Patent Application
20150284335
The present invention relates to certain benzoimidazole compounds, pharmaceutical compositions containing them, and methods of using them for the treatment of disease states, disorders, and conditions mediated by prolyl hydroxylase activity.
Cells respond to hypoxia by activating the transcription of genes involved in cell survival, oxygen delivery and utilization, angiogenesis, cellular metabolism, regulation of blood pressure, hematopoiesis, and tissue preservation. Hypoxia-inducible factors (HIFs) are key transcriptional regulators of these genes (Semenza et al., 1992, Mol Cell Biol., 12(12):5447-54; Wang et al., 1993, J Biol Chem., 268(29):21513-18; Wang et al., 1993, Proc Natl Acad Sci., 90:4304-08; Wang et al., 1995, J Biol Chem., 270(3):1230-37). Three forms of HIF-α have been described: HIF-1α, HIF-2α and HIF-3α (Scheuermann et al., 2007, Methods Enzymol., 435:3-24). Pairing of a HIFα sub-unit with HIF-1β forms a functional heterodimeric protein that subsequently recruits other transcriptional factors such as p300 and CBP (Semenza, 2001, Trends Mol Med., 7(8):345-50).
A family of highly conserved oxygen, iron, and 2-oxoglutarate-dependent prolyl hydroxylase (PHD) enzymes mediate the cells response to hypoxia via post-translational modification of HIF (Ivan et al., 2001, Science, 292:464-68; Jaakkola et al., 2001, Science, 292:468-72). Under normoxic conditions, PHD catalyzes the hydroxylation of two conserved proline residues within HIF. Von Hippel Lindau (VHL) protein binds selectively to hydroxylated HIF. The binding of VHL renders HIF a target for polyubiquitination by the E3 ubiquitin ligase complex and its subsequent degradation by the 26S proteasome (Ke et al., 2006, Mol Pharmacol. 70(5):1469-80; Semenza, Sci STKE., 2007, 407(cm8):1-3). As the affinity of PHD for oxygen is within the physiological range of oxygen and oxygen is a necessary co-factor for the reaction, PHD is inactivated when oxygen tension is reduced. In this way, HIF is rapidly degraded under normoxic conditions but accumulates in cells under hypoxic conditions or when PHD is inhibited.
Four isotypes of PHD have been described: PHD1, PHD2, PHD3, and PHD4 (Epstein et al., 2001, Cell, 107:43-54; Kaelin, 2005, Annu Rev Biochem., 74:115-28; Schmid et al., 2004, J Cell Mol Med., 8:423-31). The different isotypes are ubiquitously expressed but are differentially regulated and have distinct physiological roles in the cellular response to hypoxia. There is evidence that the various isotypes have different selectivity for the three different HIFα sub-types (Epstein et al., supra). In terms of cellular localization, PHD1 is primarily nuclear, PHD2 is primarily cytoplasmic, and PHD3 appears to be both cytoplasmic and nuclear (Metzen E, et al. 2003, J Cell Sci., 116(7):1319-26). PHD2 appears to be the predominant HIFα prolyl hydroxylase under normoxic conditions (Ivan et al., 2002. Proc Natl Acad Sci. USA, 99(21):13459-64; Berra et al., 2003, EMBO J., 22:4082-90). The three isotypes have a high degree of amino-acid homology and the active site of the enzyme is highly conserved.
The HIF target gene products are involved in a number of physiological and pathophysiological processes including but not limited to: erythropoiesis, angiogenesis, regulation of energy metabolism, vasomotor function, and cell apoptosis/proliferation. The first gene described as a HIF target was that encoding erythropoietin (EPO) (Wang et al., 1993, supra). It was recognized that a reduction in the oxygen carrying capacity of the blood is sensed in the kidney and that the kidney and liver respond by releasing more EPO, the hormone that stimulates red blood cell proliferation and maturation. EPO has a number of other important effects on non-hematopoietic cell types and has emerged as a key tissue-protective cytokine (Arcasoy, 2008, Br J Haematol., 141:14-31). Thus EPO is now implicated in wound healing and angiogenesis as well as the response of tissues to ischemic insult. Most of the enzymes involved in anaerobic glycolysis are encoded by HIF target genes and as a result glycolysis is increased in hypoxic tissues (Shaw, 2006, Curr Opin Cell Biol., 18(6):598-608). The known HIF target gene products in this pathway include but are not limited to: glucose transporters such as GLUT-1 (Ebert et al., 1995, J Biol Chem., 270(49):29083-89), enzymes involved in the break down of glucose to pyruvate such as hexokinase and phosphoglycerate kinase 1 (Firth et al., 1994, Proc Natl Acad Sci. USA, 91:6496-6500) as well as lactate dehydrogenase (Firth et al., supra). HIF target gene products are also involved in the regulation of cellular metabolism. For example, pyruvate dehydrogenase kinase-1 is a target HIF gene product and regulates the entry of pyruvate into the Kreb's cycle by reducing the activity of pyruvate dehydrogenase by phosphorylation (Kim et al., 2006, Cell Metab., 3:177-85; Papandreou et al., 2006, Cell Metab., 3:187-197). HIF target gene products are also involved in angiogenesis. For example, vascular endothelial growth factor (VEGF) (Liu et al., 1995, Circ Res., 77(3):638-43) is a known regulator of angiogenesis and vasculogenesis. HIF target gene products also function in the regulation of vascular tone and include heme oxygenase-1 (Lee et al., 1997, J Biol Chem., 272(9):5375-81). A number of HIF regulated gene products such as platelet-derived growth factor (PDGF) (Yoshida et al., 2006, J Neurooncol., 76(1):13-21), vascular endothelial growth factor (Breen, 2007, J Cell Biochem., 102(6):1358-67) and EPO (Arcasoy, supra) also function in the coordinated response to wound healing.
Targeted disruption of the prolyl hydroxylase (PHD) enzyme activity by small molecules has potential utility in the treatment of disorders of oxygen sensing and distribution. Examples include but are not limited to: anemia; sickle cell anemia; peripheral vascular disease; coronary artery disease; heart failure; protection of tissue from ischemia in conditions such as myocardial ischemia, myocardial infarction and stroke; preservation of organs for transplant; treatment of tissue ischemia by regulating and/or restoring blood flow, oxygen delivery and/or energy utilization; acceleration of wound healing particularly in diabetic and aged patients. In addition, targeted disruption of PHD is expected to have utility in treating metabolic disorders such as diabetes and obesity.
HIF has been shown to be the primary transcriptional factor that leads to increased erythropoietin production under conditions of hypoxia (Wang et al., 1993, supra). While treatment with recombinant human erythropoietin has been demonstrated to be an effective method of treating anemia, small molecule mediated PHD inhibition can be expected to offer advantages over treatment with erythropoietin. Specifically, the function of other HIF gene products are necessary for hematopoesis and regulation of these factors increases the efficiency of hematopoesis. Examples of HIF target gene products that are critical for hematopoesis include: transferrin (Rolfs et al., 1997, J Biol Chem., 272(32):20055-62), transferrin receptor (Lok et al., 1999, J Biol Chem., 274(34):24147-52; Tacchini et al., 1999, J Biol Chem., 274(34):24142-46) and ceruloplasmin (Mukhopadhyay et al., 2000, J Biol Chem., 275(28):21048-54). Hepcidin expression is also suppressed by HIF (Peyssonnaux et al., 2007, J Clin Invest., 117(7):1926-32) and small molecule inhibitors of PHD have been shown to reduce hepcidin production (Braliou et al., 2008, J Hepatol., 48:801-10). Hepcidin is a negative regulator of the availability of the iron that is necessary for hematopoesis, so a reduction in hepcidin production is expected to be beneficial to the treatment of anemia. PHD inhibition may also be useful when used in conjunction with other treatments for anemia including iron supplementation and/or exogenous erythropoietin. Studies of mutations in the PHD2 gene occurring naturally in the human population provide further evidence for the use of PHD inhibitors to treat anemia. Two recent reports have shown that patients with dysfunctional mutations in the PHD2 gene display increased erythrocytosis and elevated blood hemoglobin (Percy et al., 2007, PNAS, 103(3):654-59; A1-Sheikh et al., 2008, Blood Cells Mol Dis., 40:160-65). In addition, a small molecule PHD inhibitor has been evaluated in healthy volunteers and patients with chronic kidney disease (U.S. pat. appl. US2006/0276477, Dec. 7, 2006). Plasma erythropoietin was increased in a dose-dependent fashion and blood hemoglobin concentrations were increased in the chronic kidney disease patients.
Metabolic adaptation and preservation of tissues are jeopardized by ischemia. PHD inhibitors increase the expression of genes that lead to changes in metabolism that are beneficial under ischemic conditions (Semenza, 2007, Biochem J., 405:1-9). Many of the genes encoding enzymes involved in anaerobic glycolysis are regulated by HIF and glycolysis is increased by inhibiting PHD (Shaw, supra). Known HIF target genes in this pathway include but are not limited to: GLUT-1 (Ebert et al., supra), hexokinase, phosphoglycerate kinase 1, lactate dehydrogenase (Firth et al., supra), pyruvate dehydrogenase kinase-1 (Kim et al., supra; Papandreou et al., supra). Pyruvate dehydrogenase kinase-1 suppresses the entry of pyruvate into the Kreb's cycle. HIF mediates a switch in the expression of the cytochromes involved in electron transport in the mitochondria (Fukuda et al., 2007, Cell, 129(1):111-22). This change in the cytochrome composition optimizes the efficiency in ATP production under hypoxic conditions and reduces the production of injurious oxidative phosphorylation by-products such as hydrogen peroxide and superoxide. With prolonged exposure to hypoxia, HIF drives autophagy of the mitochondria resulting a reduction in their number (Zhang H et al., 2008, J Biol Chem., February 15 Epub ahead of print). This adaptation to chronic hypoxia reduces the production of hydrogen peroxide and superoxide while the cell relies on glycolysis to produce energy. A further adaptive response produced by HIF elevation is up-regulation of cell survival factors. These factors include: Insulin-like growth factor (IGF) 2, IGF-binding protein 2 and 3 (Feldser et al., 1999, Cancer Res. 59:3915-18). Overall accumulation of HIF under hypoxic conditions governs an adaptive up-regulation of glycolysis, a reduction in oxidative phosphorylation resulting in a reduction in the production of hydrogen peroxide and superoxide, optimization of oxidative phosphorylation protecting cells against ischemic damage. Thus, PHD inhibitors are expected to be useful in organ and tissue transplant preservation (Bernhardt et al., 2007, Methods Enzymol., 435:221-45). While benefit may be achieved by administering PHD inhibitors before harvesting organs for transplant, administration of an inhibitor to the organ/tissue after harvest, either in storage (e.g., cardioplegia solution) or post-transplant, may also be of therapeutic benefit.
