Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of

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Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases Amit Arvind Joharapurkar, Vrajesh Bhaskarbhai Pandya, Vishal J. Patel, Ranjit C Desai, and Mukul R Jain J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01686 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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Journal of Medicinal Chemistry

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Title Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases Authors

Amit A. Joharapurkar*, Vrajesh B. Pandya, Vishal J. Patel, Ranjit C. Desai, Mukul R. Jain.

Zydus Research Centre, Cadila Healthcare Limited, Sarkhej Bavla NH8A, Moraiya, Ahmedabad 382210 India

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

Chronic kidney disease, cancer, chronic inflammatory disorders, nutritional and genetic deficiency can cause anemia. Hypoxia causes induction of hypoxia-inducible factor (HIF), which stimulates erythropoietin (EPO) synthesis. Prolyl hydroxylase domain (PHD) enzyme inhibition can stabilize hypoxia-inducible factor (HIF). HIF stabilization also decreases hepcidin, a hormone of hepatic origin, which regulates iron homeostasis. PHD inhibitors represent a novel pharmacological treatment of anemia associated with chronic diseases. Many orally active PHD inhibitors like roxadustat, molidustat, vadadustat, and desidustat are in late phase clinical trials. This review discusses the role of PHD inhibitors in the treatment of anemia associated with chronic diseases.


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INTRODUCTION Anemia is a disorder of blood involving a decreased amount of red blood cells (RBC) or hemoglobin in circulation.1 Chronic kidney disease (CKD), cancer, inflammatory disorders, nutritional deficiencies, genetic disorders, and some drugs can cause anemia. Nutritional deficiency anemia is frequent in women, children, and elderly. Iron and vitamin supplementation is used to treat nutritional deficiency anemia.2 Thalassemia is a most common genetic disorder causing anemia.3 Chronic kidney disease, chemotherapy, and inflammatory diseases can reduce erythropoietin (EPO) synthesis and inefficient iron homeostasis.1,4 Treatment of anemia associated with CKD is done using EPO analogs or erythropoiesis-stimulating agents (ESA).5 However, they are associated with cardiovascular side effects and may increase the progression of malignancy in cancer patients.7 CKD patients also demonstrate iron deficiency which requires iron supplementation.8 Functional iron deficiency is a significant problem that limits the use of ESAs in most of the patients.9 Stabilization of hypoxia-inducible factor (HIF) represents a novel approach for the treatment of CKD. Under normal conditions, HIF undergoes oxidative degradation by prolyl hydroxylase domain (PHD) enzymes. Hypoxia is the major inhibitory factor for PHD activity, which stabilizes HIF, and stimulates EPO synthesis. PHD inhibition also decreases hepcidin production, which improves iron utilization.10 An increase in EPO and a decrease in hepcidin, caused by pharmacological inhibition of PHDs can efficiently cure anemia associated with chronic diseases. MECHANISM OF ERYTHROPOIESIS Erythropoiesis occurs in the bone marrow. In the bone marrow, hematopoietic stem cells (HSCs) differentiate into colony forming units-erythroid (CFU-Es) and then to erythroblasts, which enucleate to form reticulocytes.11 In the bone marrow, CFU-Es and proerythroblasts are stimulated by EPO for


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further differentiation (Figure 1). Formation of erythrocytes from reticulocytes occurs within 24-48 h, and the mature erythrocytes circulate in the blood for 120 days, before phagocytosis in the spleen, liver, and bone marrow degrades them.12 EPO-mediated signaling is necessary for the survival and differentiation of erythroid progenitors. EPO binds to specific EPO receptors (EPORs) and signals through Janus Tyrosine Kinase-2 (JAK2).11 JAK2 stimulates signal transduction and activator of transcription-5 (STAT-5), RAS–RAF–MAP kinase, and phosphoinositide-3 kinase/AKT kinase (protein kinase B).13, 14 On the other hand, erythroid progenitors undergo apoptosis by the cluster of differentiation 95 (CD95), a membrane protein of the tumor necrosis factor (TNF) receptor family, which triggers apoptosis after binding to the CD95 ligand, which is produced by mature erythroblasts. Thus, the negative feedback cycle controls the production of mature erythrocytes in blood.15 Iron is vital for hemoglobin synthesis and reticulocytes maturation. Hepcidin is a hepatic hormone that regulates iron metabolism.16 Ferroportin (FPN) is the ferrous exporter present in macrophages (reticuloendothelial cells), enterocytes, and hepatocytes. Hepcidin binds to ferroportin, and causes its degradation by internalization and lysosomal digestion. This action of hepcidin causes iron entrapment in macrophages or enterocytes, creating a functional iron deficiency in the body. Erythropoiesis and hypoxia also regulate hepcidin levels.8 Increased iron levels (as in iron loading with iron supplementation)

increases

hepcidin

production

via

Bone

morphogenetic

protein/SMAD

(BMP/SMAD) signaling. On the other hand, increased inflammation stimulates hepcidin production via IL-6/STAT signaling.8, 16

