Nonpeptidic Ligands for Peptide-Activated G Protein-Coupled

Chem. Rev. 2007, 107, 2960−3041

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Nonpeptidic Ligands for Peptide-Activated G Protein-Coupled Receptors Jade S. Blakeney, Robert C. Reid, Giang T. Le, and David P. Fairlie* Centre for Drug Design and Development, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia Received July 3, 2006

Contents 1. Introduction 2. Peptide-Activated GPCRs and Nonpeptide Ligands 2.1. Angiotensin Receptors 2.2. Bombesin Receptors 2.3. Bradykinin Receptors 2.4. Calcitonin and Calcitonin-Like Receptors 2.5. Chemokine Receptors 2.5.1. CC Chemokine Receptors 2.5.2. CXC Chemokine Receptors 2.6. Cholecystokinin and Gastrin Receptors 2.7. Complement Receptors 2.7.1. Complement C5a Receptor 2.7.2. Complement C3a Receptor 2.8. Corticotropin-Releasing Factor Receptors 2.9. Endothelin Receptors 2.10. Formyl Peptide Receptor 2.11. Galanin Receptors 2.12. Glycoprotein Hormone Receptors 2.12.1. FSH Receptor 2.12.2. LH/hCG Receptor 2.13. Gonadotropin-Releasing Hormone Receptor 2.14. Growth Hormone Secretagogue (Ghrelin) Receptors 2.14.1. Agonists of GHS Receptors 2.14.2. Antagonists of GHS Receptors 2.15. Melanin-Concentrating Hormone Receptors 2.16. Melanocortin Receptors 2.16.1. MC4R Agonists 2.16.2. MC4R Antagonists 2.16.3. Other MCR ligands 2.17. Neurokinin Receptors 2.18. Neuropeptide Y Receptors 2.18.1. Neuropeptide Y1 2.18.2. Neuropeptide Y2 2.18.3. Neuropeptide Y5 2.19. Opioid Receptors 2.20. Opioid-Like Receptor 2.21. Orexin Receptors 2.22. Oxytocin Receptor 2.23. Protease-Activated Receptors 2.24. Somatostatin Receptors 2.25. Urotensin Receptors 2.26. Vasopressin Receptors

2960 2964 2964 2966 2967 2970 2972 2972 2982 2985 2988 2988 2988 2989 2989 2992 2992 2993 2994 2995 2995 2996 2997 2999 3000 3002 3003 3006 3008 3009 3012 3012 3014 3015 3017 3021 3022 3022 3025 3026 3027 3029

* To whom correspondence should be addressed. E-mail, d.fairlie@ imb.uq.edu.au; fax, +61733462990.

3. 4. 5. 6.

Summary Abbreviations Acknowledgments References

3031 3033 3033 3033

1. Introduction There are some 850 human G protein-coupled receptors (GPCRs) currently known (http://bioinformatics2.biol.uoa.gr), making this the most abundant family of well-characterized protein receptors on the cell surface. Their function is to mediate signal transduction across the cell membrane, and they are so named because they couple intracellularly with G proteins, although it is now known that they also couple other types of intracellular proteins as well.1-5 About 60% of known GPCRs are sensory and olfactory receptors that mediate vision, taste, smell, and so forth, while the other ∼300 known GPCRs respond to extracellular stimuli like protein and peptide hormones, amino acids, biogenic amines, lipids.6,7 Consequently, GPCR proteins have become just about the most intensively studied targets for drug development, accounting for ∼30% of pharmaceuticals used in man and ∼10% of global pharmaceutical sales.8-10 Yet, such licensed human pharmaceuticals only target ∼30 (4%) of the known GPCRs, and include R1 antagonists (e.g., doxazosin, terazosin), β-agonists (salmeterol, salbutamol) and antagonists (metoprolol, atenolol), histamine antagonists (e.g., loratadine, fexofenadine, ranitidine, famotidine, cetirizine, cimetidine), 5-HT agonists/antagonists (e.g., olanzapine, respiradone, sumatriptan, ondansetron, buspirone, cisapride), and GABA agonists (gabapentin).8,9 Several hundred other human GPCRs are known to be activated by extracellular protein or peptide ligands,11 but only a few of these receptors are currently targeted by marketed drugs (Table 1).8 Such drugs include angiotensin AT1 antagonists (losartan, valsartan), endothelin antagonist (bosentan), LHRH agonists (leuprolide, goserelin), NK1 antagonist (aprepitant), oxytocin OTR antagonist (atisoban), somatostatin SSTR agonists (SRIF-14, octreotide, lanreotide), and vasopressin antagonists (conivaptan). Clearly, peptide/ protein-activated GPCRs represent a large, promising, and still under-developed source of new pharmaceutical targets for treating human diseases or medical conditions. This perspective review describes a necessarily limited selection of key small molecule nonpeptidic agonists and antagonists that have been identified to date as regulators of peptideactivated GPCRs, and outlines some of their pharmacological properties. It provides a cross section of scientific literature up to the end of 2005, in some cases through early 2006, without attempting to describe compound syntheses or the extensive patent literature.

10.1021/cr050984g CCC: $65.00 © 2007 American Chemical Society Published on Web 07/11/2007

Nonpeptidic Ligands for Peptide-Activated GPCRs

Ms. Jade Blakeney obtained a B. Biotech. (Hons) degree in drug design and development from the Institute for Molecular Bioscience in 2004 and is studying for her Ph.D. degree on GPCRs and drug design at the University of Queensland with Professor Fairlie and Dr. Reid.

Dr. Robert Reid received his B.Sc. Hons (1986) and Ph.D. (1990) degrees from the Department of Organic Chemistry, University of Sydney on the stereospecific total synthesis of natural products with M. J. Crosssley. He undertook postdoctoral studies with J. E. Baldwin at the Dyson Perrins Laboratory, Oxford University (1990−93), studying the biosynthesis of penicillins. Presently, he is a Senior Research Officer at the Centre for Drug Design and Development−Institute for Molecular Bioscience at the University of Queensland with research interests in drug design and synthesis, nonpeptidic GPCR antagonists, natural products, protease inhibitors, other enzyme inhibitors, and peptidomimetics.

GPCRs have also historically been referred to as serpentine receptors and are found on the outer cell membrane of all cells. They comprise seven transmembrane helices, with an extracellular amino-terminus and an intracellular carboxylterminus (Figure 1). GPCRs are subdivided into three major classes and three minor classes, based largely on the size of the N-terminus as well as conserved residues in the transmembrane regions (A-F classification)12,13 or are divided into five families based on their phylogenetic evolution (GRAFS classification).6,7 The A-F classification covers all GPCRs across vertebrates and invertebrates, even those GPCRs that lack human analogues (D-F subfamilies),12,13 but has been hampered by the large variation across species. The GRAFS classification focuses on mammalian GPCRs, breaking the superfamily into Glutamate, Rhodopsin, Adhesion, Frizzled/ Taste2, and Secretin families.6,7 The rhodopsin (classA), secretin (class B), and glutamate (class C) are the major classes of GPCRs. Neither classification system predicts the structural topology of the receptors, although different classes are observed to have different ligand binding sites. Class A or rhodopsin family is activated by ligands binding a crevice

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Dr. Giang Le earned his M.Sc (1996) and Ph.D. (1999) degrees in organic chemistry from the Technology University of Budapest, Hungary. He moved to Australia in 1999 to work for Alchemia, a start-up company, where his works focused on developing diverse combinatorial libraries based on carbohydrate scaffolds to generate drug leads targeting GPCRs. In 2003, he joined the Fairlie group as a Research Officer. His research interests range from design and discovery of druggable protease inhibitors, GPCR agonists and antagonists, to peptide and protein secondary structure mimetics.

Professor David P. Fairlie has B. Sc. Hons (University of Adelaide) and Ph.D. (University of New South Wales) degrees, and did postdoctoral research at Stanford University and University of Toronto. He has held research/teaching appointments in six Australian universities and led the Chemistry Group in the Centre for Drug Design & Development at University of Queensland since 1991. He holds Australian Research Council Professorial and Federation Fellowships. His research interests are in chemical synthesis (organic, medicinal, inorganic); molecular recognition; peptide and protein mimetics; nonpeptidic inhibitors of enzymes, including proteases (www.protease.net), and antagonists of GPCRs and other receptors for inflammatory disorders, viral and parasitic infections, cancers, and neurodegenerative diseases. His mechanistic studies have been on chemical reactions, biological processes, disease development, and drug action.

between transmembrane helices III, V, VI, and VII and/or in the extracellular loops. Class B or secretin are activated by peptides binding to the extracellular loops, whereas glutamate or class C receptors are activated by a ligand binding ‘venus-flytrap’ formed by the extracellular aminoterminus.6,7,13-15 The selective regulation of a GPCR is still a significant challenge in medicinal chemistry and pharmacology. The genes that produce GPCR proteins also produce multiple subtypes (isoforms) as a result of gene splicing, and these may serve to differentially regulate G protein-dependent and -independent signaling mechanisms, depending upon their tissue distribution and exposure to other factors that regulate receptor activation. GPCRs can be structurally and function-

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Table 1. Peptide-Activated GPCRs, Coupled G Proteins, Associated Diseases, and Nonpeptidic Drugs in Clinical Use

G-proteina

GPCR AT1 AT2 BB1/BB3 BB2 B1 B2

Gq/11, G11/12, Gi/o Gi, but in a different mode to other GPCRs Gq/11 Gq/11, G12/13 Gq, Gi Gq/11, G12/13, Gi, Gs

CTR CLCR CCR1

Gs

CCR2

Gi

CCR3

Gi

CCR4

Gi/o

CCR5

Gi

CCR9

Gi

CCR10

Gi/o

CXCR1/2

Gi/o

CXCR3

Gi/o

CXCR4

Gi/o

C3aR C5aR

G12/13, Gi/o, Gz G15/16, Gi/o, Gz

CCK1 CCK2

Gq/11, G12/13, Gs; Gq/11

CRF1-2 ETA ETB FPR, FPRL-1/2 GalR-1/3 GalR-2 FSHR TSHR LH/hCGR GnRH

Gs, Go, Gq/11, Gil/2, G2 Gq/11, G12/13, Gs Gq/11, G12/13, Gi/o Gi/o, Gz Gi/o Gq/11, G12/13, Gi/o Gi/o, Gs Gi/o, Gq/11, G12/13, Gs Gi/o, Gq/11, Gs Gq/11

GHSR MCHR1 MCHR2 MC1R to MC5R

Gq/11 Gi/o, Gq/11 Gq/11 Gs

NK1 NK2/3 Y1 Y2/4/5

Gq/11, G12/13, Gs Gq/11, Gs Gi/o, Gq/11 Gi/o

Gi/o, G14, G16

nonpeptidic clinical candidatesb

diseaseb Hypertension Small lung, prostate and gastrointestinal carcinomas Septic shock, pancreatitis, oedema, rhinitis, asthma, colitis, arthritis, Alzeheimer’s, pain, small cell lung cancer Paget’s disease, osteoporosis, hyperglycermia, migraine Multiple sclerosis, rheumatoid arthritis, organ transplantation, Alzeheimer’s, atherosclerosis, psoriasis, multiple myeloma Atherosclerosis, asthma, multiple sclerosis, arthritis and other inflammatory diseases Asthma, atopic dermatitis, allergic rhintis Asthma, arthritis and inflammation of the skin HIV infection, rheumatoid arthritis, multiple sclerosis, asthma, autograft rejection Crohn’s disease, inflammatory bowel disease Delayed-type hypersensitivity, psoriasis, allergic-contact Lung reperfusion injury, gout, psoriasis, cancer, rheumatoid arthritis, osteoarthritis, ARDS, COPD Rheumatoid arthritis, multiple sclerosis, hepatitis C, atherosclerosis, chronic skin reactions HIV infection, cancer, autograft rejection Asthma Rheumatoid and osteo-arthritis, inflammatory bowel disease, fibrotic conditions, systemic lupus erythematosus, ischemia/ reperfusion injury, sepsis, asthma, psoriasis, ARDS and more Pancreatic disorders, biliary colic and inflammatory bowel syndrome, pain Anxiety, Depression Hypertension, heart failure, atherosclerosis, prostate cancer Inflammation Neurogenic inflammation and pain FSH/LSH; Infertility/contraceptives; TSH - thyroid cancer Infertility, endometriosis, uterine fibroids, cancer of reproductive organs Growth hormone deficiency, obesity Obesity, memory, anxiety, depression MC4R-Obesity, diabetes, sexual dysfunction Inflammation and pain, emesis Obesity and hypertension

AT1 - Losartan, irbesartan, candesartan, valsartan, olmesartan, eprosartan, telmisartan B2 - LF16,0335 (anatibant)

CGRP - BIBN4096BS BX471, CP-481,715, AZD-8309, MLN3897, C-4462, C-6448 INCB3284 DPC168, GW766994

TAK779, UK427,857 (maraviroc), Sch-D (vicriviroc), GW873140 CCX282

SB332235, Repertaxin

T487

AMD3100 NGD-2000-1 (discontinued)

L-364,718 (devazepide-discontinued), L-365,260, YF476, Z360, T-0632 (210) R121919, SSR-125543A, NBI-35965 Bosentan, Enrasentan, Atrasentan

TAK-013, NBI42902 MK-677, capromorelin, NN703

Aprepitant, CP99994, CJ11974, GR205171



Table 1. (Continued)

GPCR

a

G-proteina

nonpeptidic clinical candidatesb

diseaseb

µ-, δ-, κreceptors

Gi/o

Pain, inflammation and gastro-mobility

OLR-1

Gi/o

OX1R + OX2R OTR PAR1 PAR2-4 SSTR1-5

Gq/11 Gi/o, Gq/11 Gq/11, G12/13, Gi/o Gq/11, Gi/o Gi/o

hUT V1R V2R V3R

Gq/11 Gq/11, G12/13 Gq/11 Gs

Anxiety, stress, drug and alcohol addictions Narcolepsy and cataplexy Preterm labor PAR1- anti-coagulation, PAR2 - cancer and inflammation Tumors, diabetic retinopathy, nephropathy and inflammation Cardiovascular disorders V1R- heart failure, arterial hypertension, atherosclerosis, dysmenorrhea V2R - heart failure, liver cirrhosis, SIADH, nephrotic syndrome, hyponatremia V3R - ACTH-tumors, social behavior

Morphine, buprenorphine, sufentanil, naloxone, naltrexone, alvimopan, loperamide, cyprodime, meperidine, fentanyl, methodone, levorphanol

L369,899 SCH-530348

V1R - Relcovaptan, V2R - OPC31260, tolvaptan, lixivaptan V1/2R - YM087

http://bioinformatics2.biol.uoa.gr. bThis review.

ally influenced by a wide range of factors, including expression levels of different subtypes, receptor dimerization, covalent modifications, phosphorylation and glycosylation of receptor and ligands, coupling with intracellular G proteins and other proteins, binding and inactivation by antagonists, inverse agonists or allosteric ligands, and binding and activation by agonists, and there is now also evidence that different agonists can effect different intracellular responses possibly due to subtly different binding locations in the receptor inducing different activation states.17-19 Historically, GPCR isoforms have been mainly discovered through structure-activity differences between small molecule agonists or antagonists. In other words, the discovery of small molecules to regulate GPCRs has paradoxically and importantly resulted in the discovery of new, and better, characterization of known GPCRs and their subtypes. The absence to date of well-defined, three-dimensional structures for these membrane-spanning proteins has made ligand discovery more difficult and more time-consuming than it is for other classes of therapeutic targets where the 3D protein structures have been determined. Currently, the only crystal structures available for GPCRs are the light-activated bovine rhodopsin in both its inactive (Figure 1b) and photoactivated states.20-23 Therefore, native peptide ligands for GPCRs have often provided the first clues for drug development, and led to many peptidomimetics constrained into GPCR-binding bioactive shapes through cyclization or other molecular constraints.11,14,24,25 Although many peptidic and peptidomimetic ligands for GPCRs have been examined in human clinical trials, and some are even marketed drugs, they are generally perceived as having limited value as therapeutics due to low oral bioavailability, poor pharmacokinetic profiles, and metabolic instability. Consequently, most efforts to produce GPCR ligands as human therapeutics have focused on discovery and development of nonpeptidic ligands. Native peptide ligands for GPCRs provide limited clues to the design of nonpeptidic ligands because they tend to bind in different ways to, or at different sites on, a GPCR. Indeed the huge variation in the sizes of the extracellular N-terminus and extracellular loops across peptide-activated

GPCRs is no doubt the result of evolutionary pressures for cells to select for specific types of extracellular protein ligands. On the other hand, most nonpeptidic ligands for GPCRs target the hydrophobic transmembrane helices of GPCRs without ever contacting their extracellular domains. As a result, the conventional path to drug discovery for GPCRs has usually involved extracting clues either from peptide fragments that bind within the transmembrane region of GPCRs, or from high-throughput often random screening of natural products and chemical libraries to find a ‘hit’ that has some activity or affinity for a particular GPCR. Hit to ‘lead’ optimization has then utilized combinatorial synthesis approaches for generating derivatives of hits, guided by structure-activity relationships obtained through measuring ligand affinities and functional potencies against GPCRs. Most lead compounds have binding affinities and functional activities against GPCRs at submicromolar concentrations. More recently, homology structural models of the conserved 7TM domain of GPCRs have been used, in conjunction with site-directed receptor mutagenesis, to pinpoint ligand binding sites and thus facilitate more rational design and lead optimization toward improved nonpeptide ligands. In principle, homology modeling has the capacity to provide information that cannot be obtained from the native ligand, through exploring different, previously unknown or overlapping, ligand binding sites on the receptor. The use of in silico or virtual screening of chemical databases in these computer models has increasingly produced valuable hits for chemical optimization and, as new software becomes available offering increased accuracy for ligand docking and scoring of complementary ligand-receptor interactions, this approach is likely to become even more effective for prioritising compounds for synthesis and pharmacological evaluation. Many thousands of compounds have been identified as regulators of GPCRs, and some useful patterns have emerged in such ligands. There is, for example, growing evidence that peptide-activated GPCRs recognize turn conformations as evidenced by structure-activity relationships in their native ligands, modified peptide fragments, cyclic peptide analogues, and turn peptidomimetics.11,14,24,26-47 Many dif-


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druggable fragments for assembling into new lead compounds, with potential for targeting GPCRs specifically and proteins in general.