PHD inhibitors are expected to be effective in preserving tissue from regional ischemia and/or hypoxia. This includes ischemia/hypoxia associated with inter alia: angina, myocardial ischemia, stroke, ischemia of skeletal muscle. There are a number of lines of experimental evidence that support the concept that PHD inhibition and subsequent elevation of HIF as a useful method for preserving ischemic tissue. Recently, ischemic pre-conditioning has been demonstrated to be a HIF-dependent phenomenon (Cai et al., 2008, Cardiovasc Res., 77(3):463-70). Ischemic pre-conditioning is a well known phenomenon whereby short periods of hypoxia and/or ischemia protect tissue from subsequent longer periods of ischemia (Murry et al., 1986, Circulation, 1986 74(5):1124-36; Das et al., 2008, IUBMB Life, 60(4):199-203). Ischemic pre-conditioning is known to occur in humans as well as experimental animals (Darling et al., 2007, Basic Res Cardiol., 102(3):274-8; Kojima I et al., 2007, J Am Soc Nephrol., 18:1218-26). While the concept of pre-conditioning is best known for its protective effects in the heart, it also applies to other tissues including but not limited to: liver, skeletal muscle, liver, lung, kidney, intestine and brain (Pasupathy et al., 2005, Eur J Vasc Endovasc Surg., 29:106-15; Mallick et al., 2004, Dig Dis Sci., 49(9):1359-77). Experimental evidence for the tissue protective effects of PHD inhibition and elevation of HIF have been obtained in a number of animal models including: germ-line knock out of PHD1 which conferred protection of the skeletal muscle from ischemic insult (Aragonés et al., 2008, Nat Genet., 40(2):170-80), silencing of PHD2 through the use of siRNA which protected the heart from ischemic insult (Natarajan et al., 2006, Circ Res., 98(1):133-40), inhibition of PHD by administering carbon monoxide which protected the myocardium from ischemic injury (Chin et al., 2007, Proc Natl Acad Sci. U.S.A., 104(12):5109-14), hypoxia in the brain which increased the tolerance to ischemia (Bernaudin et al., 2002, J Cereb Blood Flow Metab., 22(4):393-403). In addition, small molecule inhibitors of PHD protect the brain in experimental stroke models (Siddiq et al., 2005, J Biol Chem., 280(50):41732-43). Moreover, HIF up-regulation has also been shown to protect the heart of diabetic mice, where outcomes are generally worse (Natarajan et a., 2008, J Cardiovasc Pharmacol., 51(2):178-187). The tissue protective effects may also be observed in Buerger's disease, Raynaud's disease, and acrocyanosis.
The reduced reliance on aerobic metabolism via the Kreb's cycle in the mitochondria and an increased reliance on anaerobic glycolysis produced by PHD inhibition may have beneficial effects in normoxic tissues. It is important to note that PHD inhibition has also been shown to elevate HIF under normoxic conditions. Thus, PHD inhibition produces a pseudohypoxia associated with the hypoxic response being initiated through HIF but with tissue oxygenation remaining normal. The alteration of metabolism produced by PHD inhibition can also be expected to provide a treatment paradigm for diabetes, obesity and related disorders, including co-morbidities.
Globally, the collection of gene expression changes produced by PHD inhibition reduce the amount of energy generated per unit of glucose and will stimulate the body to burn more fat to maintain energy balance. The mechanisms for the increase in glycolysis are discussed above. Other observations link the hypoxic response to effects that are expected to be beneficial for the treatment of diabetes and obesity. Thus, high altitude training is well known to reduce body fat (Armellini et al., 1997, Horm Metab Res., 29(9):458-61). Hypoxia and hypoxia mimetics such as desferrioxamine have been shown to prevent adipocyte differentiation (Lin et al., 2006, J Biol Chem., 281(41):30678-83; Carrière et al., 2004, J Biol Chem., 279(39):40462-69). The effect is reversible upon returning to normoxic conditions. Inhibition of PHD activity during the initial stages of adipogenesis inhibits the formation of new adipocytes (Floyd et al., 2007, J Cell Biochem., 101:1545-57). Hypoxia, cobalt chloride and desferrioxamine elevated HIF and inhibited PPAR gamma 2 nuclear hormone receptor transcription (Yun et al., 2002, Dev Cell., 2:331-41). As PPAR gamma 2 is an important signal for adipocyte differentiation, PHD inhibition can be expected to inhibit adipocyte differentiation. These effects were shown to be mediated by the HIF-regulated gene DEC1/Stra13 (Yun et al., supra).
Small molecular inhibitors of PHD have been demonstrated to have beneficial effects in animal models of diabetes and obesity (Intl. Pat. Appl. Publ. WO2004/052284, Jun. 24, 2004; WO2004/052285, Jun. 24, 2004). Among the effects demonstrated for PHD inhibitors in mouse diet-induced obesity, db/db mouse and Zucker fa/fa rat models were lowering of: blood glucose concentration, fat mass in both abdominal and visceral fat pads, hemoglobin A1c, plasma triglycerides, body weight as well as changes in established disease bio-markers such as increases in the levels of adrenomedullin and leptin. Leptin is a known HIF target gene product (Grosfeld et al., 2002, J Biol Chem., 277(45):42953-57). Gene products involved in the metabolism in fat cells were demonstrated to be regulated by PHD inhibition in a HIF-dependent fashion (Intl. Pat. Appl. Publ. WO2004/052285, supra). These include apolipoprotein A-IV, acyl CoA thioesterase, carnitine acetyl transferase, and insulin-like growth factor binding protein (IGFBP)-1.
PHD inhibitors are expected to be therapeutically useful as stimulants of vasculogenesis, angiogenesis, and arteriogenesis. These processes establish or restore blood flow and oxygenation to the tissues under ischemia and/or hypoxia conditions (Semenza et al., 2007, J Cell Biochem., 102:840-47; Semenza, 2007, Exp Physiol., 92(6):988-91). It has been shown that physical exercise increases HIF-1 and vascular endothelial growth factor in experimental animal models and in humans (Gustafsson et al. 2001, Front Biosci., 6:D75-89) and consequently the number of blood vessels in skeletal muscle. VEGF is a well-known HIF target gene product that is a key driver of angiogenesis (Liu et al., supra). While administration of various forms of VEGF receptor activators are potent stimuli for angiogenesis, the blood vessel resulting from this potential form of therapy are leaky. This is considered to limit the potentially utility of VEGF for the treatment of disorders of oxygen delivery. The increased expression of a single angiogenic factor may not be sufficient for functional vascularization (Semenza, 2007, supra). PHD inhibition offers a potential advantage over other such angiogenic therapies in that it stimulates a controlled expression of multiple angiogenic growth factors in a HIF-dependent fashion including but not limited to: placental growth factor (PLGF), angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), platelet-derived growth factor beta (PDGFB) (Carmeliet, 2004, J Intern Med., 255:538-61; Kelly et al., 2003, Circ Res., 93:1074-81) and stromal cell derived factor 1 (SDF-1) (Ceradini et al., 2004, Nat Med., 10(8):858-64). Expression of angiopoietin-1 during angiogenesis produces leakage-resistant blood vessels, in contrast to the vessels produced by administration of VEGF alone (Thurston et al., 1999, Science, 286:2511-14; Thurston et al., 2000, Nat Med., 6(4):460-3; Elson et al., 2001, Genes Dev., 15(19):2520-32). Stromal cell derived factor 1 (SDF-1) has been shown to be critical to the process of recruiting endothelial progenitor cells to the sites of tissue injury. SDF-1 expression increased the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Furthermore inhibition of SDF-1 in ischemic tissue or blockade of CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury (Ceradini et al., 2004, supra; Ceradini et al., 2005, Trends Cardiovasc Med., 15(2):57-63). Importantly, the recruitment of endothelial progenitor cells to sites of injury is reduced in aged mice and this is corrected by interventions that increase HIF at the wound site (Chang et al., 2007, Circulation, 116(24):2818-29). PHD inhibition offers the advantage not only of increasing the expression of a number of angiogenic factions but also a co-ordination in their expression throughout the angiogenesis process and recruitment of endothelial progenitor cells to ischemic tissue.
Evidence for the utility of PHD inhibitors as pro-angiogenic therapies is provided by the following observations. Adenovirus-mediated over-expression of HIF has been demonstrated to induce angiogenesis in non-ischemic tissue of an adult animal (Kelly et al., 2003, Circ Res., 93(11):1074-81) providing evidence that therapies that elevate HIF, such as PHD inhibition, will induce angiogenesis. Placental growth factor (PLGF), also a HIF target gene, has been show to play a critical role in angiogenesis in ischemic tissue (Carmeliet, 2004, J Intern Med., 255(5):538-61; Luttun et al., 2002, Ann N Y Acad Sci., 979:80-93). The potent pro-angiogenic effects of therapies that elevate HIF have been demonstrated, via HIF over-expression, in skeletal muscle (Pajusola et al., 2005, FASEB J., 19(10):1365-7; Vincent et al., 2000, Circulation, 102:2255-61) and in the myocardium (Shyu et al., 2002, Cardiovasc Res., 54:576-83). The recruitment of endothelial progenitor cells to the ischemic myocardium by the HIF target gene SDF-1 has also been demonstrated (Abbott et al., 2004, Circulation, 110(21):3300-05). These findings support the general concept that PHD inhibitors will be effective in stimulating angiogenesis in the setting of tissue ischemia, particularly muscle ischemia. It is expected that therapeutic angiogenesis produced by PHD inhibitors will be useful in restoring blood flow to tissues and therefore the treatment of disease including but not restricted to angina pectoris, myocardial ischemia and infarction, peripheral ischemic disease, claudication, gastric and duodenal ulcers, ulcerative colitis.
PHD and HIF play a central role in tissue repair and regeneration including healing of wounds and ulcers. Recent studies have demonstrated that an increased expression of all three PHDs at wound sites in aged mice with a resulting reduction in HIF accumulation (Chang et al., supra). Thus, elevation of HIF in aged mice by administering desferrioxamine increased the degree of wound healing back to levels observed in young mice. Similarly, in a diabetic mouse model, HIF elevation was suppressed compared to non-diabetic litter mates (Mace et al., 2007, Wound Repair Regen., 15(5):636-45). Topical administration of cobalt chloride, a hypoxia mimetic, or over-expression of a murine HIF that lacks the oxygen-dependent degradation domain and thus provides for a constitutively active form of HIF, resulted in increased HIF at the wound site, increased expression of HIF target genes such as VEGF, Nos2, and Hmox1 and accelerated wound healing. The beneficial effect of PHD inhibition is not restricted to the skin and small molecule inhibitors of PHD have recently been demonstrated to provide benefit in a mouse model of colitis (Robinson et al., 2008, Gastroenterology, 134(1):145-55).
PHD inhibition resulting in accumulation of HIF is expected to act by at least four mechanisms to contribute to accelerated and more complete healing of wounds: 1) protection of tissue jeopardized by hypoxia/ischemia, 2) stimulation of angiogenesis to establish or restore appropriate blood flow to the site, 3) recruitment of endothelial progenitor cells to wound sites, 4) stimulation of the release of growth factors that specifically stimulate healing and regeneration.
Recombinant human platelet-derived growth factor (PDGF) is marketed as becaplermin (Regranex™) and has been approved by the Food and Drug Administration of the United States of America for “Treatment of lower extremity diabetic neuropathic ulcers that extend into the subcutaneous tissue or beyond, and have adequate blood supply”. Becaplermin has been shown to be effective in accelerating wound healing in diabetic patients (Steed, 2006, Plast Reconstr Surg., 117(7 Suppl):1435-1495; Nagai et al., 2002, Expert Opin Biol Ther., 2(2):211-8). As PDGF is a HIF gene target (Schultz et al., 2006, Am J Physiol Heart Circ Physiol., 290(6):H2528-34; Yoshida et al., 2006, J Neurooncol., 76(1):13-21), PHD inhibition is expected to increase the expression of endogenous PDGF and produce a similar or more beneficial effect to those produced with becaplermin alone. Studies in animals have shown that topical application of PDGF results in increased wound DNA, protein, and hydroxyproline amounts; formation of thicker granulation and epidermal tissue; and increased cellular repopulation of wound sites. PDGF exerts a local effect on enhancing the formation of new connective tissue. The effectiveness of PHD inhibition is expected to be greater than that produced by becaplermin due to the additional tissue protective and pro-angiogenic effects mediated by HIF.
The beneficial effects of inhibition of PHD are expected to extend not only to accelerated wound healing in the skin and colon but also to the healing of other tissue damage including but not limited to gastrointestinal ulcers, skin graft replacements, burns, chronic wounds and frost bite.