STIMULATION OF ERYTHROPOIESIS BY HIF-PROLYL HYDROXYLASES


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High altitude or hypoxia enhances RBC synthesis by stimulating EPO production. Hypoxia-inducible factor (HIF) is the regulator for hypoxic induction of erythropoiesis.17 There are three isoforms of HIF (HIF-1, 2, and 3), of which HIF-1 and HIF-2 regulate the vascular response to hypoxia. Specifically HIF-1 regulates the metabolic response, while HIF-2 stimulates erythropoiesis in response to hypoxia. HIF-3 is less closely related to HIF-1 and HIF-2, and its role is not yet fully understood. HIF-1 and HIF-2 consist of two subunits, namely α and ß. HIF-α is hydroxylated at two proline residues (Pro402 and Pro564 for HIF-1α, and Pro405 and Pro531 for HIF-2α) by prolyl hydroxylase (PHD). Once hydroxylated, HIF-α is recognized by Hippel-Lindau tumor suppressor protein (pVHL), which facilitates its proteasomal degradation by E3 ubiquitin ligase. Thus, E3 ubiquitin ligase does not cause degradation of HIF but rather ligates HIF thereby tagging it with a signal to carry it to proteasome for degradation. PHD enzymes require oxygen, iron, ascorbate, and 2-oxoglutarate (2-OG) for their activity (Figure 2). Oxygen deficiency (hypoxia) causes PHD inhibition and activates HIF- α, while HIF- ß is constitutively expressed.18 There are three isoforms of PHD, namely, PHD1, PHD2, and PHD3.18 The hypoxic stabilization of PHDs also follows a negative regulatory mechanism. In prolonged hypoxia, PHD3, and frequently PHD2 mRNA is increased to accelerate degradation of HIF upon reoxygenation after long-term hypoxia.18 The active sites of all isoforms of PHDs share a high-sequence homology. PHD1 shows more affinity towards HIF-2α than HIF-1α and hydroxylates it under normoxic condition. Deletion of the PHD1 gene induces hypoxia tolerance, by shifting the tricarboxylic acid cycle to the glycolytic pathway.19 Tolerance to hypoxia is due to reduced oxidative stress, and may not be due to angiogenesis, erythropoiesis, or vasodilation.20 PHD2 gene deletion accelerated RBC synthesis without a significant increase in EPO levels.21 Inactivation of PHD1 and PHD3 leads to erythrocytosis by activating the


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hepatic HIF-2α, whereas only PHD2 deficiency leads to erythrocytosis by activating the renal EPO pathway.22 Interstitial fibroblasts of kidneys express PHD enzymes. PHD2 inhibition causes HIF-2 α stabilization and generates EPO.23 Fibrosis decreases the capacity of the renal tissue to generate EPO, sequentially causing anemia.24 HIF stabilization can also cause EPO synthesis in the liver. In the kidneys and liver, HIF-2 stimulates EPO gene transcription by binding to hypoxia-responsive regulatory elements.25 Erythropoiesis requires EPO as well as iron for successful maturation of RBCs. HIF-2 induces efficient absorption as well as utilization of iron by stimulation of genes required for iron transport and metabolism (Figure 3). HIF-2 stimulates divalent metal transporter 1 (DMT1) and duodenal cytochrome b reductase 1 (Dcytb).26 Dcytb converts the ferric iron to the ferrous form (Fe2+) in the intestine. This conversion is vital since the gut lumen cannot absorb the ferric form of iron. Once the iron is converted to the ferrous form, it is taken up into enterocytes by DMT1. Ceruloplasmin is the enzyme in enterocyte that oxidizes ferrous iron back to the ferric form.16 Transferrin transports iron (in the ferric form) in plasma using the transferrin receptor.16 Heme-oxygenase-1 required for the release of iron from the reticuloendothelial system and ferroportin is the membrane iron exporter present in all major iron depots in the body.17 Although HIF has a direct role in modulation of all of these proteins involved in iron metabolism, the primary mechanism by which it indirectly stimulates efficient iron utilization is through regulation of hepcidin. Stabilization of HIF-α by oxygen deficiency, or genetic changes, or pharmacological inhibition leads to suppression of hepcidin.8 Apart from liver and kidney, the PHD-HIF system has an important role in the bone marrow. HIF activation stimulates expression of the EPO receptor and also modulates hemoglobin synthesis. Also, it modulates the differentiation and maturation of erythroid cells.27


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Peritubular interstitial cells generate EPO in the kidneys.28 With increasing hypoxia, the density of the EPO producing cells increases substantially. As a result of the increased density of EPO producing cells, , which results in increased EPO gene expression and enhanced release of EPO into the bloodstream.29 Differentiation of renal fibroblast-like cells under inflammatory stimulus results in fibrosis and, hence, decreased ability of the cells to generate and release EPO into plasma, which results in anemia.30 In the liver, GATA-4 is the major modulator for EPO production.30 It has been observed that the induction of erythropoiesis in the liver causes normalization of hemoglobin in rodents, in which anemia was induced by nephrectomy or inflammation.31 It has been observed that inactivation of all the three PHD isoforms is needed for a significant EPO synthesis from liver.32 CKD is associated with pathologies like enhanced levels of creatinine and urea in the circulation. These pathologies can modulate the HIF system, ultimately adding to the burden of anemia.33 Positive changes in renal physiology can also add to renal EPO production, as observed with diuretic treatment.34 A major population of CKD patients suffers from severe anemia. The degree of anemia in these patients is dependent on the severity of renal failure.35. However, even the fibrotic kidneys have the potential to generate EPO under a positive stimulus, which can correct anemia.35 A substantial population of CKD patients does not respond to ESA therapy due to an underlying inflammatory state. Inflammation-induced functional iron deficiency is a common reason for the unresponsiveness or resistance of CKD patients to EPO therapy.36, 37 The release of inflammatory cytokines decreases not only renal EPO production, but also suppresses efficient differentiation and maturation of erythropoietic progenitors in the bone marrow.37 Inflammation upregulates hepcidin through the STAT3 pathway or the BMP/SMAD1/5/8 pathway.8 As already discussed, increased hepcidin causes degradation of ferroportin, and causes entrapment of iron in tissue stores, resulting in a functional iron