2. Peptide-Activated GPCRs and Nonpeptide Ligands 2.1. Angiotensin Receptors

Figure 1. GPCR topology: (a) schematic representation of a GPCR, with 7 transmembrane helices (TMI-VII) connected by three extracellular (EC) and three intracellular (IC) loops; (b) crystal structure of GPCR bovine rhodopsin (pdb: 1U19).16

ferent GPCRs recognize certain common (privileged) structural fragments or scaffolds in their nonpeptidic ligands, although only a few of these have been identified to date.48-57 The combination of drug-like fragments from known bioavailable GPCR-targeting drugs might be a successful approach to obtaining drug leads for a different GPCR, though with associated risks of losing receptor selectivity. A major challenge in the design and development of druggable lead compounds for GPCRs is the effective assimilation of the voluminous data already in the literature on the nature and properties of compounds known to regulate this receptor class. To provide a more comprehensive basis for assessing the status and future prospects of GPCRdirected drug discovery, we have examined some 76 peptideactivated GPCRs and now assemble a diverse portfolio of 577 nonpeptidic ligand structures that have been reported to date to regulate them. For these ligands, we report on their chemical compositions and origins, selective affinities for GPCRs, agonist/antagonist potencies, an abbreviated summary of their most important pharmacological properties relevant to selective GPCR regulation, and their potential for the treatment of diseases. This portfolio of compounds also provides a valuable new reference guide to useful

The renin-angiotensin system (RAS) regulates physiological responses associated with maintaining blood pressure and electrolyte/fluid homeostasis and is important in hypertension and diabetes.58,59 It functions through the actions of the vasoconstrictor octapeptide angiotensin II (AII; D1RVYVHPF8), generated by proteolytic degradation of angiotensinogen. Blockage of the angiotensin II receptor at the terminal step of RAS regulates the system, irrespective of the source of angiotensin II. There are two angiotensin receptors, AT1 and AT2. Activity of AII at AT1 mediates virtually all of the physiological actions of angiotensin in the cardiovascular system. Consequently, this angiotensin receptor has been intensively studied and is one of the most advanced therapeutic targets among peptide-activated GPCRs, with nonpeptide drugs already in the marketplace. Spectroscopic studies of the peptide agonists AII and [Sar1]-AII have identified a folded hairpin structure centered at a trans His6-Pro7 peptide bond.58 There is also an observed clustering of three aromatic rings and a charge relay system involving the Tyr4-OH, His6-imidazole, and Cterminal carboxyl of Phe8.58 The conformation of these agonists was important in the rational ligand-based design of the first nonpeptide antagonists including a 1-benzylimidazole-5-carboxylic acid series, containing the prototype nonpeptide AT1 antagonist CV-2947 (1).59 Discovery of 1 in 1982 and the release of the structure of AII led to the development of three main angiotensin ligand scaffolds. The first group of compounds 2-6 all contain a benzylimidazopyridine scaffold with high receptor binding affinities (antagonist 2, L-159,282, IC50 0.3 nM (AT1); agonist 3, L-162,313, IC50 1.1 nM (AT1), 2.0 nM (AT2); agonist 4, L-163,101, IC50 13 nM (AT1), 10 nM (AT2);60 5, L-163,491, IC50 57 nM (rAT1); and 6, L-162,389, IC50 130 nM (rAT1)).61 A second group of compounds 7-14, with a biphenyl-tetrazole scaffold, are the ‘sartan’ antagonists of AT1 with high binding affinities (IC50: 7, losartan, 19 nM; 8, irbesartan, 1.3 nM; 9, candesartan, 12 nM; 10, valsartan, 8.9 nM; 11, olmesartan, 7.7 nM;62 12, SC-51,316, 0.51 nM;61 and 13, KR-31,0163, 0.52 nM).63 Compound 14 (L-158,809)61 contains both the benzyl-imidazopyridine and biphenyl-tetrazole scaffolds with an IC50 0.2 nM (rAT1) and appreciable oral bioavailability (32%) in rhesus monkeys. The biphenyl-tetrazole scaffold observed in these compounds was recently suggested to occupy a conserved privileged structure-binding site within the transmembrane region of at least 4 GPCRs.64 A third group, 15-16. is based on the imidazolyl acrylic acid scaffold and includes 15 (eprosartan, IC50 1.5 nM (hAT1))62 and 16 (SB-203,220, IC50 32 nM (rAT1)).61 Compounds 7-11, 15, and 17 are clinically used AT1 antagonists for the treatment of hypertension (Chart 1). Compounds 1-14 are usually all described as biphenylimidazoles, with the tetrazole and sulfonamide interchangeable units designed as carboxylic acid surrogates. The tetrazole and sulfonamide units lower the partition coefficient (logP) by 1-1.5 log units compared to carboxylic acid, but these substituents have different acid dissociation



Chart 1

constants (pKa’s 3.1-4.8 (tetrazoles), 9.8-10.2 (sulfonamides)65). The different acidities might influence the success of compounds in ViVo, since those containing tetrazole but not sulfonamide substituents are presently used in the clinic.

Compounds 2-14 were designed from 1 by aligning their imidazoles with that of His6 in the [Sar]-AII structure.66 Subsequent modification gave the biphenylimidazole scaffold found in most of the antihypertensive ‘sartans’ (pharmacokinetic properties in Table 2). Structure-activity relationship


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Table 2. Pharmacokinetic Properties of Angiotensin Antagonists68 c sartan

product name & producer

Dose (mg/day)

Losartan

Cozaar; Merck Sharp & Dohme

Eprosartan Candesartan cilexetil Telmisartan Irbesartan Valsartan Olmesartan

Teveten; Solvay Pharmaceuticals Atacand; AstraZeneca Micardis; Boehringer Ingelheim Avapro; Bristol-Myers Squibb Pharmaceutical Diovan, Novartis Pharmaceutical Benicar; Sankyo Pharma

a

50 mg 600 mg 8-16 mg 40 mg 150 mg 80-160 mg 20-40 mg

F% 33 13 14 40 60-80 25 26

volume of distribution

half-life (h)

L ) 34 La, E ) 12 Lb 0.22 L/kg 0.1 L/kg 6.6 L/kg 53-93 L 17 L 17 L

L ) 2 a, E ) 6b 5-9 9 18.3-23 11-15 6 13

L ) Losartan. b E ) active metabolite EXP3174 of Losartan. c Adapted from data summarized in Reference 68.

Chart 2

Chart 3

data for the tetrazole-biphenylimidazoles and homology modeling of AT1 have been used to identify a pharmacophoric model.67 The ortho-position of the tetrazole/acid function was found to be important for twisting the biphenyl groups out of planarity.67 C2 on the imidazole ring prefers a 3-4 carbon alkyl or alkene chain.59 C4 and C5 can tolerate a variety of substituents with different groups in the C4 position altering the pharmacokinetic properties of resulting antagonists.59 It is also advantageous for a hydrogen bond acceptor to occupy the C5 position, with acidic components inducing insurmountable antagonism (non-parallel rightward shift of the angiotensin concentration curve and reduced maxima). However, acidic substituents increase serum albumin binding of the antagonist, thereby introducing discrepancies between in Vitro and in ViVo assays. Fusion of C4 to C5 positions of the imidazole in ring systems is also tolerated.59 From homology modeling, one of the most significant interactions of AT1 antagonists is formed between the acid functionality (tetrazole) and Lys199 of transmembrane helix 5 in AT1.67 Hydrophobic aromatic regions were considered important for stabilizing the acid-base interaction. The model suggested an electron-rich site and two π-electron-rich sites, corresponding to oxygen or chlorine atoms and the phenyl rings or sulfonamide oxygen atoms, respectively.59 Compounds like 15 and 16 were not designed from 1, but from modeling the conformational structure of AII and from SAR data on peptides. Eprosartan (15) used a mimetic of

Tyr4, absent in the losartan-based series, and a thienylmethyl group that mimics Phe8. Modifications to, and alternative substitution patterns on, the benzyl-imidazopyridine scaffold have led to another series of antagonists including 17 (telmisartan, IC50 9.2 nM, hAT1),62 18 (L-163,017, IC50 13 nM, rAT1),61 and 19 (L746,072, IC50 97 nM, rAT1) (Chart 2).63 Angiotensin ligands 20-22 (Chart 3) do not fit into the above classes. Compound 20 is an AT1 antagonist69 containing a benzoazepinone privileged structure. Compound 21 (PD123,31970), designed from the natural product spinacine (4,5,6,7-tetrahydroimidazo[4,5-c]pyridine-6-carboxylic acid) that had micromolar affinity for the angiotensin receptor (AT2),71 is a selective AT2 antagonist with appreciable affinity (IC50 34 nM). Compound 2272 is also a selective AT2 antagonist (Ki 3 nM) designed to incorporate a γ-turn mimetic for constraining the peptide to the active angiotensin conformation.

2.2. Bombesin Receptors There are three human bombesin receptors, BB1-3. BB1 binds preferentially to neuromedin B, BB2 binds gastrinreleasing peptide, while BB3 is an orphan bombesin receptor.73 These receptors have been found to mediate a large range of biological activities, but it was their role as autocrine growth factors for tumors that catalyzed medical interest in the generation of bombesin antagonists.74 BB2 in particular



Chart 4

is highly expressed in human tumors, such as small-cell lung, prostate, and gastrointestinal carcinomas. Numerous endogenous bombesin ligand analogues have been synthesized as agonists and antagonists of BB1 and BB2. Methods of obtaining antagonist activity have included N- and C-terminal modification, amino acid substitutions and eliminations, peptide bond modification (reviewed in ref 74), and introduction of electron withdrawing groups on aromatic side-chains.75 The resulting peptidic and peptoid antagonists are highly effective (IC50 nM), but are readily degraded by protease enzymes, as is the highly selective BB2 antagonist RC-3095, [D-Tpi6, Leu13, psi[CH2NH]-Leu14]-bombesin(6-14)].76 This compound was recently found to attenuate the release of proinflammatory cytokines (TNFR, IL1β) and nitric oxide from activated macrophages in Vitro, with application to increasing survival in sepsis.77 Administration of 0.3 mg/kg, 6 h after sepsis induction, improved survival in a caecal ligation and puncture (CLP) model of sepsis and reduced acute lung injury induced by intratracheal LPS.77 This same antagonist inhibits growth of human and mouse cancers in experimental models,78 possibly by inhibiting activation of the ERK/MAPK signaling pathway.79 Intrahippocampal infusion of RC-3095 impairs memory, while activation of BB2 in the hippocampus influences emotional memory. Screening at Pfizer led to the first nonpeptide bombesin ligands, CP-70,030 23 and CP-75,998 24 (Chart 4). These compounds were specific for rat bombesin receptors with low micromolar IC50.74 Compounds 25 PD165929, and 26 PD176252, were rationally designed through the peptoid drug design strategy73 previously successfully used in developing cholecystokinin and tachykinin receptor nonpeptide ligands. The design involved identification of the smallest active bombesin fragment, an alanine scan and library search, followed by optimization. PD165929 25 is a selective hBB1 antagonist (Ki 6.3 nM), and the second generation compound PD176252 26 is a hBB1/hBB2 antagonist (KiBB1 0.17 nM, KiBB2 1 nM).73 Peptide mimetics JMV1802 (27) and JMV1716 (28), respective Ki 0.8 nM and 11 nM for BB2, contain a nonpeptide turn-inducing scaffold.80

2.3. Bradykinin Receptors There are two human kinins, bradykinin (BK, R1PPGFSPFR9) and kallidin (KD, [Lys0]-BK, K1RPPGFSPFR10) produced by proteases (kallikreins) from a precursor kininogen, which are components of the kallikrein-kinin system.81 Degradation products processed by aminopeptidases (KD converted to BK) and metabolic carboxypeptidases (removal of terminal arginine from both BK and KD) also display biological activity at bradykinin receptors, B1 and B2. The receptors have different ligand specificities: B1 prefers [des-Arg10]KD > [des-Arg9]BK ∼ KD > BK, while B2 prefers KD g BK . [des-Arg9]BK and [des-Arg10]KD. B2 is constitutively expressed, while B1 is induced by cytokines at sites of inflammation and injury.81,82 Knockout studies, selective antagonists, and antibodies have been used to identify bradykinin receptors as important targets in inflammation, pain, and cancer (e.g., septic shock, pancreatitis, edema, rhinitis, asthma, colitis, arthritis, hepatorenal syndrome, ulcerative colitis, Alzheimer’s disease, pain, and small cell lung cancer).81,83,84 It has also been noted that B1 is less sensitive to desensitisation than B2. Bradykinin and related peptides are physiological mediators of pain, inflammation, and cardiovascular homeostasis.81,83,84 Numerous peptide agonists and antagonists (with a β-turn conformation) have been reported, even as clinical candidates.83 However, nonpeptidic compounds with more favorable pharmacokinetic profiles have also been developed for these receptors. Sterling Winthrop researchers reported the first antagonist of the B2 receptor, found through highthroughput screening, and modified this hit using distance constraints matching the β-turn constrained BK to create WIN64338 (29). Compound 29 (Chart 5) is a high-affinity hB2 antagonist (affinity Ki 64 nM; pA2 8.2, Ca2+ mobilization), but contains a phosphonium salt that compromises pharmacokinetics and also has high affinity for muscarinic receptors.81,85 Researchers from Scios Nova designed potent second generation B2 antagonists, with limited peptidic elements and small size (e.g., NPC-18884, 30; hB1/B2 Ki

2968 Chemical Reviews, 2007, Vol. 107, No. 7 Chart 5

3.3 nM), which were efficacious in paw edema and had antihyperalgesic effects in mice and rats.84 Fujisawa screened angiotensin receptor inhibitor libraries for bradykinin antagonism, because of complementary roles of angiotensin and bradykinin in cardiovascular homeostasis.86 Discovery of lead compound 31 (IC50 31 µM, guinea pig ileum (GPI)) (Chart 6) led to a significant advancement in B2 antagonist design. Identification of the benzyloxy-heteroaromatic substructure led to FR167344 (32), a B2 antagonist (IC50 0.66 µM, GPI) with efficacy in mouse models of analChart 6

Blakeney et al.

gesia and pancreatic edema.86 However, FR167344 displayed cross-reactivity at muscarinic receptors. Modification of the imidazopyridine to methylquinoline gave FR173657 (33), an orally bioavailable B2 antagonist (hB2, Ki 1.4 nM).81 Substitutions at the 4-position on the quinoline ring influenced species-specific binding to the bradykinin B2 receptor.87,88 These substitutions were also used to improve solubility for i.v. formulations. It is interesting that small modifications in this region can change a B2 antagonist FR165649 (34) into the potent B2 agonist FR190997 (35; hB2 Ki 5.3 nM; in Vitro induced GPI contraction and in ViVo it causes hypotension).81 Compound 36 is also a reported B2 agonist (hB2, IC50 0.68 nM), containing a benzimidazole. Fujisawa replaced the N-methyl amide in the series with a pyrrole ring to produce a potent B2 antagonist 37 (hB2 affinity, IC50 0.26 nM; inhibition of BK induced 79% bronchoconstriction at 10 µg/kg). Other companies have also designed B2 antagonists using Fujisawa’s 2,6-dichlorobenzyloxy-quinoline scaffold. One company (Fourier) replaced the N-methyl amide in most of the Fujisawa compounds with a sulfonamide moiety, reporting a piperazine compound LF16,0335 (38: hB2, Ki 2.34 nM, HUV cells; muscarinic receptors IC50 1 mM, M1; 0.9 mM, M2)81,84 and a propylendiamine containing compound LF16,0687 (39: Ki 0.67 nM, hB2).81,84 Compound 39 is the only nonpeptidic bradykinin B2



Chart 7

antagonist currently reported in Phase II clinical trials for post-traumatic brain edema.89,90 Hoechst researchers reported antagonist 40 (Ki 1 nM) Chart 7),81 with a trifluoroalkyl substituent in place of the terminal amide in the Fujisawa series, and increased linker size compared to the typical N-methyl amide 41 (Ki 20 nM).81 Johnson & Johnson modified the Fujisawa scaffold using structural information (β-turn) from bradykinin, replacing the cis N-methyl amide with pyrrolidine to stabilize a β-turn conformation (42, B2 Ki 33 nM; had 59% inhibition at 160 µmol/kg in a graded abdominal irritant test).91 Novartis developed a structurally diverse class of B2 antagonists based on a high-throughput screening lead containing a 1-(2nitrophenyl)-4-benzylthiosemicarbazide core. Bradyzide (43) had Ki 390 nM at hB2, while 44 has an affinity Ki of 0.79 nM for hB2 in COS-7 cells and shows oral efficacy in a rat hyperalgesia model.84 American Home Products reported bradykinin receptor antagonist 45 (affinity IC50 430 nM) (Chart 8),81 GlaxoWellcome discovered a tetrahydroisoquinoline by screening against B2 (46; pKi 5.8),81 and Cortech rationally designed nonpeptide B2 antagonist CP2055 (47, IC50 55 µM) based on a peptide antagonist CP0597 with a type II′ β-turn centered at its C-terminus.81 Pfizer patented 1,4-dihydropyridines as B2 antagonists (48, IC50 0.2-10 nM).81 Merck reported two natural product bradykinin receptor antagonists. A pyrroloquinoline alkaloid (49) had activity at both BK receptors (B1, IC50 6.4 µM; B2, IC50 0.25 µM), but even higher affinity at muscarinic (IC50 90 nM) and R-adrenergic (IC50 60 nM) receptors, and a pyrrolidine fused epoxide compound L-755807 (50, B2 IC50 71 µM).81 Kam et al. designed B2 antagonists (e.g., 51, IC50 35 nM) through combinatorial synthesis using information from known B2 antagonists.92 The first reported B1 antagonist was PS020990 (52, hB1 Ki 6 nM) (Chart 9), discovered through HTS of a combinatorial chemical library.93 HTS of the Merck collection identified a dihydroquinoxalinone core for B1 antagonism (53, racemic Ki 1.4 µM for B1).94 This compound was docked in the homology model of B1 (between transmembrane helices 5 and 7); affinity was then boosted by exploiting possible binding interactions with loop 4, where a 4-ethylamine (or 4-propylamine) was attached to the anilide (54, n ) 2, Ki 2.6 nM; n ) 3, Ki 0.98 nM).94 Replacement

of 4-ethylamine with phenyl imidazoline increased putative interactions with E273 and D291 residues of B1 (55, Ki 0.034 nM, hB1; 0.05 nM, rabbit B1; 1.28 nM, dog B1; 62 nM, rat B1).94,95 The imidazoline compound (55) is more efficacious than morphine in inhibiting the spinal nociceptive reflux in rabbit hyperalgesia models and was selective for B1 over 170 other receptors, transporters, and enzymes, although it had submicromolar activity at muscarinic receptors (Ki 0.5 µM) and at a guinea pig serotonin receptor (Ki 55 nM).94,95 A benzodiazepine core B1 antagonist was also identified by Merck (56, Ki 28 nM) (Chart 10).96 Addition of a 4-pyridinyl to the piperazine, as well as a phenethyl group in place of the cyclohexane (57) improved the hB1 IC50 to 0.59 nM.96 The pharmacokinetic properties of 57 are poor with oral bioavailability of 3.4% and a half-life of 2.7 h in rats; however, it is efficacious in inhibiting carrageenaninduced hyperalgesia (80%) at 3 mg/kg, intraperitoneal injection.96 Merck further explored a third chemical series in addition to the benzodiazepine and the dihydroquinoxaline series for improved pharmacokinetic and pharmacodynamic profiles. This series was based on the 2,3-diaminopyridine core (58, Ki 0.2 µM, hB1; 59, Ki 1.2 nM, hB1), identified from screening.97 Initial optimization of the lead with three additional methyl substituents and terminal nitrile led to 59 with 100-fold increased potency at hB1;97 however, this compound had poor pharmacokinetics due to ester hydrolysis and oxidation of aryl methyl groups. Modification at these points with pyridine/phenyl replacement, halogen substitution in place of methyls, and tetrazole instead of the methyl ester gave 60 with 10-fold increased affinity (Ki 0.5 nM, hB1; 119 nM rat B1; 3 nM rabbit B1) and good pharmacokinetics (rat: F 73%, t1/2 7.8 h, CL 9 mL/(min/kg); dog: F 69%, t1/2 3.8 h, CL 2.4 mL/(min/kg)) and efficacy in complete Freund’s adjuvant induced antihyperalgesia in rabbits (at 3.1 mg/kg).97 Sanofi compound SSR240612 61 (Chart 11) was a potent B1 antagonist ([3H]KD, Ki 0.73 nM, HEK cell membranes; muscarinic M2 IC50 0.48 µM) and is selective over 1000 other receptors, channels, and enzymes.98 SSR240612 61 inhibits paw edema caused by desArg-BK at 3 and 10 mg/ kg, reduces capsaicin-induced mice ear edema (0.3-30 mg/ kg, oral), and inhibits UV-induced hyperalgesia (1-3 mg/ kg).98 Novartis utilized their screening lead, 4-alkylamino-


Blakeney et al.