Stem cells and progenitor cells are found in hypoxic niches within the body and hypoxia regulates their differentiation and cell fate (Simon et al., 2008, Nat Rev Mol Cell Biol., 9:285-96). Thus PHD inhibitors may be useful to maintain stem cells and progenitor cells in a pluripotent state and to drive differentiation to desired cell types. Stem cells may be useful in culturing and expanding stem cell populations and may hold cells in a pluripotent state while hormones and other factors are administered to the cells to influence the differentiation and cell fate.
A further use of PHD inhibitors in the area of stem cell and progenitor cell therapeutics relates to the use of PHD inhibitors to condition these cells to withstand the process of implantation into the body and to generate an appropriate response to the body to make the stem cell and progenitor cell implantation viable (Hu et al., 2008, J Thorac Cardiovasc Surg., 135(4):799-808). More specifically PHD inhibitors may facilitate the integration of stem cells and draw in an appropriate blood supply to sustain the stem cells once they are integrated. This blood vessel formation will also function to carry hormones and other factors released from these cells to the rest of the body.
Certain small molecules with Prolyl Hydroxylase antagonistic activities have been described in the literature. These include, but are not limited to, certain imidazo[1,2-a]pyridine derivatives (Warshakoon et al., 2006, Bioorg Med Chem Lett., 16(21):5598-601), substituted pyridine derivatives (Warshakoon et al., 2006, Bioorg Med Chem Lett., 16(21):5616-20), certain pyrazolopyridines (Warshakoon et al., 2006, Bioorg Med Chem Lett., 16(21):5687-90), certain bicyclic heteroaromatic N-substituted glycine derivatives (Intl. Pat. Appl. Publ. WO2007/103905, Sep. 13, 2007), quinoline based compounds (Intl. Pat. Appl. Publ. WO2007/070359, Jun. 21, 2007), certain pyrimidinetrione N-substituted glycine derivatives (Intl. Pat. Appl. Publ. WO2007/150011, Dec. 27, 2007), and substituted aryl or heteroaryl amide compounds (U.S. Pat. Appl. Publ. No.: US 2007/0299086, Dec. 27, 2007). However, there remains a need for potent prolyl hydroxylase modulators with desirable pharmaceutical properties. Certain benzoimidazole derivatives have been found in the context of this invention to have prolyl hydroxylase modulating activity.
Certain benzoimidazole derivatives have now been found to have prolyl hydroxylase modulating activity. Thus, the invention is directed to the general and preferred embodiments defined, respectively, by the independent and dependent claims appended hereto, which are incorporated by reference herein.
In one general aspect the invention relates to a compound of the following Formula (I):
wherein
X is an optionally substituted heteroaryl selected from pyrazole, pyridine, pyrimidine, and pyridazine;
A is a bond or optionally substituted methylene;
each Z is independently Carbon or Nitrogen;
n is 0-4; and
R1 is independently selected from hydrogen, halogen, nitro, —C1-4alkyl, alkoxy, cycloalkoxy, CON(Ry)(Rz), trifluoromethyl, trifluoromethoxy, N(Ry)Rz (wherein Ry and Rz are independently hydrogen, aryl, arylsulfonyl, —C1-6alkyl, and —C1-6alkenyl, or Ry and Rz may be taken together with the nitrogen of attachment to form an otherwise aliphatic hydrocarbon ring, said ring being optionally substituted and having 4 to 7 members, optionally having one carbon replaced with >O, ═N, >NH, or >N(C1-4alkyl), and optionally substituted Ar wherein said Ar is aryl or heteroaryl.
In another aspect, the invention relates to a compound of the following Formula (II):
wherein
each Z is independently Carbon or Nitrogen;
n is 0-4;
R1 is independently selected from hydrogen, halogen, nitro, —C1-4alkyl, alkoxy, cycloalkoxy, CON(Ry)(Rz), trifluoromethyl, trifluoromethoxy, N(Ry)Rz (wherein Ry and Rz are independently hydrogen, aryl, arylsulfonyl, —C1-6alkyl, and —C1-6alkenyl, or Ry and Rz may be taken together with the nitrogen of attachment to form an otherwise aliphatic hydrocarbon ring, said ring being optionally substituted and having 4 to 7 members, optionally having one carbon replaced with >O, ═N, >NH, or >N(C1-4alkyl), and optionally substituted Ar wherein said Ar is aryl or heteroaryl; and
R2 is hydrogen, hydroxymethyl or (C1-4)alkyl.
The invention also relates to pharmaceutically acceptable salts, pharmaceutically acceptable prodrugs, and pharmaceutically active metabolites of compounds of Formula (I) and/or Formula (II). In certain preferred embodiments, the compound of Formula (I) and/or Formula (II) is a compound selected from those species described or exemplified in the detailed description below.
In a further general aspect, the invention relates to pharmaceutical compositions each comprising: (a) an effective amount of a compound of Formula (I) and/or Formula (II), or a pharmaceutically acceptable salt, pharmaceutically acceptable prodrug, or pharmaceutically active metabolite thereof; and (b) a pharmaceutically acceptable excipient.
In another general aspect, the invention is directed to a method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by a prolyl hydroxylase enzyme activity, comprising administering to the subject in need of such treatment an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, pharmaceutically acceptable prodrug, or pharmaceutically active metabolite thereof.
In certain preferred embodiments of the inventive method, the disease, disorder, or medical condition is selected from: anemia, vascular disorders, metabolic disorders, and wound healing.
Additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.
The invention may be more fully appreciated by reference to the following description, including the following glossary of terms and the concluding examples. For the sake of brevity, the disclosures of the publications, including patents, cited in this specification are herein incorporated by reference.
As used herein, the terms “including”, “containing” and “comprising” are used herein in their open, non-limiting sense.
The term “alkyl” refers to a straight- or branched-chain alkyl group having from 1 to 12 carbon atoms in the chain. Examples of alkyl groups include methyl (Me, which also may be structurally depicted by the symbol, “/”), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and groups that in light of the ordinary skill in the art and the teachings provided herein would be considered equivalent to any one of the foregoing examples.
The term “cycloalkyl” refers to a saturated or partially saturated, monocyclic, fused polycyclic, or spiro polycyclic carbocycle having from 3 to 12 ring atoms per carbocycle. Illustrative examples of cycloalkyl groups include the following entities, in the form of properly bonded moieties:
A “heterocycloalkyl” refers to a monocyclic ring structure that is saturated or partially saturated and has from 4 to 7 ring atoms per ring structure selected from carbon atoms and up to two heteroatoms selected from nitrogen, oxygen, and sulfur. The ring structure may optionally contain up to two oxo groups on sulfur ring members. Illustrative entities, in the form of properly bonded moieties, include:
The term “heteroaryl” refers to a monocyclic, fused bicyclic, or fused polycyclic aromatic heterocycle (ring structure having ring atoms selected from carbon atoms and up to four heteroatoms selected from nitrogen, oxygen, and sulfur) having from 3 to 12 ring atoms per heterocycle. Illustrative examples of heteroaryl groups include the following entities, in the form of properly bonded moieties:
Those skilled in the art will recognize that the species of cycloalkyl, heterocycloalkyl, and heteroaryl groups listed or illustrated above are not exhaustive, and that additional species within the scope of these defined terms may also be selected.
The term “halogen” represents chlorine, fluorine, bromine or iodine. The term “halo” represents chloro, fluoro, bromo or iodo.
The term “substituted” means that the specified group or moiety bears one or more substituents. The term “unsubstituted” means that the specified group bears no substituents. The term “optionally substituted” means that the specified group is unsubstituted or substituted by one or more substituents. Where the term “substituted” is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system. In cases where a specified moiety or group is not expressly noted as being optionally substituted or substituted with any specified substituent, it is understood that such a moiety or group is intended to be unsubstituted.
Any formula given herein is intended to represent compounds having structures depicted by the structural formula as well as certain variations or forms. In particular, compounds of any formula given herein may have asymmetric centers and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the compounds of the general formula, and mixtures thereof, are considered within the scope of the formula. Thus, any formula given herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms, and mixtures thereof. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. Additionally, any formula given herein is intended to embrace hydrates, solvates, and polymorphs of such compounds, and mixtures thereof.
Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, 36Cl, 125I, respectively. Such isotopically labeled compounds are useful in metabolic studies (preferably with 14C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)] including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an 18F or 11C labeled compound may be particularly preferred for PET or SPECT studies. Further, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
When referring to any formula given herein, the selection of a particular moiety from a list of possible species for a specified variable is not intended to define the moiety for the variable appearing elsewhere. In other words, where a variable appears more than once, the choice of the species from a specified list is independent of the choice of the species for the same variable elsewhere in the formula.
Chemical depictions are intended to portray the compound portions containing the orientations as written.
The instant invention includes the use of compounds of Formula (I) and/or Formula (II) and pharmaceutical compositions containing such compounds thereof to treat patients (humans or other mammals) with disorders related to the modulation of the prolyl hydroxylase enzyme. The instant invention also includes methods of making such a compound, pharmaceutical composition, pharmaceutically acceptable salt, pharmaceutically acceptable prodrug, and pharmaceutically active metabolites thereof.
In preferred embodiments of Formula (I), X is selected from the group consisting of an optionally substituted pyrazole, pyridine, pyrimidine, and pyridazine. In a related embodiment, X is an unsubstituted pyrazole. In an additional embodiment, X can be an optionally substituted pyridine, pyrimidine, or pyridazine wherein said pyridine, pyrimidine, or pyridazine is substituted with one or more substituents independently selected from hydrogen, halo, hydroxyl, methyl or CF3.
In one embodiment, A is a bond or methlyene optionally substituted with hydrogen, hydroxymethyl or —C1-4alkyl. In further preferred embodiment, A is a bond.
In preferred embodiments for Formula (I) and/or Formula (II), each R1 is independently selected from hydrogen, halogen, nitro, —C1-4alkyl, alkoxy, cycloalkoxy, CON(Ry)(Rz), trifluoromethyl, trifluoromethoxy, N(Ry)Rz (wherein Ry and Rz are independently hydrogen, aryl, arylsulfonyl, —C1-6alkyl, and —C1-6alkenyl, or Ry and Rz may be taken together with the nitrogen of attachment to form an otherwise aliphatic hydrocarbon ring, said ring being optionally substituted and having 4 to 7 members, optionally having one carbon replaced with >O, ═N, >NH, or >N(C1-4alkyl), and optionally substituted Ar wherein said Ar is aryl or heteroaryl.
In preferred embodiments of Formula (I) and/or Formula (II), each R1 is independently selected from the group consisting of hydrogen, —C1-4alkyl, halo, —C1-4alkoxy, —NO2, 2-hydroxy-phenyl, 3-hydroxy-phenyl, 4-hydroxy-phenyl, trifluoromethyl, trifluoromethoxy, 3-hydroxyphenyl, 4-hydroxyphenyl, 3-benzyloxy-phenyl, 3-(2-chloro-benzyloxy)-phenyl, 3-(3-chloro-benzyloxy)-phenyl, 3-(4-chloro-benzyloxy)-phenyl, quinolin-3-yl, 4-(2-fluoro-benzyloxy)-phenyl, 4-(3-fluoro-benzyloxy)-phenyl, 4-(4-fluoro-benzyloxy)-phenyl, 4-(3-chloro-benzyloxy)-phenyl, 4-benzyloxy-3-fluoro-phenyl, 4-benzyloxy-2-fluoro-phenyl, 4-phenoxy-phenyl, benzyl-amine, 2-naphthalen-2-yl-phenyl, 3-naphthalen-2-yl-phenyl, 4-naphthalen-2-yl-phenyl, 4-(2-chloro-benzyloxy)-3,5-dimethyl-phenyl, 4-(3-chloro-benzyloxy)-3,5-dimethyl-phenyl, 4-(4-chloro-benzyloxy)-3,5-dimethyl-phenyl, 4-propoxy-phenyl, 5-chloro-2-fluoro-phenyl, benzenesulfonamide, 2-methoxy-phenyl, 3-methoxy-phenyl, 4-methoxy-phenyl, or 4-chloro-3-methyl-phenyl.