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deficiency, which cannot be corrected using oral iron supplementation. As the iron deficiency gets severe, iron regulatory proteins bind to HIF-2α, and further decrease erythropoiesis.38 This feedback mechanism makes the anemia in CKD patients difficult to treat using erythropoietin analogs or iron supplementations. Activation of HIF signaling using PHD inhibition demonstrates a unique and rational approach to correct anemia associated with chronic diseases. Due to the pharmacological inhibition of HIF-PHD, there occurs a transient induction of HIF-regulated genes. Plasma EPO generated due to this inhibition is much lower than that observed in patients after injection of recombinant erythropioetins39, suggesting that PHD inhibitors may not have the risk for cardiovascular side effects that occur due to high levels of plasma EPO. Prolyl hydroxylase inhibitors decrease hepcidin and thus reduce the functional iron deficiency in CKD patients.39, 40 Currently, six PHD inhibitors are in various phases of clinical trials for the treatment of anemia associated with CKD.41 FG-2216, the first clinically tested small molecule PHD inhibitor has shown enhanced erythropoiesis in patients with end-stage renal disease (ESRD).42 EVOLUTION OF MEDICINAL CHEMISTRY STRATEGIES FOR PHD INHIBITION Under normoxic conditions, PHDs catalyze the hydroxylation of two conserved proline residues in HIF-α. The hydroxylation of HIF-α directs it towards degradation. Since metal ions are required for the 2-OG-dependent hydroxylation, targeting or chelating metal ions was the first approach exercised for the design of PHD inhibitors. Salts of Co+2, Cu+2, and Ni+2 were used to antagonize Fe+2, a cofactor for PHD enzyme action.43 Additionally, iron chelators such as deferoxamine,44 3,4-dihydroxybenzoic acid,45 1,10-phenanthrolines,46 quercetin47 have been shown to inhibit PHD. However, these compounds


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are not specific PHD inhibitors. They also inhibit iron-dependent pathways other than PHDs and may cause undue toxicities. Synthesis of 2-OG (1) analogs was the first specific approach used for the design of PHD inhibitors. Most of the clinically advanced molecules are 2-OG derivatives. PHD enzymatic activity requires 2OG as the substrate. A cocrystal structure of 2-OG bound to the PHD2 catalytic domain (PDB: 3OUJ) has shown bidentate chelation of the Fe center by the C1 and C2 sp2 oxygen atoms, and a salt bridge between the C5 carboxylate and Arg383. The wall of the 2-OG pocket is lined with hydrophobic residues including tyrosine- 310, -303, -329, isoleucine-327, -256, methionine-299, leucine-343, tryptophan-389, alanine-301, -365, and valine-376. N-oxalylglycine (2, NOG) was the first reported 2OG mimetic molecule.48 Compound 2 shows a stronger interaction with PHD2 (PDB: 3HQR) and has an N-H group in place of the C3 methylene group in 1. This modification reduced the susceptibility of the 2-carbonyl group to nucleophilic attack by oxygen. Dimethyloxalylglycine (3, DMOG)49 is a cellpermeable precursor of NOG used extensively as a tool compound to study the in vivo pharmacology of PHD inhibitors. Several active site targeted inhibitors identified in this class have strong binding interactions with hydrophobic residues in the 2-OG pocket.50 Hydrophobic heterocycles used in the place of ferrous ion coordination fragment of 2 caused a stronger binding with PHD. A crystal structure (PDB: 2HBT, Figure 4) of isoquinoline derivative 15 (R1 = Cl, R2 = R3 = R4 = R5 = H in 4, Figure 5) bound with PHD was reported by a group at Procter and Gamble.51 Groups at Oxford University and Amgen have disclosed the crystal structure (PDB: 2G1M) of another isoquinoline derivative (R1 = R2 = R3 = R4 = H, R5 = I in 4, Figure 5).52 Three conserved iron-binding triad residues (His313, Asp315, His374) occupy half of the ligand sphere of the bound Fe (Figure 4). The other three metal coordination sites of the octahedral complex involve the bidentate binding of the isoquinoline nitrogen atom and the exocyclic carbonyl oxygen atom of the ligand and a water molecule. The


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terminal carboxylate group interacts with Arg383 by making a salt bridge and with Tyr329 in the back of the 2-OG binding pocket by a H bond interaction. A π-stacking hydrophobic interaction with Tyr310 and a H-bonding interaction of the phenolic hydroxyl group with Tyr303 are other key interactions observed for this molecule. The newer 2-OG analogs caused a specific PHD inhibition owing to a differentiated mechanism. 2-OG mimetics like 2 allowed HIF binding with PHD enzymes but blocked the formation of the Fe(IV)=O complex (PDB: 3HQR). On the other hand, larger heterocyclic inhibitors such as 15, stabilized a closed conformational structure preventing HIF substrate binding (PDB: 3HQU).53 Most of the PHD inhibitors consists of three structural features based on the ligand-protein interactions. The first characteristic is a bidentate coordination site to an iron atom. The second important feature is a carboxylic acid forming a salt bridge with the Arg383 side chain. The third attribute is a hydrogen bond acceptor for the phenolic hydroxyl of Tyr303. Figure 5 demonstrates these features. However, several potent PHD inhibitors identified through high throughput screening (HTS) do not possess a carboxylic acid group that can form a salt bridge with Arg residue.50 Chan et al. described their efforts to identify PHD inhibitors that do not contain the carboxylic acid group.54 Stroke was the primary indication of these PHD inhibitors. The lack of a carboxylic acid group helps in better CNS penetration, and hence these inhibitors were considered useful in the treatment of neurological disorders. A comprehensive review has compiled the assay procedures used to screen PHD inhibitors.55 A separate review has covered significant 2-OG derived PHD inhibitors.50 An isoquinoline scaffold (4, Figure 5)56 is widely used in the design of PHD inhibitors, especially in two clinical candidates 15 (FG-2216, Figure 6) and 16 (FG-4592, roxadustat, Figure 6) discovered by FibroGen, Inc. (San Francisco, CA, U.S.). Several research groups modified the phenyl ring of the