Chart 8

3-nitrobenzenesulfonamide (Ki 3.0 µM, B1; 1.4 µM, B2) in both their B1 and B2 receptor antagonist programs.99 The 4-amino-benzylsulfonamide was identified to be important for B1 antagonist development. Optimization of the core led to development of 62 (Ki 14 nM, B1) with good pharmacokinetics (F 42% rat, 35% dog; t1/2 161 min) for this series and was 500-fold selective for B1 over 30 other receptors, channels, and enzymes.99 A common pharmacophore, arylsulfonamide linked to amide, frequently appears in B1 antagonists reported from different research groups.

2.4. Calcitonin and Calcitonin-Like Receptors Calcitonin (CT) is a 32-residue thyroid peptide (CGNLSTCMLGTYTQFNKFHTFPQTAIGVGAP) with a C1-C7 disulfide bridge at the N-terminus. It is a component of a hormone family that includes adrenomedullin, amylin, and CGRP. CT regulates calcium in ViVo, and rhCT (miacalcin) is used in the clinic to treat Paget’s disease, osteoporosis, and hypercalcemia.100,101 There are two GPCRs, the calcitonin receptor (CTR) and calcitonin-like receptor (CLCR), but there are six active receptor complexes. The calcitonin receptor family has the unusual property of binding accessory proteins, called RAMPs (receptor activity modifying proteins) to alter the function and physiology of receptor complexes. Even uncomplexed to RAMP proteins, the calcitonin receptor can be activated by CT to regulate intracellular calcium levels, whereas the calcitonin-like receptor is non-functional in the absence of RAMPs.102 RAMPs are ∼130 amino acid proteins which traverse the

membrane once and contain a large extracellular N-terminus, thought to either alter the glycosylation level of receptors (some researchers believe glycosylation levels to be an artifact)103 or the conformation of the GPCR that defines the agonist-binding site. Although the exact role of RAMPs is still unclear, it is known that RAMP-1 or -3 is required to complex to CTR to create amylin receptors. Amylin is found in the β-cell islet of the pancreas, being co-secreted with insulin in response to glucose.102 Amylin acts as a vasodilator; however, its most important role has been attributed to its ability to reduce tissue responses to insulin.102 The calcitonin gene-related neuropeptide (CGRP) is a powerful vasodilator released from sensory nerves in the central and peripheral nervous system. Injections of this peptide increase blood flow for several hours. CGRP requires RAMP-1 to complex to CLCR for a responsive receptor to be formed. It couples with heterotrimeric G-proteins Gs/Gq to promote activation of adenylyl cyclase and phospholipase C, stimulating intracellular calcium release on the molecular level, and physiologically acting as a potent vasodilator. There are two isoforms of CGRP, released from the central and peripheral sensory neurons.102 CGRP has been implicated in the pathophysiology of migraine headaches,104,105 where it has been associated with vasodilation of cerebral blood vessels and activation of the trigeminovascular system. CGRP has also been implicated in type 2 diabetes (CGRP receptor activation impairs glycogen synthesis and enhances glycogenolysis, glycolysis, and lactate production in muscles), inflammation, and congestive heart failure.106 CGRP receptor

Nonpeptidic Ligands for Peptide-Activated GPCRs Chart 9

antagonists in clinical trials have validated CGRP involvement in the pathology of migraines. Adrenomedullin (AM), a circulating vasodilator expressed by a number of cells, including vascular cells, requires RAMP-2 or -3 to complex CLCR to form its functional receptor.102 Although all the receptors contain different complexing proteins, they are not entirely selective. For example, calcitonin receptor can respond to salmon CT g human CT g amylin, CGRP > AM; amylin receptors are activated by salmon CT g amylin g CGRP > human CT > AM; CGRP receptor is activated by CGRP > AM g Chart 10


amylin g salmon CT; AM1 receptor (CLCR + RAMP2) is activated by AM . CGRP > amylin > salmon CT; AM2 (CLCR + RAMP3) activate by AM g CGRP > amylin > salmon CT). Small molecule antagonists have only been reported for the CGRP receptor. The first nonpeptidic CGRP receptor antagonist, BIBN4096BS (63 olcegepant) (Chart 12) was developed through modification of a dipeptide mimetic R1-[3,5-dibromo-Tyr]-Lys-R2 (64, IC50 17 µM; 65, IC50 40 µM).107 Nonpeptidic CGRP antagonists like 63 inhibit vasodilation, as well as neuronal transmission,108 and these properties may be responsible for their benefits in clinical trials on migraine sufferers. Structure-activity relationships conducted on the R1-[3,5-dibromo-Tyr]-Lys-R2 analogues found that the Nterminal (R1) functionality was best when aromatic and separated from the 3,5-dibromo-Tyr moiety, whereas the C-terminal portion preferred a rigid negative polarized group linked to a pyridine.107 These cumulative modifications resulted in 63 (pA2 11.1; IC50 14 pM),105,107 which reverses CGRP-mediated vasodilation of human cerebral vessels (in Vitro), inhibits neurogenic vasodilation in a surrogate animal model of migraine pathophysiology, and has completed phase I and II human trials for migraine treatment as an iv formulation.107 One of the experimental difficulties using 63 was its pronounced species selectivity (100-fold more selective for hCGRP receptor than rat receptor). This species selectivity was attributed to low homology across specific RAMP-1 proteins, with W74 in humans and primates and K74 in rats.105 Species selectivity was not observed in SB(+)273779 (66 membrane binding IC50 0.31 µM; cyclase IC50 0.39 µM) (Chart 13).106 SB(+)273779 66 was developed after identification of a benzanilide compound with CGRP antagonist activity (67 SB211973 binding IC50 1.93 µM) in the SmithKlineBeecham chemical collection.106 Enantiomerically pure SB273779 66 reversed >90% of CGRP inhibition of insulin-stimulated glucose uptake at 3 µM, as well as SB273779 66 significantly reduced CGRP dose-related hypotension.106 Most other CGRP antagonists were shortened versions of 63, including 68 (membrane binding affinity Ki 56.9 nM),105 and conformationally restricted version 69 (n ) 0, Ki 3.6 µM; n ) 1 Ki >5 µM).109 A novel orally bioavailable CGRP antagonist 70 (Ki 21nM, 125I-hCGRP;


Chart 12

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PI3Kγ (phosphoinositide 3-kinaseγ) by the Gβγ subunit is crucial for chemokine-regulated recruitment of leukocytes in Vitro and in ViVo. This kinase is an important regulatory signaling protein in specialized immune cells, macrophages, neutrophils, mast cells, T-cells, and platelets.111 At the molecular level, chemokines typically have little sequence identity but are folded by two characteristic disulfide bonds into the same monomeric shape, consisting of three β strands, a C-terminal helix, and a flexible N-terminal region thought to be important for receptor activation.115 They are classified by their characteristic pattern of conserved N-terminal cysteines as R (C-X-C), β (CC), γ (C), and δ (C-3X-C).116 Chemokines are also further divided by expression level. Constitutively expressed chemokines have roles in development and lymph node architecture, and inducible chemokines have roles in inflammation and other diseases.117 Among the most important chemokines in inflammation are CXCL8 (IL-8), CCL2 (MCAF/MCP-1), CCL5 (RANTES), and CCL3 (MIP-1). Chemokines bind to about 20 GPCRs on all types of leukocytes (Table 3). They also bind to surface glycosaminoglycans (GAGs). The GAG binding sites are specific for each chemokine and are hypothesised to be involved in building chemotactic gradients.112,115 Both chemokines and their GPCRs have a certain level of redundancy, with overlapping receptor-ligand profiles. This leads to problems in antagonist design.112 The validation of chemokine receptors as therapeutic targets remains elusive, relying on mRNA and antibody detection of ligands and receptors in patient samples, together with mouse knockouts and animal models of disease.111,112,114,115 While these characteristics have helped to identify roles for chemokines in inflammatory and autoimmune diseases, cancer, and HIV, they are not reliable because of differing chemokine networks, levels of redundancy, and pronounced species selectivity of designed antagonists. Different chemokine profiles in animals and unexpected toxicity profiles of the compounds (with interactions at cardiac potassium channel hERG, inhibition of certain P450s, and activity at certain biogenic amine receptors) have frequently been problems with clinical development of chemokine antagonists.118,119

2.5.1. CC Chemokine Receptors IC50 78nM, cAMP; F 36% rat, 83% dog, 29% rhesus) has recently been developed from a benzodiazepinone HTS hit (Ki 4800nM).110

2.5. Chemokine Receptors Chemotactic cytokines (chemokines) are very important components for immunological surveillance. They comprise a network of at least 40 secreted proteins of 60-130 amino acids, and were first identified in the 1990s.111,112 They have diverse functions, such as regulating cell adhesion and chemotaxis, controlling chemotactic migration and mobilization of leukocytes, activating cytokine networks, altering expression of adhesion molecules, increasing cell proliferation, regulating angiogenesis and organogenesis, promoting viral-target cell interactions, increasing hematopoiesis, stimulating mucus production, directing Th1/2 polarization, increasing tumor growth and metastasis, and activating the innate immune system.113,114 Activation of chemokinebinding GPCRs leads to release of heterotrimeric GRβγ subunits that initiate downstream signaling. Activation of

2.5.1.1. CCR1. CCR1 was the first discovered CC chemokine receptor, using sequence information from a conserved region identified in chemotactic GPCRs, C5a, fMLP, CXCR1 and 2, as well as PCR technology, receptor binding and functional assays of the CCL5 (RANTES) and CCL3 (MIP-1R) chemokines.121,122 This chemokine receptor is located on chromosome 3p21 as a single exon open reading frame encoding a 355 residue GPCR.122 CCR1 is expressed on a broad range of cell types: monocytes/macrophages (especially during monocyte maturation into macrophages), mast cells, natural killer cells, polymorphonuclear leukocytes, and lymphocytes as well as astrocytes and neurons.121,123-125 CCR1 responds to a number of endogenous, high-affinity (EC50 100 nM).121,124,125 CCR1 is involved in trafficking of immune cells and Th1/Th2 cytokine balance.121 CCR1 has been associated with the pathology of multiple sclerosis (mouse EAE models and patient samples), rheumatoid arthritis (role of CCL5 and



Chart 13

Table 3. Chemokine Receptors and Ligands120 a receptor

chemokinesb

CCR1

CCL3 (MIP-1R), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL14a (HCC-1), CCL15 (HCC-2), CCL23 (MPIF-1); antagonist CCL4 (MIP-1β) CCR2 CCL2 (MCP-1), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL16 (HCC-4); antagonists CCL11 (eotaxin), CCL26 (eotaxin-3) CCR3 CCL11 (eotaxin), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL15 (HCC-2), CCL24 (eotaxin-2), CCL26 (eotaxin-3), CCL28 (MEC); antagonists CXCL10 (IP10), CXCL9 (Mig), CXCL11 (I-TAC) CCR4 CCL22 (MDC), CCL17 (TARC), CCL3 (MIP-1R), CCL5 (RANTES), CCL4 (MIP-1β) CCR5 CCL3 (MIP-1R), CCL4 (MIP-1β), CCL5 (RANTES), CCL8 (MCP-2), CCL11 (eotaxin), CCL14a (HCC-1), CCL16 (HCC-4); antagonist CCL7 (MCP-3) CCR6 CCL20 (LARC) CCR7 CCL19 (ELC, MIP-3b), CCL21 (SLC) CCR8 CCL1 (I-309), CCL4 (MIP-1β), CCL16 (HCC-4), CCL17 (TARC) CCR9 CCL25 (TECK) CCR10 CCL27 (Eskine, ALP, CTACK), CCL28 (MEC) CXCR1 (IL8Ra) CXCL6 (GCP-2), CXCL8 (IL8) CXCR2 (IL8Rb) CXCL1 (GROR), CXCL2 (GROβ), CXCL3 (GROg), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL7 (NAP-2), CXCL8 (IL8) CXCR3 CXCL9 (Mig), CXCL10 (IP10), CXCL11 (I-TAC); antagonists CCL11 (eotaxin), CCL7 (MCP-3) CXCR4 CXCL12R & β (SDF-1R & SDF-1β) CXCR5 CXCL13 (BLC, BCA-1) CXCR6 CXCL16 (SR-PSOX) CX3CR1 CX3CL1 (Fractalkine) XCR1 XCL1 R & β (lymphotactin R & β) a Table adapted from data summarized in Reference 120. b Endogenous antagonists in italics, agonists not italicized.

a clinical trial by Pfizer), organ transplantation (implicated by CCL5 involvement and CCR1 mouse knockout), Alzheimer’s disease, atherosclerosis, psoriasis, and multiple myeloma (through CCL5 and CCL3 involvement).121-126 Berlex has disclosed numerous studies of the CCR1 antagonists, BX513 (71) and BX471 (72) (Chart 14), identified through high-throughput binding assays. BX513 (71 Ki 40 nM, CCL3; 60 nM, CCL5) or 2,2-diphenyl-5-(4chlorophenyl-4-hydroxypiperidin-yl)valeronitrile belongs to a family of 4-hydroxypiperidine compounds that are functional CCR1 antagonists of Ca2+ mobilization and chemotaxis.121,122,124,125 BX513 71 has 250-fold selectivity for CCR1

over other chemokine receptors tested, even CCR5 for which CCL5 and CCL3 are also high-affinity ligands, but it has significant cross-reactivity for neurotransmitter biogenic amine receptors.121,122 The ammonium salt of this compound is 10-fold more potent,123 and structure-activity data for this series indicate that chain length is related to the potency and species selectivity at CCR1. Optimization of 71 for GPCR selectivity, while keeping potency, proved difficult so this series was abandoned. Compounds exemplified by BX471 (72 IC50 1 nM, CCL3; 6 nM, CCL5) were also identified in initial screening, with BX471 72 being 10 000-fold more selective for CCR1 than


Blakeney et al. Chart 16

Chart 15

28 other GPCRs,122 and with good pharmacokinetic properties (60% oral bioavailability in dogs,127 t1/2 3 h123). In ViVo studies established protective roles for BX471 72 in heart transplant models and in an EAE model of multiple sclerosis. There is difficulty in interpreting the in ViVo data, because of species selectivity problems (rat Ki 121 nM, CCL3) requiring higher doses in rats.127 BX471 72 is in Phase 1 clinical trials for Alzheimer’s disease and Phase II for multiple sclerosis, psoriasis, and eczema.119 Takeda patented a potent CCR1 antagonist (73 IC50 3-10 nM, HEK cells) (Chart 15) similar to BX513 71 but with the nitrile replaced by piperidinyl urea or cyclohexyl urea.121 Banyu developed a series of CCR1 and CCR3 antagonists using tricyclic amide scaffolds attached to piperidine quaternary ammonium salts, with varying cyclic alkane/alkene groups. This series of compounds were antagonists of CCR1 and CCR3 at low nanomolar concentrations.121-123,125 Optimization for chemokine receptor selectivity involved attaching different halogens to the tricyclic scaffold (xanthene-9carboxamide), as well as cycloalkyls, to the piperidine. One such compound, 1-cycloheptenylmethyl-1-ethyl-4-(2,7dichloroxanthene-9-carboxamido)-piperidinium iodide (74 UCB35625), had high affinity (IC50 0.9 nM, hCCR1; IC50 5.8 nM, mCCR1), potency (hCCR1, IC50 0.73 nM, CCL3;Ca2+ mobilization), and 100-fold greater selectivity over CCR3 (IC50 93.7 nM) and other chemokine receptors (IC50 > 1 µM).125 However, some doubt has been cast about the mechanism and selectivity of action, with allosteric modulation proposed for this compound.128 It is suspected that the ammonium formulation of these compounds may have reduced oral bioavailability and caused rapid elimination,121,122,127 although no in ViVo data was reported.

Pfizer has patented low molecular weight heterocyclic amine derivatives as CCR1 antagonists.117,121,125 Initial leads identified through high-throughput screening117 contained a hydroxethylene peptide isostere more commonly found in protease inhibitors and had modest receptor affinity (75 affinity IC50 2.3 µM, CCL3; chemotaxis IC50 0.84 µM, CCL3 and 0.63 µM, CCL5) (Chart 16).129 Heterocyclic components such as benzofuran and quinoxaline and alternative Cterminal amines have been reported,121 most being heterocyclic amides attached to 5-benzyl hydroxethylene peptide isosteres, with different alkyl groups next to the terminal amide (position 2) and alternative substituents on the benzyl ring (position 5), although alternative groups on the heterocyclic amide benzyl fragment have been reported.125 Pfizer’s lead clinical candidate CP-481,715 (76 CCR1 IC50 74 nM, CCL3) competitively antagonizes CCL3-induced calcium mobilization, leukocyte chemotaxis, and CD11b up-regulation and is 100-fold selective for CCR1 over ≈40 other GPCRs.117 CP-481,715 (76) is in Phase II clinical trials for rheumatoid arthritis.119 AstraZeneca have a CCR1 antagonist in Phase I clinical trials for rheumatoid arthritis and COPD, AZD-8309 (structure not disclosed). The company has filed patents covering indolecarboxylates 77 (Chart 17), and benzylpiperidinyl-4methylbenzylamines.122 Millenium has a CCR1 antagonist MLN3897 (structure not disclosed) in Phase I clinical trials for multiple sclerosis, psoriasis, and rheumatoid arthritis with reported IC50 of 3.4 nM for blocking CCL3 chemotaxis, t1/2 of 3 h in rats, 35% oral bioavailability in rat, and 100% oral bioavailability in dog.130 Millenium’s antagonists incorporate a substituted pyridylbenzoxepine scaffold (78).130 Natural product CCR1 antagonists have been identified in Chinese herbal medicines (79 affinity IC50 2.6 µM, CCL3; 3.6 µM, CCL5).125 Schering has patented CCR1 antagonists, covering piperazine derivatives like 80 (Ki 1 nM, CCR1 in competition with CCL3).125 Merck reported two CCR1 antagonists in Phase II clinical trials, C-4462 for rheumatoid arthritis and C-6448 for multiple sclerosis, antagonist 81 (Chart 18) (IC50 0.9 nM, binding affinity for hCCR1 in the 125I-CCL3 displacement assay) had good in ViVo pharmacokinetics as well as efficacy in a murine model of collagen-induced arthritis.130 Chemocentryx patented two classes of CCR1 antagonists, azaheterocyclic derivatives and piperazine derivatives, the best being CCX-469 (82 binding affinities IC50 0.6 µM (NSO cells), 2 µM (HEK-293 cells)) and compound 83 (IC50 < 500 nM) which showed efficacy in a rat model of collagen-induced


Chart 18

arthritis.130 Novartis patented a series of piperazine derivates like 84 as CCR1 antagonists with activities 0.1-1000 nM.130

