mmol) , and stirred for 12h at room temperature under N2. The DMF was removed in vacuo, and the compound was partitioned between EtOAc and H20. The ethyl acetate layer was washed with H20 and brine. The organics were dried over MgS04, filtered and concentrated to a white solid. The white solid was redissolved/suspended in CH2C12 and stirred for lhr. Hexanes then were added to precipitate the desired product as a white solid. The product then was filtered, washed with hexanes, and dried overnight in vacuo . Pure desired product (4-bromobenzyl sulfanyl-N-hydroxyethyl acetamide) was recovered (46.8 g, 77% yield) .
Figure imgf000070_0001
3rd step:
4-Bromobenzyl sulfanyl-N-hydroxyethyl acetamide (22.9 g, 75.3 mmol) was dissolved in MeOH (50 ml) . Acetone (150 ml) was added, then the reaction mixture was placed in an ice bath. To this reaction mixture, oxone (115.5 g, 188.25 mmol) in 577 ml of H20 was added dropwise over 30 minutes. After addition of the oxone solution was complete, a thick white slurry formed and the reaction mixture was allowed to warm to room temperature and stir for 22h. Water (300 ml) then was added, and the mixture was stirred for lh. The slurry then was filtered and the solid was washed with 3x50 ml water. The resulting white powder was dried in vacuo to remove the H20. The resulting powder was stirred in hot MeOH for 15 minutes. The mixture was filtered hot, and mother liquor was concentrated in vacuo to yield the product (4-bromophenylmethanesulfony1-N-hydroxy- ethyl acetamide) as flat shiny white crystals (21.3 g, 85% yield) .
Figure imgf000071_0001
General Procedure:
Suzuki Coupling; (High Throughput)
4-Bromophenylmethanesulfonyl-N-hydroxy- ethyl acetamide (50 mg, 0.15 mmol), palladium acetate (1 mg, 0.0046 mmol), triphenylphosphine attached to solid support (3 mmol/g) (4.5 mg, 0.0135 mmol), and arylboronic acid (0.15 mmol) were added to a mixture of DMF (1.5 ml) and 2M potassium carbonate (0.5 ml). The mixture was heated at 80°C for 12 hours, then filtered and purified by reverse phase HPLC to yield the desired biphenyl product. Example 4 was prepared using 3 , 4 , 5-trimethoxyphenyl boronic acid.
Figure imgf000071_0002
Example 4 Preparation of Example 5 (Biphenylsulfonamides)
Figure imgf000072_0001
2,2, 2 -Trifluoroethanesulfonic acid (4 -bromophenyl) amide
4-Bromoaniline (1.0 g, 5.81 mmol) was dissolved in 25 ml dry methylene chloride, followed by the addition of 1.4 ml of pyridine. The reaction solution then was cooled to 0°C upon which (0.71 ml, 6.39 mmol) of 2 , 2 , 2-trifluoroethanesulfonyl chloride was added dropwise. The reaction was complete after one hour at 0°C. The solvent was removed under re- duced pressure, and the residue was partitioned between ethyl acetate and water, extracted two times, dried over magnesium sulfate, filtered, and concentrated to dryness to yield 1.88 g of product.
Figure imgf000072_0002
(4-Bromophenylsulfamoyl) acetic acid
2 , 2 , 2-Trifluoroethanesulfonic acid (4- bromophenyl) amide (1.88 g, 5.91 mmol) was dissolved in 12 ml of dioxane followed by the addition of 60 ml of a IM NaOH solution. The reaction was complete after stirring at room temperature for 16 hours. The solution then was cooled to 0°C, and 25 ml of methylene chloride was added followed by the drop- wise addition of concentrated aqueous HCl until the pH of the solution was acidic. The product was extracted with methylene chloride, dried over magnesium sulfate, and concentrated to an orange solid. The product was used without further purification.
Figure imgf000073_0001
2- (4-Bromophenylsulfamoyl) -N- (2 -hydroxyethyl) acetamide
(4-Bromophenylsulfamoyl) acetic acid (1.83 g, 6.22 mmol) was dissolved in dry dioxane, followed by the addition of pyridine (1.3 ml, 18.66 mmol), and (2.1 g, 7.46 mmol) of trifluoroacetic acid pentafluorophenyl ester. The reaction mixture was allowed to stir for thirty minutes, upon which ethanolamine (1.12 ml, 18.66 mmol) was added. The reaction was complete after two hours. The solvent was removed under reduced pressure, and the product was purified by flash chromatography using an ethyl acetate/hexanes gradient from 10% to 70%. Purified product (2.1 g) was recovered.
Figure imgf000074_0001
Example 5
N- (2 -Hydroxyethyl)-2-(3',4',5l-trimethoxybiphenyl-4 - ylsulfamoyl) acetamide
To a tube charged with 2- (4 -bromophenyl - sulfamoyl ) -N- (2 -hydroxyethyl) acetamide (70.0 mg, 0.0208), Pd(0Ac)2 (2.8 mg, 0.0124 mmol), triphenyl- phosphine polymer supported (6.24 mg, 0.0187 mmol), and K2C03 (57.4 mg, 0.416 mmol) in 500 ml of H20 was added 3 , 4 , 5-trimethoxyboronic acid (49 mg, 0.23 mmol) in 2 ml of DMF. The tube was sealed with a cap and heated at 85°C for 3 hours. The reaction mixture then was syringe filtered, purified on re- verse phase HPLC, and concentrated to dryness via Speed Vac .
Preparation of Aryl/Heteroaryl Substituted Derivatives
2 -Fluorophenyl Substituted
Step 1
Figure imgf000075_0001
4-Bromo-2-fluorobenzyl bromide (5 g, 18.7 mmol) was dissolved in DMF (25 ml) . Potassium carbonate (5.15 g, 37.3 mmol) and methyl thiogly- colate (1.68 ml, 18.7 mmol) were added, and the mixture was stirred at room temperature for 12 hours . The DMF was removed in vacuo and the compound was partitioned between EtOAc and H20. The EtOAc layer was washed with H20 and brine. The organics were dried over MgS04 , filtered, and concentrated to a yellow oil that was used without further purification.
Step 2 :
Figure imgf000075_0002
The sulfide (18.7 mmol) obtained in Step 1 was dissolved in acetic acid (80 ml) . Potassium permanganate (5.9 mg, 37.4 mmol) dissolved in water (100 ml) was added dropwise over a period of 30 minutes, and the reaction was stirred at 25°C for an additional 2 hours. To the purple solution, NaHS203 was added to remove the color, and water (70 ml) was added until the product precipitated as a white solid that was filtered and dried under vacuum to yield the pure sulfone.
Step 3
Figure imgf000076_0001
The ester prepared in Step 2 (18.7 mmol) was dissolved in dioxane (50 ml) . Sodium hydroxide solution (IN, 50 ml) was added to the dioxane solution and the mixture was stirred at 25°C for 1 hour. The mixture was acidified with cone. HCl and extracted with ethyl acetate (3 x 50 ml) . The organics were dried over MgS04, filtered, and concentrated to a white solid that was used without further purification. Step 4
Figure imgf000077_0001
The carboxylic acid obtained in Step 3
(18.7 mmol) was dissolved in 20 ml of dried CH2C12 and 5 ml of pyridine. Pentafluorophenol trifluoroacetate (4.82 ml, 28.05 mmol) was added, and the reaction was stirred for 1 hour at 25°C. Ethanol- amine (3.4 ml, 56.1 mmol) was added, and stirring was continued for an additional 12 hours. The mixture was diluted with water (50 ml) and extracted with CH2C12 (3 x 50 ml) . The organics were dried over MgS04, filtered, and concentrated. The mixture was purified by column chromatography (ethyl ace- tate/hexane) to yield 1.5 g of the aryl bromide as a white solid (yield 23% for 4 steps) .
Step 5
Figure imgf000077_0002
Example 6 4 -Bromo-2 - fluorophenylmethanesulfonyl-N- hydroxyethyl acetamide (50 mg, 0.141 mmol), palladium acetate (1 mg, 0.0046 mmol), triphenylphosphine attached to solid support (3 mmol/g) (4.5 mg, 0.0135 mmol), and trimethoxyphenyl boronic acid (29.9 mg, 0.141 mmol) were added to a mixture of DMF (1.5 ml) and 2M potassium carbonate (0.5 ml) . The mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 6 (16 mg, 97% purity) .
Other Heterocvcles
The synthetic route from 5-bromo-2-thio- phenecarboxaldehyde to 5-bromothienylmethanesulfonyl -N-hydroxyethyl acetamide also was used to prepare the thiophene, furan, and pyridine heterocycles, with the exception that furans used a modified version of Step 2. The starting material for the pyridine example was 2-chloro-5-methylcarboxypyri- dine .
Step 1:
Figure imgf000078_0001
To a suspension of sodium borohydride (992 mg, 26.2 mmol) in THF (25 ml) at 0°C was added a solution of 5-bromo-2-thiophenecarboxaldehyde (5 g, 26.2 mmol) dropwise. The reaction was stirred at 25 °C for 2 hours then poured over water (25 ml) and diluted with ether. The two layers were separated, and the aqueous layer extracted with ether (2 x 30 ml) . The combined ether solutions were dried with MgS04, filtered, and evaporated to give a dark oil that was used without further purification.
Step 2:
Figure imgf000079_0001
Phosphorus tribromide (1.25 ml, 13.1 mmol) was added dropwise to a solution of 2-bromo-5-hy- droxymethylthiophene from Step 1 in dichloromethane (52.4 ml) below 10°C. The solution was stirred for 2h at 20°C, then washed with ice-water, dried, and evaporated to give a white solid that was used without further purification.
Substituted furans were iodinated according to the following protocol :
Furfuryl alcohol (1 g, 5.71 mmole) was added to a solution of methyl triphenoxyphosphonium iodide (3.1 g, 6.85 mmole) in DMF (20 mL) , and allowed to react under nitrogen for 30 minutes at room temperature. This solution was used in step 3 without further work up. Step 3
Figure imgf000080_0001
N- (hydroxyethyl) -2 -mercapto acetamide (5.3 g, 39.3 mmol) and 2-bromo-5-bromomethylthiophene (26.2 mmol) from Step 2 were combined in DMF (30 ml) with K2C03 (10.8 g, 78.6 mmol), and stirred for 12h at room temperature under N2. The DMF was removed in vacuo, and the compound was partitioned between EtOAc and H20. The EtOAc layer was washed with H20 and brine. The organics were dried over MgS04, filtered, and concentrated to a yellow oil that was used without further purification.
Step 4 :
Figure imgf000080_0002
The compound obtained in Step 3 was dissolved in acetic acid (53 ml) and peroxide (30% in water, 78.6 mmol, 9 g) , then the solution was heated at 80°C for 2 hours. The solvent was removed in vacuo and the solid residue was triturated with ethyl acetate, water, and ether to give 1.6 g (yield 18%, 4 steps) of the final pure product. These intermediates were used in the general Suzuki reaction described above to prepare the following Examples 7 to 11.
Figure imgf000081_0001
Example 7
Figure imgf000081_0002
Example 8
CH,
Figure imgf000081_0003
Example 9
Example 10
Figure imgf000082_0002
Example 11
Preparation of Example 12 (Substituted Oxadiazoles)
1st step:
Figure imgf000082_0003
The mixture of 4- (trifluoromethyl) benz- amidoxime (135 mg, 0.66 mmol) and chloroacetyl chloride (1 ml) was refluxed for 30 minutes. The excess acid chloride was removed, and the residue was dissolved in ethyl acetate and treated with sodium carbonate solution. The organics were dried over MgS04, filtered, and concentrated to a solid residue that was used without further purification.
2nd step:
Figure imgf000083_0001
N- (hydroxyethyl) -2 -mercapto acetamide (134 mg, 0.99mmol) and chloromethyl oxadiazole prepared in Step 1 (0.66 mmol) were combined with DMF (2 ml) and K2C03 (274 mg, 1.98 mmol), and stirred for 12h at room temperature under N2. The DMF was removed in vacuo, and the compound was partitioned between EtOAc and H20. The EtOAc layer was washed with H20 and brine. The organics were dried over MgS04, filtered, and concentrated to a residue that was carried to next step without purification.