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isoquinoline core with 5,6-membered bicyclic, tricyclic, and heterocyclic rings.50 Monocyclic pyridine derivatives (represented by general structure 5) were developed by Akebia Therapeutics.57 Oxygen atom from carbonyl group of the quinolone (e.g., 6) and monocyclic pyridone (e.g., 7) derivatives was useful for Fe chelation.50 The phenyl ring of the quinolone core underwent modification in these compounds for optimizing the activity.50 The compounds discussed above contain a glycinamide side chain attached to the ring containing the heteroatom that chelates iron. Modifications where the iron chelating heteroatom is present in another ring fused with a central core, were also reported. GSK has reported some such compounds like quinolines (X = CH in 8),58 quinoxalines (X = N in 8)59 with 6-membered ring fusion and benzimidazole derivatives (e.g., 9 and 10)60, 61 with a 5-membered ring fusion. In compounds denoted by 10, an additional cyclic ring fused with the central core has replaced the phenolic OH. It has the potential to produce symmetrical analogs which offer two chelation binding modes. Janssen Pharmaceuticals reported benzimidazole derivatives represented by 11.62 An X-ray cocrystal structure with one of the compounds (R1 = R4 = H, R2 = R3 = Cl) with PHD2 (PDB: 3OUI) has shown that the NH group from the imidazole ring interacts with Tyr303. In earlier compounds, this purpose was served by the key phenolic OH. The other nitrogen of the imidazole ring is involved in Fe chelation with carbonyl group from the glycinamide side chain. Cyclic modification of the amide group in pyrazole derivatives caused strong iron chelation (12). JNJ-42041935 (R1 = R4 = H, R2 = OCF3, R3 = Cl in 12, Figure 5) is a pan-PHD inhibitor whose pharmacological profile has been reported.63 Expansion of the benzimidazole core in 12 by insertion of “C=O” group into the C−N bond allowed a H-bond interaction with Tyr303 as in a series of quinazolinones (13).64 JNJ-42905343 (R1 = R4 = H, R2 = 2,6-dimethylphenoxy, R3 = F in 13) increased serum iron and erythropoietin in rats.65 In PGPS (peptidoglycan-polysaccharide)-treated rats, administration of JNJ-42905343 for 28 days corrected


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functional iron deficiency (FID) and anemia, an effect attributed to the increased expression of iron reductase Dcytb and iron transporter protein DMT1 in the duodenum. Human recombinant EPO (rhEPO) did not affect Dcytb and DMT1 and was not effective in correcting anemia in the PGPS model.65 The aminoquinoxaline derivatives 14 demonstrate a significant modification in which a substituted amino group replaced the carbonyl group that forms a H bond with Tyr303.66 As discussed above, inhibitors derived from 2-OG possess a carboxylic acid group as a critical pharmacophore, while non-carboxylic acid-based PHD inhibitors are also reported (mainly identified through HTS). In this review, we have classified them as carboxylic acid-based PHD inhibitors, non-carboxylic acid PHD inhibitors and even in the third category where they have demonstrated a non-conventional binding mode especially with iron. CARBOXYLIC ACID-BASED PHD INHIBITORS The first optimized structural modification of 2-OG derived chemotypes resulted in 15 (FG-2216, Figure 6). FibroGen advanced this molecule to Phase II trials in 2005.67 Compound 15 caused enhanced erythropoiesis in preclinical studies as well as in patients. The erythropoiesis was higher in nephric dialysis patients than in anephric patients, implying that 15 induced EPO production in malfunctioning kidneys. However, following the death of one subject due to fulminant hepatitis in the trial, clinical development of 15 was suspended.68 Another clinical candidate is 16 (roxadustat, FG4592), an analog of FG-2216 with an additional 7-phenoxy substituent and methyl group in place of the chloro in 15.69 Compound 16 is currently in Phase III clinical trials. Zhejiang Beta Pharma Inc. also reported the polymorphic forms of 16.70 Fibrogen reported PHD1 selective isoquinoline derivatives for therapeutic indications such as muscle degeneration, colitis, and inflammatory bowel disease (IBD) exemplified by 17-20.71 This patent application describes data for 184 compounds that show selective inhibition of PHD1 over PHD2. The


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prototype compound 17 was nonselective, and selectivity was enhanced by chain elongation, e.g., 18, which was five-fold selective for PHD1, although further elongation did not improve the selectivity. Branching the alkyl chain by introducing a methyl group in 19 made it forty-fold selective for PHD1 over PHD2. New branching with alkyl, aryl and aralkyl groups at both the amino and carboxylic acid terminals did not improve selectivity. Compound 20, with dimethyl branching and methoxy substituent on phenyl ring, has shown excellent activity and forty-fold selectivity (half maximal inhibitory concentration IC50 PHD1 = 0.24 µM, PHD2 = 9.4 µM). Shenyang Sunshine Pharmaceuticals in two separate publications reported 5-hydroxy-1,7 naphthyridine derivatives exemplified by 21 and 22. Compounds 21 and 22 are aza analogs of isoquinoline derivatives of Fibrogen (e.g., 16) substituted with aryl/heteroaryl and aryloxy/heteroaryloxy groups, respectively.72-73 These compounds were evaluated for EPO induction and PHD2 inhibitory activity in preclinical assays and compared with 16, where compounds 21 and 22 have shown superior activity in of reticulocyte production. Procter and Gamble invented monocyclic pyridines exemplified by 23 and 24 and later licensed to Akebia therapeutics.74 Compound 23 (also known as vadadustat) is now undergoing late phase clinical trials for the treatment of anemia associated with CKD. A fluoro analog 24 (AKB6899) has the potential to treat cancer.75 It was shown to be a stabilizer of HIF-2α via inhibition of PHD3, which enhances the production of suppressor of vascular endothelial growth factor receptor-1 (sVEGFR-1) but not vascular endothelial growth factor (VEGF) from granulocyte-macrophage-colony-stimulating factor (GM-CSF)-stimulated monocytes and macrophages. Compound 24 suppresses angiogenesis and provides a potential method for treating cancer. Compound 24 caused selective up-regulation of HIF-2α but did not affect VEGF. An increase in HIF-2α also enhanced sVEGFR-1 production in human peripheral blood monocytes when stimulated with GM-CSF in the presence of 24. Recent reports indicate the polymorphic forms of 23 and a process for the preparation of 23, 24 and analogs.76, 77