In another embodiment of Formula (I) and/or Formula (II), individual R1 comprises an electron withdrawing group. In a further embodiment, said electron withdrawing
group is in the X position according to the formula
In preferred embodiments of Formula (II), R2 is hydrogen, hydroxymethyl, or —C1-4alkyl.
In certain preferred embodiments, the compound of Formula (I) or Formula (II) is selected from the group consisting of:
The invention includes also pharmaceutically acceptable salts of the compounds of Formula (I), preferably of those described above and of the specific compounds exemplified herein, and methods of treatment using such salts.
A “pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base of a compound represented by Formula (I) that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, S. M. Berge, et al., “Pharmaceutical Salts”, J Pharm Sci., 1977, 66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Examples of pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of patients without undue toxicity, irritation, or allergic response. A compound of Formula (I) may possess a sufficiently acidic group, a sufficiently basic group, or both types of functional groups, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
If the compound of Formula (I) contains a basic nitrogen, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as mandelic acid, citric acid, or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, a sulfonic acid, such as laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, any compatible mixture of acids such as those given as examples herein, and any other acid and mixture thereof that are regarded as equivalents or acceptable substitutes in light of the ordinary level of skill in this technology.
If the compound of Formula (I) is an acid, such as a carboxylic acid or sulfonic acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide, alkaline earth metal hydroxide, any compatible mixture of bases such as those given as examples herein, and any other base and mixture thereof that are regarded as equivalents or acceptable substitutes in light of the ordinary level of skill in this technology. Illustrative examples of suitable salts include organic salts derived from amino acids, such as glycine and arginine, ammonia, carbonates, bicarbonates, primary, secondary, and tertiary amines, and cyclic amines, such as benzylamines, pyrrolidines, piperidine, morpholine, and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
The invention also relates to pharmaceutically acceptable prodrugs of the compounds of Formula (I), and treatment methods employing such pharmaceutically acceptable prodrugs. The term “prodrug” means a precursor of a designated compound that, following administration to a subject, yields the compound in vivo via a chemical or physiological process such as solvolysis or enzymatic cleavage, or under physiological conditions (e.g., a prodrug on being brought to physiological pH is converted to the compound of Formula (I)). A “pharmaceutically acceptable prodrug” is a prodrug that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to the subject. Illustrative procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.
Exemplary prodrugs include compounds having an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues, covalently joined through an amide or ester bond to a free amino, hydroxy, or carboxylic acid group of a compound of Formula (I). Examples of amino acid residues include the twenty naturally occurring amino acids, commonly designated by three letter symbols, as well as 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.
Additional types of prodrugs may be produced, for instance, by derivatizing free carboxyl groups of structures of Formula (I) as amides or alkyl esters. Examples of amides include those derived from ammonia, primary C1-6alkyl amines and secondary di(C1-6alkyl) amines. Secondary amines include 5- or 6-membered heterocycloalkyl or heteroaryl ring moieties. Examples of amides include those that are derived from ammonia, C1-3alkyl primary amines, and di(C1-2alkyl)amines. Examples of esters of the invention include C1-7alkyl, C5-7cycloalkyl, phenyl, and phenyl(C1-6alkyl) esters. Preferred esters include methyl esters. Prodrugs may also be prepared by derivatizing free hydroxy groups using groups including hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, following procedures such as those outlined in Adv. Drug Delivery Rev. 1996, 19, 115. Carbamate derivatives of hydroxy and amino groups may also yield prodrugs. Carbonate derivatives, sulfonate esters, and sulfate esters of hydroxy groups may also provide prodrugs. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group may be an alkyl ester, optionally substituted with one or more ether, amine, or carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, is also useful to yield prodrugs. Prodrugs of this type may be prepared as described in J Med Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including ether, amine, and carboxylic acid functionalities.
The present invention also relates to pharmaceutically active metabolites of the compounds of Formula (I), which may also be used in the methods of the invention. A “pharmaceutically active metabolite” means a pharmacologically active product of metabolism in the body of a compound of Formula (I) or salt thereof. Prodrugs and active metabolites of a compound may be determined using routine techniques known or available in the art. See, e.g., Bertolini, et al., J Med Chem. 1997, 40, 2011-2016; Shan, et al., J Pharm Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev Res. 1995, 34, 220-230; Bodor, Adv Drug Res. 1984, 13, 224-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen, et al., eds., Harwood Academic Publishers, 1991).
The compounds of Formula (I) and their pharmaceutically acceptable salts, pharmaceutically acceptable prodrugs, and pharmaceutically active metabolites of the present invention are useful as modulators of PHD in the methods of the invention. As such modulators, the compounds may act as antagonists, agonists, or inverse agonists. “Modulators” include both inhibitors and activators, where “inhibitors” refer to compounds that decrease, prevent, inactivate, desensitize or down-regulate PHD expression or activity, and “activators” are compounds that increase, activate, facilitate, sensitize, or up-regulate PHD expression or activity.
The term “treat” or “treating” as used herein is intended to refer to administration of an active agent or composition of the invention to a subject for the purpose of effecting a therapeutic or prophylactic benefit through modulation of prolyl hydroxylase activity. Treating includes reversing, ameliorating, alleviating, inhibiting the progress of, lessening the severity of, or preventing a disease, disorder, or condition, or one or more symptoms of such disease, disorder or condition mediated through modulation of PHD activity. The term “subject” refers to a mammalian patient in need of such treatment, such as a human.
Accordingly, the invention relates to methods of using the compounds described herein to treat subjects diagnosed with or suffering from a disease, disorder, or condition mediated by Prolyl Hydroxylase, such as: Anemia, vascular disorders, metabolic disorders, and wound healing. Symptoms or disease states are intended to be included within the scope of “medical conditions, disorders, or diseases.”
In a preferred embodiment, molecules of the present invention are useful in the treatment or prevention of anemia comprising treatment of anemic conditions associated with chronic kidney disease, polycystic kidney disease, aplastic anemia, autoimmune hemolytic anemia, bone marrow transplantation anemia, Churg-Strauss syndrome, Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome, graft versus host disease, hematopoietic stem cell transplantation, hemolytic uremic syndrome, myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpura Schoenlein-Henoch, refractory anemia with excess of blasts, rheumatoid arthritis, Shwachman syndrome, sickle cell disease, thalassemia major, thalassemia minor, thrombocytopenic purpura, anemic or non-anemic patients undergoing surgery, anemia associated with or secondary to trauma, sideroblastic anemia, anemic secondary to other treatment including: reverse transcriptase inhibitors to treat HIV, corticosteroid hormones, cyclic cisplatin or non-cisplatin-containing chemotherapeutics, vinca alkaloids, mitotic inhibitors, topoisomerase II inhibitors, anthracyclines, alkylating agents, particularly anemia secondary to inflammatory, aging and/or chronic diseases. PHD inhibition may also be used to treat symptoms of anemia including chronic fatigue, pallor and dizziness.
In another preferred embodiment, molecules of the present invention are useful in the treatment or prevention of diseases of metabolic disorders, including but not limited to diabetes and obesity. In another preferred embodiment, molecules of the present invention are useful in the treatment or prevention of vascular disorders. These include but are not limited to hypoxic or wound healing related diseases requiring pro-angiogenic mediators for vasculogenesis, angiogenesis, and arteriogenesis
“Modulators” include both inhibitors and activators, where “inhibitors” refer to compounds that decrease, prevent, inactivate, desensitize or down-regulate PHD expression or activity, and “activators” are compounds that increase, activate, facilitate, sensitize, or up-regulate PHD expression or activity.
In treatment methods according to the invention, an effective amount of a pharmaceutical agent according to the invention is administered to a subject suffering from or diagnosed as having such a disease, disorder, or condition. An “effective amount” means an amount or dose sufficient to generally bring about the desired therapeutic or prophylactic benefit in patients in need of such treatment for the designated disease, disorder, or condition. Effective amounts or doses of the compounds of the present invention may be ascertained by routine methods such as modeling, dose escalation studies or clinical trials, and by taking into consideration routine factors, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the compound, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician. An example of a dose is in the range of from about 0.001 to about 200 mg of compound per kg of subject's body weight per day, preferably about 0.05 to 100 mg/kg/day, or about 1 to 35 mg/kg/day, in single or divided dosage units (e.g., BID, TID, QID). For a 70-kg human, an illustrative range for a suitable dosage amount is from about 0.05 to about 7 g/day, or about 0.2 to about 2.5 g/day.
Once improvement of the patient's disease, disorder, or condition has occurred, the dose may be adjusted for preventative or maintenance treatment. For example, the dosage or the frequency of administration, or both, may be reduced as a function of the symptoms, to a level at which the desired therapeutic or prophylactic effect is maintained. Of course, if symptoms have been alleviated to an appropriate level, treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.
In addition, the agents of the invention may be used in combination with additional active ingredients in the treatment of the above conditions. The additional compounds may be co-administered separately with an agent of Formula (I) or included with such an agent as an additional active ingredient in a pharmaceutical composition according to the invention. In an exemplary embodiment, additional active ingredients are those that are known or discovered to be effective in the treatment of conditions, disorders, or diseases mediated by PHD enzyme or that are active against another targets associated with the particular condition, disorder, or disease, such as an alternate PHD modulator. The combination may serve to increase efficacy (e.g., by including in the combination a compound potentiating the potency or effectiveness of a compound according to the invention), decrease one or more side effects, or decrease the required dose of the compound according to the invention.
The compounds of the invention are used, alone or in combination with one or more other active ingredients, to formulate pharmaceutical compositions of the invention. A pharmaceutical composition of the invention comprises: (a) an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, pharmaceutically acceptable prodrug, or pharmaceutically active metabolite thereof; and (b) a pharmaceutically acceptable excipient.
A “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of a compound of the invention and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Delivery forms of the pharmaceutical compositions containing one or more dosage units of the compounds of the invention may be prepared using suitable pharmaceutical excipients and compounding techniques now or later known or available to those skilled in the art. The compositions may be administered in the inventive methods by oral, parenteral, rectal, topical, or ocular routes, or by inhalation. The preparation may be in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations, or suppositories. Preferably, the compositions are formulated for intravenous infusion, topical administration, or oral administration. A preferred mode of use of the invention is local administration of PHD inhibitors particularly to sites where tissue has become or has been made ischemic. This may be achieved via a specialized catheter, angioplasty balloon or stent placement balloon.
For oral administration, the compounds of the invention can be provided in the form of tablets or capsules, or as a solution, emulsion, or suspension. To prepare the oral compositions, the compounds may be formulated to yield a dosage of, e.g., from about 0.05 to about 100 mg/kg daily, or from about 0.05 to about 35 mg/kg daily, or from about 0.1 to about 10 mg/kg daily.
Oral tablets may include a compound according to the invention mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating.
Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, compounds of the invention may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the compound of the invention with water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.
Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.
The active agents of this invention may also be administered by non-oral routes. For example, the compositions may be formulated for rectal administration as a suppository. For parenteral use, including intravenous, intramuscular, intraperitoneal, or subcutaneous routes, the compounds of the invention may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms will be presented in unit-dose form such as ampules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation. Illustrative infusion doses may range from about 1 to 1000 μg/kg/minute of compound, admixed with a pharmaceutical carrier over a period ranging from several minutes to several days.