3rd step:
Figure imgf000083_0002
Example 12 The sulfide prepared in Step 2 (0.66 mmol) was dissolved in acetic acid (1.3 ml) . Water peroxide (30%, 255 μl, 1.98 mmol) was added, and the mixture was heated at 80°C for 2 hours. The solvent was evaporated, the sample dissolved in DMSO (2 ml), and purified by reverse phase chromatography to yield Example 12 (4.7 g, 100% purity).
Preparation of Example 13
1st step: See, V.N. Yazovenko et al . , Tetrahedron, 46 (11 ) , pp. 3941-3952 (1990).
Sodium carbonate (5.3 g, 50 mmol) was added to a solution of hydroxylamine hydrochloride
(6.9 g, 99.29 mmol) in 25 ml of water. Then chloro- acetonitrile (7.5g, 99.3 ml) was added over 15 minutes at 30°C. After completion of the reaction, the mixture was extracted with ether and dried over MgS04. Removal of the solvent yielded 6.5g of a brown oil that was used without purification.
Step 2 :
Figure imgf000084_0001
A mixture of chloroacetamidoxime (100 mg, 0.92 mmol) and 3 , 4-dimethoxybenzoyl chloride (185 mg, 0.92 mmol) was heated with a heatgun for 10 minutes. The residue was dissolved in ethyl acetate, and treated with sodium carbonate solution. The organics were dried over MgS04, filtered, and concentrated to a solid residue that was used without further purification.
Step 3 :
Figure imgf000085_0001
N- (hydroxyethyl) -2-mercaptoacetamide (187 mg, 1.38 mmol) and chloromethyl oxadiazole prepared in step 2 (0.92 mmol) were combined with DMF (2 ml) and K2C03 (381 mg, 2.76 mmol) , and stirred for 12h at room temperature . The DMF was removed in vacuo and the compound was partitioned between EtOAc and H20. The EtOAc layer was washed with H20 and brine. The organics were dried over MgS04, filtered, and concentrated to a residue that was used without further purification.
Step 4 :
Figure imgf000085_0002
Example 13
The sulfide prepared in Step 3 (0.92 mmol) was dissolved in acetic acid (1.84 ml) . Water per- oxide (30%, 313 μl, 2.76 mmol) was added and the mixture was heated at 80°C for 2 hours. The solvent was evaporated, the sample dissolved in DMSO (2 ml) , and purified by reverse phase chromatography to yield Example 13 (6.8 mg, 95% purity) .
Reverse Suzuki Chemistry
Preparation of N- (2 -Hydroxyethyl) -2- [4- (4, 4, 5, 5- tetramethyl- [1, 3, 2] dioxaborolan-2-yl) -phenylmethane sulfonyl] acetamide
Figure imgf000086_0001
A flask charged with PdCl2 (dppf) (0.874g, 1.07mmol), KOAc (10.51g, 107.13mmol), bis (pina- colato) diboron (10.88g, 42.85mmol), and 4-bromo- phenylmethanesufonyl-N-hydroxyethyl acetamide
(12. Og, 35.71mmol) was flushed with nitrogen. DMF (200 ml) was added, and the reaction mixture was heated for 16 hours at 80 °C. The reaction mixture was concentrated in vacuo, reconstituted in 100 ml of H20, and filtered through fluted filter paper. The filtrate then was extracted with EtOAc four times. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to a viscous dark yellow oil. The product was triturated with EtOAc/hexanes to provide
7.0 g of pure material as a white solid. General Procedure for Reverse Suzuki Reaction
To a tube charged with Pd(OAc)2 (0.06 eq) , triphenylphosphine polymer supported (3mmol P/g resin) (0.09 eq) , and aryl bromide (1.0 eq) was added N- (2 -hydroxyethyl) -2- [4- (4,4,5, 5-tetramethyl - [1,3,2] dioxaborolan-2-yl)phenylmethanesulfonyl] - acetamide (1.0 eq) in 1.6 ml of DMF, followed by the addition of K2C03 (2.0 eq) in 400 ml of H20. The tube was sealed with a cap, then heated at 85°C for 3 hours. The reaction next was syringe filtered, purified via reverse phase HPLC, and concentrated to dryness via Speed-Vac.
Figure imgf000087_0001
Example 14 was prepared by this method from 8-hydroxy-2-bromonaphthalene :
Figure imgf000087_0002
Example 14 Preparation of Monomers and of Compounds Using the Reverse Suzuki Method
Preparation of 3-Bromo-6-methoxyphenol
To 6.0 g (43.2 mmol) of 3 -amino- 6 -methoxy- phenol in 36 ml of H2S04/l8ml MeOH/60 ml H20 at 0°C was added 3.3 g (47.5 mmol) of NaN02 in 25 ml of H20 over 30 min. The reaction mixture was allowed to stir 45 min., and 3.5 g (12.1 mmol) of Cu2Br2 in 60 ml H20/12 ml of HBr was added over 20 min. The mixture was warmed to reflux for 1.5h, cooled to ambi- ent temperature, extracted with four portions of ether, dried over MgS04, filtered, and concentrated in vacuo . Column chromatography (10% EtOAc/hexanes) provided 3.3 g as a white solid.
Figure imgf000088_0001
4-Methoxy-3 - (4-methyl) benzyloxybromobenzene
To a tube charged with a stir bar was added of 5-bromo-2-methoxyphenol (150 mg, 0.739 mmol), 4-methylbenzyl bromide (410 mg, 2.22 mmol), and potassium carbonate (203mg, 1.48mmol), and suspended in 2 ml dry acetone. The tube was sealed with a cap, then heated to reflux for 20h. The re- action mixture then was filtered, and concentrated in vacuo . The crude product was used without further purification.
Preparation of Example 15
To a tube charged with of N- (2 -hydroxyethyl) -2- [4- (4,4,5, 5 -tetramethyl- [1 , 3 , 2] dioxabor- olan-2-yl) phenylmethanesulfonyl] acetamide (282 mg, 0.739 mmol), Pd(OAc)2 (9.9 mg, 0.044 mmol), triphen- ylphosphine polymer supported (22.2 mg, 0.066 mmol), and K2C03 (203 mg, 1.48 mmol) in 500 ml of H20 was added 5-bromo-2-methoxyphenoxy benzyl in 2 ml of DMF. The tube was sealed with a cap, then heated at 85 °C for 3 hours. The reaction mixture was then syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac.
Figure imgf000089_0001
Preparation of 5 -Bromo- 2 , 3 -dimethoxybenzaldehyde
Figure imgf000090_0001
Sodium bicarbonate (10.26 g, 122.2 mmol) was added to a stirring solution of 2 , 3-dimethoxybenzaldehyde (20 g, 120.4 mmol) in 85.5 ml dry CC14. This suspension was cooled in an ice bath during the addition of 6.3 ml of bromine over a lh period. The reaction was stirred for four hours, then allowed to stir for 15h at ambient temperature. The reaction mixture was washed twice with dilute sodium bisufite solution and once with water. The solvent was evaporated under reduced pressure, and the residue was recrystallized from 30 ml of a 3:1 ligroin (90°C- 120°C) /benzene mixture, to yield 12.0 g of pure product as a white solid.
Preparation of 5-Bromo-2.3-dimethoxybenzoic acid
Sodium hydroxide (2.3 g, 57.0 mmol) in 25 ml H20 was slowly added to a suspension of 5-bromo- 2 , 3-dimethoxybenzaldehyde (2.9 g, 12.0 mmol) in a solution of silver nitrate (4.8 g, 28.25 mmol) in 50 ml of H20. The reaction then was heated to reflux for lh, filtered, and the filtrate was acidified with cone. HCl. The product was recrystallyzed from H20 to yield 2.04 g of pure material as a white solid.
Figure imgf000091_0001
4 i _ [ (2-Hydroxyethylcarbamoyl)methanesulfonylmethyl] ■ 4, 5-dimethoxybiphenyl-3 -carboxylic acid benzylamide (Example 16)
Figure imgf000091_0002
Preparation of N-Benzyl-5-bromo-2, 3- di ethoxybenzamide
5-Bromo-2 , 3 -dimethoxybenzoic acid (100 mg,
0.383 mmol) was dissolved in 1ml dry CH2C12, followed by the addition of 3 ml dry DMF, 2 ml of S0C12 (2.0M in CH2C12) was added and the reaction mixture was allowed to stir for lh at room temperature under a nitrogen atmosphere. The reaction mixture then was concentrated under reduced pressure and coevaporated three times with THF. The acid chloride residue then was reconstituted in 2 ml dry dioxane and added to a solution of benzylamine (205 mg, 1.92 mmol) in 160 ml of TEA. After 1 hour, the reaction was com- plete by LC/MS . The reaction mixture was syringe filtered to remove HCl salts, and concentrated in vacuo .
Preparation of Example 16
To a tube charged with N- (2 -hydroxyethyl) - 2- [4- (4,4,5, 5-tetramethyl- [1, 3 , 2] dioxaborolan-2-yl) - phenylmethanesulfonyl] acetamide (147 mg, 0.383 mmol), Pd(0Ac)2 (5.1 mg, 0.023 mmol), triphenylphos- phine polymer supported (11.5 mg, 0.0344 mmol), and K2C03 (106 mg, 0.766 mmol) in 500ml of H20 was added N-benzyl-5-bromo-2 , 3 -dimethoxybenzamide in 2 ml of DMF. The tube was sealed with a cap, then heated at 85 °C for 3 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac.
Preparation of 5-Bromo-2 , 3-dimethoxybenzyl alcohol
Figure imgf000092_0001
5 -Bromo-2 , 3-dimethoxybenzaldehyde (1.5 g, 6.12 mmol) was added to a suspension of sodium boro- hydride (0.232g, 6.12mmol) in 25 ml dry THF. After 1 hour, TLC and LC/MS confirmed complete conversion to the alcohol. The reaction was quenched over cold water and extracted four times with EtOAc. The organics were dried over MgS04, filtered, and concentrated in vacuo to yield 1.3g as a white solid.
Preparation of Example 17
Figure imgf000093_0001
Example 17
5-Bromo-2 , 3-dimethoxybenzyl alcohol (85 mg, 0.344 mmol) in 1.5 ml dry DMF was added to freshly washed sodium hydride (28 mg, 0.688 mmol) suspended in 1.0 ml dry DMF. After 1 hour, benzyl- bromide (118 mg, 0.688 mmol) was added to the re- action mixture, and allowed to stir for 2h, after which the reaction was quenched with 2 ml of water and extracted twice with EtOAc. The organics were combined, dried over MgS04, filtered, and concentrated in vacuo to a viscous oil. The crude mater- ial was 85% pure LC/MS (TIC, UV 220 nm and 254 nm) , and was used without further purification.
To a tube charged with N- (2 -hydroxyethyl) 2- [4- (4, 4, 5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) phenylmethanesulfonyl] acetamide (132 mg, 0.344 mmol), Pd(OAc)2 (5 mg, 0.021 mmol), and triphenyl- phosphine polymer supported (10.3 mg, 0.031 mmol) was added 5-bromo-2 , 3-dimethoxybenzyloxymethylben- zene in 2 ml DMF followed by the addition of (95 mg, 0.688 mmol) K2C03 in 400 ml H20. The tube was sealed with a cap, then heated for 3 hours at 85 °C. The reaction mixture then was syringe filtered, and purified on HPLC reverse phase.
Sulfone Analogs
Figure imgf000094_0001
Preparation of 5-Bromo-2-methoxybenzenesulfonyl fluoride '
5-Bromo-2-methoxybenzenesulfonyl chloride (3.83 g, 13.43 mmol) was dissolved in 38 ml dry acetonitrile followed by the addition of potassium fluoride (1.6 g, 26.86 mmol) and 18-crown-6 ether
(71 mg, 0.269 mmol) . The reaction was sealed with a septum and stirred under a nitrogen atmosphere at ambient temperature for 16 hours. The reaction mixture then was diluted with 120 ml of water to pre- cipitate the product. The product was collected by filtration and dried to yield 3.5 g of the product as a white solid.