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Recently, both of these compounds were shown to be useful for the treatment of ocular diseases,78 followed by two disclosures studying formulation and dosing regimens for both the compounds79 and the synthesis of deuterium-enriched analogs .80 In the same monocyclic pyridine class, China Pharmaceutical University reported alkynyl pyridines as PHD inhibitors (e.g., 25).81 Compounds in this series possess a substituted alkynyl group on the pyridine ring of 23 and 24. Compound 25 inhibited PHD2 with an IC50 of 13.7 nM. Another modification of the pyridine class was reported by Shenyang Sunshine Pharmaceuticals in the form of diaryl ether analogs of compounds 23 and 24, in which a phenoxy group replaced the aryl substituent (e.g., 26, PHD2 IC50 = 15 µM). Mitsubishi Tanabe reported pyrazolopyrimidine derivatives exemplified by 27-29.82 These compounds are the heterocyclic modification of phenyl ring of 13 (Figure 5) described earlier by Janssen. In this disclosure, PHD2 and PHD3 enzymatic inhibition data of more than five hundred compounds are reported, along with EPO production measured in a cell-based assay. The structure-activity modifications involved the use of branched alkyl, cycloalkyl, and aralkyl groups on the pyrazole ring. Compound 27, with a bulky bis (4-chlorophenyl) methane group, inhibited PHD2 with an IC50 value of 0.02 µM and PHD3 with 2.6 µM demonstrating 130-fold selectivity for PHD2. The inhibition of PHDs correlated with a forty-fold increase in EPO, whereas compound 28 with a naphthyl substituent (PHD2 IC50 = 0.012 µM, PHD3 IC50 = 0.18 µM) has been shown to increase EPO induction by more than a hundred-fold. Compounds with substituents on the other nitrogen, as described in 29, have usually been found to have a negligible effect on EPO induction despite higher potency for PHD inhibition. Merck has reported bis-carboxamide derivatives exemplified by 30, 31, 32, and 33.83, 84 Compound 30 was a potent PHD2 inhibitor (IC50 1 mM. The contribution of Zn+2 to PHD3 inhibition was found to be non-significant. A mechanistic investigation led to the understanding that it is an allosteric inhibitor of PHD3.

LEARNINGS FROM THE CLINICAL TRIALS OF PROLYL HYDROXYLASE INHIBITORS The first rationally designed PHD inhibitor that reached clinical trials in ESRD patients for the treatment of anemia was 15. A modified analog 16 (roxadustat) is currently in the late stage of Phase III clinical trials. Earlier trials of 16 were carried out on patients with non-dialysis dependent chronic kidney disease (ND-CKD) and dialysis-dependent CKD. In these trials, 16 improved anemia (defined as hemoglobin increase ≥1 g/dL) and maintained hemoglobin levels (defined as hemoglobin not falling more than 0.5 g/dL).39 It has also improved EPO release and decreased hepcidin in clinical trials, which can be considered the first proof of principle for PHD inhibitors in late-stage clinical trials, without a significant safety concern. Compound 16 could be the first molecule in the PHD class to reach the clinic for the treatment of anemia associated with chronic kidney disease patients. It has a half-life of 12 h after oral dosing. When 16 was administered in doses of either 1.0 or 2.0 mg/kg twice weekly (BIW), or three times weekly (TIW), endogenous EPO release started within 4h which reached a peak at 10 h after dosing. The increase in EPO was reversible since endogenous EPO levels returned to baseline a day after administration. Maximum serum EPO levels after administration of 1 mg/kg roxadustat were 115 mU/mL, which was significantly lower than those achieved by intravenous injection of recombinant erythropoietin. After six weeks of treatment, 16 has shown a significant decrease in hepcidin levels, which has translated into reducing functional iron deficiency. After these initial trials, 16 has undergone clinical trials in more than 1000 patients.39 Apart from EPO, HIF activation causes VEGF stimulation that can lead to angiogenesis and induce tumor progression.


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However, despite the induction of erythropoiesis, 16 does not promote tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer.132 Compound 16 is a pan-PHD inhibitor (which inhibits all the three PHD isoforms), and the dosing frequency was mostly bi-weekly or tri-weekly doses ranging from 1 mg/kg to 2 mg/kg. It is expected to enter the clinical use in late 2018. Compound 34 or daprodustat (GSK1278863) is currently undergoing Phase III clinical trials.133 In a double-blind, placebo-controlled trial in anemia patients on hemodialysis (HD), 34 increased hemoglobin in a dose-related manner after four weeks of treatment. A dose-dependent increase in plasma EPO concentration was observed up to 8 mg dose, where the response was saturated. Serum EPO levels increased beyond 500 mIU/mL in the patients treated with 10 mg of 34, though VEGF did not change. However, daprodustat affects cardiac repolarization. Statistically significant decreases in the ∆QTc were observed by daprodustat treatment, along with higher incidences of gastrointestinal adverse events.134 Compound 23 (vadadustat/AKB-6548), is currently in Phase III clinical trials. In Phase-I trials, 23 (900 mg) increased the peak serum concentration of EPO to 32.4 mU/mL at 18h after treatment in healthy adults.135 In a phase II study, 23 treatment for six weeks has significantly increased hemoglobin in a dose-dependent manner, and also increased iron-binding capacity in the blood. It was associated with a decrease in ferritin and hepcidin concentration, indicating a significant improvement in iron homeostasis for efficient erythropoiesis. There were no changes in blood pressure, VEGF, C-reactive protein, or total cholesterol.135 A transient decrease in mean arterial blood pressure and a mild transient increase in serum uric acid level was observed in a four weeks dose escalation study. 136