For topical administration, the compounds may be mixed with a pharmaceutical carrier at a concentration of about 0.1% to about 10% of drug to vehicle. Another mode of administering the compounds of the invention may utilize a patch formulation to affect transdermal delivery.
Compounds of the invention may alternatively be administered in methods of this invention by inhalation, via the nasal or oral routes, e.g., in a spray formulation also containing a suitable carrier.
Exemplary compounds useful in methods of the invention will now be described by reference to the illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. Unless otherwise specified, the variables are as defined above in reference to Formula (I). Reactions may be performed between the melting point and the reflux temperature of the solvent, and preferably between 0° C. and the reflux temperature of the solvent.
Referring to Scheme A, imidazoles A1 are coupled with appropriate amines using a peptide coupling reagent such as HATU in the presence of a base such as DIPEA to provide A2. When Z═CO2R, exposure to aqueous base or acid provides A3.
An alternative method for preparing A2 is shown in Scheme B. Brominated imidazoles B1 (prepared as described in Scheme A) are protected with a suitable reagent such as di-tert-butyl-dicarbonate to provide B2. Coupling reaction with boronic acids or similar organic nucleophile in the presence of a organotransition metal catalyst such as PdCl2(dppf) and a base such as CsF affords B3. Removal of the protective group is effected by exposure to an acid such as trifluoroacetic acid to give A2.
A method to obtain certain benzoimidazoles A1 that are not commercially available or previously known is shown in Scheme C. Known imidazole C1 is coupled to appropriate acid chlorides or sulfonyl chlorides in the presence of a base such as DIPEA to give C2. Hydrolysis is achieved using a base such as LiOH in THF/H2O, or aqueous acid providing A1.
An alternative method to obtain C2 is shown in Scheme D. Subjecting C1 to reductive amination conditions with appropriate aldehydes in the presence of a reducing agent such as sodium triacetoxyborohydride provides C2.
Referring to Scheme E, imidazoles E1 are protected using a suitable reagent such as SEMCl or MEMCl in the presence of a base such as NaH or DIPEA to afford E2. Heating E2 with pyrazoles in the presence of a base such as Cs2CO3 provides E3. Deprotection occurs under acidic conditions to give E4. Hydrolysis when B═CO2R is effected with a base such as LiOH in THF/H2O or aqueous acid to deliver E5.
An alternative preparation of E3 is shown is Scheme F. Brominated imidazoles F1 (prepared as described in Scheme E) are subjected to an organotransition metal mediated coupling reaction, such as a Suzuki reaction, with appropriate organic nucleophile, such as a boronic acid, in the presence of a catalyst such as PdCl2(dppf) and a base such as CsF to supply E3.
Referring to Scheme G, in one embodiment, protected 2-bromobenzoimidazoles G1 can be treated with organic nucleophiles such as boronic acids or esters G2 under organotransition metal coupling reaction conditions such as the Suzuki reaction to provide G3. When protecting groups such as MEM or SEM are used, deprotection can be effected with acid to provide G4. Hydrolysis when Y═CO2R can be effected with a base such as LiOH in THF/H2O or aqueous acid to provide G5. As an alternative method, G6 can be coupled with 2-halopyridine or 2-halopyrimidine or 2-halopyridazine derivatives such as G7 under Suzuki reaction conditions to provide G3. In yet another alternative method, G4 can be accessed by coupling G8 with G9 under oxidative cyclization conditions.
Referring to Scheme H, in one embodiment H1 can be reacted with a reagent such as CU in THF to provide the cyclic urea H2. Treatment of H2 with a chlorinating agent such as POCl3 at moderate to high temperatures can then provide H3. Protection of imidazoles H3 can be achieved using a suitable reagent such as SEMCl or MEMCl in the presence of a base such as NaH or DIPEA to provide H4. In one embodiment, heating H4 with substituted pyrazoles in the presence of a base such as Cs2CO3 can then provide H5. Deprotection can be carried out under acidic conditions to yield H6. Hydrolysis when A or B=CO2R can be effected with a base such as LiOH in THF/H2O or aqueous acid to provide H7. In another embodiment H3 can be coupled with pyrazoles to provide H6 without use of protecting groups.
Referring to Scheme I, hydrolysis of ester I1 can be carried out with a base such as LiOH in THF/H2O or aqueous acid to provide I2. In one embodiment, I2 can be homologated using well-known procedures such as the Arndt-Eistert reaction to provide I3. Deprotection under acidic conditions can then provide I4.
Referring to Scheme J, in one embodiment J1 can be coupled with organic nucleophiles such as boronic acids or esters J2 under organotransition metal catalyzed coupling reactions such as the Suzuki reaction to provide J3. Deprotection under acidic conditions can then provide J4. Hydrolysis of the ester moiety can be effected with a base such as LiOH in THF/H2O or aqueous acid to provide J5. In another embodiment, J6 can also be coupled with J7 to provide J3.
Compounds prepared according to the schemes described above may be obtained as single enantiomers, diastereomers, or regioisomers, by enantio-, diastero-, or regiospecific synthesis, or by resolution. Compounds prepared according to the schemes above may alternately be obtained as racemic (1:1) or non-racemic (not 1:1) mixtures or as mixtures of diastereomers or regioisomers. Where racemic and non-racemic mixtures of enantiomers are obtained, single enantiomers may be isolated using conventional separation methods known to one skilled in the art, such as chiral chromatography, recrystallization, diastereomeric salt formation, derivatization into diastereomeric adducts, biotransformation, or enzymatic transformation. Where regioisomeric or diastereomeric mixtures are obtained, single isomers may be separated using conventional methods such as chromatography or crystallization.
For starting materials requiring stereospecific amino acid chemistry, these materials were purchased as preferred stereospecific enantiomers which retained their specificity throughout the synthesis reactions.
The following examples are provided to further illustrate the invention and various preferred embodiments.
In obtaining the compounds described in the examples below and the corresponding analytical data, the following experimental and analytical protocols were followed unless otherwise indicated.
Unless otherwise stated, reaction mixtures were magnetically stirred at room temperature (rt). Where solutions were “dried,” they were generally dried over a drying agent such as Na2SO4 or MgSO4. Where mixtures, solutions, and extracts were “concentrated”, they were typically concentrated on a rotary evaporator under reduced pressure.
Thin-layer chromatography (TLC) was performed using Merck silica gel 60 F254 2.5 cm×7.5 cm 250 μm or 5.0 cm×10.0 cm 250 μm pre-coated silica gel plates. Preparative thin-layer chromatography was performed using EM Science silica gel 60 F254 20 cm×20 cm 0.5 mm pre-coated plates with a 20 cm×4 cm concentrating zone. Normal-phase flash column chromatography (FCC) was performed on silica gel (SiO2) eluting with 2 M NH3 in MeOH/DCM, unless otherwise noted.
Reversed-phase HPLC was performed on a Hewlett Packard HPLC Series 1100, with a Phenomenex Luna C18 (5 μm, 4.6×150 mm) column. Detection was done at X=230, 254 and 280 nm. The gradient was 10 to 99% acetonitrile/water (0.05% trifluoroacetic acid) over 5.0 min with a flow rate of 1 mL/min. Alternatively, HPLC was performed on a Dionex APS2000 LC/MS with a Phenomenex Gemini C18 (5 μm, 30×100 mm) column, and a gradient of 5 to 100% acetonitrile/water (20 mM NH4OH) over 16.3 min, and a flow rate of 30 mL/min.
Mass spectra (MS) were obtained on an Agilent series 1100 MSD equipped with a ESI/APCI positive and negative multimode source unless otherwise indicated. Nuclear magnetic resonance (NMR) spectra were obtained on Bruker model DRX spectrometers. The format of the 1H NMR data below is: chemical shift in ppm downfield of the tetramethylsilane reference (apparent multiplicity, coupling constant J in Hz, integration). Chemical names were generated using ChemDraw Version 6.0.2 (CambridgeSoft, Cambridge, Mass.).
According to Scheme A, triethylamine (0.77 mL, 5.5 mmol) was added dropwise to a mixture of 1H-benzoimidazole-2-carboxylic acid (0.20 g, 1.2 mmol), glycine methyl ester hydrochloride (0.17 g, 1.4 mmol), HATU (0.57 g, 1.5 mmol), and DMF (10 mL). The reaction was allowed to proceed for 16 h at 23° C. Water (25 mL) was added, and the resulting precipitate was collected and dried (0.18 g, 61%). MS (ESI/CI): mass calcd. for C11H11N3O3, 233.2; m/z found, 234.1 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 13.29 (s, 1H), 9.23 (t, J=6.1 Hz, 1H), 7.75 (d, J=7.2 Hz, 1H), 7.55 (d, J=7.26 Hz, 1H), 7.31 (m, 2H), 4.08 (d, J=6.1 Hz, 2H), 3.68 (s, 3H).
A solution of LiOH.H2O (0.090 g, 2.1 mmol) and H2O (2 mL) was added to a mixture of [(1H-benzoimidazole-2-carbonyl)-amino]-acetic acid methyl ester (0.10 g, 0.43 mmol) and THF (5 mL). The resulting mixture was stirred rapidly for 30 min, then the THF was removed in vacuo. A solution of 1M HCl (3 mL) was added, and the resulting precipitate was collected to provide the titled compound (0.085 g, 90%). MS (ESI/CI): mass calcd. for C10H9N3O3, 219.2; m/z found, 220.0 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 12.40-12.90 (broad s, 2H), 9.06 (t, J=6.10, 6.10 Hz, 1H), 7.64 (s, 1H), 7.31 (m, 2H), 3.99 (d, J=6.14 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H11N3O3, 233.2; m/z found, 234.1 [M+H]+. 1H NMR (600 MHz, DMSO-d6): 13.31 (s, 1H), 12.78 (s, 1H), 9.00 (d, J=7.6 Hz, 1H), 7.75 (d, J=7.7 Hz, 1H), 7.55 (d, J=7.7 Hz, 1H), 7.31 (m, 2H), 4.53-4.46 (m, 1H), 1.46 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C10H7C12N3O3, 287.0; m/z found, 288.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 13.66 (s, 1H), 12.76 (s, 1H), 9.23 (t, J=6.1 Hz, 1H), 8.05 (s, 1H), 7.75 (s, 1H), 3.97 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H9C12N3O3, 302.1; m/z found, 300.0 [M−H]−. 1H NMR (400 MHz, DMSO-d6): 12.30-14.40 (br. s, 2H), 9.14 (d, J=7.6 Hz, 1H), 7.90 (br. s, 2H), 4.44-4.53 (m, 1H), 1.45 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C10H8N4O6, 264.05; m/z found, 265.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): resonances assignable to major tautomer, 14.02 (s, 0.65H), 12.78 (s, 1H), 9.34 (br t, J=6.0 Hz, 1H), 8.64 (br s, 1H), 8.25 (dd, J=8.9, 1.8 Hz, 1H), 7.74 (d, J=8.8, 1 H), 3.99 (d, J=6.4 Hz, 2H); resonances assignable to minor tautomer, 14.07 (s, 1H), 12.78 (s, 1H), 9.34 (br t, J=6.0 Hz, 1H), 8.39 (br s, 1H), 8.18 (d, J=8.8, 1H), 7.97 (d, J=9.2, 1 H), 3.99 (d, J=6.4 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C12H7F6N3O3, 355.2; m/z found, 354.0 [M−H]−. 1H NMR (400 MHz, DMSO-d6): 1H NMR (600 MHz, DMSO-d6): 14.39 (s, 1H), 12.83 (s, 1H), 9.16 (s, 1H), 8.15 (s, 1H), 7.94-7.93 (m, 1H), 4.03 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C13H9F6N3O3, 369.2; m/z found, 368.0 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 9.06 (d, J=7.6 Hz, 1H), 8.18 (s, 1H), 7.91 (s, 1H), 4.54 (q, J=7.23 Hz, 1H), 1.48 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C10H8IN3O3, 345.1; m/z found, 343.9 [M−H]−. 1H NMR (500 MHz, DMSO-d6, mixture of tautomers): 13.50 (s, 0.51H, major tautomer), 13.41 (s, 0.48H, minor tautomer), 12.73 (s, 1H), 9.16 (t, J=6.0 Hz, 1H), 8.11 (s, 0.51H, major tautomer), 7.87 (s, 0.48H, minor tautomer), 7.61 (d, J=8.7 Hz, 0.48H, minor tautomer), 7.58 (s, 1H), 7.39 (d, J=8.7, 0.51H, major tautomer), 3.97 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C10H8BrN3O3, 297.0; m/z found, 298.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6, mixture of tautomers): 13.55 (m, 1H), 12.75 (br s, 1H), 9.16 (br s, 1H), 7.98-7.45 (m, 3H), 4.01 (d, J=6.0 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H10FN3O3, 251.1; m/z found, 252.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, mixture of tautomers): 9.05 (d, J=7.6 Hz, 1H), 7.71-7.67 (m, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.24-7.19 (m, 1H), 4.54-4.50 (m, 1H), 1.48 (d, J=7.2 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H10FN3O3, 251.1; m/z found, 252.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, mixture of tautomers): 9.04 (d, J=7.6 Hz, 1H), 7.69 (br s, 1H), 7.44 (br s, 1H), 7.22 (t, J=9.2 Hz, 1H), 4.53-4.51 (m, 1H), 1.48 (d, J=7.2 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H11N3O4, 249.1; m/z found, 250.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, mixture of tautomers): 13.22 (br s, 1H), 12.75 (br s, 1H), 9.02 (t, J=6.0 Hz, 1H), 7.65-6.94 (m, 3H), 3.99 (d, J=5.6 Hz, 2H), 3.84 (s, 3H).