Figure imgf000095_0001
3-Bromobenzenesulfonyl fluoride
3-Bromobenzenesulfonyl chloride (2.0 g,
7.84 mmol) was dissolved in 25 ml dry acetonitrile followed by the addition of potassium fluoride (909 mg, 15.68 mmol) and 18 -crown-6 ether (42 mg, 0.157 mmol) . The reaction was sealed with a septum, then stirred under a nitrogen atmosphere at ambient temperature for 16 hours. The reaction mixture then was diluted with 120 ml of water and extracted three times with EtOAc. The organics were combined, dried over anhydrous magnesium sulfate, filtered, and con- centrated in vacuo, to yield 1.6 g of product as a clear oil .
Preparation of Example 18
Figure imgf000095_0002
4-Bromo-2-cyclohexanesulfonyl-l-methoxybenzene
To a dry 25 ml round bottom flask equipped with a stir bar was added 5-bromo-2-methoxybenzene- sulfonyl fluoride (100 mg, 0.372 mmol). The flask was sealed with a septum and the contents dissolved in 7 ml of dry acetonitrile. A 2.0M solution (0.560 ml) of cyclohexane magnesium chloride was added dropwise to the stirring solution at ambient temper- ature under a nitrogen atmosphere. After 3 hours the reaction was complete, then quenched with 3 ml water and extracted three times with EtOAc. The organics were combined, dried over MgS04, filtered, and concentrated in vacuo to yield 36 mg of product as a yellow oil.
Preparation of 2- (3 ' -Cyclohexanesulfonyl-4 ' -methoxy- biphenyl-4-ylmethanesulfonyl) -N- (2 -hydroxyethyl) - acetamide (Example 18)
To a tube charged with (41.5 mg, 0.108 mmol) of N- (2 -hydroxyethyl) -2- [4- (4,4, 5, 5-tetrameth- yl- [1,3,2] dioxaborolan-2-yl) phenylmethanesulfonyl] acetamide (1.5 mg, 0.0065 mmol), Pd(0Ac)2 (3.2 mg, 0.0097 mmol), triphenylphosphine polymer supported (45 mg, 0.324 mmol), and K2C03 in 500 μl of H20 was added 4 -bromo-2-cyclohexanesulfonyl-1 -methoxybenzene in 2 ml of DMF. The tube was sealed with a cap and heated at 85°C for 3 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac. Secondary Alcohols from Griqnard Reagents
Preparation of Example 19
Figure imgf000097_0001
5-Bromo-2-methoxybenzaldehyde (100 mg, 0.465 mmol) was placed in a dry flask, sealed with a septum under a nitrogen atmosphere, and dissolved in 7 ml of dry THF. The reaction mixture was cooled to 0°C, and a solution (0.200 ml) of pentylmagnesium bromide (0.65 mmol, 3.0M) in Et20 as added dropwise. The reaction mixture was allowed to warm to room temperature, and after one hour, the reaction was complete. The mixture was quenched with a saturated solution of ammonium chloride and extracted three times with ethyl acetate. The organics were combined, dried over MgS04, filtered, and concentrated to dryness in vacuo . The desired product (99 mg) was obtained as a clear oil and used without further purification.
To a tube charged with of N- (2 -hydroxyethyl) -2- [4- (4,4,5, 5 -tetramethyl- [1,3,2] dioxaborolan-2- yl) phenylmethanesulfonyl] acetamide (165 mg, 0.429 mmol), Pd(0Ac)2 (1.5 mg, 0.0065 mmol), triphenyl- phosphine polymer supported (3.2 mg, 0.0097 mmol), and K,C03 (118.4 mg, 0.858 mmol) in 500 ml of H20 was added 1- (5-bromo-2-methoxyphenyl) ethanol (99 mg) in 2 ml of DMF. The tube was sealed with a cap, then heated at 85°C for 3 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac.
Preparation of 3- (2-Imidazolyl) -bromobenzenes
Figure imgf000098_0001
5-Bromo-2 , 3-dimethoxybenzaldehyde (100 mg, 0.408 mmol), ammonium acetate (314 mg, 4.08 mmol), and 2 , 3-butanedione (52 mg, 0.608 mmol) were added to a tube, dissolved in 3 ml of acetic acid, then heated to 100°C for 2h. The solvent was removed under reduced pressure, and the residue was coevap- orated three times with water to remove excess acetic acid. The residue was dried under reduced pressure and was used without any further purification.
Figure imgf000098_0002
Preparation of Example 20
To a tube charged with of N- (2 -hydroxyethyl )-2- [4 -(4, 4, 5, 5 -tetramethyl- [1,3,2] dioxaborolan-2 - yl) phenylmethanesulfonyl] acetamide (156 mg, 0.408 mmol), Pd(0Ac)2 (1.5 mg, 0.0065 mmol), triphenyl- phosphine polymer supported (3.2 mg, 0.0097 mmol), and K2C03 (113 mg, 0.816 mmol) in 500 ml of H20 was added 2- (5-bromo-2 , 3-dimethoxyphenyl) -4 , 5 -dimethyl - lH-imidazole (127 mg, 0.408 mmol) in 2 ml of DMF. The tube was sealed with a cap, then heated at 85°C for 3 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac .
Analogs Via N-Arylation
Figure imgf000099_0001
Figure imgf000099_0002
Example 21
Preparation of Example 21
To a tube charged with N- (2 -hydroxyethyl) 2- [4 -(4, 4, 5, 5 -tetramethyl- [1,3,2] dioxaborolan-2 -yl) phenylmethanesulfonyl] acetamide (50 mg, 0.131 mmol), 4-phenylimidazole (19 mg, 0.131 mmol), Cu(0Ac)2 (36 mg, 0.197 mmol), and molecular seives, was added 2 ml DMF and 21 ml of pyridine. The tube was loosely sealed with a cap, then heated at 85°C for 16 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac.
The following Example 22 was prepared using a similar synthetic sequence.
Figure imgf000100_0001
Example 22
4-Cyano Analogs
Preparation of 4-Bromo-2-bromomethylbenzonitrile
4-Bromo-2-methylbenzonitrile (5.0 g, 25.5 mmol) was dissolved in 90 ml CC14. To this solution was added dibenzoyl peroxide (433 mg, 1.79 mmol) and NBS (4.53 g, 25.5 mmol) . The reaction vessel was fitted with a condenser, and the reaction mixture was heated to reflux under a nitrogen atmosphere for 2.5 hours. The reaction mixture then was filtered hot and concentrated in vacuo to a thick yellow oil. The product was purified via column chromatography using a gradient of 2% EtOAc/98% hexanes to yield 4.8 g of pure product as a white solid.
Figure imgf000101_0001
Preparation of 4-Bromo-2-bromomethyl-l- chlorobenzonitrile
4-Bromo-l-chloro-2-methylbenzene (5.0 g, 24.4 mmol) was dissolved in 90 ml CC14. To this solution was added dibenzoyl peroxide (414 mg, 1.71 mmol) and NBS (4.3 g, 24.4 mmol) . The reaction vessel was fitted with a condenser, and the reaction mixture heated to reflux under a nitrogen atmosphere for 2.5 hours. The reaction mixture then was filtered hot and concentrated in vacuo to a thick pale oil . The product was purified via column chromatography using a gradient of 2% EtOAc/98% hexanes to yield 5.0 g of pure product as a white solid.
Figure imgf000101_0002
Preparation of 2-Benzyloxymethyl-4-bromobenzonitrile
To a tube charged with a stir bar was added a solution of 4-bromo-2-bromomethylbenzo- nitrile (120 mg, 0.436 mmol) in 2 ml dry THF, followed by the addition of benzyl alcohol (99 ml, 0.96 mmol) . The reaction mixture was cooled to 0°C upon which KHMDS (2.21 ml, 1.13 mmol) was added over a 30-minute period. The reaction mixture then was allowed to warm to room temperature. After 2 hours, the reaction was quenched with saturated aqueous ammonium chloride, and extracted three times with EtOAc. The organics were combined and dried over MgS04, filtered, and concentrated in vacuo to a dark oil used without further purification.
Figure imgf000102_0001
Preparation of 2- (3 ' -Benzyloxymethyl-4 ' -cyano- biphenyl-4-ylmethanesulfonyl) -N- (2 -hydroxyethyl) acetamide (Example 23)
To a tube charged with N- (2 -hydroxyethyl] 2- [4- (4,4,5, 5 -tetramethyl- [1,3,2] dioxaborolan-2 - yl) henylmethanesulfonyl] acetamide (192 mg, 0.500 mmol), Pd(OAc)2 (6.7 mg. 0.03 mmol), triphenylphos- phine polymer supported (15 mg, 0.045 mmol), and K2C03 (138 mg, 1.00 mmol) in 500 μl of H20 was added 2-benzyloxymethyl-4-bromobenzonitrile in 2 ml of DMF. The tube was sealed with a cap, then heated at 85°C for 3 hours. The reaction mixture then was syringe filtered, purified on reverse phase HPLC, and concentrated to dryness via Speed Vac.
Preparation of Example 24
Step 1: Chem . Pharm . Bull , 35 (2 ) , pp. 660-667 (1987)
Figure imgf000103_0002
A formic acid solution (6 ml) containing 5-bromo-2, 3-dimethoxybenzaldehyde (1 g, 4 mmol) was added to performic acid (HCOOOH) prepared from 85% formic acid (HCOOH) (2.6 ml) and 30% H202 (0.92 g) at 5°C, then the solution was left in a refrigerator overnight. After addition of Na2S203 (0.4 g) and water (6 ml) , the reaction was extracted with ether (2 x 10 ml) . To the ethereal solution was added to IM NaOH until basic pH (about 30 ml) and the solution was stirred for 2 hours at 25 °C. The water was acidified with cone. HCl, then extracted with ethyl acetate (3 x 30 ml) . The organic phase was evaporated, and the residue purified by column chromatography (hexanes : ethyl acetate 4/1) to give the phenol as a colorless oil (620 mg, 66% yield) .
Step 2:
Figure imgf000104_0001
To a solution of phenol prepared in Step 1 (50 mg, 0.21 mmol) in acetone (1 ml) were added anhydrous K2C03 (59 mg, 0.43 mmol) and benzyl bromide (76.3 ml, 0.64 mmol) . The reaction mixture was stirred at 60°C for 12 hours. The suspension was filtered using a disposable filter device (0.45 mM PTFE membrane) . The solvent was evaporated, the crude residue dissolved in DMSO, and purified by reverse phase HPLC to give l-benzyloxy-5-bromo-2 , 3- dimethoxybenzene (39 mg, 56% yield) as a white solid. Step 3
Figure imgf000105_0001
Example 24
Preparation of Example 24
Boronic acid, pinacol ester (46 mg, 0.12 mmol), palladium acetate (1 mg, 0.0046 mmol), tri- phenylphosphine attached to solid support (3 mmol/g) (4.5 mg, 0.0135 mmol), and l-benzyloxy-5-bromo-2 , 3- dimethoxybenzene (39 mg, 0.12 mmol) were added to a mixture of DMF (1.5 ml) and 2M potassium carbonate (0.5 ml) . The mixture was heated at 80° C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 24 (3 mg, 100% purity) .
Sulfonamides from Sulfonyl Chlorides
Preparation of Example 25
Step 1: Preparation of Sulfonyl Chlorides (Soon- Kyoung et al . , Arzneim Forsch, 46 (10) , pp. 966-971 (1986) )
Figure imgf000105_0002
4-Bromotoluene (7.2 ml, 58 mmol) was added to chlorosulfonic acid (7.72 ml, 116 mmol) at 10°C slowly and the mixture was stirred for 1 hour at 10 °C. The reaction was poured over ice and extracted with CH2C1, (3 x 40 ml) . The organic phase was dried over MgS04 , filtered, and evaporated to give 4 g (yield 25%) of the sulfonyl chloride as viscous oil that crystallizes upon standing.