Chemical Reviews, 2007, Vol. 107, No. 7 2975 Chart 19

2.5.1.2. CCR2. The CCR2 gene encodes two alternatively spliced receptors, CCR2a and CCR2b, splicing occurring in the C-terminus of the receptors.131 The predominant CCR2b receptor is expressed on monocytes/macrophages, basophils, and activated T memory lymphocytes.123 This receptor is activated by a number of endogenous peptide ligands, including CCL2 (MCP-1), CCL7 (MCP-3), CCL8 (MCP2), CCL13 (MCP-4), CCL16 (HCC-4), and HIV-Tat,120 with CCL2 having the highest affinity, efficacy, and selectivity for CCR2. Two endogenous antagonists of CCR2 are also present in humans, CCL11 (eotaxin) and CCL26 (eotaxin3). Gene knockout studies in animals, neutralizing antibodies, and antagonists of this receptor and CCL2 have suggested roles for CCR2 in atherosclerosis, asthma, multiple sclerosis, arthritis, and other inflammatory diseases.123,127,131,132 Several pharmaceutical companies have reported small molecule antagonists of CCR2. So far only one CCR2 antagonist (INCB3284, undisclosed structure) has been reported to have completed Phase I clinical trials for rheumatoid arthritis and delayed type hypersensitivity.112 Incyte has patented 3-aminopyrrolidine derivatives (WO2004050024) and 3-cycloalkylaminopyrrolidine derivatives (WO2005060665) as potential CCR2/CCR5 antagonists. Roche has developed CCR2 antagonists from highthroughput screening using binding and chemotaxis assays to identify initial hits, resulting in 10 structurally diverse chemical classes with affinity (IC50 5 µM-30 nM).131 Only two of these classes, spiropiperidines 85 (Chart 19) and 2-carboxy-pyrroles 86, were deemed drug-like. Further SAR led to spiropiperidine derivatives as CCR2 antagonists, while 2-carboxy-pyrroles were found relatively intolerant of large substituents with the best of the series developed remaining around 5 µM activity.131 The spiropiperidine derivatives however were observed to have a very specific pharmacophore requiring a basic tertiary nitrogen (N-alkyl piperidines) orthogonal to the 1,4-dihydro-benzo[d][1,3]oxazin-2-one. The arylethyl attached to the piperidine tolerated a wide range of nonpolar substitutions.127,131 It was proposed that the basic nitrogen of the piperidine made an ionic interaction with Glu291 of CCR2. This acidic residue was not required for CCL2 binding to the receptor.127 The spiropiperidine compounds have not successfully advanced to clinical trials because of high cross-reactivity with biogenic amine receptors. The lead compound RS-21825 (85) was originally designed as an R-adrenergic receptor antagonist (IC50 11.4 µM, CCR2),131 which was optimized to RS-504393 (87 CCR2: IC50 89 nM (affinity), 330 nM (chemotaxis blockade); R1-adrenergic receptor: IC50 72 nM).125,131 Introduction


Chart 21

of a 3-fluoro substituent in RS-511336 (88) increased affinity 3-fold to 33 nM (for CCR2);121,127 however, the compound still had biogenic amine receptor activity. CCR2 antagonists by Teijin included a series of modified glycine derivatives (89 IC50 54 nM) (Chart 20) and a series based on diphenyl linked to a heterocyclic ring by an alkyl chain (lead compound 90 IC50 9-13 µM).121,133 Addition of a hydroxyl group increased affinity (91 IC50 4 µM, hCCR2), and further addition of a second hydroxyl group in the paraposition of the diphenylmethane further increased hCCR2 affinity (92 IC50 721 nM).133 SmithKline Beecham identified indolepiperidine derivatives with a five-carbon chain linking the piperidine to an amide, developing the screening lead (93 IC50 5.3 µM for CCR2) (Chart 21)125 into SB-282241 (94 IC50 50 nM);125 however, this compound still had a C5 chain and thus many rotatable bonds. Further optimization of the piperidine

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component and introduction of conformational restraints to remove the C5 linker gave 95 (IC50 40 nM) with 1000-fold selectivity over 5-HT and dopamine receptors.124,125 AstraZeneca reported the development of CCR2 antagonists N-benzylindole-2-carboxylic acid (IC50 1.7 µM).124,125,134 1-(3,4-Dichloro-benzyl)-5-fluoro-1H-indole-2-carboxylic acid was the best compound of this series (96 IC50 56 nM, CCL2 displacement assay; 312 nM, CCL2 induced chemotaxis) and is selective over 40 other GPCRs (Chart 22).134 LeukoSite/ Parke-Davies reported a compound that inhibited MCP-1 induced chemotaxis (97 IC50 5.5 µM).121,123 Merck has patented cyclopentanyl modulators (98) of CCR2, and Dai Ichi Seiyaku Co., Ltd have patented a series of benzoxazine derivative (99 IC50 31 nM, CCL2 displacement assay).125 Johnson and Johnson identified 2-mercaptoimidazoles from high-throughput screening; their lead inhibited calcium mobilization (100 IC50 200 nM), and their best compound 101 reported after optimization had a pIC50 of 8.1.135 2.5.1.3. CCR3. The CCR3 chemokine receptor is important in eosinophil-dependent inflammation,125 with a high percentage of eosinophil migration due to CCR3.127 Thus, CCR3 has been associated with asthma, atopic dermatitis, and allergic rhinitis. The gene for this receptor is on chromosome 3p21.31, like many of the CC chemokine receptors, but while CCR3 is primarily expressed on eosinophils, it is also detected on Th2 lymphocytes, basophils, mast cells, platelets, and dendritic cells.136 CCR3 is activated in response to CCL11 (eotaxin), CCL24 (eotaxin-2), CCL26 (eotaxin-3), CCL5 (RANTES), CCL8 (MCP2), CCL7 (MCP3), CCL13 (MCP4), CCL15 (HCC-2), CCL28 (MEC), and HIV Tat.120 After activation, the receptor is internalized, and eosinophils are unable to respond to the same ligand.137 Two nonpeptidic CCR3 antagonists have been reported in clinical trial, Bristol-Myers Squibb compound DPC168 (102) (Chart 23) and GlaxoSmithKline compound GW766994 (103), evaluated against allergic rhinitis and asthma in Phases I and II, respectively.111,119 Bristol-Myer Squibb started their CCR3 antagonist development from a 4-benzylpiperidinylalkylurea series (104 CCR3 IC50 10 nM; 5HT2A Ki 193 nM; 105 CCR3 IC50 5 nM).138 Modification gave a 3-benzylpiperidinyl-alkylurea series with higher selectivity for CCR3 (106 CCR3 IC50 55 nM; 5HT2A Ki 913 nM; 107 CCR3 IC50 41 nM; 5HT2A Ki 2.9 µM; 108 CCR3 IC50 19 nM; 5HT2A Ki 2.3 µM; 109 CCR3 IC50 0.7 nM, inhibited Ca2+ mobilization and chemotaxis with IC50 values of 0.76 and 0.4 nM).138 Altering the size of the piperidine cycle by reducing carbon bonds reduced activity for CCR3. Introduction of an 8-azabicyclo[3.2.1]octane system in place of the piperidine was tolerated and conferred high selectivity with 4-fluoro-benzyl(8-aza-bicyclo[3.2.1]octane) in the equatorial position linked to a 3-acylphenylurea by a propyl group having an CCR3 IC50 7.4 nM, while having a Ki of 6.4 µM at 5HT2A receptors.138 The pharmacophore of these compounds was further explored by altering the linkage between the 4-benzylpiperidine and phenylurea units. The propyl group (CCR3 IC50 10 nM; 5HT2A Ki 193 nM) was exchanged with a phenyl group (CCR3 IC50 125 nM), a phenethyl group (CCR3 IC50 2.6 nM; 5HT2A Ki 11 nM), an aminomethylbenzamide group (CCR3 IC50 1.0 nM; 5HT2A Ki 5.6 µM), and cyclohexyl group as observed in DPC-168 (102 CCR3 IC50 2 nM; inhibition of Ca2+ mobilization and chemotaxis IC50 values of 8 and 0.034 nM, respectively).139 The aromatic moiety attached to the urea tolerates 5- and 6-membered heterocyclic



Chart 22

Chart 23

rings (CCR3 affinity and calcium inhibition IC50 e 1 nM and chemotaxis IC50 < 1 nM). The direction of the piperidine can also be reversed as seen in the phenethylpiperidine-3alkylurea series (110 CCR3 IC50 0.7 nM; IC50 27 nM, Ca2+ mobilization; IC50 30 nM, chemotaxis).140 Banyu identified potent xanthene-9-carboxamide CCR1/ CCR3 antagonists (mentioned above in CCR1 74). Key elements identified during SAR studies of the xanthene-9carboxamide series, two hydrophobic elements (accounting for the xanthene and cyclic alkyl moieties), a carbonyl hydrogen bond acceptor, and a basic nitrogen for electrostatic interactions with CCR3, were used to develop a new series of CCR3 antagonists.141 The series was designed on 2-(benzothiazolethio)acetic acid 1-benzyl-4-aminopiperidine (111

CCR1 IC50 7.1 µM; CCR3 IC50 0.75 µM) (Chart 24),141 with a 3-benzylpiperidine linkage leading to decreased selectivity of the compounds (CCR1 IC50 1 µM; CCR3 IC50 4.2 µM).142 An amine substituent on the 2-(mercapto-benzothiazole)acetamide as well as 3,4-dichloro substituents on the Nbenzylpiperidine increased both the affinity and selectivity of these compounds (112 CCR1 IC50 1.9 µM; CCR3 IC50 2.3 nM).141 SmithKline Beecham developed a series of phenylalaninederived CCR3 antagonists with their lead molecule 113 (Chart 25) identified from high-throughput screening having IC50 of 535 nM.125 Solution-phase optimization identified 1-naphthoyl and 4-nitrophenylalanine for increased potency (114 IC50 5 nM); however, these compounds still contained


Chart 25

a labile ester that could potentially be cleaved in ViVo, and thus, SmithKline Beecham examined different heterocycles as ester bioisosteres. Of the heterocyclic mimics of esters, only dihydrooxazole was active (115 IC50 0.6 µM, Ca2+ mobilization). Substitution of 2-amino-N-phenyl-propionaChart 26

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mide in place of the ester maintained affinity (116 IC50 5 nM) and antagonist potency (116 IC50 15 nM, eosinophil chemotaxis) for CCR3.125 Schering Plough identified a CCR3 antagonist through high-throughput screening (117 CCR3, Ki 403 nM) (Chart 26), with initial optimization involving replacement of the 2-methoxybenzamide with 2-tolylacetamide (118 Ki 66 nM).143 Further modification, with 3,4-dichloro substitutions on the benzylpiperidine and quinoline-6-carboxamide (119 Ki 30 nM) in place of 2-tolylacetamide and additional hydroxymethyl group on the piperidine of the benzylpiperidine, generated the most potent compound of the series (120 affinity hCCR3 Ki 3.3 nM, mCCR3 Ki 606 nM, rCCR3 Ki 2.9 µM; inhibition of Ca2+ mobilization and eosinophil chemotaxis with Ki values of 9.2 and 160 nM, respectively). Addition of a carbonyl to the benzylpiperidine changes the compound into a CCR3 agonist (121 Ki 36 nM; %Emax (GTPγS) 128%). The direction of the bipiperidine component can be reversed without effecting compound affinity (122 affinity Ki 23 nM; inhibition of Ca2+ mobilization and chemotaxis Ki values of 213 and 136 nM, respectively). Addition of 3-methyl to the piperidine of the benzylpiperidine also generated agonist activity (123 Ki 7.3 nM; %Emax (GTPγS) 96%).144 AstraZeneca (124), Roche (125, IC50 0.21 µM), and others (126, 0.6-10 µM) have also reported CCR3 antagonists.125 2.5.1.4. CCR4. The chemokine receptor CCR4 is important for the migration of T lymphocytes to and through the thymus during T lymphocyte maturation and differentiation. It is particularly associated with CD4+ Th2 lymphocytes, although it can also be expressed on Th1 lymphocytes in the presence of CXCR3.124 Its association with Th2 lymphocytes correlates to its pathophysiology in Th2 driven conditions such as asthma, arthritis, and inflammation of the skin, validated using neutralizing antibodies against CCR4 and high-affinity endogenous selective ligands CCL22


(MDC) and CCL17 (TARC).124,145,146 CCR4 can also be activated by HHV8 vMIP-III, CCL3 (MIP-1R), CCL5 (RANTES), and CCL4 (MIP-1β).120 Four companies have reported development of CCR4 antagonists. Array Biosciences reported two series of compounds varying the central ring systems of thiazolidinones and lactams.145,147 Screening a thiazolidinone-based diversity library resulted in an initial lead 2-aryl-thiazolidinone (mixture of diastereomers) containing a terminal morpholine and a naphthalene group (affinity IC50 2.4 µM-mixture, IC50 0.8 µM-trans diastereomer).145 The most potent compound identified by SAR studies of this lead was (R,R)-2-(2,4dichlorobenzyl)-thiazolidinone attached to a terminal piperidine and (S)-1-methyl-naphthalene group (127 affinity IC50 110 nM, inhibited CCL22 induced chemotaxis IC50 600 nM as well as inhibited CCL17 induced chemotaxis IC50 300 nM) (Chart 27).145 Lactam CCR4 antagonists were designed by switching the core thiazolidinone structure for lactams, to improve metabolic stability and to simplify synthetic approaches for the antagonist series.147 The (R,R)-2-(2,4dichlorophenyl)-butyrolactam with terminal piperidine and 2-methyl-3-chloroanilide appendages was the most potent compound of this series (128 affinity IC50 90 nM, and inhibition of CCL22 and CCL17 driven chemotaxis with IC50 300 and 100 nM, respectively). This potent lactam was 100fold more selective for CCR4 over 5 other chemokine receptors.147 Bristol-Myers Squibb reported CCR4 antagonists containing quinazoline, quinoline, and isoquinoline derivatives from compound screening; however, these compounds were also cytotoxic. Structural modifications were made to isolate CCR4 activity from cytotoxicity. Removal of the fused phenyl ring from the quinoline leads was well-tolerated as Chart 28


well as 2,4-diamino substituents on the new pyridine/ pyrimidine scaffolds. The most potent CCR4 antagonist was N4-(2,4-dichlorobenyl)-N2-(4-diethylamino-1-methyl-butanyl)pyrimidine-2,4-diamine (129 affinity IC50 0.27 µM, inhibition of chemotaxis (IC50 2 µM)) (Chart 28) with no cytotoxicity. Modification of the side chain of 129 gave potent CCR4 antagonist 130 effective in a murine allergic inflammation model (affinity IC50 50 nM; Ca2+ IC50 10 nM; ED50 30 mg/ kg in murine allergic lung inflammation model).148 Another CCR4 antagonist series was reported in patents from Ono Pharmaceuticals and AstraZeneca.149,150 Ono report a series of aryl pyrimidine sulfonamides with 131 reported to inhibit Ca2+ mobilization (IC50 0.13 µM) and suppress TNFR production induced by LPS stimulation, and 132 inhibiting CCL22-induced chemotaxis (IC50 10 nM) and suppressing ear swelling in murine DTH model.150 AstraZeneca reports N-pyrazinyl-thienylsulfonamides as chemokine antagonists.149 2.5.1.5. CCR5. CCR5 responds functionally to CCL3 (MIP-1R), CCL4 (MIP-1β), and CCL5 (RANTES),151 as well as to CCL8 (MCP-2), CCL11 (eotaxin), CCL14a (HCC-1), CCL16 (HCC-4), and R5 HIV-1 gp120120 and has high homology with CCR2b (76%). CCL7 (MCP-3) is a endogenous antagonist for CCR5. CCR5 is predominantly known for its expression on macrophages and Th1 polarized T lymphocytes.136 It has been reported to be expressed on monocytes, dendritic cells, microglial, astrocytes, and aortic smooth muscle.136 CCR5 effects are mediated through coupling with GRi heterotrimeric protein, which activate adenylyl cyclase and phospholipase C, produce IP3 and mobilize calcium ions. gp120 of HIV also binds to CCR5 and activates intracellular signaling events, but these are not necessary for viral entry into host cells.152 It is speculated that activation of CCR5 signaling enhances viral replication. It is known that CCR5 is not an essential chemokine receptor for human development as a natural, non-functional polymorphism exists in the general population. This polymorphism encodes truncated CCR5 through a 32bp deletion (∆32 CCR5), which in homozygous form greatly protects individuals against HIV infection and heterozygous form slows HIV progression.127,151 CCR5 mediates leukocyte recruitment in rheumatoid arthritis, multiple sclerosis, and asthma;136 activation of CCR5 has also been implicated in autograft136 rejection with homozygous ∆32-CCR5 conferring protection. However, it is the role of CCR5 as a key mediator of HIV infection that has generated most interest, with a large number of patents and literature reported for small molecule antagonists, as well as compounds in clinical trials.111,112,119 Takeda reported the first CCR5 antagonist, TAK-779 (133) (Chart 29), which has high-affinity ([125I]CCL5 competition IC50 1.4 nM) and potent antiviral activity (membrane fusion


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Chart 29

Chart 30

IC50 0.87 nM; HIV-1 replication IC50 1.4 nM);152 however, it was not selective (IC50 27 nM, CCR2b)152 and exhibited poor oral bioavailability because of its polar quaternary ammonium salt. TAK-779 133 was designed from two HTS hits, a quaternary salt and a phosphonium salt (134, IC50 390 nM; 135, IC50 620 nM).153 Benzosuberene conversion to 2,3-dihydrobenzo[b]oxepine and attachment variation at 4-methylphenyl of 133 have been described.152 To generate an orally active compound, the 4-amino tetrahydropyran was replaced by an [(N-containing heteroaryl)methyl]sulfinyl moiety. Cumulative modifications led to 136 and 137, which are potent CCR5 antagonists (136 IC50 1.7 nM; 137 IC50 1.6 nM), potent antivirals (membrane fusion 136 IC50 2.8 nM; 137 IC50 7.7 nM), and orally active (136 AUC0-24h 75.1 µg/mL; 137 AUC0-24h 22.4 µg/mL after 10 mg/(kg/po)).154 Takeda also developed another CCR5 antagonist, TAK-220 (138 affinity IC50 3.5 nM in competition with CCL5) that inhibits viral replication (IC50 0.55-1.7 nM).155 Merck patented a number of small molecule CCR5 antagonists, mainly based on the 1-amino-(2S)-2-aryl-4-

(piperidine-1-yl)-butane, 1,3,4-trisubstituted pyrrolidine, and cyclopentane templates.152 The affinity of the initial screening hit for the 1-amino-(2S)-2-aryl-4-(piperidine-1-yl)-butane series, 139 (Chart 30) (affinity IC50 35 nM; antiviral IC95 6-12 µM), was increased 3-fold by simply removing the 4-Cl from the 3,4-dichlorophenyl group (140 affinity IC50 10 nM; antiviral IC95 1.5 µM).125 Alteration of the moiety attached to the piperidine greatly affected anti-HIV activity (141 affinity IC50 0.1 nM; antiviral IC95 3 nM HeLa (BAL) cells, 300 nM).152 Merck replaced the 1-amino-(2S)-2-aryl-butane of 140 with 1,3,4trisubstituted pyrrolidines (143 IC50 0.8 nM)125 and explored the conformation about the amide bond by replacing the pyrrolidine with bicyclic isoazolidine (144 IC50 0.13 nM).125 The pyrrolidine series had a side activity at hERG K+ channel and relatively low oral bioavailability, the best compound being 145 (affinity IC50 1.2 nM, antiviral IC90 11 nM + 50% serum; 33% bioavailability in rats).156