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Compound 70 (molidustat/BAY85-3934) is a pan-PHD inhibitor, which is currently in Phase II clinical trials. The dose of 5–50 mg of 95 has resulted into a dose-dependent increase in serum EPO levels, with a peak of 39.8 mU/mL, at 12 h after a single dose of 50 mg in healthy humans, when compared to 14.8 mU/mL for placebo.137 Compound 36 (desidustat/ZYAN1), is a pan-PHD inhibitor in Phase II clinical trials. Preclinical profile of 36 indicates that it has a potential to treat anemia associated with CKD and chemotherapy-induced anemia at equivalent doses. It has demonstrated hematinic potential by combined effects on EPO release and efficient iron utilization owing to hepcidin suppression.138 Its pharmacokinetics is minimally affected in patients with CKD.139 In Phase I clinical trials conducted in Australia and India, single (10–300 mg) and multiple doses (100–300 mg) of 36 in healthy subjects was found to be safe and well-tolerated. Administration of 36 was associated with a dose-related increase in Cmax and AUC. Serum EPO concentrations showed a trend of dose-response, as well.93 Based on the t½, pharmacodynamic activity, and lack of drug accumulation, this compound is suitable for alternate day dosing in the clinic, which is currently in Phase II clinical trials. There are a few other compounds like 54 (JTZ-951), JNJ-42905343, and DS-1093. However, no significant development activities have been published for these compounds. CONCLUSION The discovery of prolyl hydroxylase enzyme inhibitors introduced new therapeutic options in the field of anemia associated with chronic diseases. More than a decade after the development of the first molecule that reached clinical trials, the understanding of prolyl hydroxylase inhibitors has increased extensively, with the conclusive evidence that inhibition of PHD could provide an exciting approach for the treatment of anemia associated with CKD. Many safe and effective PHD inhibitors followed the


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discovery of 15. These PHD inhibitors consist of a 2-oxoglutarate scaffold. These compounds blocked the interaction between 2-OG and prolyl hydroxylase and also stabilized the closed conformation of the PHD-inhibitor complex, which inhibited the substrate binding for degradation of HIF. Thus, carboxylic acid-based PHD inhibitors were discovered that incorporated an isoquinoline ring and which were optimized to the most advanced clinical candidate 16. Akebia Therapeutics has developed monocyclic pyridine derivative

23, GSK developed pyrimidine-trione derivative 34, and Cadila Healthcare

developed N-alkoxyquinolone derivative 36, which maintains the carboxylic acid chain intact. Bayer took a different approach to the development of a non-carboxylic acid based moiety, 70. Compound 70 achieved two-fold iron chelation using the ring nitrogen atoms of the pyrazole core and the adjacent pyrimidine ring, with an added interaction of the triazole moiety with the 2-OG binding residue Arg383. These compounds have been developed for a once daily or an alternate day (thrice a week) dosing regimen. The alternative day dosing regimen was designed to address the compliance issue in dialysis patients, where the drug administration needs to be in synchrony with a dialysis routine. On the other hand, an overly long half-life may invite safety issues due to prolonged stabilization of HIF. The Merck group’s optimization of 63 highlighted the importance of a structure-activity approach balancing potency, reducing side effects, and achieving the desired half-life owing to increased metabolism of the compound. PHD inhibitors stimulate renal and hepatic erythropoiesis, along with the promotion of efficient iron utilization. They induce a transient increase in the expression of HIF-regulated genes, including renal and hepatic EPO, and maintain a physiological concentration of EPO in blood. The peak EPO concentration after a clinically useful dose of 16, 23, 34, 36, and 70 have resulted in EPO levels of 32.4-500 mU/mL in circulation, which is many-fold lower than those achieved by recombinant erythropoietin administration. Thus PHD inhibitors are a safer therapeutic option for the treatment of



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anemia associated with CKD. An underestimated advantage of PHD inhibitors lies in their positive modulation of iron homeostasis under anemic conditions. CKD and other inflammatory disorders are associated with increased hepcidin. By suppression of hepcidin, PHD inhibitors improve functional iron deficiency, which may reduce the need for iron supplementation (Figure 11). Loss of function mutations in humans and PHD gene knockout data in mice have indicated that inhibition of PHDs can be useful in some indications related to hypoxia and vascular responses, although diseases other than anemia associated with CKD have yet to establish clinical proof of concept. Studies in PHD knockout mice suggest that the effect of PHD inhibitors is dependent on the extent of activation of the different isoforms of PHDs (PHD1, 2, and 3). It also depends on how the erythropoiesis in the kidney and the liver are differentially affected. On the other hand, PHD2 inhibition alone is enough to deliver a clinically relevant erythropoiesis response from the kidneys. However, to achieve a significant erythropoiesis response from the liver (as in anephric patients), simultaneous inhibition of PHD1, PHD2, and PHD3 are necessary. On the other hand, HIF activation has a widespread effect on the body, which causes angiogenesis and oncogenesis due to vascular responses to hypoxia. Hence, PHD inhibitors may arouse a safety concern in situations like diabetic nephropathy or malignancies. However, the induction of erythropoiesis by PHD inhibition has been reported not to increase tumor progression in VEGF-sensitive models of cancer. If translated to humans, this phenomenon may be useful to position PHD inhibitors for the treatment of chemotherapyinduced anemia in cancer patients. The majority of the PHD inhibitors were designed based on a 2-OG mimetic approach using in vitro and in vivo screening, although some have been discovered by an HTS approach. The in vitro assays used for the testing of compounds are not uniform across the laboratories, mostly due to the use of