Figure imgf000106_0001
4-Chloroaniline-3-sulfonic acid (9 g, 43.2 mmol) was dissolved in 30 ml sulfuric acid, 18 ml methanol, and 60 ml water at 0°C. Then a solution of NaN02 (3.3 g, 47.5 mmol) in 25 ml water was added dropwise over a period of 30 minutes. The mixture was stirred at this temperature for 45 minutes.
Cupric bromide (3.5 g, 12.1 mmol) dissolved in water (60 ml) then was added dropwise, followed by slow addition of HBr (12 ml) . The resulting solution was heated at reflux for 1.5 hours. The reaction was extracted with ethyl acetate (5 x 30 ml) . The organic extracts were dried with MgS04, filtered, and concentrated in vacuo to give 300 mg of 5-bromo-2- chlorosulfonic acid as a yellow solid. Step 2
Figure imgf000107_0001
5-Bromo-2-methylphenylsulfonyl chloride (48 mg, 0.176 mmol) was dissolved in dried dioxane (1 ml). Triethylamine (73.6 ml, 0.53 mmol) and 1,4- dioxo-8-azaspiro [4.5] decane (22.5 ml, 0.176 mmol) were added, and the mixture was stirred at 25°C. After 3 hours, the suspension was filtered using a disposable filter device (0.45 mM PTFE membrane) and the solvent was evaporated. The crude residue was used step without further purification.
Step 3 :
Figure imgf000107_0002
Example 25
Preparation of Example 25
Sulfonamide prepared in Step 2 was dis- solved in DMF (1.5 ml) . Boronic acid, pinacol ester (67 mg, 0.176 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenylphosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added. The mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield product example 25 (36.5 mg, 92% purity).
4-Cyano-3-mercaptobrombenzenes
Preparation of Example 26
Step 1:
Figure imgf000108_0001
4-Bromo-2-fluorobenzonitrile (100 mg, 0.5 mmol) and sodium thiophenolate (0.5 mmol) were sus- pended in 1 ml of THF, and the mixture was stirred at this temperature for 12 hours. The reaction was diluted with ether (10 ml) and washed with water (2x5 ml) . The organic layer was dried with MgS04, filtered, and concentrated in vacua . The crude res- idue was used without further purification.
Step 2
Figure imgf000109_0001
The sulfide prepared in Step 1 (42 mg, 0.166 mmol) was dissolved in DMF (1.5 ml) . Boronic acid, pinacol ester (63 mg, 0.166 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenylphosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added. The mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 26 (9.2 mg, 100% purity) .
Examples 27 and 28 (Thiazolidinones)
Preparation of Example 27
Step 1
Figure imgf000110_0001
Benzylamine (1.08 mmol), 3-bromo-4-meth- oxybenzaldehyde (1.08 mmol), and molecular sieves 3A were dissolved in dioxane (1.5 ml) and heated at 80°C for 2 hours. Mercaptoacetic acid (225.3 μl , 3.24 mmol) was added, and the mixture was heated for an additional 12 hours. The reaction was filtered, then diluted with ethyl acetate (3 ml) and washed with sat. sodium carbonate (2 ml) . The two layers were separated and the aqueous layer extracted with ethyl acetate (5 ml) . The combined organic solu- tions were dried with MgS04, filtered, and evaporated to give a solid that was used without further purification. Step 2 :
Figure imgf000111_0001
The thiaxolidinone prepared in Step 1 (48.5 mg, 0.155 mmol) was dissolved in DMF (1.5 ml) , Boronic acid, pinacol ester (60 mg, 0.155 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenyl- phosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added. The mixture was heated at 80°C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 27 (9.4 mg, 97.1% purity) .
- I ll Preparation of Example 28
Step 1 :
Figure imgf000112_0001
Cyclohexylamine (1.08 mmol), 3-bromobenz- aldehyde (186 μl , 1.08 mmol), and molecular sieves 3A were dissolved in dioxane (1.5 ml) , then heated at 80°C for 2 hours. Mercaptoacetic acid (225.3 μl , 3.24 mmol) was added, and the mixture heated for an additional 12 hours. The reaction was filtered, then diluted with ethyl acetate (3 ml) and washed with aqueous sat. sodium carbonate (2 ml) . The two layers were separated and the aqueous layer extracted with ethyl acetate (5 ml) . The combined organic solutions were dried with MgS04, filtered, and evaporated to give a solid that was carried to the next step without further purification.
Step 2
Figure imgf000113_0001
The thiazolidinone prepared in Step 1 (0.72 mmol) was dissolved in acetic acid (1.5 ml) . Water peroxide (30%, 247 μl , 2.16 mmol) was added and the mixture was stirred at 25°C for 12 hours. The solvent was evaporated and the thiazolidinone sulfoxide was used without further purification.
Step 3
Figure imgf000113_0002
The thiaxolidinone sulfoxide prepared in
Step 2 (48.5 mg, 0.155 mmol) was dissolved in DMF (1.5 ml) . Boronic acid, pinacol ester (60 mg, 0.155 mmol), palladium acetate (1.2 mg, 0.0053 mmol), tri- phenylphosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added. The mixture was heated at 80°C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 28 (14.4 mg, 93.1% purity) .
Preparation of Example 29 (Styrenes)
Step 1 :
Figure imgf000114_0001
Diethyl 4-methylbenzyl phosphonate (169 mg, 0.697 mmol) was added to a suspension of sodium hydroxide (NaH) (33 mg, 0.81 mmol) in THF (2 ml) at 0°C. The mixture was stirred at room temperature for lh, cooled to 0°C then 5-bromo-2-methoxybenz- aldehyde (100 mg, 0.465 mmol) was added. The reaction mixture was stirred at 0°C for lh, quenched with saturated NH4C1 (1.5 ml), and the aqueous phase was washed with ether (2 x 2 ml) . The combined organic extracts were dried over MgS04, then concentrated to give a residue that was used without further purification. Step 2 :
Figure imgf000115_0001
Example 29
Preparation of Example 29
The crude aryl bromide prepared in Step 1 was dissolved in 6 ml of DMF, and 2 ml were used in the reverse Suzuki reaction (about 0.155 mmol). Boronic acid, pinacol ester (60 mg, 0.155 mmol), palladium acetate (1.2 mg, 0.0053 mmol), triphenyl- phosphine attached to solid support (3 mmol/g) (5.3 mg, 0.0158 mmol), and 2M potassium carbonate (0.5 ml) were added. The mixture was heated at 80 °C for 12 hours, then filtered and purified by reverse phase HPLC to yield Example 29 (4.3 mg, 100% purity) . Preparation of Example 30 (3-Alkoxy-4-cyanobiphenyl Analogs)
J. Org. Chem . , 63 , pp. 9594-9596 (1998)
Figure imgf000116_0001
Alcohol (0.46 mmol, 2 eq) was weighed into a tube followed by the addition of 4-bromo-2-fluoro- benzonitrile (0.23 mmol, 46 mg) dissolved in 0.5 mL of dry THF. Potassium bis-trimethylsilyl amide (KHMDS) (0.7 mL of 0.5 M in toluene, 0.35 mmol, 1.5 eq) was added dropwise with vortexing. After reacting for at least 30 min., 2 mL of IM potassium bisulfate were added, followed by the addition of 2 mL of ether. The mixture was shaken or vortexed, then the phases were allowed to separate. The aqueous phase was removed by pipette, and the organic phase was concentrated under vacuum to give the crude product that was used without further purification.
Preparation of Example 30
Step 1: J. Org. Chem . , 63 , pp. 9594-9596 (1998).
4-Bromo-2- (2-cyclopentyl-2-phenylethoxy)benzonitrile
Figure imgf000117_0001
2-Cyclopentyl-2-phenylethanol (0.35 mmol, 1.5 eq) was weighed into a tube followed by the addition of 4-bromo-2 -fluorobenzonitrile (0.23 mmol, 46 mg) dissolved in 0.5 mL of dry THF. Potassium bis-trimethylsilyl amide (KHMDS) (0.7 mL of 0.5 M in toluene, 0.35 mmol, 1.5 eq) was added dropwise with vortexing. After reacting for at least 30 min., 2 mL of IM potassium bisulfate was added followed by the addition of 2 L of ether. The mixture was shaken or vortexed and the phases were allowed to separate. The aqueous phase was removed by pipette, and the organic phase was concentrated under vacuum to give the crude product that was used without further purification. Step 2
Figure imgf000118_0001
Boronic acid, pinacol ester (70 mg, 0.18 mmol), palladium acetate (1 mg, 0.0046 mmol), tri- phenylphosphine attached to solid support (3 mmol/g) (4.5 mg, 0.0135 mmol), and 4-bromo-2- (2-cyclopentyl- 2 -phenyl -ethoxy) benzonitrile (0.18 mmol) were added to a mixture of DMF (1.6 ml) and 2M sodium carbonate (0.4 ml) . The mixture was heated at 80°C for 12 hours, then filtered and about 50% purified by reverse phase HPLC to yield Example 30 (2.7 mg, 98% purity) .
- l l ϊ
Preparation of 4-bromo-2, 6-difluorobenzonitrile (Example 31)
Step 1 : Mol . Cryst . Liq. Cryst . , 172 , pp. 165-189 (1989)
Figure imgf000119_0001
4-Bromo-2 , 6-difluorophenylamine (15 g, 72 mmol) was mechanically stirred with 70 L of concentrated 1:1 sulfuric acid/water. The suspension was heated until a solution was obtained, followed by cooling to -10°C with a salt/ice bath. A solution of sodium nitrite (8.28 g, 120 mmol) in water (20 mL) was added dropwise to the stirring aniline such that the temperature was maintained below -5°C. The mixture was stirred for 2.5h after the addition was complete .
A solution of potassium cyanide (23.4 g, 360 mmol) in water was added dropwise to a stirred solution of copper (II) sulfate pentahydrate (23.2 g, 94 mmol) in water (50 mL) and ice (30 g) . Sodium bicarbonate (72 g) , cyclohexane (300 mL) , and glacial acetic acid (30 mL) were added followed by warming to solution to 50°C. The cold diazonium salt solution was added to the vigorously stirring cyanide in portions. The reaction was cooled after 0.5h and the phases separated. The aqueous phase was extracted twice with ether, and the combined organics were washed sequentially with water, 1 M sodium hydroxide, and water. The organics were dried with anhydrous magnesium sulfate and removed under reduced pressure. The resulting solid was crystallized twice from ethanol to yield 6 g of product .
Step 2:
4-bromo-2-fluoro-6-methoxybenzonitrile
Figure imgf000120_0001
4-Bromo-2-fluoro-6-methoxybenzonitrile (2 g, 9.2 mmol) was dissolved in THF (10 mL) , followed by the dropwise addition of 2.1 mL of 21% sodium methoxide (10 mmol) in methanol. After reacting for lh, IM potassium bisulfate (20 mL) and ether (20 mL) were added, the phases separated, and the organic layer dried and concentrated. LC/MS indicated a mixture of starting material, and mono and disubsti- tution. The crude material was chromatographed on silica using a four-step gradient of hexanes/ethyl acetate (0% ethyl acetate, 5%, 10%, and 15%) to yield 1.1 g of 4-bromo-2-fluoro-6-methoxybenzo- nitrile .
Step 3 :
4-Bromo-2- (2-cyclopentyl-2 -phenyl-ethoxy) -6- methoxybenzonitrile
Figure imgf000121_0001
2 -Cyclopentyl -2 -phenylethanol (0.35 mmol,
66 mg, 1.5 eq) was weighed into a tube followed by the addition of 4-bromo-2-fluoro-6-methoxybenzonitrile (0.23 mmol, 53 mg) dissolved in 0.5 mL of dry THF. Potassium bis-trimethylsilyl amide (KHMDS) (0.7 mL of 0.5 M in toluene, 0.35 mmol, 1.5 eq) was added dropwise with vortexing . After reacting for at least 30 min. , 2 mL of IM potassium bisulfate was added followed by the addition of 2 mL of ether. The mixture was shaken or vortexed, then the phases were allowed to separate. The aqueous phase was removed by pipette, and the organic phase was concentrated under vacuum to give a crude product that was used without further purification. Step 4
Figure imgf000122_0001
Example 31
Preparation of Example 31
Boronic acid, pinacol ester (70 mg, 0.18 mmol), palladium acetate (1 mg, 0.0046 mmol), tri- phenylphosphine attached to solid support (3 mmol/g) (4.5 mg, 0.0135 mmol), and 4-bromo-2- (2 -cyclopentyl- 2-phenylethoxy) -6-methoxybenzonitrile (72 mg, 0.18 mmol) were added to a mixture of DMF (1.6 ml), and 2M sodium carbonate (0.4 ml) . The mixture was heated at 80°C for 12 hours, then filtered, and about 50% purified by reverse phase HPLC to yield Example 31 (5.2 mg, 99% purity).