Chart 31

Chart 32

Pfizer also reported piperidine-based analogues, starting their CCR5 program by screening their compound library and subjecting hits through logP and ligand efficacy/druglike characteristics (Lipinski’s rules) filters. Of five compounds identified with CCR5 activity, UK-107,543 (146, IC50 0.04 µM, CCL3157,158) (Chart 31) was chosen for chemical optimization but was later found to inhibit CYP4502D6. Chemical optimization of this lead involving replacement of 2-methyl-imidazopyridine with 2-methyl-benzimidazole, the piperidine with 8-aza-bicyclo[3.2.1]octane, and altering the diphenyl substituent generated UK-396,794 (147) with potent activity IC90(AV) 0.9 nM, but poor pharmacokinetics.157 Altering the methyl-benzylimidazole to 3,5-dimethyl-1,2,4triazole led to UK427,857 (148, antiviral IC90 0.5 nM, human t1/2 3 h, human %F 10-25),157,158 which has completed Phase II clinical trials for HIV infections. Schering Plough has also designed piperidine and piperazine containing CCR5 antagonists. Initial screening identified a 1,4′-bipiperidinyl compound that also had high affinity for the muscarinic receptors (149 CCR5 Ki 1 µM; M2 Ki 1.3 nM) (Chart 32).125,159 Removal of the methoxyphenyl sulfone and exchange of the fluoronaphthalene for 2,4-dimethylpyridine generated Sch-350634 (150), which had better CCR5 selectivity (CCR5 IC50 7 nM,CCL5; M2 IC50 250 nM) and oral bioavailability (dog %F 33).125,152 However, oxidative metabolism was observed to occur at the nitrogen of the pyridine; therefore, the next next generation contained the pyridine-N-oxide as well as an oxime in place of the methylene between the piperidine and 4-bromophenyl, creating SCH-351125 (Sch-C 151) the Schering Plough clinical lead (CCR5 affinity IC50 2.1 nM,CCL5; M2 IC50 n/a).125,152 Sch-C 151 with its good pharmacokinetics (oral bioavailability 52% monkey; half-life 6 h) was used in a ‘proof-ofconcept’ clinical trial and reduced the viral RNA titers in HIV-infected patients at doses of 100 mg twice daily. However, Sch-C 151 was withdrawn from clinical trials because of observed QTc prolongation.124 Schering Plough developed a backup clinical candidate, SCH-417690,(SchD, vicriviroc, 152) which structure had a 1,4′-piperidinyl-

piperazine core, an ether in place of the oxime in Sch-C 151 and a pyrazine attached to the piperidine by an amide bond. The pharmacodynamic and pharmacokinetic properties of 152 were excellent with a CCR5 affinity IC50 of 2.5 nM, antiviral activity IC90 ranging from 0.45 to 18 nM, and 100% oral bioavailability in rats and 89% in monkeys. In experimental models, no QTc prolongation was observed.130 Sch-D (152) is currently in phase II clinical trials.130 GlaxoSmith Kline and Ono Pharmaceuticals are together developing the CCR5 antagonist AK602/ONO4128/ GW873140 (153) (Chart 33).160 This compound is developed on a spirodiketopiperazine scaffold. SAR data gathered on the lead compound E913 (154)161 explored different attachments to the piperidine and diketopiperazine. Information from two most potent compounds of the first generation spiroketopiperazine scaffold EP913 (154 affinity IC50 2 nM, CCL3; anti-viral IC50 30-60 nM; cytotoxicity index CC50 51.8 µM)161 and E917 (155 affinity IC50 9 nM, CCL3; antiviral IC50 60-160 nM; cytotoxicity index CC50 10.8 µM)161 were used to design second generation GW873140 (153), which is currently in phase II clinical trials. GW873140 is a potent CCR5 antagonist (blocks CCL3 binding CCR5 with a Kd of 3 nM) with an anti-viral inhibitory constant (IC50) of 0.2-0.6 nM.160 It only has a plasma half-life of 29 min, but takes 9 h for 50% of GW873140 to be removed from CCR5 at cell surfaces.160 It is predominantly metabolized at CYP4503A4.162 GlaxoSmith Kline has patented other templates as CCR5 antagonist structures, including (3-ether-4methoxyphenyl)(biaryl)amides (156).152 Several other CCR5 antagonists have been reported. Compound 157 (Chart 34) was developed by Novartis by combining a lead fragment identified in a HTS (158 IC50 64 nM)163 and the 4-methylpiperidinyl-carbonyl-3-(2,4-dimethylpyridine-N-oxide) from Sch-C (151). It was believed that the fragment identified in the HTS hit could be a surrogate for benzyloxime-4-piperidinyl portion of Sch-C (151).163 Compound 157 had nanomolar affinity at hCCR5 (IC50 2.9 nM) and inhibited calcium mobilization (IC50 24 nM).163 Three CCR5 antagonists have been isolated from nature (159


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compound 162 (Chart 35) has inhibitory activity of Ca2+ mobilization and chemotaxis 99% of PGE2 production at 10 µM).171 Dompe´’s clinical Chart 40

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candidate repertaxin 182 designed on (R)-ketoprofen inhibited chemotaxis induced by CXCL8 (IC50 1 nM, CXCR1 IC50 1 nM; CXCR2 IC50 100 nM in transfected cells), while it did not affect LPS-induced prostaglandin E2 production at 10 µM (indicating no COX activity at this concentration).171 It is currently in Phase II clinical trials for prevention of reperfusion injury. Neither ketoprofen or repertaxin affects CXCL8 (IL-8) binding to CXCR1 or CXCR2; these compounds only affect downstream effects of receptor activation, thus, they are noncompetitive allosteric blockers of these receptors.171-173 2.5.2.2. CXCR3. The CXCR3 receptor is expressed predominantly on activated T lymphocytes (also found on natural killer cells), responds to the chemokines CXCL9 (Mig), CXCL10 (IP10), and CXCL11 (1-TAC/IP9),120 and has been observed on infiltrating T cells in rheumatoid arthritis, multiple sclerosis, in hepatitis C-infected livers, atherosclerosis, and chronic skin reactions.174,175 CXCR3 antagonist T-487 (undisclosed structure) developed by Chemocentryx & Tularik/Amgen is being evaluated in Phase II trials for psoriasis and rheumatoid arthritis.111,112,136 Berlex reported the development of 4-aryl-[1,4]-diazepane ethyl ureas identified from high-throughout screening. Compound 183 (Chart 40) had good affinity (IC50 60 nM), inhibited



Chart 41

chemotaxis (IC50 100 nM), and was selective for CXCR3 over 14 other GPCRs at 10 µM.175 Storelli and researchers presented structure-activity data on decanoic acid (2dimethylamino-ethyl)-{1-[3-(4-fluoro-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-y]-ethyl}-amide (184 IC50 3.2 µM, CXCL10 displacement).174 Replacement of the 4-fluoro substituent with a 4-cyano group increased affinity 3-fold (185 IC50 0.93 µM).174 Merck reported a number of natural products with CXCR3 activity (IC50 0.1 - 15 µM), including cyclic peptides, duramycin (IC50 100 nM), three roselipins (186 IC50 15-41 µM), three diasgenin glycosides (187 IC50 0.43-3 µM), and a novel 3-alkyl pyridinium alkaloid (188 IC50 0.69 µM).176 From the T487 series, Heise et al. studied NBI-74330 (189 IC50 3.6 nM, [125I]-CXCL11 displacement; IC80 30 nM and 1 nM against CXCL10- and CXCL11induced calcium flux, respectively; IC50 of 10.8 and 9.3 nM inhibiting [35S]GTPγS induced by 0.3 nM of CXCL11 and 300 nM of CXCL300, respectively; IC50 3.9 nM inhibiting CXCL11-induced chemotaxis).177 2.5.2.3. CXCR4. The receptor for stromal cell-derived factor (SDF-1R (CXCL12R) and SDF-1β (CXCL12β)), CXCR4 is involved in normal cardiac development, hematopoiesis, and homing of hematopoietic stem cells. It is expressed in developing vascular endothelial cells, B-cell lymphopoiesis, and bone marrow myelopoiesis. CXCR4, along with CCR5, is a key co-receptor for HIV-1, particularly of T-tropic HIV. CXCR4 has thus been implicated as a good target in HIV, cancer (CXCL12 agonist activity at CXCR4 may direct tumor cell metastasis), transplantation (mobilization of CD34+ cells), as well as showing efficacy in collagen-induced arthritis and airway hyperreactivity in allergic mice.178-180 It was discovered soon after the identification of HIV as the causative agent in AIDS that polyoxometalates reduce viral replication. In the progress of finding safer metal chelates than polyoxometalates, AnorMED examined commercially available cyclams (1,4,8,11-tetraazacyclotetradecane) for antiviral activity. An impurity in one sample, AMD1657 190 (Chart 41), was identified to have antiviral activity against HIV-1 (III-B) and HIV-2 (ROD) (190 EC50 0.144 and 1.01 µM, respectively).180 The tethering unit between cyclam moieties was extended first with alphatic

linkage with no effect (AMD 2763 191 antiviral activity EC50 0.248 µM, HIV-1(IIIB); HIV-2(ROD) EC50 0.248 µM).180 Next a 1,4-phenylene-bis(methylene) linkage was examined, AMD3100 (192 antiviral activity EC50 5 ng/mL; selectivity >100 000, cytotoxicity CC50 > 500 µg/mL).180 Time course experiments established that the bicyclams targeted the fusion/uncoating stage of the viral replication cycle, further analysis identifying CXCR4 as the cell co-receptor for T-lymphotropic HIV strains targeted. AMD3100 192 has some efficacy in models of HIV infection, collagen induced arthritis, ovarian cancer, non-Hodgkin’s lymphomas, and mobilization of stem cells in patients undergoing bone marrow transplant, completing Phase I/II clinical trials as an iv formulation since it lacked oral bioavailability. The most potent bicyclams incorporated pyridine (193 antiviral activity at HIV-1 (IIIB) and HIV-2(ROD) EC50 values of of 0.8 and 1.6 nM, respectively).180 One of the 1,4,8,11-tetraazacyclotetradecane can be replaced to obtain oral bioavailability (194 antiviral activity EC50 6-12 nM, HIV-1(IIIB); 12.3 nM, HIV-2(ROD)); however, this compound had cardiovascular side effects.123 KRH-1636 (195) is the only other nonpeptidic compound reported with antiviral activity at CXCR4 (EC50 19.3 nM; CC50 406.2 µM).181

2.6. Cholecystokinin and Gastrin Receptors Cholecystokinin (CCK) is a peptide hormone present in both the gastrointestinal tract and the central nervous system.182,183 CCK exists as a variety of bioactive peptides of varying lengths, including CCK-22, CCK-33, CCK-39, CCK-58, sulfated and unsulfated CCK-8 [DY(SO3H)MGW5MDF8-NH2], CCK-7, CCK-5, and CCK-4, all derived from a 115 residue precursor, prepro-CCK. Gastrin [pEGPWLEEEEEAY(SO3H)GWMDF-NH2] is a related peptide hormone that shares the same bioactive C-terminal pentapeptide as CCK, but differs in the position of sulfation. There are two GPCRs for CCK-peptides; CCK1 (or CCKA receptor) is about 500-fold more selective for CCK over gastrin and is found in the periphery, while CCK2 (or CCKB/G receptor) has comparable affinity for both CCK and gastrin and is mainly located in the central nervous system.182,183 These peptides have a variety of physiological functions, including those mediated via CCK1, such as


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Chart 42

Chart 43

regulation of satiety, pancreatic enzyme secretion, gastric emptying, and duodenal, colonic and gall bladder motility;182,183 and those mediated through CCK2, such as regulation of pain, memory, and anxiety. CCK is currently used as a diagnostic agent for detecting gall bladder and pancreatic disorders. Opioid administration and neural injury are believed to increase CCK concentrations; these peptides have anti-opioid actions, and antagonists have analgesic effects and regulate nociception. A large number of peptides,11,182 peptoids,184 and peptidomimetics11,182 based on a β-turn in CCK-4 and CCK-8, have been made and found to have high affinity, efficacy, and selectivity for CCK1 versus CCK2 and are reviewed elsewhere.11,182,184,185 Among glutamate analogues developed as potent and selective CCK antagonists were proglumide, lorglumide, loxiglumide (CR1505), dexloxiglumide (CR2017), itriglumide (CR2945), A-64718, A-65186, and a variety of analogues that are described in detail elsewhere.182 Most of these antagonists were active at submicromolar concentrations or below and some had sufficient bioavailability for clinical studies for pancreatic disorders, biliary colic, and inflammatory bowel syndrome.182 However, due to their poor pharmacokinetic profiles and low bioavailability, their principal uses today are as pharmacological tools in cellular and animal studies. For example, a local nociceptive effect of peripheral CCK-8 was recently reported186 to be significantly reduced by 100 mg of either proglumide (4-benzoylamino-5-dipropylamino-5-oxopentanoic acid), a nonselective

CCK receptor antagonist; or CCK1R antagonist lorglumide ((()-4-[(3,4-dichlorobenzoyl)amino]-5-(di-n-pentylamino)5-oxopentanoic acid), or CCK2R antagonist CR-2945 (b[2-[(2-(8-azaspiro-[4,5]-dec-8-ylcarbonyl)-4,6-dimethylphenyl)amino]-2-oxoethyl]-(R)-1-naphthalenepropanoic acid, itriglumide). Peptidic CCK1 antagonists have also recently been reported to have antiproliferative effects in HT-29 colon cancer cells, while agonists have been shown to stimulate growth of colon cancer.187 An even larger number of nonpeptidic antagonists of CCK1 and CCK2 are now known and are well-described in detail elsewhere.182,185 Here, we select just a few examples to illustrate representative structures (196-213). Two less peptidic compounds are the partial agonist 196 (PD-134,308) (Chart 42) with a type II′ β-turn constraint (IC50 4.7 nM (CCK1), >10 000 (CCK2)),188 and the R-methyl tryptophan derivative 197 ((IC50 4300 nM (CCK1), 1.8 nM (CCK2)) which mimics tetrapeptide CCK-4.189 A landmark discovery was a compound from microbial fermentation, asperlicin 198 (IC50 1400 nM (CCK1), >10 nM (CCK2)), which was the first nonpeptidic antagonist of CCK1. Compound 198 (Chart 43) has been modified into a wide variety of selective antagonists of CCK1 and CCK2, with an especially large series of benzodiazepine and quinazolidinone derivatives.182 Among the many nonpeptidic ligands for CCK1R are agonist SR146,131 (199), antagonists SR27,897 (200 IC50 > 0.58 nM (CCK1), 489 nM (CCK2)) and L-364,718 (devazepide, 201 IC50 0.08 nM (CCK1), 270 nM



Chart 44

(CCK2)), and benzodiazepine agonist 202 (EC50 150 nM, pancreatic amylase secretion; binding affinity IC50 61 nM) and antagonist 203 (IC50 490 nM).190 Compound 201 was discontinued in Phase II clinical trials for gastric motility, but the results of a Phase II trial for pain are unknown. The structure of the receptor binding sites for such agonists and antagonists in relation to the mechanism of receptor activation/inactivation is still under intensive investigation using structure-activity relationship data in conjunction with receptor mutagenesis.190,191 Hundreds of other small molecule ligands have been reported for CCK2, and most often they have been derived from a screening hit identified from high-throughput assays of compound libraries. Among more studied antagonists are 1,4-benzodiazepines 204 (L-365,260) (Chart 44) and 205 (YF476, also known as YM-220), 1,5-benzodiazepine 206 (Z360), quinazolinone/indole 207 (LY-202769), quinazolinone 208, indol-2-ones 209 (AG-041R) and 210 (T-0632), indole 211 (JB93182), benzimidazole 212 (JB95008), and substituted imidazoles (e.g., 213).182,185 Compound 204 (IC50 280 nM (CCK1), 2.0 nM (CCK2)) was developed into a large number of early CCK2 antagonist leads,182 most of which had low oral bioavailability except for 205 and 206, and was itself examined in Phase II clinical trials for anxiety/drug dependence (discontinued) and pain. Compound 205 (IC50 502 nM (CCK1), 0.11 nM (CCK2)) potently inhibited gastrin-induced acid secretion in dogs and rats after iv, po, or sc administration, reversed omeprazole- or gastrin-induced hypergastrinemia, and cell proliferation in ulcerated gastric mucosa, and entered Phase I clinical trials for gastroesophageal reflux.

CCK2 selective antagonists 207 (IC50 >10 000 nM (CCK1), 9.3 nM (CCK2)) and 208 (IC50 >7,100 nM (CCK1), 14 nM (CCK2)) were derived from aperlicin (198). Compound 207 was found to require flexibility between its quinazolinone and indole components, while a bulky isopropyloxyl substituent at the meta position of the 3-phenyl group was important as was the corresponding dimethylamino substituent in the quinazolinone 208. Oral administration of 208 (22% orally bioavailable) at 1 mg/kg to rats produced anxiolytic activity in an X-maze test, but was not as effective as 207 at 0.1 mg/kg and showed low water solubility. The 1,3,3-trisubstituted indol-2-ones 209 (IC50 555 nM (CCK1), 1.1 nM (CCK2)) and 210 (IC50 0.24 nM (CCK1), 5600 nM (CCK2)) are highly receptor-selective antagonists derived from screening leads. Compound 209 potently inhibited gastrin-induced acid secretion, was more effective than 204 in stress and ulcer models, was more effective than 205 in inhibiting gastrin-stimulated histidine decarboxylase activation in rats, and showed some promise in cartilage repair/regeneration. On the other hand, the structural analogue 210, which is a selective CCK1 antagonist, inhibits rat/mouse/dog pancreatic enzyme release at single digit nanomolar concentrations, rabbit gallbladder smooth muscle contractions (pA2 8.5), prevents acute pancreatitis in rat/mouse models such as caerulein-induced pancreatitis, and is in Phase I trials for treating pancreatic disorders. The orally active bicyclic heteroaromatic compounds 211 and 212 were potent and selective CCK2 antagonists, but were not particularly orally bioavailable (1% for 212) due to biliary excretion. Orally active imidazole analogues like 213 had comparable potency and receptor


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Chart 45

selectivity, but improved oral bioavailability (23%) and reduced elimination.185 In summary, a large number of small molecule ligands with selectivity for CCK1 or CCK2 have now been developed, and at least 18 compounds have proceeded to clinical trials for conditions like pancreatic disorders, gastric disorders, anxiety, and pain.182 Most synthetic efforts in recent years have focused on optimizing for selectivity and pharmacokinetic profiles, and culminated in more orally bioavailable compounds. At the very least, these compounds will allow better resolution of the physiological roles of CCK and gastrin in ViVo. A broader implication of these studies is the interchangeability of scaffolds within some of these compounds, leading to the concepts of scaffold hopping192 and privileged structures193 that are now being applied to the creation of new ligands for many other GPCRs.

2.7. Complement Receptors In response to infection and injury, the complement network of plasma proteins is activated and produces complement factors C5a and C3a.194 Such proteins are called anaphylatoxins, and they have potent proinflammatory properties that are mediated through binding to rhodopsinlike GPCRs. There are two known receptors for C5a (C5aR or CD88, C5L2195) and one for C3a (C3aR), with no crossreactivity between them. They occur on both endothelial and epithelial cells, being particularly prevalent on neutrophils, monocytes, macrophages, eosinophils, mast cells, bronchial epithelial cells, smooth muscle cells, vascular endothelial cells, and hepatocytes, where their expression correlates with cell activation and induced synthesis of proinflammatory mediators.196 C5a is much more potent than C3a in causing phagocyte chemotaxis, degranulation, spasmogenesis, and cell adhesion, and both receptors couple with pertussis toxinsensitive Gi, insensitive Gz, and G16 proteins.