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different active fragment proteins of HIF and PHD. On the other hand, the in vivo screening using hematocrit as the marker of erythropoiesis has successfully translated to the clinic for most of the clinically advanced compounds. HIF signaling is a complicated process which affects many vital pathways including glucose and lipid metabolism, angiogenesis, and vascular metabolism. The role of specific PHD isoforms or HIF-α isoforms in the regulation of these different pathways is not yet adequately delineated. Hence, it is difficult to distinguish the toxicities of PHD inhibitors that are related to the mechanism and those which are off-target. Still, PHD inhibitors are safe in CKD patients. However, the effect in long-term clinical studies using the markers of tumor progression, angiogenesis, and cardio-metabolic abnormalities are necessary in order to expand the therapeutic use of PHD inhibitors to condition like chemotherapy-induced anemia and anemia associated with aging.


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AUTHOR INFORMATION *Corresponding Author: Amit A. Joharapurkar Email: [email protected] Phone: +912717665555 NOTES

All the authors are employees or consultants of Zydus Research Centre, a unit of Cadila Healthcare Limited, which develops PHD inhibitors. The authors have no other conflicts of interest to report. The authors are grateful to Mr. Pankaj R. Patel for his guidance.

BIOGRAPHIES

Amit A. Joharapurkar received his Ph.D. in 2005, after which, he followed a career in research at the Zydus Research Centre, Ahmedabad, India, where he has led pharmacology efforts for several programs in the metabolic disorders therapeutic area, including the discovery of PHD inhibitors. He is the recipient of the CDRI Award for Excellence in Drug Research-2018. His scientific interests include obesity, diabetes, dyslipidemia, and anemia. Vrajesh B. Pandya is a medicinal chemist at Zydus Research Centre, Cadila Healthcare Limited, Ahmedabad, India. He obtained his Ph.D. degree from The Maharaja Sayajirao University of Baroda, Vadodara, India. His work in medicinal chemistry focused on novel compounds for the treatment of thrombotic and related disorders. He has contributed to the discovery and development of PHD inhibitors at Zydus Research Centre. Vishal J. Patel received his Ph.D. in Pharmacology from Kadi Sarva Vishwavidyalaya, India. His Ph.D. project focused on the development of coagonist of glucagon-derived peptide hormones as useful


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tools in the treatment of metabolic disorders. He has worked as a pharmacologist in Torrent Research Centre, India, and joined Zydus Research Centre in 2007. In Zydus Research Centre, he is involved in screening and development of new chemical entities for obesity, dyslipidemia, and anemia programs. He is the recipient of Wiley-Journal of Diabetes Young Investigator Award in 2013, and Keystone Symposia Future of Science Fund scholarship in 2016, Ranjit C. Desai was a Senior Vice President and Head of Chemistry (July 2012-Dec 2017) at Zydus Research Centre, Cadila Healthcare Ltd, Ahmedabad, India. He is the inventor of desidustat, a PHD inhibitor currently in Phase 2 clinical trials. Dr. Desai received his Ph.D. from the MS University of Baroda, India working with Dr. Sukh Dev at the Malti-Chem Research Centre. After working as a postdoctoral associate at Clemson University, Purdue University and the University of Montreal, he worked at Sanofi-Winthrop, Hoechst Celanese, and Merck Research Laboratories. Currently, he is a Senior Consultant at Zydus Research Centre and runs projects in the metabolic diseases and anti-bacterial area. Mukul R. Jain is a Senior Vice President at Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad, India. Here, Dr. Jain leads the preclinical development of new drugs. Dr. Jain obtained his M.Pharm. and

Ph.D. from Nagpur University, India. After completing Ph.D., he worked at

Wockhardt and Ranbaxy Research Centers before moving to the University of Florida at Gainesville as a Postdoc Associate. He worked as Assistant Professor of Pharmacology in National Institute of Pharmaceutical Education and Research, Mohali, India, before joining Zydus Research Centre in 2000. His group at Zydus Research Centre has developed several NCEs and biologics that are currently undergoing clinical development. Dr. Jain is a member of several scientific associations. ABBREVIATIONS USED



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AUC, area under curve; EPO, erythropoietin; ESA, erythrocyte-stimulating agents; PHD, prolyl hydroxylase domain; HIF, hypoxia-inducible transcription factor; RBC, red blood cells; CKD, chronic kidney disease; HSC, hematopoietic stem cells; CFU-E, colony forming units-erythroid; EPOR, EPO receptors; rhEPO, human recombinant EPO; JAK2, Janus Tyrosine Kinase-2; STAT, signal transduction and activator of transcription; AKT kinase, protein kinase B, CD95, cluster of differentiation 95; TNF, tumour necrosis factor; FPN, ferroportin; BMP/SMAD, Bone morphogenetic protein/SMAD; IL-6, interleukin 6; ERFE, erythroferrone; pVHL, Hippel-Lindau tumor suppressor protein; 2-OG, 2-oxoglutarate; TRF, transferrin; DMT1, divalent metal transporter 1; Dcytb, duodenal cytochrome b; TRFR, transferrin receptor; VEGF, vascular endothelial growth factor; NOG, Noxalylglycine; DMOG, dimethyloxalylglycine; IBD, inflammatory bowel disease; GM-CSF, Granulocyte-macrophage colony-stimulating factor; VEGFR-1, vascular endothelial growth factor receptor-1; sVEGFR-1, suppressor of vascular endothelial growth factor receptor-1; IC50, half maximal inhibitory concentration; EC50, Half maximal effective concentration; CP4H, collagen prolyl hydroxylase; FIH, factor inhibiting HIF; HTS, high throughput screening; HTRF, Homogeneous Time Resolved Fluorescence; hERG, Human ether-a-go-go-related gene; PK, pharmacokinetic; PCV, packed cell volume; rhEPO, human recombinant erythropoietin; LDH, Lactate dehydrogenase; SAR, structure–activity relationship; PAMPA, Parallel artificial membrane permeability assay; CNS, central nervous system; MDR, multidrug resistance protein ; FDA, U S Food and Drug Administration; BIW, twice weekly; TIW, three times weekly; ESRD, End stage renal disease; ND-CKD, Non-dialysis dependent chronic kidney disease; HD, hemodialysis