Preparation of Example 32
Example 32 was prepared according to procedure set forth for the preparation of a 3-alkoxy- 4-cyanobiphenyl using 4-fluoro-3-nitrobromobenzene as the starting aryl bromide.
Figure imgf000123_0001
Example 32
Example 33 (Napthylated Analogs)
Step 1:
Figure imgf000123_0002
N- (Hydroxyethyl) -2-mercapto acetamide (136 mg, 1.0 mmol) and l-bromo-2-bromomethylnaphtalene
(150 mg, 0.5 mmol) were combined in DMF (2 ml) with K2C03 (207 mg, 1.5 mmol) and stirred for 12h at room temperature . The DMF was removed in vacuo and the compound was partitioned between EtOAc with H20. The EtOAc layer was washed with H20 and brine. The organics were concentrated to a residue that was used without further purification. Step 2
Preparation of Example 33
Figure imgf000124_0001
Example 33
The sulfide prepared in Step 1 (0.25 mmol) was dissolved in acetic acid (0.5 ml) . Water peroxide (30%, 88 μl, 0.75 mmol) was added and the mixture was heated at 80°C for 2 hours. The solvent was evaporated, the sample dissolved in DMSO (2 ml) , and purified by reverse phase HPLC to yield Example 33 (23.3 mg, 98.5% purity).
Example 34 (N-Alkylsulfonamides)
Step 1:
Figure imgf000124_0002
N- (Hydroxyethyl) -2 -mercapto acetamide (10 g, 73.97 mmol) and l-bromo-2-chloroethane (9.23 ml, 110.96 mmol) were combined in DMF (25 ml) with K2C03 (20.4 g, 147.94 mmol) and stirred for 12h at room temperature . The DMF was removed in vacuo, and the compound was partitioned between EtOAc with H20. The EtOAc layer was washed with H20 and brine. The organics were concentrated to a residue that was dissolved in acetic acid (148 ml) . Water peroxide (30%, 25.9 ml, 221.9 mmol) was added, and the mixture was heated at 80 °C for 2 hours. The solvent was evaporated in vacuo and the compound was partitioned between EtOAc with H20. The EtOAc layer was washed with H20 and brine. The organics were concentrated to a residue used without further purification.
Step 2
Figure imgf000125_0001
Example 34
Preparation of Example 34
Chloride from Step 1 (lg, 4.43 mmol), K2C03
(1.8 g, 13.3 mmol), and 1-aminoindane (0.7g, 5.22 mmol) were suspended in 10 ml of DMF, then stirred at room temperature for 12h. The mixture was filtered and to 1.5 ml of this DMF solution were added 176 mg of 2-naphthalene sulfonyl chloride and K2C03 (225 mg, 1.63 mmol). The mixture resulting was stirred at room temperature for 12h. The reaction was filtered and purified by reverse phase HPLC to give Example 34 (0.4 mg, 100% purity) .
Compounds of the present invention were tested for an ability to inhibit PDE3B. The ability of a compound to inhibit PDE3B activity is related to the IC50 value for the compound, i.e., the concentration of inhibitor required for 50% inhibition of enzyme activity. The IC50 value for compounds of the present invention were determined using recombinant human PDE3B.
The in vi tro phosphodiesterase activity inhibitory ICS0 values were determined by measuring the inhibition as a function of the concentration of the test compound over the range of 0 to 1 mM. The IC50 values of the compounds tested in the aforementioned assay ranged from about 0.01 μM to about 10 μM.
The compounds of the present invention typically exhibit an IC50 value against recombinant human PDE3B of less than about 10 μM, and preferably less than about 5 μM, and more preferably less than about 1 M. The compounds of the present invention typically exhibit an IC50 value against recombinant human PDE3B of less than about 1000 nM, and often less than about 100 nM. To achieve the full advantage of the present invention, a present PDE3B inhibitor has an IC50 of about 700 pM (picomolar) to about 10 μM . The IC50 values for the compounds were determined from concentration-response curves typically using concentrations ranging from 0.1 pM to 500 μM . Tests against other PDE enzymes using stan- dard methodology, as described in Loughney et al . , J. Biol . Chem . , 271 , pp. 796-806 (1996), also showed that compounds of the present invention are highly selective for PDE3B. The production of recombinant human PDEs and the ICS0 and EC50 determinations can be accomplished by well-known methods in the art. Exemplary methods are described as follows:
EXPRESSION OF HUMAN PDEs
Expression in Baculovirus-Infected Spodoptera fucripe-rda (Sf9) Cells
Baculovirus transfer plasmids were constructed using either pBlueBacIII (Invitrogen) or pFastBac (BRL-Gibco) . The structure of all plasmids was verified by sequencing across the vector junc- tions and by fully sequencing all regions generated by PCR. Plasmid pBB-PDElA3/6 contained the complete open reading frame of PDE1A3 (Loughney et al . , J. Biol . Chem . , 271 , pp. 796-806 (1996)) in pBlueBacIII. Plasmid Hcam3aBB contained the complete open reading frame of PDE1C3 (Loughney et al .
(1996)) in pBlueBacIII. Plasmid pBB-PDE3A contained the complete open reading frame of PDE3A (Meacci et al., Proc . Na tl . Acad . Sci . , USA, 89 , pp. 3721-3725
(1992)) in pBlueBacIII. Plasmid pFB-PDE3B contains the complete open reading frame of PDE3B (Miki et al., Genomics, 36, pp. 476-485 (1996)).
Recombinant virus stocks were produced using either the MaxBac system (Invitrogen) or the FastBac® system (Gibco-BRL) according to the manu- facturer's protocols. In both cases, expression of recombinant human PDEs in the resultant viruses was driven off the viral polyhedron promoter. When using the MaxBac® system, virus was plaque purified twice in order to insure that no wild type (occ+) virus contaminated the preparation. Protein expression was carried out as follows. Sf9 cells were grown at 27°C in Grace's Insect culture medium (Gibco-BRL) supplemented with 10% fetal bovine serum, 0.33% TC yeastolate, 0.33% lactalbumin hydro- lysate, 4.2 mM NaHC03, 10 μg/ml gentamycin, 100 units/ml penicillin, and 100 μg/ml streptomycin. Exponentially growing cells were infected at a multiplicity of approximately 2 to 3 virus particles per cell and incubated for 48 hours. Cells were collected by centrifugation, washed with nonsupple- ented Grace's medium, and quick-frozen for storage.
Expression in Saccharomyces cerevisiae (Yeast)
Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C, PDE4D, PDE5 , PDE7 , PDE8 , PDE9, and PDE10 was carried out similarly to that described in Example 7 of U.S. Patent No. 5,702,936, incorporated herein by reference, except that the yeast transformation vector employed, which is derived from the basic ADH2 plasmid described in Price et al . , Methods in Enzymology, 185, pp. 308-318 (1990) , incorporated yeast ADH2 promoter and termi- nator sequences and the Saccharomyces cerevisiae host was the protease-deficient strain BJ2-54 deposited on August 31, 1998 with the American Type Culture Collection, Manassas, Virginia, under accession number ATCC 74465. Transformed host cells were grown in 2X SC-leu medium, pH 6.2, with trace metals, and vitamins. After 24 hours, YEP medium- containing glycerol was added to a final concentration of 2X YET/3% glycerol. Approximately 24 hr later, cells were harvested, washed, and stored at -70°C.
CALMODULIN PURIFICATION
Calmodulin used for activation of the PDE1 enzymes was purified from bovine testes essentially as described by Dedman et al . , Methods in Enzymology, 102, pp. 1-8 (1983) using the Pharmacia Phenyl -Sepharose® procedure.
IMMOBILIZATION OF CALMODULIN ON AGAROSE
Calmodulin was immobilized on BioRad Affi- Gel® 15 per manufacturer's instructions.
HUMAN PHOSPHODIESTERASE PREPARATIONS
Phosphodiesterase Activity Determinations
Phosphodiesterase activity of the preparations was determined as follows. PDE assays utilizing a charcoal separation technique were performed essentially as described in Loughney et al . (1996). In this assay, PDE activity converts [32P] cAMP or [32P]cGMP to the corresponding [32P] 5 ' -AMP or [32P]5'-GMP in proportion to the amount of PDE activity present. The [32P]5'-AMP or [32P]5'-GMP then was quantitatively converted to free [32P] phosphate and unlabeled adenosine or guanosine by the action of snake venom 5 ' -nucleotidase . Hence, the amount of [32P] phosphate liberated is proportional to en- zyme activity. The assay was performed at 30°C in a 100 μL reaction mixture containing (final concentrations) 40 mM Tris HCl (pH 8.0) , 1 μM ZnS04, 5 mM MgCl2, and 0.1 mg/ml bovine serum albumin (BSA) . Alternatively, in assays assessing PDEl-specific activity, incubation mixtures further incorporated the use of 0.1 mM CaCl2 and 10 μg/ml calmodulin. PDE enzyme was present in quantities that yield <30% total hydrolysis of substrate (linear assay conditions) . The assay was initiated by addition of sub- strate (1 mM [32P] cAMP or cGMP) , and the mixture was incubated for 12 minutes. Seventy- five (75) μg of Crotalus atrox venom then was added, and the incubation was continued for 3 minutes (15 minutes total) . The reaction was stopped by addition of 200 μL of activated charcoal (25 mg/ml suspension in 0.1 M
NaH2P04, pH 4) . After centrifugation (750 X g for 3 minutes) to sediment the charcoal, a sample of the supernatant was taken for radioactivity determination in a scintillation counter and the PDE activity was calculated.
Inhibitor analyses were performed similarly to the method described in Loughney et al . , J". Biol . Chem . , 271 , pp. 796-806 (1996), except both cGMP and cAMP were used, and substrate concentra- tions were kept below 32 nM, which is far below the Km of the tested PDEs. Purification of PDE1A3 from SF9 Cells
Cell pellets (5 g) were mixed with 10 ml of Lysis Buffer (50 mM MOPS pH 7.5 , 2 mM dithio- threitol (DTT) , 2 mM benzamidine HCl, 5 μM ZnS04,
0.1 mM CaCl2, 20 μg/ml calpain inhibitors I and II, and 5 μg/ml each of leupeptin, pepstatin, and apro- tinin) at room temperature. The cells were lysed by passage through a French® pressure cell (SLM- Aminco®, Spectronic Instruments, Inc., Rochester NY) . The resultant lysate was centrifuged in a Beckman ultracentrifuge using a type T180 rotor at 45,000 rpm for 1 hr. The supernatant was recovered and filtered through a 0.2 μm filter. This filtrate was applied to a 2.6 X 90 cm column of SEPHACRYL®
S-300 equilibrated in Column Buffer A (Lysis Buffer containing 100 mM NaCl, and 2 mM MgCl2) . The column flow rate was adjusted to 1 mL/min and fractions of 7 ml were collected. Active fractions were pooled and supplemented with 0.16 m g of calmodulin. The enzyme was applied overnight at a flow rate of 0.2 mL/min to an ACC-1 agarose immunoaffinity column as described in Hansen et al . , Methods in Enzymology 159, pp. 453-557 (1988) . The column was washed with 5 volumes of Column Buffer B (Column Buffer A without NaCl) and followed by 5 volumes of Column Buffer C (Column Buffer A containing 250 mM NaCl) . The column was eluted with Column Buffer D (50 mM MOPS pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benz- amidine HCl, 100 mM NaCl, 20 μg/ml calpain inhibitors I and II, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin) by applying one column volume at 0.1 mL/min, stopping flow for 1 hour, and then continuing elution at the same flow rate. Fractions of 0.5 ml were collected. Fractions displaying activity were pooled, and first dialyzed against dialysis buffer containing 25 mM MOPS pH 7.5, 100 mM NaCl, 10 μM ZnS04 , 1 mM CaCl2, 1 M DTT, and 1 mM benzamidine HCl. A subsequent dialysis against dialysis buffer containing 50% glycerol was performed prior to quick-freezing the sample with dry ice and storage at -70°C. The resultant prepara- tions were about 10 to 15% pure by SDS-PAGE. These preparations had specific activities of about 5 to 20 μmol cAMP hydrolyzed per minute per milligram protein.