2.7.1. Complement C5a Receptor C5a is a 74-residue peptide with an N-terminal helix bundle (residues 1-64) and a C-terminal activation domain (residues 65-74). Activation of C5aR decreases intracellular cAMP but elevates intracellular Ca2+, PI3, and Ras/Raf/MAP kinase activity197 and regulates cytokine and chemokine gene expression and apoptosis.198,199 The C5aR gene knockout in mice inhibits formation of collagen-induced arthritis, blocks chemotaxis and recruitment of neutrophils and macrophages, and inhibits formation of TNFR, IL-1β, IL-6, and other

inflammatory mediators that initiate disease.200 Since C5a or its receptor is implicated in the pathogenesis of a wide range of diseases (e.g., rheumatoid and osteoarthritis, inflammatory bowel disease, fibrotic conditions, systemic lupus erythematosus, ischemia/reperfusion injury, sepsis, various forms of shock, asthma, psoriasis, gingivitis, atherosclerosis, multiple sclerosis, meningitis, pancreatitis, lung injury, burn injuries, tissue graft rejection, ARDS, and neurodegenerative diseases such as Alzheimer’s disease), selective antagonists of C5aR could have a diverse range of therapeutic properties. Peptidic and nonpeptide antagonists of C5aR are known.201-203 Cyclic peptide Ac-Phe-[Orn-Pro-D-Cha-TrpArg] (3D53), with IC50 30 nM for human PMNs, has been used as a model (insurmountable) antagonist204-206 to demonstrate efficacy in many disease models (e.g., ischemia/ reperfusion, arthritis, and inflammatory bowel disease)204,207 and has completed Phase II clinical trials for rheumatoid arthritis and psoriasis. More orally bioavailable nonpeptidic antagonists have also been developed. Initial nonpeptides had moderate affinity for human C5aR, such as aminoquinolines (e.g., 214, 215) (Chart 45) and phenylguanidines (e.g., 216, EC50 0.8 µM).208 A hydantoin (217) has been reported as a high affinity (EC50 20 nM) full agonist, while W-54011 (218) is the most potent reported209 competitive nonpeptidic C5aR antagonist ([125I]hC5a IC50 2.2 nM), inhibiting intracellular Ca2+ mobilization, chemotaxis and generation of reactive oxygen species (IC50 3.1, 2.7, and 1.6 nM, respectively). Among other nonpeptidic C5a antagonist structures are two series related to Jerini JSM7431 (preclinical) and Neurogen NGD 2000-1 (Phase II clinical trials for asthma and rheumatoid arthritis discontinued), analogue NDT9520492 (219) having been disclosed.210 It is worth noting that the IC50 values reported for competitive reversible antagonists W54011 and NDT952492 are biased by the low nanomolar concentrations of C5a that they were measured against, and neither compound is as effective as the insurmountable cyclic peptide antagonist 3D53 at higher C5a concentrations.

2.7.2. Complement C3a Receptor C3a is a 77-residue peptide with similar structure to C5a and similar pharmacological properties. C3a is probably best linked as a prospective pharmaceutical target of asthma. There are two known nonpeptidic C3a antagonists. The less potent antagonist is the 1,3-diiminoisoindoline 220 (Chart 46) with weak affinity for C3aR (IC50 1.7 µM).211 This compound mimics the important C-terminal arginine of C3a and inhibits calcium mobilization (IC50 2 µM) and wheal


formation (inflammatory response) in ViVo. Its structure suggests no target selectivity. A more potent antagonist (IC50 200 nM) of C3aR is the dipeptide surrogate SB290157 (221).212 It inhibits rhC3ainduced Ca2+ mobilization (IC50 7-30 nM) on RBL-2H3 cells, inhibits C3a chemotaxis of mast cells in Vitro, and blocks C3a-mediated contraction of rat arteries in ViVo. It had anti-inflammatory activity in animal models of airway neutrophilia and arthritis.212 Although the compound did not antagonize C5aR or five other chemotactic receptors, it was toxic to cells consistent with its predicted lack of selectivity and displayed significant agonist activity.213,214

2.8. Corticotropin-Releasing Factor Receptors Two known GPCRs (CRF1 and CRF2) are currently thought to be the targets for the neuropeptide hormones Corticotropin Releasing Factor (CRF, CRH) and the structurally related urocortins (Ucn I-III), which mediate stressinduced physiological and behavioral changes. These receptors have sequence identity with other GPCRs for secretin, calcitonin, and glucagons.11,215 Whereas CRF-1 binds CRF and UcnI, CRF2 binds only urocortins.215,216 These peptides are released into the CNS, expressed in peripheral tissues including skin and mast cells,217 and exhibit proinflammatory and anti-inflammatory actions. They regulate many physiological processes, particularly inhibiting gastric emptying, promoting bowel motility,218 and inflammatory skin conditions (e.g., psoriasis and atopic dermatitis)217 that are induced by stress. Brain penetrant nonpeptidic antagonists (e.g., 222226) (Chart 47) have been developed for CRF receptors to enable treatment of anxiety, depression, substance abuse, and other CNS associated diseases.219 However, the proinflamChart 47


matory actions of CRF in the GI tract suggest that antagonists might also be useful in treating other stress-related disorders such as ulcerative colitis and inflammatory bowel conditions/ diseases, while antiinflammatory actions of urocortins and CRF2 antagonists might be useful in treating preterm labor and other GI tract inflammatory diseases. CP-154526 (222, R ) Me) and antalarmin (222, R ) H) are potent, brain-penetrable, selective nonpeptidic CRF1 antagonists with efficacy in stress-induced CNS and peripheral disorders,220 suggesting use in psychiatric disorders. Compound 222 (Ki 6 nM, CRF1) has low oral bioavailability but has therapeutic effects in preclinical models of anxiety, depression, and substance abuse220 and attenuates peripheral antinociceptive effects of CRF in Wistar rats with Freund’s complete adjuvant-induced hind paw inflammation, an effect also seen for the selective CRF2 antagonist astressin 2B.221 CRF releases opioid peptides from immune cells and exerts peripheral antinociceptive effects by activating opioid receptors on peripheral sensory nerve endings. Two highly selective, noncompetitive, orally bioavailable antagonists with nanomolar affinity for human CRF1 are DMP696 (223) and the pyrazolopyrimidine derivative DMP904 (224).222 They block adenylyl cyclase activity in cells expressing CRF1, inhibit ACTH release from rat pituitary corticotropes, display anxiolytic activity at 1-3 mg/ kg over 14 days in rats, bind >50% CRF1 receptors in the brain, and have good pharmacokinetic profiles.222 The structural analogue R-121919 (225, NBI-30775; Neurocrine Biosciences, Inc.) was the first clinical candidate, with similar potency (Ki 3 nM, CRF1) and selective antagonism of CRF1, and high oral bioavailability and good pharmacokinetics due to the pyridyl substituent conferring water solubility.219 Clinical results suggested efficacy for anxiety and depression, but there was some hepatotoxicity. Thiazole analogue SSR125543A (226, Ki 2 nM, CRF1) and the water soluble NBI35965 (Ki 4 nM, CRF1) are examples of orally active, CRF1selective antagonists being evaluated for stress-induced anxiety and depression, and they may also have antiinflammatory activity.

2.9. Endothelin Receptors Endothelins (ET-1, 2, 3) are potent vasoactive peptides acting at ETA and ETB GPCRs. They are 21-residue peptides with 2 intramolecular disulfide bridges and are produced by cleavage of inactive precursor peptides termed big ET-1,2,3 by the metalloproteases, endothelin converting enzymes ECE-1,2,3. Prominently known for contractile activity on vascular networks, they have been established clinically and/ or experimentally in the treatment of hypertension, heart


Blakeney et al.

Chart 48

Chart 49

failure, and atherosclerosis.223,224 In addition, endothelins have wide-ranging physiological actions on cardiovascular, renal, pulmonary, and gastrointestinal tract systems, including important roles in inflammation. The first nonpeptidic endothelin antagonist was reported by Hoffmann-La Roche in 1993.225 Their intention was to identify nonspecific antagonists of all endothelin receptors to determine the role of ET-1 in models of focal vasoconstriction. Screening of compounds, for their ability to inhibit [125I]-ET-1 binding to a human placenta membrane preparation, led to the discovery of a class of arylsulfonamido pyrimidines that with further synthetic modifications afforded Ro-46-2005 (227) (Chart 48), a moderately potent antagonist of both ETA (human vascular smooth muscle cells) and ETB (rat aortic endothelial cells), with IC50 values of 220 nM and 1 µM, respectively. Compound 227 was also confirmed to be a competitive antagonist in functional assays of both ETA and ETB and was extremely selective for ET in not preventing contractions caused by angiotensin II, serotonin, noradrenaline, potassium chloride, or prostaglandin F2R at concentrations of 30 µM that were achievable following oral administration (F ∼ 30%). Because of the modest potency of 227, other analogues were investigated including 228 (Bosentan)226 that achieved considerable gains in affinity at both ETA and ETB receptors (Ki 6.5 and 343 nM, respectively, in CHO cells expressing the receptors separately). Bosentan 228 inhibited the pressor response of co-administered ET-1 and was as effective as enalapril in humans with hypertension. Bosentan 228 was the first drug approved by the FDA to treat pulmonary arterial hypertension (PAH), a pathological condition associated with high levels of ET-1 in the lung. Further optimization of 228 was achieved by introduction of the 2-pyridinecarbamoyl group and 2-aza-4-isopropyl modifications to the arylsulfonamide resulting in 229 which was almost equipotent with ET-1 for the ETA receptor and also well-balanced toward ETB (IC50 0.7 and 5 nM, respectively, in receptor expressing CHO cells).227 Screening at SmithKline Beecham identified the 1,3diphenylindene-2-carboxylic acid 230 (Chart 49) as a weak antagonist of cloned hETA receptors with virtually no affinity

for ETB (Ki 6.5 and >100 µM, respectively). The diarylindene system was oxidatively labile and difficult to obtain pure; however, a similar trans-diarylindane system was equipotent and more manageable. Potent analogues were rationally designed after alignment of 230 with the solution structure of ET-1, determined by NMR, which suggested that the two phenyl rings present in 230 could be mimicking a hydrophobic region mapped by the side chains of Tyr13, Phe14, and Trp21 and that the 2-carboxylic acid group could mimic either Asp18 or the important C-terminal carboxyl group of ET-1. Consequently, a second carboxylic acid group was tethered to the phenyl ring, and addition of a methylenedioxy ring and other alkoxy groups that probably reflect the electron rich nature of Tyr and Trp rings culminated in 231 with high affinity for ETA (Ki 0.2 nM) and ETB (Ki 18 nM).228,229 Although effective in several animal models of disease including hypertension, when administered iv, 231 suffered from poor oral bioavailability (F ∼ 4%), which prompted the search for more suitable analogues. The outcome was 232 with only slightly reduced affinity ETA (Ki 1.1 nM), greater selectivity over ETB (Ki 111 nM), and oral bioavailability (F ) 66%). A pharmacophoric analysis of known nonpeptidic ET antagonists at Abbot laboratories concluded that a pyrrolidine ring might replace the indane ring present in the SmithKline Beecham compounds 230-232.230 A series of highly potent ET antagonists were described in which ETA/ETB selectivity was controllable. Compound 233 (Chart 50) was first to be proposed as a potential clinical candidate with good activity for ETA receptors and 860-fold selectivity over ETB receptors (IC50 0.22, 190 nM, respectively). It was observed that if the direction of the amide bond in 233 was transposed, the selectivity declined dramatically. Examination of other acid mimics led to the sulfonamide 234, a potent but relatively nonselective antagonist in CHO cells (IC50 0.78 nM, hETA; 0.28 nM, hETB). Compound 234 was also shown to be a functional antagonist of rat ETA (IC50 0.48 nM) and human ETB (IC50 1.28 nM), by measurement of ETR mediated hydrolysis of inositol phosphates. It showed good pharmacokinetic parameters in rats (F 54%, T1/2 5 h).231 Further development of selective ETA antagonists from this



Chart 50

Chart 51

Chart 52

series resulted in 235, with dramatically improved activity and selectivity over ETB (IC50 0.16 and 6200 nM, respectively, selectivity A/B 38,700). Improved metabolic stability was also observed with the replacement of the methoxy group (de-methylates) by methyl, and methylenedioxyphenyl (CP450 inhibition) fragment with dihydrobenzofuran resulting in good pharmacokinetics (F 72%, T1/2 4.7 h, AUC 12.5 µg (h mL)) after oral dosing (10 mg/kg) to rats.232 Researchers at Merck recognized similarities between endothelin and angiotensin II receptors and their agonists, in particular their preference for a C-terminal carboxylic acid and the importance of key aromatic residues. This observation prompted the selective screening of known AII antagonists that offered a rich assortment of aromatic scaffolds with acidic groups and uncovered the R-aryl phenoxyacetic acid derivative 236 (Chart 51) with weak but comparable ETA and ETB receptor affinities (IC50 11, 31 µM, respectively).233 Simplification of the highly substituted phenyl ring by incorporating the known, favorable methylenedioxyphenyl group afforded almost 300-fold gain in ETA affinity. Next the carboxylic acid group was converted to a series of acylsulfonamides to circumvent potential metabolism via O-acyl-glucuronidation in future in ViVo studies, but also as an additional site for SAR investigation. Highly substituted benzenesulfonamides were also able to fulfill the role of one propyl group from the central ring, and the 4-isopropylbenzenesulfonamido compound 237 emerged as the most potent antagonist in the series (IC50 38 nM, ETA; 230 nM, ETB).

Interestingly, 237 also retained substantial AII antagonist activity (IC50 13 nM, AT1; 32 nM, AT2) suggesting that such pharmaceutical agents may be useful for treating hypertension caused by elevated levels of angiotensin II and endothelins. Replacing imidazopyridine with a simple carboxylic acid resulted in the highly potent and selective antagonist 238 (Ki 0.062 nM, ETA; 2.25 nM, ETB) that was also devoid of activity at both AT1 and AT2 receptors.234 In ViVo, 238 inhibited the pressor response to exogenously administered big ET-1 in ferrets and rats and protected against ET-1 induced lethality in mice with a duration of action >12 h following 3 mg/(kg/po). Screening at Bristol-Meyers Squibb for compounds that inhibit [125I]-ET-1 binding to vascular smooth muscle A10 cells identified several known antibacterial sulfa drugs, including sulfisoxazole 239 (Chart 52) as weak ETA antagonists (IC50 0.78 µM); however, this did not translate into functional antagonism determined by increase in intracellular Ca2+ concentration. Regardless, SAR studies were conducted revealing that the sulfonamide NH was essential for activity and the methyl groups on the isoxazole ring were also important for potency. Alkylation of the 4-amino group conferred some functional antagonism, which prompted the investigation of a series of naphthalenesulfonamides of which the 5-dimethylamino derivative 240 was found to be reasonably potent (binding IC50 150 nM, functional antagonism 520 nM). Additionally, 240 was orally active and had a long duration of action in a DOCA-salt rat model of hyperten-


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Chart 53

sion.235 The isoxazole ring of 240 could also be replaced by a variety of nitrogen-containing 6-membered heterocycles, in particular pyrazines, as exemplified by 241 (IC50 8 nM) that were also orally active. At 10 mg/kg 241 suppressed the pressor response of infused big ET-1 for at least 5 h in conscious rats.236 The dansyl group present in 240 and 241 was also replaced by a variety of substituted biphenyls affording improved ETA potency, for example, compound 242 (IC50 0.5 nM) which was also orally active.237 Compound 243 (Chart 53) was representative of a series of 3-thiophenesulfonamides incorporating the aminoisoxazole unit and a second hydrophobic fragment linked via an amide bond which had low nanomolar affinity for ETA in Vitro (IC50 3 nM). In ViVo activity of 243 was compromised by poor oral bioavailability (F 8%) and a short serum half-life (T1/2 2.5 h) that was probably caused by metabolic cleavage of the amide bond. An investigation of suitable replacements for the amide discovered other linkers including an acetyl unit resulting in 244 which retained high potency and selectivity for ETA but had remarkably improved oral bioavailability (F 50-60% rat, 90-100% dog) and serum half-life (T1/2 6.7 h). Compound 244 was effective in a rat model of acute hypoxia-induced pulmonary hypertension when administered either iv or orally (ED50 0.5 and 1 mg/ kg, respectively).238 Screening the BASF compound collection against ETA expressed in CHO cell membranes led to discovery of 245 (Ki 160 nM, ETA; 4.7 µM, ETB), originally designed as a herbicide. A series of analogues were prepared to remove one asymmetric center and improve affinity, including 246 (Ki 6 nM, ETA; 1 µM, ETB). The simple unsubstituted diphenyl arrangement was optimal as further substitution led to reduced activity. Variation of the methoxy group to larger alkyl, or hydroxy, was also detrimental. Substituents on the heterocycle affected binding affinity with several subnanomolar analogues identified; however, 246 showed the greatest duration of in ViVo activity following oral administration. The (S)-enantiomer of 246 was 50 times more potent than the (R)-enantiomer. In a rat model of ET-1-induced sudden death, 246 administered orally at 30 mg/kg afforded complete protection lasting 4 h, and at 100 mg/kg, complete protection was extended to 8 h.239

2.10. Formyl Peptide Receptor N-Formyl peptides were widely thought to have evolved to mediate trafficking of phagocytes to sites of bacterial invasion or tissue damage. N-Formyl peptides were the first structurally defined chemotactic factors and were introduced into the bloodstream through eruption of the mitochondrial membrane or from invading pathogens. These peptides activate N-formyl receptors, formyl-peptide receptor (FPR), and its variants FPRL-1 and FPRL-2, which induce signaling

events through the dissociation of GR and GβGγ subunits, activation of CD38, and of the protein kinase C-dependent pathway.240 However, there has been only one well-reported FPR nonpeptide ligand, a quinazolinone partial agonist (247 EC50 1.4 µM, Ca2+ mobilization from human neutrophils) (Chart 54) with less potency than reported peptidic agonist ligands (WKYMVm, EC50 4 nM), but its pro/anti-inflammatory activity has not yet been reported.241 Chart 54

2.11. Galanin Receptors The neuroendocrine peptide 29-residue galanin (30-residue in human) was discovered in 1983, from porcine intestine, and is associated with neurogenic inflammation and pain.242,243 To date, three galanin receptors, GalR1-3, have been identified and cloned. Galanin and its receptors are expressed widely in the central and peripheral nervous systems, including the dorsal root ganglion cells. Galanin receptor 1 is upregulated on colonic epithelial cells in DSS-induced murine colitis, increasing fluid secretion. They are implicated in regulation of many important physiological processes such as recognition, emotion control, learning, feeding behavior, and insulin and growth hormone release.242,243 Recent studies also indicated their involvement in modulation of pain and seizure control. For example, in pathological inflammatory conditions, neuropeptides are released to inhibit trafficking of nociceptive input through galanin receptor 1; thus, agonists could be used to treat inflammatory pain.244,245 Despite having been discovered over 20 years ago, there are only a few agonists and antagonists of moderate potency for galanin receptors, and this has hampered elucidation and validation of the roles of galanin in physiological and pathophysiological states. SAR studies identified residues Trp2, Asn5, and Tyr9 as important pharmacophoric groups. This information has been successfully exploited to design combinatorial libraries targeting galanin receptors.246-249 Two nonpeptidic agonists, galnon (248) and galmic (249) (Chart 55) were discovered with moderate potency. Galnon was shown to displace 125I-galanin from the cell membrane with Ki 2.9 µM, and inhibited adenylate cyclase activity (25% at