According to Scheme B, triethylamine (0.57 g, 10 mmol), 4-(dimethylamino)pyridine (0.06 g, 0.5 mmol), and (Boc)2O (2.2 g, 10.3 mmol) were added to a solution of [(5-bromo-1H-benzoimidazole-2-carbonyl)-amino]-acetic acid methyl ester (prepared in a manner analogous to EXAMPLE 1, step A) (1.6 g, 5.1 mmol) and CH2Cl2 (30 ml). The resulting mixture was stirred at 23° C. for 1 hr. The solution was then concentrated and the residue was chromatographed (15:85 EtOAc/hexanes) to produce the titled compounds (2.3 g, 110%). MS (ESI/CI): mass calcd. for C16H18BrN3O5, 411.0; m/z found, 412.0 [M+H]+.
[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (0.046 g, 0.06 mmol) was added to a mixture of cesium fluoride (0.19 g, 1.2 mmol), 3-(3′-chlorobenzyloxyl)phenylboronic acid (0.22 g, 0.75 mmol), 5-bromo-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylic acid tert-butyl ester and 6-bromo-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylic acid tert-butyl ester (0.26 g, 0.63 mmol) and DME (5 mL) in a sealable tube. The reaction mixture was stirred at 80° C. for 3 h, then the mixture was allowed to cool and was diluted with EtOAc (50 ml) and filtered. The filtrate was concentrated and the residue was chromatographed (85:15 EtOAc/hexanes) to produce the titled compounds (0.22 g, 63%). MS (ESI/CI): mass calcd. for C29H28ClN3O6, 549.2; m/z found, 550.1 [M+H]+.
TFA (0.45 g, 3.9 mmol) was added to a solution of 5-[3-(3-chloro-benzyloxy)-phenyl]-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylic acid tert-butyl ester, 6-[3-(3-chloro-benzyloxy)-phenyl]-2-(methoxycarbonylmethyl-carbamoyl)-benzoimidazole-1-carboxylic acid tert-butyl ester (0.22 g, 0.39 mmol) and CH2Cl2 (2 ml). The mixture was stirred for 1 hr and was neutralized with sat. NaHCO3. The resulting precipitate was collected to afford the titled compound (0.12 g, 67%). MS (ESI/CI): mass calcd. for C24H20ClN3O4, 449.1; m/z found, 450.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.78-12.68 (m, 1H), 9.24 (d, J=1.1 Hz, 1H), 8.06-7.53 (m, 4H), 7.52-7.37 (m, 4H), 7.36-7.24 (m, 2H), 7.02 (d, J=6.7 Hz, 1H), 5.23 (s, 2H), 4.09 (d, J=6.1 Hz, 2H), 3.68 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1, Step B. MS (ESI/CI): mass calcd. for C23H18ClN3O4, 435.1; m/z found, 436.1[M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.37 (d, J=13.6 Hz, 1H), 9.06 (d, J=5.8 Hz, 1H), 8.09-7.53 (m, 4H), 7.53-7.36 (m, 4H), 7.36-7.24 (m, 2H), 7.09-6.95 (m, 1H), 5.23 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C23H18ClN3O4, 435.1; m/z found, 436.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.41 (d, J=12.9 Hz, 1H), 9.12 (d, J=5.5 Hz, 1H), 8.09-7.18 (m, 9H), 7.04 (t, J=6.3, Hz, 1H), 5.27 (s, 2H), 3.98 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C23H18ClN3O4, 435.1; m/z found, 436.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.36 (d, J=12.6 Hz, 1H), 9.07 (dd, J=14.4, 6.0 Hz, 1H), 8.07-7.19 (m, 10H), 7.01 (t, J=6.2, 6.2 Hz, 1H), 5.21 (s, 2H), 4.00 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C23H18FN3O4, 419.1; m/z found, 420.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.37 (d, J=13.2 Hz, 1H), 9.08 (s, 1H), 8.12-7.22 (m, 9H), 7.21-7.12 (m, 1H), 7.02 (d, J=7.4 Hz, 1H), 5.24 (s, 2H), 4.00 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C23H18FN3O4, 419.1; m/z found, 420.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.36 (d, J=13.7 Hz, 1H), 9.35-8.77 (m, 1H), 8.09-7.16 (m, 10H), 7.03 (t, J=6.1, 6.1 Hz, 1H), 5.25 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C22H17N3O4, 387.1; m/z found, 388.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 9.14 (s, 1H), 7.82 (s, 1H), 7.77-7.68 (m, 3H), 7.60 (dd, J=8.5, 1.7 Hz, 1H), 7.43 (dd, J=8.5, 7.4 Hz, 2H), 7.23-7.14 (m, 1H), 7.14-7.04 (m, 4H), 3.99 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C23H18FN3O4, 419.1; m/z found, 420.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.45 (d, J=25.0 Hz, 1H), 9.28-8.91 (m, 1H), 8.09-7.56 (m, 3H), 7.50 (d, J=7.1 Hz, 2H), 7.42 (t, J=7.3, 7.3 Hz, 2H), 7.39-7.31 (m, 1H), 7.25-7.07 (m, 2H), 6.97-6.85 (m, 1H), 5.23 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C19H14N4O3, 346.1; m/z found, 347.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 9.37 (d, J=2.2 Hz, 1H), 9.13 (t, J=6.1, 6.1 Hz, 1H), 8.81 (d, J=1.8 Hz, 1H), 8.21-8.06 (m, 3H), 7.90-7.78 (m, 3H), 7.77-7.63 (m, 1H), 4.02 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C23H17Cl2N3O4, 469.1; m/z found, 470.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.41 (d, J=17.1 Hz, 1H), 9.12 (d, J=6.1 Hz, 1H), 8.06-7.52 (m, 6H), 7.53-7.38 (m, 3H), 7.37-7.27 (m, 1H), 5.30 (s, 2H), 3.99 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.40 (br. s, 1H), 13.36 (br. s, 1H), 9.06-9.15 (m, 1H), 7.34-7.95 (m, 9H), 4.87 (s, 2H), 3.99 (d, J=6.07 Hz, 2H), 2.33 (s, 6H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.36 (br. s, 1H), 9.07 (br. m, 1H), 6.95-8.10 (m, 7H), 4.00 (d, J=6.09 Hz, 1H), 3.84 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C17H15N3O4, 446.4; m/z found, 447.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.45 (d, J=7.60 Hz, 1H), 12.2-13.0 (br. s, 1H), 9.05-9.2 (m, 2H), 7.15-8.20 (m, 11H), 4.51 (d, J=5.9 Hz, 2H), 4.00 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C20H15N3O3, 345.4; m/z found, 346.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.41-13.52 (m, 1H), 9.09-9.20 (m, 1H), 8.26 (br. s, 1H), 7.60-8.10 (m, 7H), 7.45-7.60 (m, 2H), 4.00 (d, J=6.1 Hz, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C19H19N3O4, 353.4; m/z found, 354.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 12.90-13.80 (br s, 1H), 9.08-9.16 (m, 1H), 7.50-8.10 (m, 3H), 7.10-7.45 (m, 3H), 6.91-6.97 (m, 1H), 6.45-6.64 (br. m, 1H), 3.96-4.06 (m, 4H), 2.54-2.57 (m, 2H), 1.72-1.83 (m, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C17H14ClN3O4, 343.8; m/z found, 344.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.35-13.53 (br. m, 1H), 9.00-9.22 (m, 1H), 7.46-8.42 (m, 6H), 4.00 (d, J=6.1 Hz, 2H), 2.42 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 13. MS (ESI/CI): mass calcd. for C16H11ClFN3O4, 347.7; m/z found, 348.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.45-13.53 (m, 1H), 9.13-9.22 (m, 1H), 7.35-7.96 (m, 6H), 3.98 (d, J=6.1 Hz).
According to Scheme C, DIPEA (0.45 mL, 2.6 mmol) was added to a solution of 5-amino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.200 g, 1.04 mmol) in THF (5 mL) at 0° C., followed by benzoyl chloride (0.154 g, 1.09 mmol). After 2 h, the reaction was quenched with water (6 mL), the THF was evaporated, and the resulting aqueous layer extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (15 mL), dried, and concentrated to yield the desired product (0.272 g, 89%). MS (ESI/CI): mass calcd. for C16H13N3O3, 295.10; m/z found, 296.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 13.50 (s, 0.3H), 13.44 (s, 0.7H), 10.41 (s, 0.7H), 10.33 (s, 0.3H), 8.30 (d, J=2.0 Hz, 0.7H), 8.26 (d, J=1.2 Hz, 0.3H), 8.01-7.96 (m, 2H), 7.76-7.70 (m, 1H), 7.63-7.51 (m, 4H), 3.96-3.94 (m, 3H).
LiOH.H2O (0.23 g, 5.4 mmol) was added to a solution of 5-benzoylamino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.25 g, 0.85 mmol) and THF (6 mL) at rt, followed by water (2 mL). After stirring for 1 h the THF was evaporated and HCl (1 M, 10 mL) was added. The resulting precipitate was dried to yield the desired compound (0.210 g, 88%). MS (ESI): mass calcd. for C16H11N3O3, 281.08; m/z found, 282.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 10.42 (s, 1H), 8.30 (d, J=1.2 Hz, 1H), 7.99 (dt, J=6.8, 1.6 Hz, 2H), 7.71-7.52 (m, 5H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C17H14N4O4, 338.10; m/z found, 339.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 10.36 (s, 1H), 9.08 (t, J=6.0 Hz, 1H), 8.27 (t, J=1.2 Hz, 1H), 8.00 (t, J=1.3 Hz, 1H), 7.98 (t, J=1.8 Hz, 1H), 7.69-7.49 (m, 5H), 3.98 (d, J=6.1 Hz, 2H).