Purification of PDE1B from S . cerevisiae
Yeast cells (50 g) were thawed by mixing with 100 ml glass beads (0.5 mM, acid washed) and 200 ml Buffer A at room temperature. Buffer A con- sisted of 50 mM MOPS pH 7.5, 1 mM DTT, 2 mM benzamidine HCl, 0.01 mM ZnS04, 5 mM MgCl2, 20 μg/ml calpain inhibitors I and II, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin. The mixture was cooled to 4°C, transferred to a Bead-Beater®, and the cells lysed by rapid mixing for 6 cycles of 30 seconds each. The homogenate was centrifuged for 15 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at 9,000 rpm and 4°C. The supernatant was recovered and centrifuged in a Beckman XL-80 ultracentrifuge using a TI45 rotor at 36,000 rpm for 45 minutes at 4°C. The supernatant was recovered and PDE1B was precipitated by the addition of solid ammonium sulfate (0.33 g/ml supernatant) while stirring in an ice bath and maintaining the pH between 7.0 and 7.5. This mixture then was centrifuged for 22 minutes in a Beckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000 X g) . The supernatant was discarded and the pellet was dissolved in 100 ml of buffer B (50 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mM ZnS04, 2 mM MgCl2, 2 mM CaCl2, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin) . The pH and conductivity were corrected to 7.5 and 15-20 milli- Siemens (mS) , respectively. This solution was loaded onto a 20 ml column of calmodulin-Agarose that had been equilibrated with 10 column volumes of Buffer B at a rate of 1 mL/min. The flow-through was reapplied to the column at least 5 times. The column was washed with 5 volumes of Buffer B, 5 volumes of buffer B containing 250 mM NaCl, and 2 volumes of Buffer B without NaCl again. Elution was accomplished by applying one volume of Buffer C (50 mM MOPS pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benzamidine HCl) at 0.33 mL/min, then stopping flow for 1 hour before continuing the elution. Fractions of about 4 ml were collected and assayed for PDE activity. Active fractions were pooled and concentrated to a volume of 5 mL, using an Amicon ultra- filtration system. The concentrate was then applied to a 320 ml Sephacryl® S-300 column (1.6 X 150 cm) that had been equilibrated with at least 2 volumes of Buffer D (25 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mM ZnS04, 2 mM CaCl2, and 100 mM NaCl) . The column was developed at a flow rate of 1 mL/min (11 cm/hr) , and 5 ml fractions were collected. The activity peak was pooled and dialyzed overnight against Buffer D containing 50% glycerol. The purified enzyme was frozen on dry ice and stored at -70°C. The resultant preparations were about >90% pure by SDS-PAGE. These preparations had specific activities of about 10 to 30 μmol cGMP hydrolyzed per minute per milligram protein.
Purification of PDE1C31 from Sf9 Cells
Frozen cell pellets (2 x 1010) cells were resuspended in Lysis Buffer (50 mM Na MOPS (pH 7.2), 1 mM dithiothreitol, 2 mM benzamidine HCl, 5 μM ZnS04, 20 μg/ml calpain inhibitors I and II, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin) . The mixture was sonicated twice for 30 seconds and the cells were lysed in a French® pressure cell
(SLM-Aminco®, Spectonic Instruments, Inc.) at 20,000 psi (4°C) . The lysate was centrifuged at 100,000 x g for 45 minutes. The supernatant was recovered and filtered with a 0.2 μm unit containing a glass fiber prefilter. The filtered supernatant was exchanged in Buffer A (50 mM Na MOPS (pH 7.2), 1 mM dithiothreitol, 2 mM benzamidine HCl, 5 μM ZnS04, 5 mM MgCl2 and 100 mM NaCl) using a Sephadex G25 column. This material was loaded onto a Cibacron blue Seph- arose column that had been equilibrated with at least 10 volumes of Buffer A. The column was washed with 10 volumes of Buffer A and eluted with Buffer B (50 mM Na MOPS (pH 7.2) , 1 mM dithiothreitol, 2 mM benzamidine HCL, 5 μM ZnS04 , 100 mM NaCl and 1-5 mM cAMP) . Fractions containing the PDE activity were pooled and concentrated by filter centrifugation. The resultant material was exchanged into a Buffer C (50 mM Na MOPS (pH 7.2), 2 mM dithiothreitol, 2 mM benzamidine HCl, 10 μM ZnS04, 2 mM CaCl2, and 200 mM NaCl) using a Sephadex G25 column and an equal volume of glycerol was added. Aliquots were quick frozen in dry ice and stored at -70°C.
Purification of PDE2A from S . cerevisiae
Frozen yeast cell pellets from strain YI34 (lOg, stored at -70°C) were allowed to thaw on ice in 25 ml of Lysis Buffer (50 mM MOPS, pH 7.2 , 1 mM EDTA, 1 mM EGTA, 0.1 mM DTT, 0.1 mM 4-(2-amino- ethyl) benzenesulfonyl fluoride (AEBSF) , 1 μg/ml of pepstatin, leupeptin, aprotinin, calpain inhibitors I and II, and 2 mM benzamidine) . Cells were lysed by three passages through a French® pressure cell (SLM-Aminco® , Spectronic Instruments) . The lysate was centrifuged at 36,000 rpm in a Beckman Ultracen- trifuge rotor Type 45Ti for 60 minutes at 4°C. The supernatant was separated from sediment and passed through a 15 ml Epoxy-cGMP Sepharos® resin at 4°C two times at about 0.5 mL/min. The column subsequently was washed with 45 ml of Wash Buffer 1 (50 mM MOPS, pH 7.2, 0.1 mM EDTA, 0.1 mM DTT). Following this wash, the column was washed with 45 ml of Wash Buffer 2 (Wash Buffer 1 containing 0.5 M NaCl) . Following this salt wash, the column was washed with 15 ml of Wash Buffer 3 (Wash Buffer 1 containing 0.25 M NaCl) . The column was transferred to room temperature and allowed to warm. Approximately 25 ml of Elution Buffer (Wash Buffer 3 containing 10 mM cGMP, maintained at room temperature) was applied to the column and the effluent was collected in 2 ml fractions. Small aliquots of each of the fractions were diluted 20-fold in PBS containing 5 mM MgCl2 to allow hydrolysis of the competing ligand and to aid detection of PDE2 activity. Active fractions were passed through a Pharmacia PD-10® gel filtration column to exchange into Wash Buffer 3. This exchanged pool was diluted 50% v/v with sterile 80% glycerol and stored at -20°C. The resultant preparations were greater than 85% pure as judged by SDS- PAGE with subsequent staining of protein by Coomassie R-250. These preparations had specific activities of about 150 to 250 μmol cGMP hydrolyzed per minute per milligram protein.
Preparation of PDE3A and PDE3B from Sf9 Cells
Cells (2 x 1010) were suspended in Lysis Buffer containing 50 mM MPOS pH 7.5, 2 mM DTT-, 2 mM benzamidine HCl, 5 μM ZnS04, 0.1 mM CaCl2, 20 μg/ml calpain inhibitors I and II, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin. The mixture was sonicated twice for 30 seconds and the cells were lysed in a French® pressure cell (SLM-Aminco® , Spectronic Instruments) at 4°C. The lysate was centrifuged 100,000 x g for 45 minutes. The pellet was washed once in Lysis Buffer and suspended in 46 ml Lysis Buffer with a Dounce homogenizer. Aliquots were stored at -70°C. These preparations had specific activities of about 1 to 2 nmol cAMP hydrolyzed per minute per milligram protein. Preparation of PDE4B2 from S . cerevisiae
Yeast cells (150 g of yeast strain YI23 harboring HDTJN2.32) were thawed by mixing with 100 ml glass beads (0.5 mM, acid washed) and 150 ml Lysis Buffer (50 mM MOPS pH 7.2 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, 5 μg/ml each of pepstatin, leupeptin, aprotinin, calpain inhibitors I and II) at room temperature. The mixture was cooled to 4°C, transferred to a Bead-Beater®, and the cells lysed by rapid mixing for 6 cycles of 30 seconds each. The homogenate was centrifuged for 22 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at 9,000 rpm and 4°C. The supernatant was recovered and centrifuged in a Beckman XL- 80 ultra- centrifuge using a TI45 rotor at 36,000 rpm for 45 minutes at 4°C. The supernatant was recovered and PDE4B was precipitated by the addition of solid ammonium sulfate (0.26 g/ml supernatant) while stir- ring in an ice bath and maintaining the pH between 7.0 and 7.5. This' mixture was then centrifuged for 22 minutes in a Beckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000 X g) . The supernatant was discarded and the pellet was dissolved in 200 ml of Buffer A (50 mM MOPS pH 7.5 , 5 mM MgCl2, 1 mM DTT, 1 mM benzamidine HCl, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin) . The pH and conductivity were corrected to 7.5 and 15-20 mS, respectively. The resuspended sample was loaded onto a 1.6 X 200 cm column (25 mL) of Sigma Cibacron Blue Agarose-type 300 equilibrated in Buffer A. The sample was cycled through the column 4 to 6 times over the course of 12 hours . The column was washed in succession with 125 to 250 ml of Buffer A, 125 to 250 ml of Buffer A containing 1.5 M NaCl, and 25 to 50 ml of Buffer A. The enzyme was eluted with 50 to 75 ml of Buffer E (50 mM Tris HCl pH 8 , 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and 20 mM cAMP) and 50 to 75 ml of Buffer E containing 1 M NaCl. The PDE activity peak was pooled, and precipitated with ammonium sulfate (0.4 g/ml enzyme pool) to remove excess cyclic nucleotide. The precipi- tated proteins were resuspended in Buffer X (25 mM
MOPS pH 7.5, 5 μM ZnS04 , 50 mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desalted via gel filtration on a Pharmacia PD-10® column per manufacturer's instructions. The enzyme pool was dialyzed over- night against Buffer X containing 50% glycerol.
This enzyme was quick- frozen in a dry ice/ethanol bath and stored at -70°C.
The resultant preparations were about >90% pure by SDS-PAGE. These preparations had specific activities of about 10 to 50 μmol cAMP hydrolyzed per minute per milligram protein.
Purification of PDE5A from S . cerevisiae
Cell pellets (29 g) were thawed on ice with an equal volume of Lysis Buffer (25 mM Tris HCl, pH 8, 5 mM MgCl2, 0.25 mM DTT, 1 mM benzamidine, and 10 μM ZnS04) . Cells were lysed in a Microfluid- izer® (Microfluidics Corp.) using nitrogen at 20,000 psi. The lysate was centrifuged and filtered through 0.45 μm disposable filters. The filtrate was applied to a 150 ml column of Q SEPHAROSE® Fast- Flow (Pharmacia) . The column was washed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl,, 0.25 mM DTT, 10 μM ZnS04) and eluted with a step gradient of 125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mM NaCl in Buffer A. Active fractions from the linear gradient were applied to a 180 ml hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane (pH 6.8), 1 mM MgCl2, 0.25 mM DTT, 10 μM ZnS04, and 250 mM KCI) . After loading, the column was washed with 2 volumes of Buffer B and eluted with a linear gradient of 0-125 mM potassium phosphate in Buffer B. Active fractions were pooled, precipitated with 60% ammonium sulfate, and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mM NaCl, 0.5 mM DTT, and 10 μM ZnS04) . The pool was applied to a 140 ml column of SEPHA- CRYL® S-300 HR and eluted with Buffer C. Active fractions were diluted to 50% glycerol and stored at -20°C.