Chart 55

10 µM) comparable to that of galanin (36% at 10 µM), suggesting its agonist-like properties.246,248,249 Administration of galnon to mice or rats elicited anticonvulsant activity, reduced opiate withdrawal signs, and suppressed appetite. The galmic agonist was designed on a rigid oxazole-based macrocycle. It displayed weak binding affinity (Ki 34 µM) against Gal-R1 and was found to cross the blood-brain barrier and mimic the biological activity of galanin.247,249 High-throughput screening of corporate compound collections was also employed to obtain alternative leads for galanin receptors. Scott and colleagues optimized a dithiin1,1,4,4-tetraoxide hit by variation of the aromatic substitutions, resulting in identification of submicromolar antagonist 250.250,251 This compound bound to Gal-R1 (Ki 190 nM) and behaved as a galanin antagonist in functional assays. However, the reactive vinyl sulfone moiety rendered it unattractive from a drug discovery point of view. Another antagonist, the spirocoumarone Sch202596 (251) was discovered from a large library of samples derived from fungal and bacterial fermentations.249,252 Sch202596 modestly inhibited Gal-R1 (IC50 1.7 µM) under in Vitro conditions. Recently, a series of Gal-R3 antagonists were disclosed in the patent literature, including the indol-2-one derivative 252 which was reported to have high affinity (Ki 15 nM) for GalR3.249 Another indole derivative antagonist, SNAP-37889 (253) also exhibits high affinity toward GAL-R3 (Ki 17.4 nM) with high selectivity against other galanin receptors (Ki > 10 000 nM).253 In functional assay, SNAP-37889 concentration-dependently shifts rightward the effect-dose curves of galanin with a KB of 29 nM. When administered orally, SNAP-37889 produced acute and chronic efficacy in rat models of anxiety and depression in a dose-related manner (3-30 mg/kg). SNAP-37889 also showed anxiolytic-like activity in rats (3-10 mg/kg/i.p.), reduced stress-induced hyperthermia in mice (0.3-30 mg/(kg/po)) and anxietyrelated vocalizations of guinea pig pups after maternal separation (3-30 mg/(kg/po)).253,254

2.12. Glycoprotein Hormone Receptors The family of glycoprotein hormones consists of four proteins, follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), and thyroid-stimulating hormone (TSH), all of which exist as a noncovalent heterodimer of a conserved R subunit and a hormone-specific β subunit.255,256 There are 3 GPCRs, one each for FSH (FSHR) and TSH (TSHR) which exert their physiological effects through different GPCRs, and one GPCR (LH/hCGR) which binds to both LH and hCG as they have almost identical sequences, the β-subunit of hCG having 31 additional amino acids at the C-terminus. The specificity of these hormones is thought to be governed by recognition of hormone specific β-subunits. However, recent crystal structure data indicates that the common R-subunit also participates in interactions between hormone and receptorbinding domain and jointly mediates specificity.257 The glycoprotein hormone receptors are a rather unique GPCR family. They are characterized by an unusually large N-terminus, with 350-400 residues consisting of a number of leucine rich repeating (LRR) units. The LRRs exist as parallel β-strands forming a concave face that serves as the binding surface for the hormones.255,256 The gonadotropins, FSH, hCG, and LH, occupy central roles in reproduction, and are used clinically to treat infertility. Both FSH and LH are produced in the pituitary gland following activation of the GnRH receptor. FSH stimulates follicular maturation in females and triggers spermatogenesis in males. Agonists targeting the FSH receptor could be useful therapeutics for treating infertility, while antagonists could find applications as a novel class of contraceptive drugs. Binding of luteinizing hormone (LH) to LH/hCG receptor stimulates Theca or Leydig cells to generate testosterone; thus, modulators of LH/hCG receptor could provide benefits similar to that of the FSH receptor. TSH, on the other hand, is responsible for regulating the synthesis and secretion of thyroid hormones and is valuable as a diagnostic in thyroid cancer treatment.


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Chart 56

2.12.1. FSH Receptor The crystal structure of FSH bound to the extracellular binding domain of its receptor (FSHR) has been determined recently to 2.9 Å.257 Although the interacting protein surfaces involve 2600 Å2 of buried surface area, they could provide clues for designing small molecules to interact with the FSH receptor. In 2002, Arey and colleagues reported the discovery of the first nonpeptide FSHR antagonist using a radioligand competitive assay to screen large compound libraries.258 They identified diazonaphthylsulfonic acid derivative (254) (Chart 56) as an inhibitor (IC50 5.4 µM) of [125I]-hFSH binding to hFSHR expressed on 3D2 cells. Additional experiments revealed that 254 interacted with the extracellular domain of FSHR in a noncompetitive manner. However, it was capable of inhibiting FSH-induced cAMP accumulation in a concentration-dependent fashion and completely inhibited ovulation in adult cycling rats at 100 mg/(kg/i.p.). Other bissulfonate derivatives were moderately potent FSHR antagonists, the best being compound 255 [IC50 2.5 (radioligand competitive binding) and 0.9 µM (cAMP accumulation)].259 Recently, a series of tetrahydroquinoline derivatives were reported as potent FSHR antagonists,260 following discovery of a lead 256 (IC50 4.4 µM) through high-throughput screening. Preliminary SAR studies demonstrated that the

1-acetyl, 4-phenyl, and 6-amino groups all influenced potency. Surprisingly, acylation of the 6-amino moiety with an aromatic carboxylate switched the full agonist activity of 256 to full antagonist activity with 50-100-fold improvement in potency. Variation of the acyl groups at this position revealed that moderately large aromatic-hydrophobic groups were well-tolerated and contributed significantly to tight binding. Quantitative SAR studies also suggested the presence of a hydrophobic binding pocket to accommodate these bulky groups.261 The most potent compounds exhibit single digit nanomolar antagonism (IC50 7 nM for racemic 257). Interestingly, when resolved only one enantiomer was found to be active; the configuration for compound 258 (IC50 ) 4 nM) was tentatively assigned (R) based on vibrational circular dichroism studies. In an ex ViVo assay, at 10 µM, antagonist 258 significantly inhibited follicle growth that was stimulated by FSH. Encoded combinatorial libraries have proven to be powerful for generating hits for numerous drug targets. Employing a CHO-hFSHR-luciferase assay, Guo et al. identified novel biphenyl-derived agonists of hFSHR.262 From three parallel libraries of >300 synthetic compounds made to explore SAR and to optimize low micromolar hits, several potent agonists were found (e.g., EC50 13 nM, 259; 1.2 nM, 260). In the


cAMP accumulation assay, these agonists were able to activate FSHR with 47-85% efficacy relative to human FSH. Maclean and colleagues utilized an encoded split-pool combinatorial library of 42 000 thiazolidone derivatives to rapidly optimize full agonists of FSHR,263 such as 261 (EC50 32 nM) from 262 (EC50 5-10 µM). However, the thiazolidone scaffold of these compounds is prone to isomerize, making isolation and preparation of the more active (2S,3R) cis isomers difficult. Replacing the sulfur with a carbon atom, effectively solved this problem, converting the thiazolidone ring to a chemically more stable γ-lactam,264 leading to potent agonist 263 (EC50 25 nM).

2.12.2. LH/hCG Receptor High-throughput screening programs at Organon produced a thienopyrimidine compound 264 (EC50 1.4 µM)265 (Chart 57) as an LH receptor-selective agonist. Optimization of this lead revealed that meta substitution of the phenyl ring is beneficial for activity. The small -SMe group proved optimal, since enlarging it to S-pentyl or replacing it with alkylated amino groups leads to inactive compounds. Hydrolysis of the ester group also caused several fold loss in activity, whereas introduction of a moderately large hydrophobic substitutent at this position through amidation furnished much more potent compounds (e.g., 265, EC50 20 nM). The N3 atom of the pyrimidine also seemed to be important, as substituting it with a carbon atom delivered a series of less potent thienopyridine derivatives. In Vitro agonist 265 induced testosterone production (EC50 430 nM), while administration to immature, FSH-primed female mice at 50 mg/(kg/po) resulted in 40% ovulating animals.

2.13. Gonadotropin-Releasing Hormone Receptor The decapeptide gonadotropin-releasing hormone, or luteinizing hormone-releasing hormone (pGlu-His-Trp-SerTyr-Gly-Leu-Arg-Pro-Gly-NH2: GnRH) plays a crucial role in the biology of the reproductive system. It stimulates differential release of the gonadotropins FSH and LH from the pituitary gland.266 Two separate genes encode two forms of GnRH receptor (type I and type II). Currently, only expression of type I receptor has been verified; the gene encoding type II receptor carries a frame-shift and an internal stop codon, suggesting that it is silenced in humans.267 Many peptidic GnRH derivatives are used clinically as GnRH receptor agonists or antagonists to treat infertility, endometriosis, uterine fibroids. and cancer of reproductive organs.266,268 The bioactive conformation of gonadotropin is believed to be a type II′ β turn centered at Gly6,266 so replacing Gly with D-amino acids led to stabilization of the β-turn and the discovery of GnRH receptor superagonists. While GnRH receptor antagonists exert their pharmacological effects through competing with GnRH and blocking receptor activation, clinical administration of GnRH agonists and superagonists achieves therapeutic efficacy by down-regulat-


ing the GnRH receptor after an initial surge (known as a ‘flare’) of gonadotropin and gonadal hormones.266,268 Development of an orally bioavailable GnRH antagonist is therefore highly desirable, not only to address the cumbersome administration of peptidic agents, but also to avoid the unpleasant ‘flare’ effects of the agonists. One of the earliest examples of a nonpeptidic GnRH antagonist was the antifungal ketaconazole 266 (Chart 58) , which exhibits weak binding affinity (IC50 2 µM) and antagonist activity (pA2 4.19) against GnRH receptor.268 Researchers at Takeda reported the first orally active GnRH receptor antagonist, based on ‘directed screening’ of chemical libraries that produced a thienopyridinone scaffold as a mimic of the (Tyr5-Gly6-Leu7) β-turn of GnRH.269 It was found that an aminomethyl group, which presumably mimics the Arg8 in the 3-position, is important for receptor binding. Further optimization led to 267 (IC50 0.2 nM), which suppressed the plasma level of LH in cynomolgus monkeys in a timedependent manner after oral administration.268,270 In a related compound, a bicyclic thienopyrimidinone scaffold resulted in potent antagonist TAK-013 268 (IC50 0.10 nM) (Chart 59), which is in a Phase II clinical trial for treating endometriosis and uterine fibroids.268,270 Other fused 5- and 6-membered ring systems have also resulted in highly active antagonists of GnRH receptor (e.g., pyrazolopyrimidinone 269, imidazolopyrimidinone 270, triptamine 271).268,270,271 The basic amino group was essential for antagonist potency. Pyrazolo derivatives (e.g., 269) were not stable under acidic conditions due to the retro-Mannich reaction involving the basic amino group, but introducing a strong electron-withdrawing fluoro substitutent somewhat improved acid stability (269 Ki 9 nM). Replacing a carbon with a nitrogen atom in the ring afforded the imidazolopyrimidinone core, conferring complete acid stability to the ligands. Further substitution of the hydrolyzable ester group with a hydrophobic aromatic moiety increased plasma stability of antagonist 270 (Ki 11 nM). Merck reported tryptamine derivatives as potent GnRH receptor antagonists. After the discovery of the core through screening of in-house compound collections, exhaustive optimization and SAR studies identified compound 271 as a potent GnRH antagonist that was orally efficacious in reducing LH plasma level in castrated male rats. High-throughput screening of Merck compound libraries led to the quinolone core, decoration with basic amino and hydrophobic aromatic groups at appropriate positions furnishing the highly potent 272 (IC50 0.44 nM) with functional antagonism at 1 nM.268,270,271 Despite subnanomolar potencies, GnRH receptor antagonists built around bicyclic systems are of high molecular


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Chart 59

Chart 60

weight, often in excess of 500 Da. Recently, several smaller (monocyclic) scaffolds were used to address the issue of high molecular weight. Starting from the imidazolopyrimidinone series, it was reasoned that deletion of the imidazole ring might be well-tolerated. New uracil derivatives like 273 (IC50 0.5 nM) (Chart 60) retained the excellent potency of 270, while having lower molecular weight,272,273 but were metabolically unstable. When incubated with human microsomes, 273 undergoes dealkylation to a less active metabolite. Tucci et al.274,275 elegantly addressed this problem by preparing novel uracil derivatives, in which the N-substituent was moved to the nearby R-carbon atom of the amino group, leading to very active compounds such as NBI 42902 (274, Ki 0.56 nM, functional IC50 3.0 nM) which was selected for clinical development to treat endometriosis and uterine fibroids.275,268 The same research group also reported the use of a triazine-trione scaffold in place of uracil, the best compound being 275 (Ki 2 nM).276 Other scaffolds, such as diazepine, benzimidazole, oxoindole, azaindole, quinazolines, pydridinone, pyridazinone, and furan have all been claimed to be useful in generating GnRH receptor antagonists.268,270,271

2.14. Growth Hormone Secretagogue (Ghrelin) Receptors Over 25 years ago, certain opioid peptide derivatives were reported to display growth hormone releasing activity.277-279 Subsequently, a potent hexapeptide, GRHP-6 was found to

be active both in ViVo and in Vitro. Further investigations revealed that this compound acts through a pathway independent of the growth hormone releasing hormone receptor.280 These findings provided the impetus for searching for small molecule, drug-like compounds that were able to mimic the intriguing GH releasing actions of GRHP-6. Compounds of such activity were termed Growth Hormone Secretagogues (GHSs), and were believed to be promising drug candidates to treat growth hormone deficiency and to rejuvenate the GH/IGF-1 axis in fragile aging people.280 The radiolabeled [35S]MK-0677, a potent GHS, was exploited to identify and characterize a receptor for this class of ligands.280 The GHS receptor is a GPCR that is expressed widely in hypothalamus, hippocampus, pituitary gland, and many peripheral tissues including pancreas, intestine, stomach, kidney, heart, lung, and adipose tissues.277-279,281 Two types of GHS receptors have been identified. Peptidic and nonpeptidic GHSs mediate their biological activity through GHSR1a. The other GHS receptor, type 1b, is also encoded by the same gene as type 1a, but as the result of alternate processing, it consists of only five transmembrane helices, TM 1-5. Biological functions of the GHS-R1b are yet to be defined. In a classic example of reverse pharmacology, the endogenous ligand for GHSR, ghrelin, was discovered last, more than two decades after the description of the first peptidic GHS.277 Adenosine was also shown to be partial agonist of GHS receptor, though it interacts via a binding



Chart 61

pocket other than that for ghrelin and synthetic GHS analogues280 and does not stimulate GH release. Ghrelin is a 28-residue peptide. Its Ser3 side chain is modified by an octanoyl ester group, which is essential for GH releasing activity at GHS-R1a.277-279,281 Ghrelin is mainly produced in the stomach, where it was first discovered, but its expression has been identified in many other organs such as duodenum, colon, pancreas, hypothalamus, pituitary gland, kidney, lung, and placenta. Similar to synthetic GHS analogues, ghrelin stimulates strong and dose-dependent release of growth hormone.277-279,281 Ghrelin is a strong appetite stimulant, its administration resulting in increased food intake and fat mass in mice. However, ghrelin or GHSR knockout mice developed normally with size, growth rate, food intake, and body composition similar to those of wildtype mice.277-279,281 Apart from important roles in the regulation of food intake and energy homeostasis, ghrelin/ GHSR is also implicated in insulin secretion, gastric acid secretion, stimulation of gastric motility, and protective functions in cardiovascular systems.277-279,281 Small molecule GHSR agonists are proposed to have therapeutic potential in treating GH deficiency diseases, to improve appetite in people suffering eating disorders, and to restore a youthful physiological GH profile in aging people.277-281 Recently, it has emerged that blocking the effects of ghrelin by antighrelin immunoglobulin C, anti-sense GHS-R mRNA, or peptidic GHS-R antagonists led to reduced body weight and decreased food intake.282 Therefore, antagonists of GHS-R might be potential agents for treatment of obesity.282,283

2.14.1. Agonists of GHS receptors The original hexapeptide GHSs, such as GHRP-6 (Hiswere potent agonists of GHS-R (EC50 10 nM), but exhibited low in ViVo stability and poor oral bioavailability. Drug development efforts to improve on the characteristics of these hexapeptides led to the discovery of many truncated and heavily modified small peptides, peptidomimetics, and small molecules having agonistic activity.280,284,285 Smith and colleagues from Merck pioneered the search for small molecule GHS-R agonists.280,284 SAR information on GHRP-6 showed that a basic amine in position 1 and aromatic residues at positions 2, 4, and 5 were preferred for potency. This knowledge guided selection of nonpeptide compounds, containing appropriate pharmacophores, from the corporate library for screening in functional assays monitoring GH release.280,284 A benzolactam hit (EC50 3 µM) was identified and readily optimized to furnish 276 (Chart 61) as the first potent, peptidomimetic agonist of GHS-R (EC50 60 nM). Further investigations revealed that derivatizing the free amino group could be beneficial for potency. It was also found that the tetrazole group, which leads to zwitterion formation, could be replaced by an amide moiety. Incorporating such modifications, D-Trp-Ala-Trp-D-Phe-Lys-NH2),284

compound 277 exhibited 20-fold increased potency (EC50 3 nM) and improved oral bioavailability compared to 276.280,284 The same group also screened compounds having the proposed ‘privileged structure’ spiroindanylpiperidine ring system. Compound 278, comprising four diastereomers, was identified as a lead (EC50 50 nM), although it lacked oral bioavailability. Substitution of the ureidoquinuclidine by aminoisobutyric acid (Aib) and utilizing D-tryptophan resulted in improvement of both potency and bioavailability (279, EC50 14 nM) (Chart 62). Further optimization led to MK-677 (280) and a close analogue (281, L-163,255), both of which were used extensively in animal models. This series of GHS-R agonists incorporated a sulfonamide group in the spiroindanylpiperidine privileged structure, while the Dtryptophan was replaced by O-benzyl-D-serine (280, MK0677) or D-phenethyl-alanine (281, L-163,255). Compound 280 displayed high agonist potency (EC50 1.3 nM), stimulated GH release at 0.125 mg/(kg/po) in dogs, had excellent oral bioavailability (60%), and entered clinical trials. Oral administration of 280 stimulated GH secretion dosedependently with a threshold dose of 5 mg, and daily treatment of elderly hip fracture patients with 280 led to 84% increase of IGF-I serum level. In healthy obese males, 280 caused increase in lean mass, suggesting potential use of GHS-R agonists for treating cachexia patients.280,284 Agonist 281 exhibited comparable potency (EC50 1.5 nM), stimulated thymic functions in old mice, and acclerated recovery of dogs from limb immobilization.280,284 Other potent GHS-R agonists were obtained by varying 278-281; examples included ligands 282, 283, 284, 285, and 286 having EC50 6.3, 1.9, 0.7, 3.0, and 1.6 nM, respectively.280,284,285 Caprino et al. applied a pharmacokinetic-oriented screening strategy to identify derivatives of agonists 280 and 286 that possessed good intestinal absorption in rodents.286,287 A pyrazolinone-piperidine ring system was found to be a suitable substitution for the spiroindanylpiperidine moiety. Capromorelin 287 (Chart 63) and its analogue 288 displayed Ki 7 and 5 nM for displacement of [125I]-ghrelin and induced GH release in pituitary cells with EC50 3 nM; and ED50 values in rats were 0.05 and 0.1 mg/kg, respectively.286,287 Both ligands had excellent oral bioavailability (44% and 77%), with t1/2 of 1.3 and 3.7 h in dogs, respectively. Both compounds stimulated GH release in dogs after oral administration and were selected as candidates for treatment of frailty. Other examples of effective isosteres of spiroindanylpiperidine included substituted imidazoles and 2,3-dihydroisothiazole.285,288,289 LY444711 (289)285,288 (Chart 64) was a potent GHS-R1a agonist, displacing radiolabeled ghrelin (Ki 10.6 nM) and stimulating Ca2+ mobilization (EC50 1.2 nM), with oral bioavailability (10% rat, 37% beagle dog), and when administrated chronically, it stimulated food intake and adiposity in rats. SAR studies of serine-Aib dipeptide and