According to Scheme C, DIPEA (0.45 mL, 2.6 mmol) was added to a solution of 5-amino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.200 g, 1.04 mmol) and THF (5 mL) at 0° C., followed by benzenesulfonyl chloride (0.193 g, 1.09 mmol). After 3.5 h the reaction was quenched with water (5 mL), the THF was evaporated, and the resulting aqueous layer was extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (10 mL), toluene (2 mL) was added, and the solution was concentrated to yield the titled compound (0.335 g, 97%). MS (ESI/CI): mass calcd. for C15H13N3O4S, 331.06; m/z found, 332.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 13.36 (br s, 1H), 10.47 (br s, 1H), 7.73 (d, J=7.6 Hz, 2H), 7.62-7.50 (m, 4H), 7.34 (br s, 1H), 7.07 (br s, 1H), 3.91 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 29, Steps B-C. MS (ESI/CI): mass calcd. for C16H14N4O5S, 374.07; m/z found, 375.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 10.24 (s, 1H), 9.03 (t, J=6.2 Hz, 1H), 7.73-7.71 (m, 1H), 7.70 (t, J=1.8, 1H), 7.61-7.44 (m, 4H), 7.33 (d, J=1.8 Hz, 1H), 7.04 (dd, J=8.8, 2.0 Hz, 1H), 3.94 (d, J=6.1 Hz, 2H).
According to Scheme D, Benzaldehyde (0.11 mL, 1.0 mmol) and NaBH(OAc)3 (0.309 g, 1.46 mmol) were added to a solution of 5-amino-1H-benzoimidazole-2-carboxylic acid methyl ester (0.200 g, 1.04 mmol) and 1,2-dichloroethane (4 mL). After 8 h, additional benzaldehyde (0.010 mL, 0.10 mmol) was added; after an additional 15 h, NaBH(OAc)3 (0.221 g, 1.04 mmol) was added to the reaction. After 5 h the reaction was quenched with sat. aq. NaHCO3 (10 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (15 mL), dried, and concentrated. The resulting residue was chromatographed (35-75% EtOAc/hexanes) to yield the titled compound (0.268 g, 91%). MS (ESI/CI): mass calcd. for C16H15N3O2, 281.12; m/z found, 282.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 12.78 (s, 1H), 7.44-7.36 (m, 3H), 7.33 (t, J=7.6 Hz, 2H), 7.23 (t, J=7.2 Hz, 1H), 6.78 (d, J=8.5 Hz, 1H), 6.56 (t, J=5.5 Hz, 1H), 6.39 (s, 1H), 4.30 (d, J=5.8 Hz, 2H), 3.87 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 29, Step B-C. MS (ESI/CI): mass calcd. for C17H16N4O3, 324.12; m/z found, 325.1 [M+H]+. 1H NMR (400 MHz, CD3OD): 7.62 (d, J=6.8 Hz, 1H), 7.47-7.28 (m, 5H), 7.20-7.08 (m, 2H), 4.51 (s, 2H), 4.17 (s, 2H).
According to Scheme E, a mixture of NaH (60% dispersion in oil, 0.40 g, 9.8 mmol) and THF (10 mL) was cooled to 0° C., then solid 2-chlorobenzoimidazole (1.0 g, 6.5 mmol) was added portion wise over 10 min. The resulting mixture was stirred at 0° C. for 1 h, then 2-trimethylsilylethoxymethyl chloride (1.5 mL, 8.5 mmol) was added. The reaction mixture was allowed to warm to 23° C. and was stirred 16 h. The mixture was carefully poured over ice (ca. 200 g) and then was extracted with Et2O (3×100 mL). The combined organic extracts were dried and concentrated, and the residue was chromatographed (1:99 to 15:85 EtOAc/hexanes) to provide the titled compound, which has been previously described: PCT Int. Appl. (2005), 465 pp. CODEN: PIXXD2 WO 2005012297 A1 20050210 CAN 142:219286 AN 2005:120926.
A mixture of 2-chloro-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazole (0.34 g, 1.2 mmol), ethyl pryazole-4-carboxylate (0.24 g, 1.7 mmol), cesium carbonate (0.78 g, 2.4 mmol), and anhydrous DMF (2.5 mL) was stirred at 100° C. for 5 h. The mixture was allowed to cool to 23° C. and was diluted with EtOAc, then filtered through a pad of silica gel. The resulting solution was concentrated and the residue was chromatographed (5:95 to 40:60 EtOAc/hexanes) to providing the titled compound (0.36 g, 77%). 1H NMR (500 MHz, CDCl3): 8.88 (s, 1H), 8.18 (s, 1H), 7.77-7.69 (m, 1H), 7.60-7.50 (m, 1H), 7.40-7.30 (m, 2H), 6.03 (s, 2H), 4.34 (q, J=7.1 Hz, 2H), 3.57-3.50 (m, 2H), 1.37 (t, J=7.1, Hz, 3H), 0.87-0.80 (m, 2H), −0.11 (s, 9H).
A solution of HCl and dioxane (4M, 2 mL, 8 mmol) was added to a mixture of 1-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrrole-3-carboxylic acid ethyl ester (0.30 g, 0.78 mmol) and EtOH (4 mL). The reaction mixture was heated to reflux for 30 min, then cooled to 23° C. Et2O was added (20 mL), and the mixture was cooled to 0° C. for 10 min. The resulting precipitate was collected by filtration and washed well with Et2O to afford the titled compound (0.18 g, 91%). MS (ESI/CI): mass calcd. for C13H12N4O2, 256.3; m/z found, 257.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 8.96 (s, 1H), 8.33 (s, 1H), 7.56 (s, 2H), 7.28-7.21 (m, 2H), 4.30 (q, J=7.1 Hz, 2H), 1.32 (t, J=7.1 Hz, 3H)
A solution of LiOH and H2O (1.0 M, 1.0 mL, 1.0 mmol) was added to a mixture of 1-(1H-benzoimidazol-2-yl)-1H-pyrrole-3-carboxylic acid ethyl ester hydrochloride (0.040 g, 0.16 mmol) and THF (2.0 mL), and the reaction mixture was stirred at 23° C. for 16 h. The THF was removed in vacuo and then aqueous HCl (1.0 M, 2 mL, 2 mmol) was added at 0° C. The resulting precipitate was collected and washed with water to give the titled compound (0.033 g, 90%). MS (ESI/CI): mass calcd. for C11H8N4O2, 228.2; m/z found, 229.0 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 13.32 (s, 1H), 13.00-12.86 (br. s, 1H), 8.90 (d, J=0.6 Hz, 1H), 8.28 (d, J=0.6 Hz, 1H), 7.64 (d, J=4.6 Hz, 1H), 7.49 (d, J=5.5 Hz, 1H), 7.28-7.20 (m, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C11H6Cl2N4O2, 297.1; m/z found, 296.0 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 14.18-12.52 (br. s, 2H), 8.89 (d, J=0.5 Hz, 1H), 8.31 (d, J=0.5 Hz, 1H), 7.80 (s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C12H7F3N4O2, 296.2; m/z found, 295.0 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 14.44-12.32 (br. s, 2H), 8.94 (d, J=0.5 Hz, 1H), 8.33 (d, J=0.5 Hz, 1H), 7.96-7.83 (br. s, 1H), 7.75 (br. d, 1H), 7.58 (dd, J=8.49, 1.41 Hz, 1H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C11H6ClFN4O2, 280.7; m/z found, 279.0 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 14.21-12.25 (br. S, 2H), 8.88 (d, J=0.6 Hz, 1H), 8.30 (d, J=0.6 Hz, 1H), 7.81-7.67 (br. s, 1H), 7.65-7.52 (br. s, 1H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C13H12N4O2, 256.3; m/z found, 257.1 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 13.16-12.81 (m, 2H), 8.85 (d, J=0.6 Hz, 1H), 8.25 (d, J=0.6 Hz, 1H), 7.43-7.21 (br. s, 2H), 2.31 (s, 6H)
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C11H7BrN4O2, 306.0; m/z found, 307.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 8.82 (d, J=0.5 Hz, 1H), 8.22 (s, 1H), 7.67 (d, J=1.2 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.32 (dd, J=8.5, 1.9 Hz, 1H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C12H10N4O3, 258.2; m/z found, 259.1[M+H]+. 1H NMR (500 MHz, DMSO-d6, tautomeric mixture): 13.16 (s, 1H), 12.91 (s, 1H), 8.84 (s, 1H), 8.26 (s, 1H), 6.83-7.54 (m, 3H), 3.80 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C12H6ClF3N4O2, 330.7; m/z found, 329.0 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 13.90-14.50 (br. s, 1H), 12.75-13.45 (br. s, 1H), 8.95 (s, 1H), 8.36 (s, 1H), 7.72 (s, 1H), 7.70 (s, 1H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C13H12N4O4, 288.3; m/z found, 289.1 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 8.81 (s, 1H), 8.25 (s, 1H), 7.09 (s, 2H), 3.80 (s, 6H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C13H12N4O2, 256.3; m/z found, 257.2 [M+H]+. 1H NMR (500 MHz, DMSO-d6, tautomeric mixture): 12.60-13.30 (br. m, 2H), 8.83-8.90 (m, 1H), 8.23-8.29 (m, 1H), 7.0-7.35 (m, 2H), 2.47 (s, 3H), 2.33 (s, 3H).
Prepared in the same manner analogous to EXAMPLE 32. MS (ESI/CI): Mass calcd. for C12H7F3N4O3 312.0. m/z found: 313.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 8.91 (s, 1H), 8.31 (s, 1H), 7.83-7.41 (m, 2H), 7.30-7.21 (m, 1H).
According to Scheme F, [1,1′-Bis(diphenylphosphino)ferrocene]dichloro palladium (0.12 g, 0.16 mmol) was added to a mixture of cesium fluoride (0.33 g 2.2 mmol), 3-(3′-chlorobenzyloxyl)phenylboronic acid (0.37 g, 1.3 mmol), 1-[5-Bromo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylic acid ethyl ester and 1-[6-Bromo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-benzoimidazol-2-yl]-1H-pyrazole-4-carboxylic acid ethyl ester (mixture of regioisomers prepared in a manner analogous to EXAMPLE 31, Step A. MS (ESI/CI): Mass calcd. for C19H25BrN4O3Si, 464.1; m/z found, 465.1), (0.5 g, 1.1 mmol), and DME (5 ml) in a sealable tube. The reaction was stirred at 80° C. After 3 h, the mixture was cooled to rt, then was diluted with EtOAc (50 ml) and filtered. The filtrate was concentrated and the residue was chromatographed (15:85 EtOAc/hexanes) to yield the titled compounds as a regioisomeric mixture (0.47 g, 72%). MS (ESI/CI): mass calcd. for C32H35ClN4O4Si, 602.2; m/z found, 603.2 [M+H]+.