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Figure 1

Figure 1. The stages of erythropoiesis. The process of erythropoiesis occurs in the bone marrow. Erythropoiesis is the process of proliferation and differentiation of erythroid progenitors into reticulocytes and erythrocytes (RBC). The common myeloid progenitors also known as stem cells, form RBC, white blood cells, and platelets. Stem cells are differentiated into early-stage burst-forming unit-erythroid (BFU-E) and then later stage colony-forming unit-erythroid (CFU-E) progenitors. CFUEs differentiate into proerythroblasts, which then undergo mitosis to form erythroblasts. The erythroblast is basophilic, polychromatic, and orthochromatic. The erythroblast denucleates to become a reticulocyte, which enters the bloodstream. Reticulocytes mature to become RBCs after shedding cellular organelles such as mitochondria, Golgi apparatus, and endoplasmic reticulum. A stage of erythropoiesis from BFU-E to erythroblast differentiation requires EPO. Maturation of RBCs from erythroblasts requires iron. Figure 2


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Figure 2. PHD-HIF mechanism of enzyme action. At normal oxygen concentration (normoxia), prolyl hydroxylase domain-containing (PHD) enzymes (PHD1, PHD2 or PHD3) are active. Active PHDs hydroxylate HIF-α residues. Once hydroxylated, HIF-α is recognized by Hippel-Lindau tumor suppressor protein (pVHL), which facilitates its proteasomal degradation by E3 ubiquitin ligase. Thus, E3 ubiquitin ligase does not cause degradation of HIF but rather ligates HIF thereby tagging it with a signal to carry it to proteasome for degradation. For this reaction, PHDs require 2-oxoglutarate (2-OG) as a substrate and iron (Fe) as a co-factor. On the other hand, in hypoxic conditions, or by pharmacological inhibition of PHDs, HIF-α is protected from degradation and heterodimerizes with HIF-ß. The HIF complex is then translocated to the nucleus and stimulates EPO gene expression. Stimulation of EPO gene expression under hypoxia or PHD inhibition translates into efficient erythropoiesis. HIF complex is also involved in the regulation of angiogenesis, glucose metabolism, lipid metabolism and vasodilation.


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Figure 3

Figure 3. Iron metabolism and the role of hepcidin. Absorption of iron mainly occurs in the intestine. The non-heme iron in the ferric form is converted in the ferrous form by duodenal cytochrome b (Dcytb). Divalent metal transporter 1 (DMT1) facilitates the transfer of iron into enterocytes. Hepcidin is a regulator of iron homeostasis that downregulates DMT1 and limits iron absorption. Ferritin is the soluble and nontoxic form of iron in plasma. Ferritin is the form of stored iron. Iron in the ferrous state enters into blood circulation with the help of the transport protein ferroportin. Hephastin converts ferrous iron to ferric iron which then binds to transferrin (TRF) and circulates as TRF bound ferric iron. It is taken up into the metabolic tissues such as macrophage or the liver by transferrin receptor (TRFR). TRF-bound ferric iron is then released free into the cell. Inside the cell, DMT1 converts ferric iron into


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ferrous iron, after which it is stored as ferritin or released from the cell by ferroportin. Hepcidin inhibits ferroportin, by which it can block iron uptake into cellular stores, and decreases iron in circulation. Like hephastin, ceruloplasmin present in blood converts ferrous iron into the ferric form. The ferric form binds to TRF and circulates in the blood. TRF-bound ferric iron is taken up by reticulocytes for hemoglobin synthesis, which then mature into RBCs.


Figure 4

Figure 4. Structure of a 247-residue C-terminal catalytic domain of PHD2 bound to 15 (PDB: 2HBT).


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Figure 5

Figure 5: Chemotypes derived from 2-OG.


Figure 6

Figure 6: Carboxylic acid-based PHD inhibitors (Part 1).


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Figure 7

Figure 7: Carboxylic acid-based PHD inhibitors (Part 2).


Figure 8

Figure 8: Non-carboxylic acid PHD inhibitors (Part 1).


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Figure 9

Figure 9: Non-carboxylic acid PHD inhibitors (Part 2).


Figure 10

Figure 10. PHD inhibitors with the non-conventional binding mode.


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Figure 11

Figure 11: Mechanism of action of HIF-PHD inhibitors. The chronic inflammatory condition or kidney disease increases the release of inflammatory cytokines and decreases erythropoietin release. Inflammatory cytokines stimulate hepcidin synthesis. Increased levels of hepcidin downregulate DMT1, Dcytb and block ferroportin, and inhibit iron absorption. Hepcidin degrades ferroportin in macrophages and the liver, which leads to reduced iron availability in the blood, and increases intracellular iron. The decrease in EPO and increased hepcidin inhibit hemoglobin synthesis and the maturation of RBCs, resulting in anemia. PHD inhibitors stabilize HIF and stimulate EPO release and decrease hepcidin levels, and thus stimulate efficient erythropoiesis.


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Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of

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