The resultant preparations were about 85% pure by SDS-PAGE. These preparations had specific activities of about 3 μmol cGMP hydrolyzed per minute per milligram protein.
Preparation of PDE7 from S . cerevisiae
Cell pellets (126 g) were thawed and resuspended at room temperature for about 30 minutes with an equal volume of Lysis Buffer (50 mM Tris HCl, pH 8, 1 mM EDTA, 1 mM DTT, 50 mM NaCl, 2 mM benzamidine HCl, and 5 μg/ml each of pepstatin, leupeptin, and aprotinin) . The cells were lysed at 0-4°C with the aid of glass beads (125 mL) in a Bead-Beater® for 6 X 30 second cycles. The lysate was centrifuged and filtered through 0.45 μm disposable filters. The filtered extract (178 mL) was distributed into 4 ml aliquots, quick-frozen with dry ice, and stored in a freezer at -70°C. These preparations were stable to several cycles of freezing and thawing and had specific activities of about 50 to 100 pmol cAMP hydrolyzed per minute per milligram protein.
Preparation of PDE8A1 from S . cerevisiae
Frozen cell pellets (2.2 g) were mixed with 2.2 mL of Lysis Buffer (50 mM Tris-CI (pH 8.5),
2 μM ZnCl2, 1 mM DTT, 2 mM benzamidine, 20 μg/ml Calpain inhibitors I and II, 0.1 M NaCl, and 5 μg/ml each of pepstatin A, leupeptin, and aprotinin) . After thawing, the mixture was distributed into two
30 mL Corex tubes containing 2 mL acid-washed glass beads (Sigma) each. The tubes were vortexed three times for 30 seconds with a 2.5 minute cool down period between each cycle. The beads were allowed to settle, and the resulting homogenate was centrifuged at 45,000 rpm for 25 minutes in a Beckman TL-100. The supernatant was collected, flash frozen in dry ice/ethanol, and stored at -70°C until use.
Preparation of PDE9A3 from S. cerevisiae
Frozen cell pellets (7.8 g) were resus- pended in 8 L of Lysis Buffer (25 mM Tris (pH 8.0), 5 mM EDTA, 0.1 mM DTT, and 10 μg/ml each of pepstatin, leupeptin, and aprotinin) . The mixture was chilled on ice and lysed by passing through a French® pressure cell (SLM-Amino®, Speetonic Instruments) at 20,000 psi. The lysate was centrifuged at 25,000 x g for 20 minutes at 4°C. The supernatant was removed, and an equal volume 80% glycerol was added prior to storage at -20°C. Also see Example 4 of PCT Publication WO 99/42596, published August 26, 1999.
Preparation of PDE10A1 from S . cerevisiae
Frozen cell pellets (5 x 1011 cells) were resuspended in Lysis Buffer (50 mM MOPS (pH 7.2), 1 mM dithiothreitol, 2 mM benzamidine HCl, 5 μM ZnS04 , 20 μg/ml calpain inhibitors I and II, and 5 μg/ml each of leupeptin, pepstatin, and aprotinin) . The mixture was sonicated twice for 30 seconds and the cells were lysed in a French® pressure cell (SLM- Aminco®, Spectronic Instruments) at 20,000 psi (4°C) . The lysate was centrifuged at 100,000 x g for 45 minutes. The supernatant was recovered and filtered with a 0.2 μm unit containing a glass fiber prefilter. The filtered extract was aliquotted and stored at -70°C. Also see Example 5 of Loughney U.S. Patent No. 6,133,007.
Biological Data
Compounds of the present invention were found to exhibit an ICS0 value of less than about 10 μM, typically less than about 1 μM (i.e., 1000 nM) , and often less than about 100 nM. In vi tro test data for representative compounds of the invention is summarized in the following table.
Figure imgf000142_0001
Figure imgf000143_0001
To further illustrate the potency and PDE3B selectivity of a compound of the present in- vention, Table 1 contains the ICS0 value vs. PDE3B for Examples 1-34 (left column) , and the ratio of IC50 (vs. PDE3A) to IC50 (vs. PDE3B) (right column) . The potency of the inhibitors is illustrated in the IC50 value vs. PDE3B, which is about 8.5 μM or less for each example. For some examples, the IC50 value vs. PDE3B is about 0.01 μM (i.e., 10 nM) .
Table 1 further shows the PDE3B inhibition selectivity of compounds of the present invention. The selectivity of the inhibitors for PDE3B is illustrated in the ICS0 value ratio, which shows a strong selectivity for PDE3B over PDE3A, i.e., up to a factor of about 30, and typically a factor of at least about 5 to about 20.
The data in Table 1 allows the selection of a very potent or a very selective PDE3B inhibitor, or a PDE3B inhibitor that exhibits both potency and selectivity. The choice of a particular compound for use in therapy is related to several factors, including the toxicity, physical properties, and pharmacological properties of the compound, the condition or disease to be treated, and the intended route of administration. The selectivity of a compound of the present invention to inhibit PDE3B over PDE3A and other PDE enzymes is illustrated in the following Table 2. Table 2 shows that the compounds of Examples 4, 35, and 36 are potent inhibitors of PDE3B, i.e., have an IC50 value vs. PDE3B of 0.085, 0.59, and 1.05 μM, respectively. Table 2 also shows that the compounds are selective inhibitors of PDE3B over PDE3A, i.e., have a ratio of IC50 (vs. PDE3A) to IC50 (vs. PDE3B) of 14.1, 23.7, and 12.6, respectively. Table 2 further shows a selective inhibition of PDE3B over other listed PDE isoforms by a factor of 141 to 8129 for Example 4, 23.7 to 347.5 for Example 35, and 28.6 to 952.4 for Example 36.
Figure imgf000145_0001
Figure imgf000145_0002
Lipolytic Properties of Selective PDE3B Inhibitors
Adipocytes store excess energy in the form of triglyceride. Synthesis of adipocyte triglycer- ide is regulated hormonally, and is a crucial aspect of body weight regulation. The breakdown of triglyceride to glycerol and free fatty acids is known as lipolysis. Agents that induce lipolysis are expected to have utility in the treatment of obesity. PDE3 inhibitors have been known to induce lipolysis both in vi tro and in vivo (P.B. Snyder, Emerging Therapeutic Targets, 3 , pp. 587-599 (1999)). Because adipocytes express the PDE3B isoform (M. Taira et al . , J". Biol . Chem . , 268, pp. 18573-18579 (1993)), the effect of selective PDE3B inhibitors on lipolysis in cultured human adipocytes was tested.
Methods and Materials
Culture of human adipocytes: Human adipocyte were purchased from Zen-Bio, Inc. (Research Triangle Park, NC) . The cells were derived from surgical specimens of subcutaneous adipose tissue by collagenase digestion. The cells were grown to con- fluence in 96-well tissue culture plates, and induced to differentiate into adipocytes by addition of a medium containing adipogenic and lipogenic hormones (see Hauner et al . , J". Clin . Invest . , 84 , pp. 1663-1670 (1989) for a representative protocol) . After differentiation, the cells were maintained in a medium consisting of DMEM/Ham's F-10 Medium (1:1), 15 mM HEPES (pH 7.4) supplemented with 3% FCS, 33 μM Biotin, 17 μM pantothenate, 100 nM human insulin, 1 μM dexamethasone , 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Maintenance Medium) . The cells were maintained at 37°C in a 5% C02 atmosphere and were fed every 2 to 3 days by removing 100 μl of culture supernatant and replacing it with 100 μl of fresh, prewarmed Maintenance Medium.
Lipolysis assay: Adipocytes were treated with inhibitors that were diluted to the appropriate concentration in a diluent consisting of DMEM/Ham's F-12 (1:1 v/v), 15 mM HEPES supplemented with 33 μM biotin, 17 μM pantothenate, and 4% BSA. Because the inhibitor stocks were dissolved in 100% DMSO, a vehicle control containing the same final concentra- tion of DMSO as in the drug dilutions also was tested. Each test condition was performed in triplicate. Cells were incubated with inhibitors or vehicle for 6 hours at 37°C in an atmosphere containing 5% C02, after which supernatants from the treated cells were collected and stored at -20°C until ready for analysis.
Lipolysis was measured by determining the concentration of glycerol (a breakdown product of triglyceride) in the culture supernatants. The assay was performed in a 96-well format as follows. Culture supernatant (100 μl) was mixed with an equal volume of GPO Trinder Reagent A (Sigma Diagnostics, Inc., St. Louis, MO). The reaction was allowed to proceed for 15 minutes at room temperature and the absorbance of the samples was determined at 540 nm. The value of a blank (containing only diluent) was subtracted from each sample. The mean and the standard error of the mean were determined for each set of three replicates and these values were expressed as a percentage of the value for the vehicle control. EC50 values were determined by fitting the data to a four-parameter logistic dose-response model using Table Curve 2D (version 4) software (SSPS, Inc., Birmingham, UK).
Results
The following nine selective PDE3B inhibitors of the present invention were tested for induction of lipolysis in cultured human adipocytes. Each of these nine compounds had an IC50 value vs. recombinant human PDE3B of less than 100 nM (see Table 3) . The selectivity for inhibition of PDE3B over PDE3A of these nine compounds ranged from about three-fold to about 14-fold. The EC50 of each compound for induction of lipolysis was determined by incubating cultured human adipocytes in the presence of various concentrations of drug and measuring the amount of glycerol released into the culture medium during a six-hour incubation. The results were expressed as a percentage of the response of cells treated with vehicle alone (0.33% DMSO). Each data point corresponded to the mean of three replicate samples . Test compounds :
Figure imgf000149_0001
Example 30
Figure imgf000149_0002
Example 37
Figure imgf000150_0001
Example 24
Figure imgf000150_0002
Example 38
Figure imgf000151_0001
Example 23
Figure imgf000151_0002
Example 4
Figure imgf000151_0003
Example 39
Figure imgf000152_0001
Example 40
Figure imgf000152_0002
Example 41
In particular, lipolysis (vs. % control) for the nine tested compounds over a concentration range of 0.01 to 100 μM varied from 0% to 100% at
0.01 μM to 950% to 1300% at 100 μM. The EC50 values derived from the dose-response curves are summarized in Table 3, and ranged from 1 μM to 27 μM.
Figure imgf000153_0001
Additional compounds of the present invention are illustrated in following pages 153 through 191 of Appendix A.
Obviously, many modifications and variations of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof, and, therefore, only such limitations should be imposed as are indicated by the appended claims .