The titled compound was prepared in a manner analogous to EXAMPLE 32, Steps C-D. MS (ESI/CI): mass calcd. for C24H17ClN4O3 444.1; m/z found, 445.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) 8.93 (d, J=0.5 Hz, 1H), 8.32 (s, 1H), 7.84-7.79 (m, 1H), 7.68-7.63 (m, 1H), 7.60-7.55 (m, 2H), 7.52-7.37 (m, 4H), 7.36-7.27 (m, 2H), 7.03 (dd, J=7.8, 2.1 Hz, 1H), 5.26 (s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C24H17ClN4O3, 444.1; m/z found, 445.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) 8.91 (s, 1H), 8.30 (s, 1H), 7.80 (s, 1H), 7.72-7.60 (m, 2H), 7.59-7.48 (m, 2H), 7.48-7.36 (m, 3H), 7.36-7.25 (m, 2H), 7.02 (dd, J=8.1, 1.9 Hz, 1H), 5.27 (s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C24H17ClN4O3, 444.1; m/z found, 445.1 [M+H]+. 1H NMR (600 MHz, DMSO-d6). 8.91 (s, 1H), 8.30 (s, 1H), 7.98-7.50 (m, 5H), 7.50-7.44 (m, 2H), 7.43-7.35 (m, 1H), 7.35-7.22 (m, 2H), 7.00 (s, 1H), 5.22 (s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C24H18N4O3, 410.1; m/z found, 411.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) 8.99 (s, 1H), 8.38 (s, 1H), 8.02-7.28 (m, 11H), 7.09 (dd, J=8.1, 1.9 Hz, 1H), 5.31 (s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C24H18N4O3, 410.1; m/z found; 411.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) 9.00 (s, 1H), 8.39 (s, 1H), 7.81-7.39 (m, 10H), 7.20 (d, J=8.8 Hz, 2H), 5.26 (s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C18H11F3N4O2, 372.1; m/z found, 373.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6). 8.91 (s, 1H), 8.31 (s, 1H), 8.11-7.79 (m, 3H), 7.78-7.52 (m, 4H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): Mass calcd. for C17H10Cl2N4O2 372.0. m/z found: 373.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 9.01 (s, 1H), 8.41 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.81 (d, J=1.2 Hz, 2H), 7.74 (d, J=8.4 Hz, 1H), 7.69 (dd, J=8.5, 1.7 Hz, 1H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H11N3O3, 233.2; m/z found, 234.1 [M+H]+. 1H NMR (600 MHz, DMSO-d6): 13.31 (s, 1H), 12.78 (s, 1H), 9.00 (d, J=7.6 Hz, 1H), 7.75 (d, J=7.7 Hz, 1H), 7.55 (d, J=7.7 Hz, 1H), 7.31 (m, 2H), 4.46-4.53 (m, 1H), 1.46 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H11N3O3, 249.2; m/z found, 250.1 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 8.74 (d, J=8.0 Hz, 1H), 7.66-7.72 (m, 2H), 7.34-7.40 (m, 2H), 4.51-4.56 (m, 1H), 3.89-3.95 (m, 2H), 3.81-3.86 (m, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C13H9F6N3O3, 369.2; m/z found, 368.0 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 12.20-15.00 (br. s, 1H), 9.08 (d, J=7.6 Hz, 1H), 8.18 (s, 1H), 7.92 (s, 1H), 4.55 (q, J=7.3 Hz, 1H), 1.48 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H9Cl2N3O3, 302.1; m/z found, 301.9 [M−H]−. 1H NMR (500 MHz, DMSO-d6): 13.45-13.90 (br. s, 1H), 12.60-13.00 (m, 1H), 9.13 (d, J=7.6 Hz, 1H), 8.03 (br. s, 1H), 7.76 (br. s, 1H), 4.44-4.55 (m, 1H), 1.45 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 1. MS (ESI/CI): mass calcd. for C11H10IN3O3, 359.1; m/z found, 357.9 [M−H]−. 1H NMR (500 MHz, DMSO-d6, tautomeric mixture): 13.34-13.51 (br. m, 1H), 12.76 (s, 1H), 9.04 (d, J=7.2 Hz, 1H), 7.85-8.16 (br. m, 1H), 7.35-7.65 (br. m, 2H), 4.44-4.52 (m, 1H), 1.44 (d, J=7.3 Hz, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C12H6BrF3N4O2, 374.0; m/z found, 375.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 13.69 (br s, 1H), 9.09 (s, 1H), 7.79 (br s, 1H), 7.55 (br s, 1H), 7.43 (dd, J=8.4, 1.6 Hz, 1H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C12H5Cl2F3N4O2, 365.1; m/z found, 363.0 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 13.25-14.30 (br. s, 2H), 9.10 (s, 1H), 7.87 (br. s, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 32. MS (ESI/CI): mass calcd. for C13H11BrN4O2, 334.0; m/z found, 335.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): 13.21 (br s, 1H), 12.77 (br s, 1H), 7.73 (br s, 1H), 7.51 (br s, 1H), 7.36 (dd, J=8.4, 1.6 Hz, 1H), 2.98 (s, 3H), 2.46 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 32 and purified by preparatory HPLC. MS (ESI/CI): mass calcd. for C13H11Cl2N4O2, 325.2; m/z found, 327.1 [M+H]+. 1H NMR (500 MHz, DMSO-d6): 7.79 (s, 2H), 2.98 (s, 3H), 2.46 (s, 3H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C17H12N4O3, 320.3; m/z found, 321.1 [M+H]+. 1H NMR (600 MHz, DMSO-d6): 12.52-13.80 (br. s, 1H), 9.25-10.05 (br s, 1H), 8.84 (s, 1H), 8.25 (s, 1H), 7.43-7.80 (m, 5H), 6.86 (d, J=8.6, 2H).
The titled compound was prepared in a manner analogous to EXAMPLE 43. MS (ESI/CI): mass calcd. for C17H12N4O3, 320.3; m/z found, 321.1 [M+H]+. 1H NMR (600 MHz, DMSO-d6): 12.50-13.56 (br. m, 2H), 9.54 (br. s, 1H), 8.91 (s, 1H), 8.30 (s, 1H), 7.45-7.88 (br. m, 3H), 7.26 (t, J=7.8 Hz, 1H), 7.04-7.14 (m, 2H), 6.75 (dd, J=8.0, 1.7 Hz, 1H).
The titled compound was prepared in a manner analogous to Example 32. MS (ESI/CI): mass calcd. for C11H7ClN4O2, 262.0; m/z found, 263.0 [M+H]+. 1H NMR (400 MHz, CD3OD, tautomeric broadening): 8.89 (s, 1H), 8.17 (s, 1H), 7.67-7.44 (m, 2H), 7.26 (dd, J=8.6. 1.9 Hz, 1H).
The titled compound was prepared in a manner analogous to Example 32. MS (ESI/CI): mass calcd. for C11H7ClN4O2, 334.0; m/z found, 335.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6, tautomeric broadening): 13.51-12.68 (m, 2H), 8.88 (s, 1H), 8.29 (s, 1H), 7.80-7.40 (m, 1H), 2.56 (s, 3H), 2.40 (s, 3H).
The human PHD2 expression construct containing amino acids 181-417 of GenBank Accession ID NM—022051 was cloned into a pBAD vector (Invitrogen), incorporating both an N-terminal histidine tag and a Smt3-tag, both of which are cleaved by Ulp1. Protein production was achieved by expression in BL21 cells grown in Terrific Broth containing 100 μg/ml ampicillin. Cell cultures were inoculated at 37° C. and grown to an OD600 of 0.8. Cultures were induced with 0.1% arabinose and grown overnight at 20° C. with continuous shaking at 225 rpm. Cells were then harvested by centrifugation and stored at −80° C. Cell pellets were suspended in Buffer A (50 mM Tris-HCl pH 7.2, 100 mM NaCl, 100 mM L-arginine, 1 mM TCEP, 0.05% (w/v) NP-40, 50 mM imidazole) followed by the addition of lysozyme and benzonase. Cells were lysed by sonication and the lysate was cleared by centrifugation (15,000 rpm, 90 min, 4° C.). The protein was purified by nickel affinity chromatography using a HisTrap Crude FF column (GE Healthcare). Samples were eluted in Buffer A with a 50-200 mM imidazole gradient. Cleavage of the Smt tag with Ulp1 protease was achieved via overnight incubation with dialyzing against Buffer A. The PHD2181-417 sample was then passed over a second HisTrap Crude FF column (GE Healthcare) to remove uncleaved protein. The flow-through was then dialyzed into 50 mM MES pH 6.0, 1 mM TCEP, 5 mM NaCl for ion exchange chromatography on a HiTrap SP Cation Exchange column (GE Healthcare). The PHD2181-417 protein was eluted with a 0-0.2 M NaCl gradient. Fractions were pooled for further purification by size exclusion chromatography over a Superdex 75 Size Exclusion Column (GE Healthcare). Final protein was concentrated to 4 mg/ml and dialyzed in 10 mM PIPES pH 7.0, 100 mM NaCl, 0.5 mM TCEP. The protein was determined to have a purity of >95% by gel electrophoresis.
The PHD enzymatic assay was performed in 0.5 ml of reaction mixture containing the following: purified PHD2181-417 polypeptide (3 μg), synthetic HIF-1α peptide comprising residues [KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLS] (10 μM, California Peptide Research Inc., Napa, Calif.), and [5-14C]-2-oxoglutaric acid (50 mCi/mmol, Moravek Chemicals, Brea, Calif.) in reaction buffer (40 mM Tris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM DTT, 1 mM ascorbate) for 10 minutes. The reaction was stopped by addition of 50 μl of 70 mM H3PO4 and 50 μl of 500 mM NaH2PO4, pH 3.2. Detection of [14C]-succinic acid was achieved by separating from [5-14C]-2-oxoglutaric acid by incubating the reaction mixture with 100 μl of 0.16 M DNP prepared in 30% perchloric acid. Next, 50 μl of unlabeled 20 mM 2-oxoglutaric acid/20 mM succinic acid, serving as carrier for the radioactivity, was added to the mixture, and was allowed to proceed for 30 minutes at room temperature. The reaction was then incubated with 50 μl of 1 M 2-oxoglutaric acid for 30 additional minutes at room temperature to precipitate the excess DNP. The reaction was then centrifuged at 2800×g for 10 minutes at room temperature to separate [14C]-succinic acid in the supernatant from the precipitated [14C]-dinitrophenylhydrazone. Fractions of the supernatant (400 μl) were counted using a beta counter (Beckman Coulter, Fullerton, Calif.). Inhibition of PHD2181-417 activity was measured as a decrease in [14C]-succinic acid production. The IC50 values were estimated by fitting the data to a three-parameter logistic function using GraphPad Prism, version 4.02 (Graph Pad Software, San Diego, Calif.).
Hep-3B cells (ATCC, Manassas, Va.) were plated in 96-well plates at 20,000 cells per well in 100 μl of DMEM containing 10% fetal bovine serum, 1% non-essential amino acids, 50 IU/mL of penicillin and 50 μg/mL of streptomycin (all cell culture reagents from Invitrogen, Carlsbad, Calif.). Twenty-four hours after plating, compounds were added and incubated for an additional 24 hours. All compounds were tested under saturating conditions with final compound concentrations at 100 μM, with the exception of ex. #50, 51, and 53. These three compounds were tested at 10 μM. Fifty microliters of the supernatant was then transferred to a human Hypoxia assay kit (Meso-Scale Discovery, Gaithersburg, Md.). Erythropoietin in the supernatant was detected according to the manufacturer's instructions as follows. EPO detection plates were blocked with 3% BSA in PBS overnight and 50 μl of the supernatant was incubated at room temperature in an orbital shaker for 2 h. Twenty-five microliters of 0.5 μg/ml anti-EPO detection antibody was added for 2 hours at room temperature in an orbital shaker. After 3 washes in PBS, 150 μl of 1× read buffer is added and the plate is then read on the MSD SECTOR instrument. Data was analyzed by determining the percent of EPO secretion in the presence of 10 μM or 100 μM compound relative to an assay control compound, 7-[(4-Chloro-phenyl)-(5-methyl-isoxazol-3-ylamino)-methyl]-quinolin-8-ol.
While the invention has been illustrated by reference to exemplary and preferred embodiments, it will be understood that the invention is intended not to be limited to the foregoing detailed description.
| Number | Date | Country | |
|---|---|---|---|
| 61048531 | Apr 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14282901 | May 2014 | US |
| Child | 14732332 | US | |
| Parent | 12990104 | Oct 2010 | US |
| Child | 14282901 | US |