APPENDIX A
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001

Claims

19:
WHAT IS CLAIMED IS:
A compound having a formula
(R°) -m2-x
Figure imgf000193_0001
wherein R°, independently, is selected from the group consisting of halo, C.-6alkyl, C2_salkenyl,
Figure imgf000193_0002
, C3_8cycloalkyl , C3_8heterocycloalkyl , aryl, heteroaryl, C(=0)Ra, OC(=0)Ra, C(=0)ORa, C^- alkyleneNRaRb, C1_4alkyleneORa, C(=0)NRaRb, C(=0)OC1.3- alkyleneC(=0)Ra, 0Ra, NRaRD, NRaC1_4alkyleneNRaR , NRaC(=0)Rb, NRaC(=0)NRaR , N (S02C1.4alkyl) 2 , NRa(S02C1_4- alkyl) , nitro, trif luoromethyl , trif luoromethoxy, cyano, C1_3alkyleneCN, SRa, S02Ra, S02NRaRb, N02, and
■
Figure imgf000193_0003
or two R° groups are taken together to form a 5- or 6-membered nonaromatic ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur;
R1 is C-L.galkyl or halo;
R2 is selected from the group consisting of hydrogen, Chalky! , aryl, heteroaryl, C1_3alkylene- aryl , C1_3alkyleneheteroaryl , C3_8cycloalkyl , C3_8heter- ocycloalkyl , and
- CHC ( =0 ) OCH 3
CH20H
Y is selected from the group consisting of CH(R4), CH2CH(R4), CH(R4)CH2, NRC, C (=0) (CH2) j..2S (CH2) 0-2. O(CH2)0_4, NRcC(=0) (CH2)0_2, and SO2NRa(CH2)0_2, or Y is null;
R3 and R4, independently, are selected from the group consisting of hydrogen,
Figure imgf000194_0001
aryl, heteroaryl, and halo;
X is selected from the group consisting of hydrogen, OH, 0C1_3alkyl, cycloalkyl, CH(Rc)CH2OH, CH2CH(Rc)0H, NRaR\ NHCH (Rc) CH20H, NHCH2CH (Rc) OH, CH(RC)CH2NH2, CH2CH(RC)NH2, NHC(=0)Ra, NHC (=S) NHRa, NHC(=0)ORa, NHC(=0)C(=0)NRaRb, and NHC(=0)NHRa, or X is a bond between NR2 and an atom of ring A or ring B, or R2 and X are taken together to form an optionally substituted 5- or 6-membered nonaromatic ring containing one to three heteroatoms selected from oxygen, sulfur, and nitrogen;
A is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, or A is null ;
B is aryl or heteroaryl and is selected from the group consisting of an optionally substituted 5- or 6-membered aromatic ring and optionally substituted fused bicyclic and polycyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
Ra and Rb, independently, are selected from the group consisting of hydrogen, Chalky!, aryl, arylC1_3alkyl, C^alkylenearyl , heteroaryl, hetero- arylC-^alkyl, C1_3alkyleneheteroaryl , haloC1_6alkyl , C1_3alkoxyC1_6alkyl , C3_8cycloalkyl , C1_3alkyleneC3_8- cycloalkyl, C3_Bheterocycloalkyl , C^jalkyleneOC^- alkylenearyl, C1_4alkyleneOC1_3alkyl , C1_4alkyleneSi- (C1_3alkyl) 3, and C1_3alkyleneheterocycloalkyl , or Ra and Rb are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and optionally substituted with
Figure imgf000195_0001
C1_3alkylenearyl , and C(=0)NRaRb;
Rc is selected from the group consisting of hydrogen, aryl, heteroaryl, C1_6alkyl, C3_8cycloalkyl , C3_8heterocycloalkyl , C1_3alkylenearyl , C1_3alkyleneC3_8- heterocycloalkyl , C1_3alkyleneheteroaryl , arylC^- alkyl, and heteroarylC1_3alkyl ; n is 0, 1, 2 , 3 , or 4 ; p is 0 or 1; and pharmaceutically acceptable salts or solvates thereof.
2. The compound of claim 1 having a structure
Figure imgf000196_0001
The compound of claim 1 having a structure
Figure imgf000196_0002
The compound of claim 1 having a structure
Figure imgf000196_0003
5. The compound of claim 1 wherein n is 0, or R°, independently, is selected from the group consisting of C._6alkyl, aryl, heteroaryl, CN, ORa, C^alkyleneOR3, C(=0)ORa, C1_4alkyleneNRaRb, OC(=0)Ra, C(=0)Ra, S02NRaRb, NRaRb, C3_8cycloalkyl , C(=0)NRaRb,
CH3
Figure imgf000197_0001
and
Figure imgf000197_0002
or two R° groups are taken together to form with the B ring,
Figure imgf000197_0003
wherein q is 1 or 2 , and G, independently, is C(Ra)2, 0, S, or NRa .
6. The compound of claim 5 wherein
Figure imgf000198_0001
d from the group consisting of
>
Figure imgf000198_0002
, and
Figure imgf000198_0003
7. The compound of claim 1 wherein A and B, independently, are selected from the group consisting of phenyl, furanyl , thienyl, pyrrolyl, oxa- zolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl , 1 , 2 , 3-oxadiazolyl , 1 , 2 , 3 -triazolyl ,
1, 3, 4-thiadiazolyl, 1 , 2 , 4-oxadiazolyl , 1,2,5-oxadi- azolyl, 1 , 3 , 4-oxadiazolyl , 1, 2 , 3 , 4-oxatriazolyl , 1 , 2 , 3 , 5-oxatriazolyl , pyridinyl , pyridazinyl, pyrirnidinyl, pyrazinyl, 1 , 3 , 5-triazinyl , 1,2, 4-triazinyl, and 1, 2 , 3-triazinyl .
8. The compound of claim 1 wherein B is selected from the group consisting of indolizinyl, indolyl , isoindolyl, benzo [b] furanyl , benzo [b] thienyl , lH-indazolyl , benzimidazolyl , benzthiazoyl, purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, indenyl , pyrenyl , and naphthyl .
9. The compound of claim 1 wherein p is 0; R2, R3, and R4 each are hydrogen; and Rc is hydrogen or C1_3alkyl .
10. The compound of claim 1 wherein S (O)0_2 is S02.
11. The compound of claim 1 wherein Y is CH(R4) or NRC.
12. The compound of claim 11 wherein R3 is hydrogen, R4 is hydrogen, and Rc is hydrogen.
13. The compound of claim 1 wherein X is selected from the group consisting of hydrogen, OH, OC1.3alkyl/ CH2CH20H, NH2, NHCH2CH2OH, and CH2CH2NH2.
14. The compound of claim 1 wherein X is a bond between NR2 and an atom of ring A or ring B .
15. The compound of claim 1 wherein R2 and X are taken together to form a 5- or 6-membered nonaromatic ring.
16. The compound of claim 1 wherein Y is null.
17. The compound of claim 1 wherein Ra and Rb are taken together to form a 5- or 6-membered nonaromatic ring.
18. The compound of claim 1 wherein R° is selected from the group consisting of chloro, methyl, methoxy, benzyloxy, C (=0)NHCH2C6H5, CH2OCH2- C6H5, CN, S02N(CH3)2,
CH3
Figure imgf000201_0001
and
Figure imgf000201_0002
Rx is hydrogen; R2 is selected from the group consisting of hydrogen, Chalky1 , and aryl; S (O) 0_2 is S02; Y is CH(R4); R3 and R4, independently, are hydrogen, C1_3alkyl, phenyl, fluoro, or chloro; X is CH2CH2OH or CH2CH2NH2; A is selected from the group consisting of
Figure imgf000201_0003
Figure imgf000201_0004
Figure imgf000202_0001
N .
//
O— N
, and
Figure imgf000202_0002
and B is selected from the group consisting of
Figure imgf000202_0003
R£
Figure imgf000203_0001
Figure imgf000203_0002
Figure imgf000203_0003
Figure imgf000203_0004
Figure imgf000203_0005
Figure imgf000204_0001
Figure imgf000204_0002
Figure imgf000204_0003
Figure imgf000204_0004
, and
Figure imgf000204_0005
9. A compound having a structure
Figure imgf000205_0001
Figure imgf000205_0002
Figure imgf000205_0003
Figure imgf000206_0001
Figure imgf000206_0002
Figure imgf000206_0003
Figure imgf000206_0004
Figure imgf000207_0001
and pharmaceutically acceptable salts and solvates thereof.
20. A compound having a structure as set forth in Appendix A, and pharmaceutically acceptable salts or solvates thereof .
21. A compound selected from the group consisting of
Figure imgf000208_0001
Figure imgf000208_0002
Figure imgf000208_0003
Figure imgf000209_0001
Figure imgf000209_0002
Figure imgf000209_0003
Figure imgf000209_0004
Figure imgf000210_0001
Figure imgf000210_0002
Figure imgf000210_0003
Figure imgf000211_0001
Figure imgf000211_0002
Figure imgf000211_0003
Figure imgf000211_0004
Figure imgf000212_0001
Figure imgf000212_0002
Figure imgf000212_0003
Figure imgf000213_0001
Figure imgf000213_0002
H3C
Figure imgf000213_0003
Figure imgf000213_0004
Figure imgf000214_0001
Figure imgf000214_0002
Figure imgf000215_0001
Figure imgf000215_0002
Figure imgf000215_0003
and pharmaceutically acceptable salts or solvates thereof.
22. A compound of claim 1, 19, 20, or 21 having an IC50 value versus human recombinant PDE3B of about 10 μM or less.
23. A compound of claim 1, 19, 20, or 21 having an IC50 value versus human recombinant PDE3B of about 1 μM or less .
24. A compound of claim 1, 19, 20, or 21 having an IC50 value versus human recombinant PDE3B of about 700 picomolar to about 1,000 nM.
25. A compound of claim 1, 19, 20, or 21 having a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) of at least 5.
26. A compound of claim 1, 19, 20, or 21 having a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) of at least 10.
27. A compound of claim 1, 19, 20, or 21 having a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) of at least 20.
28. A compound of claim 1, 19, 20, or 21 having a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) of at least 30.
29. The compound of claim 22 wherein the compound has a ratio of IC50 (vs . human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) at least 5.
30. The compound of claim 23 wherein the compound has a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) at least 5.
31. The compound of claim 23 wherein the compound has a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) at least
15.
32. The compound of claim 22 wherein the compound has a ratio of ICS0 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) at least 20.
33. The compound of claim 24 wherein the compound has a ratio of IC50 (vs. human recombinant PDE3A) to IC50 (vs. human recombinant PDE3B) at least 5.
34. A pharmaceutical composition comprising a compound of claim 1, 19, 20, or 21, together with a pharmaceutically acceptable diluent or carrier.
35. A method of treating a male or female animal for a condition where inhibition of PDE3B is of a therapeutic benefit comprising administering to said animal an effective amount of a pharmaceutical composition comprising a compound of claim 1, 19, 20, or 21, and a pharmaceutically acceptable diluent or carrier.
36. The method of claim 35 wherein the condition is selected from the group consisting of obesity, diabetes, hyperlipidemia, an inflammatory disease, an autoimmune disorder, and organ transplant rejection.
37. A method of treating a condition where inhibition of PDE3B is of therapeutic benefit, in a human or a nonhuman animal body, comprising administering to said body a therapeutically effective amount of a compound of claim 1, 19, 20, or 21.
38. The method of claim 37 wherein adverse side effects associated with inhibition of PDE3A are eliminated or minimized.
39. The method of claim 37 wherein the condition is obesity or diabetes.
40. A method of treating a male or female animal suffering from a condition where inhibition of PDE3B is of a therapeutic benefit comprising administering a therapeutically effective amount of (a) a compound of claim 1, 19, 20, or 21, and (b) a second therapeutically active ingredient used in treatment of the condition.
41. The method of claim 40 wherein (a) and (b) are administered simultaneously, separately, or sequentially.
42. The method of claim 40 wherein the condition is obesity or diabetes.
43. A method of claim 42 wherein the second therapeutically active ingredient is a PDE4 inhibitor.
44. A kit for the treatment of obesity comprising a compound of claim 1, 19, 20, or 21, or a composition containing the same, packaged with instructions for administration of the compound, or composition, to a mammal to promote weight loss.
45. A method of inducing lipolysis in mammalian cells comprising contacting the mammalian cells with a compound of claim 1, 19, 20, or 21 at a concentration effective to stimulate lipolysis in the cells.
46. A method of inducing weight loss in a mammalian subject comprising administering to the mammalian subject a compound of claim 1, 19, 20, or 21 at a concentration effective to promote weight loss in the mammalian subject.
47. A method according to claim 46 wherein the mammalian subject is a human subject.
48. A method according to claim 47 wherein the subject is obese.
49. A method of treating a male or female animal for a condition where inhibition of PDE3A and PDE3B is of a therapeutic benefit comprising administering to said animal an effective amount of a pharmaceutical composition comprising a compound of claim 1, 19, 20, or 21, and a pharmaceutically acceptable diluent or carrier.
50. The method of claim 49 wherein the condition is selected from the group consisting of intermittent claudication, atherosclerosis, glomernlar nephritis, coronary restenosis, and thrombocythermin .
51. A method of preventing pregnancy comprising administering to said body a therapeutically effective amount of a compound of claim 1, 19, 20, or 21.
PCT/US2002/005624 2001-03-05 2002-02-26 Selective pde3b inhibitors and use of the same in therapy WO2002070469A2 (en)

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