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Chart 62

Chart 63

dihydroisothiazole conjugates289 showed that 2,6-dihalogen substitution of the benzyl ring, which was assumed to mimic the octanoyl group of ghrelin, was favorable for agonist potency (290, EC50 1.1 nM). The length of the linker between the phenyl ring and the R-carbon of serine was found to be already optimal, but replacing the oxygen atom with methylene afforded modest improvement (291, EC50 0.1 nM). Most modifications of the Aib portion were not welltolerated, with the exception of hydroxylation when several fold increase in potency was achieved (292, EC50 0.7 nM).289 Starting from the pentapeptide ipamorelin, Novo Nordisk developed a highly N-methylated tripeptide derivative (293, NN703) (Chart 65) with good in ViVo potency and oral bioavailability.290 Compound 293 stimulated GH release from Chart 64

rat pituitary cell with EC50 of 2.7 nM; however, in a binding assay using COS-7 cell membranes and [35S]MK-0677, it displayed only moderate affinity (Ki 50 nM). In beagle dogs, 293 had oral bioavailability of 30-35%, and a dosedependent GH release was observed after oral administration. In accord with the physiological effects of ghrelin discussed above, an oral dose of 100 µmol/kg of 293 resulted in significant increase in body weight compared to saline treatment in rats. In a clinical trial, 293 was well-tolerated up to 6 mg/kg in healthy males and induced robust and dosedependent GH release.291 Modifications of 293, such as truncating the C-terminal amide group, introducing hydrazinyl or sulfonamido groups, and varying aromatic substitutions furnished a number of highly potent GHS-R agonists.285,292,293 For example, compounds 294-296 were either equipotent to, or better than, 293 and had oral bioavailability in beagle dogs of 20-40%.285,292,293 2-Oxoindolines are another class of GHS-R agonists.294-296 Screening of an in-house chemical library uncovered a structurally novel indolin-2-one hit, with initial modifications leading to 297 (EC50 380 nM) (Chart 66). Extensive SAR studies improved the activity and clogP (5.12) of compound 297. The combination of a small 4-CF3 and hydrophilic 6-carboxamide substitutents on the indole ring was found to be beneficial for potency. The bulky naphthyl ring could also be replaced with smaller 2-chloro-phenyl, resulting in the


discovery of SM-130686 (298, clogP 3.83). This compound was able to displace [35S]-280 (IC50 1.2 nM) and induced GH release (EC50 3.0 nM) from rat pituitary cells. However, the maximal GH releasing response of 298 was about half of ghrelin, suggesting that 298 was a partial agonist at GHSR1a. In rats, the oral bioavailability of 298 was 28%, and its repetitive administration led to significant increases in lean body mass. SAR studies of the 2-chloro-phenyl ring detailed the effects of substitutions on potency.296 The optimal 2,4-dichloro-phenyl derivative 299 was reported to Chart 66


be 10-fold more active (IC50 0.02 nM) than ghrelin, though it remained a partial agonist in a functional assay (EC50 17 nM). Several other series of GHS-R agonists were identified from high-throughput screening and computational approaches.285,297,298 When a small database of peptidic and nonpeptidic growth hormone secretagogues was used, a 3D pharmacophore model consisting of two aromatic rings, a protonated amino group, and a proton acceptor was developed, and subsequently used to screen for potential GHS-R agonists from 3D databases.285,299 Optimization of hits was facilitated by a site-dependent fragment QSAR procedure to furnish low nanomolar ligands, exemplified by benzothiazepinone 300 (S-37435, EC50 1 nM) (Chart 67). In another report, researchers at Bristol-Myers Squibb described optimization of a HTS lead (301: EC50 11.7 nM) using a similar pharmacophore model of a hydrophobic region (benzyl), a basic amine, and a hydrogen bond acceptor (carbamate group).285,297 The tert-butyl carbamate (Boc) and the bisdiisopropylamino groups were optimal, while replacing the benzyl substituent with a shorter phenyl group provided a 20-fold more potent agonist 302 (EC50 0.46 nM). The single enantiomer 302 (undetermined stereochemistry) was shown to increase plasma GH levels by 10-fold in anesthetized rats.

2.14.2. Antagonists of GHS Receptors For GHS receptors, relatively few nonpeptidic antagonists have been reported in the literature.282,283,300-302 Benzoazepinone derivative 303 (Chart 68) was the first reported nonpeptidic antagonist, but its activity or SAR data was not disclosed. Abbott researchers reported development of isoxazole and tetralin carboxamide derivatives as new classes of GHS-R antagonists, for use in exploring the potential for treating obesity. Isoxazole lead 304 was found in a highthroughput screening program. Optimization studies revealed that modifications of the aniline ring or the diethyl substituents of the amino group reduced potency. The 2,6-dichloro substitutions on the 3-phenyl ring also proved optimal. However, introducing bulky, hydrophobic groups into the 5-methyl moiety seemed to improve the potency significantly (304, IC50 180 nM; 305, 8 nM). To increase solubility of 305 for icv administration, the aniline was replaced with trans 1,4-diaminocyclohexane, which slightly decreased antagonist potency (306, IC50 54 nM).283,300,301 On the basis of the requirement for 2,6-dichloro substitution for potency, it was


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Chart 68

Chart 69

proposed that the dihedral angle between the phenyl ring and the amide group was a determinant, and that the isoxazole ring served to project these two groups in appropriate positions. This assumption led to development of new antagonists containing the tetralin carboxamide core,283,302 such as 307 (IC50 1.4 µM and ghrelin-induced Ca2+ mobilization). Introducing a substituent on the R-carbon of the amide carbonyl increased activity and stability of tetralin carboxamide congeners (308, IC50 27 nM; 309, 33 nM),282,283,302 with reasonable oral bioavailability (18% and 21%, respectively, in rats). In contrast to the isoxazole series, changing the aniline ring to trans 1,4-diaminocyclohexane to improve aqueous solubility had a significant negative effect on the potency.

2.15. Melanin-Concentrating Hormone Receptors Melanin concentrating hormone (MCH) is a 19-residue peptide cyclo-(7,16)-[DFDMLRCMLGRVYRPCWQV] containing a disulfide bridged cyclic decapeptide with a twoturn helix at the N-terminus and a cyclic loop.303 Two MCH specific GPCRs are known (MCHR1 and 2). MCH is found in the brain, is expressed in the hypothalamus, pituitary, adrenal, and CNS, and is an important regulator of body weight and energy balance.304 Injected MCH causes rodents to eat more and significantly increase their body weight.305 MCH gene overexpression makes mice susceptible to insulin resistance and obesity,306 while MCH and MCHR1 gene knockout mice are leaner and have elevated metabolic rates.307,308 The physiological importance of MCHR2 is not yet clear; thus, most interest in MCH has focused on its appetite-stimulating properties and the potential of MCHR1

antagonists for treating obesity, although this receptor is also thought to be involved in memory, depression and anxiety.309-311 The first reported MCHR1 antagonists were aminotetraline 310 (T-226296)312 and 311 (SNAP-7941)313 (Chart 69) which reduced food intake after MCH injection to rats, the latter being reported to have antidepressant, anxiolytic and anorectic effects in rats. Researchers at Abbott (312),314 (Chart 70) Schering-Plough (313)315 and 7TM Pharma (314)316 have reported substituted benzamides (e.g., 312-314) as MCHR1 antagonists with some evidence for efficacy after oral dosing to feeding mice. A similar urea-based MCHR1 antagonist 315 (IC50 2.2 nM (MCHr1), 101 nM (Ca2+ release))317 (Chart 71) was reported to have been developed from Abbott’s antagonist, N-2pyrrolidinylethyl indazole 316 (IC50 5.9 nM (MCHR1), 393 nM (Ca2+ release)),318 through in silico virtual screening using an electrostatic similarity matching algorithm. Abbott researchers discovered the indole-based antagonist 317 (Chart 72), a 6-substituted 2-pyridinylethylindole, from high-throughput screening against MCHR1 obtained from human neuronal IMR-32 cells. This moderately potent antagonist (IC50 0.3 µM (MCHR1), IC50 1.8 µM (Ca2+ release)) showed significant levels in plasma (AUC 1.16 µg/ (h mL)) and brain (AUC 0.67 µg/(h g)) after oral administration at 10 mg/kg to DIO mice. Optimization led to higher affinity ligands with enhanced antagonist potency against MCH-induced Ca2+ release, good plasma and CNS penetration after oral delivery, and efficacy in blocking weight gain in rodents. Notable compounds included indole 318 (IC50 22 nM (MCHR1), IC50 470 nM (Ca2+ release); AUC 1.37



Chart 70

Chart 71

µg/(h mL) (plasma), 0.35 µg/(h g) (brain) after 10 mg/(kg/ po)), benzimidazole 319 (IC50 161 nM (MCHR1), IC50 > 10 µM (Ca2+ release); AUC 0.94 µg/(h g) (plasma), 0.24 µg.hr/g (brain) after 10 mg/kg/po), indazoles 320 (IC50 1.4 nM (MCHR1), IC50 0.011 µM (Ca2+ release); AUC 2.12 µg/ (h g) (plasma), 1.33 µg/(h g) (brain) after 10 mg/(kg/po)), and 321 (IC50 41.4 nM (MCHR1), IC50 0.287 µM (Ca2+ release); AUC 2.09 µg/(h g) (plasma), 0.214 µg/(h g) (brain) after 10 mg/(kg/po)). In particular, compound 320, with a brain concentration 12 h after 10 mg/(kg/po) of 29 ng/g, was receptor-selective for MCHR1 over MCHR2, somatostatin, and opioid receptors, and at 10 or 30 mg/(kg/b.i.d.) dosedependently decreased body weight of DIO mice without appetite suppression over a 2 week observation period.314 A large number of 2- and 4-aminoquinolines have been reported by various research groups as potent MCHR1 antagonists. Devita et al.319,320 reported 2-aminoquinolines like 322 (Chart 73) and 4-aminoquinolines like 323. Clarke et al.321,322 reported 2-amino-6-acylamino-quinolines as potent MCHR1 antagonists; for example, the virtual screening lead Chart 72

324 (IC50 55 nM (MCHR1), MW 425, cLogP 4.47, PSA 64.5 Å2), from which a number of optimized analogues like 325 (whole cell binding : IC50 5.9 nM (MCHR1), 4.5 µM (MCHR2), solubility 80 µM)309 have been derived. Abbott researchers323 reported 2-amino-8-alkoxy quinolines as MCHR1 antagonists, starting from screening lead A-224940 (326, IC50 91 nM (MCHR1); 1.7 µM (Ca2+ release)) and conversion to 327 (R ) CN, Cl, OCF3; IC50 0.4-6.0 nM (MCHR1); 3-24 nM (Ca2+ release); AUC 328 (R ) CN) 424 (R ) OCF3) ng/(h g) (plasma), 11 (R ) CN) 68 (R ) OCF3) ng/(h g) (brain) after 10 mg/(kg/po) in DIO mice) and 328 (IC50 0.9 nM (MCHR1); 8 nM (Ca2+ release)). Amino piperidine coumarin antagonists of MCHR1 have also been reported by Abbott researchers.324 Optimization of a screening hit resulted in compounds like 329 (IC50 4 nM (MCHR1) (Chart 74), IC50 44 nM (Ca2+ release); AUC 0.98 µg/(h g) (plasma), 0.43 µg/(h g) (brain) after 10 mg/ (kg/po)), and 330 (IC50 2 nM (MCHR1), IC50 28 nM (Ca2+ release); AUC 6.4 µg/(h g) (plasma), 4.2 µg/(h g) (brain) after 10 mg/(kg/po)) with some cross-reactivity at micromolar concentrations for nonselective adrenergic/opiate/dopamine receptors. Compound 330 was an effective inhibitor of weight gain when given at 30 mg/(kg/b.i.d.) to DIO mice over a 24 day period, but failed to show a promising therapeutic index in beagle dogs, due to insufficiently high drug levels in the brain and cardiovascular side effects. Substituted quinazolines have been found to be potent and selective antagonists of MCHR1.325 Compound screening led to identification of 331 (A129330, IC50 140 nM (MCHR1), 2.7 nM (Y5), 7.7 nM (R2A)) (Chart 75) as an MCHR1 antagonist, which was subsequently optimized for potency and receptor selectivity by altering the spacer between quinazoline and sulfonamide units, changing the trans-1,4-


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Chart 73

Chart 74

biaryldiamides like 335.328 This group also develeped benzylpiperidines from high-throughput screening (e.g., 336) into 400-fold more potent and receptor-selective MCHR1 antagonists like 337 (Ki 5 nM).329

2.16. Melanocortin Receptors

Chart 75

cyclohexane to cis, followed by removing the sulfonamide and changing substituents on the benzamide ring. Among derivatives was [N-[cis-4-cyclohexyl)-3,4-difluorobenzamide hydrochloride], 332 (ATC0175, IC50 3.4 nM (MCHR1), 2700 nM (Y5), 260 nM (R2A)), which has been reported to have anxiolytic and antidepressant effects in rats at 1-10 mg/ (kg/po) in swimming and maze tests.326 Among many compounds developed as MCHR1 antagonists by Schering-Plough is a large series of biaryl ureas (e.g., 333, IC50 8.9 nM, MCHR1) (Chart 76) and biaryl diamides (e.g., 334, IC50 3.7 nM, MCHR1), the former containing a mutagenic biarylaniline327 removed through development of

The melanocortin peptide hormones, R-, β-, and γ-melanocyte-stimulating hormones (R-, β-, γ-MSH) and adrenocorticotropin hormone (ACTH) have a diverse range of biological functions. They are produced in tissues specifically from pro-opiomelanocortin prohormone (POMC), thus, providing a measure of control over functional activity which is mediated through five GPCRs (MC1R to MC5R).330 Beside endogenous agonists, there also exist MCR endogenous antagonists, agouti (AGP) and agouti-related protein (AGRP). All natural agonist hormones contain a conserved sequence of His-Phe-Arg-Trp (HFRW), which is essential for their agonist activities, and this motif provided a focus for the early drug discovery toward ligands for melanocortin receptors.331 Understanding of the physiological roles of MC receptors has progressed tremendously in the past decade, due to advances in gene targeting and cloning technologies as well as the availability of new endogenous peptide agonists, antagonists, and their derivatives as physiological probes. MC1R plays a pivotal role in pigmentation, its variation being responsible for different shades of hair and skin color in humans, and it has important properties in the inflammatory response. On the other hand, MC2R in the adrenal cortex and adipose tissues responds to ATCH by regulating production of corticoids. MC3R and MC4R are expressed centrally and are keys to the complex regulation of energy homeostasis. Recently, there has been a great deal of drug discovery for MC4R, with >15 published papers in 2005 alone reporting on development of small molecule ligands for this receptor. MC4R agonists are believed to have therapeutic potential as anti-obesity and anti-diabetic agents and possibly for treating sexual dysfunction, while MC4R antagonists could potentially be used to manage cachexia and anorexia.332 The role of MC5R is less clear at present, but its pattern of expression suggests involvement in exocrine gland function.



Chart 76

Chart 77

2.16.1. MC4R Agonists Structure-activity relationship studies of the melanocortin hormones and their truncated derivatives led to the hypothesis that the important ‘message’ sequence (HFRW) adopts a β-turn active conformation.331 Using a thioether cyclized scaffold, Bondebjerg and colleagues constructed libraries of compounds to mimic the side chains of the FRW motif in the β-turn, producing low-affinity agonists like 337 (EC50 8.7 µM, MC4R) (Chart 77) without MCR selectivity.333 A series of ureas was also prepared to mimic the FWK sequence; the best agonists are exemplified by 338 (EC50 400 nM, MC1R, MC4R)331 but ranged in potency from high nanomolar to low micromolar for MC1R and MC3RMC5R. A tetrahydropyran scaffold that projected side chains of D-Phe-Arg-Trp led to compounds with low micromolar receptor affinities but with only partial or no agonist activities.334 Amgen researchers used the NMR-derived solution structure335 of the highly active Ac-Nle-cyclo[AspPro-D-Phe-Arg-Trp-Lys]-NH2 to create MC4R agonists with various ring systems decorated by amino acid side-chain mimetics (e.g., 339, IC50 7.7 nM, EC50 4 nM, MC4R). Truncation revealed that all three side chain mimetics were important for receptor binding. In a landmark publication, Merck researchers disclosed compound 340 (Chart 78) as the first example of a small molecule, highly potent, and selective agonist for MC4R.336 Recognizing that pharmacophores of peptidic MC agonists and growth hormone secretagogue agonists were similar, directed-screening was used to identify MC4R hits from inhouse libraries. A spiroindanyl piperidine moiety, proposed to mimic the Ala-Trp dipeptide, is present in 341 (IC50 108 nM, MC4R) but little or no agonist activity was observed. Substitution of the large 2-naphthylalanine by phenylalanine gave a full agonist 342 (EC50 500 nM) and further optimiza-

tion led to the more potent MC4R agonist 340 (IC50 1.2 nM, radioligand binding; EC50 2.1 nM, CHO cells), which had several hundred fold selectivity over other MC receptors. I.v. administration of 340 to rats significantly reduced food intake and increased erectogenic activity by up to 60%. Other groups have successfully screened focused libraries of compounds containing GPCR privileged structures to find MC4R ligands. The dipeptide (R)-Tic-(R)-(4-Cl)Phe motif of compound 340 appears repeatedly in numerous hits, and remains within many potent agonists. It is proposed that the dipeptide is the ‘address element’ which is linked to the hydrophobic GPCR-privileged structure at the C-terminus.337 Modification of the latter had the greatest impact on potency and selectivity of agonists. Table 4 Chart 78


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Table 4. Structure-Activity Relationships of MC4R Agonists 343-350

summarizes activities and selectivities of key compounds from SAR studies involving the ‘message’ C-terminal motif. It is evident that substituted piperazines are especially favored as the amine for amidation with the Tic-(4-Cl)Phe dipeptide. The 4-amino group of the piperazine ring is usually appended with a hydrophobic aryl ring that is

further decorated with ortho substituents. An additional basic group or an N-azole ring seems to be necessary for high potency. The activities of agonists 343-350 are in the low nanomolar range; however, the first reported example 340 is still the most potent and most selective MCR4 agonist reported.


The (R) configuration of the 4-chloro-phenylalanine is essential for tight binding and potent activation, since IC50 for (S) isomers increased several hundred fold compared to 340, and even at 10 µM, the cell stimulation was only partial.336 On the other hand, the requirement for chirality of the tetrahydroisquinone (Tic) is not as stringent. The (S)Tic containing diastereomer (351) of 340 (Chart 79) had comparable activity (IC50 0.5 nM, EC50 8.7 nM). Amgen identified the related 352 as a potent MC4R agonist, with excellent selectivity over MC3R but only 21-fold less active at MC5R.342 Optimization of the sulfonamido moiety revealed that large benzyl and phenyl sulfonic acids slightly reduced binding. However, small N-substituents gave potent agonists (EC50 0.1 nM) like 353. Interestingly, propyl, cyclopropylmethyl, or aminoethyl did not greatly alter potency (EC50 0.44 nM (354), 0.58 nM (355), 0.1 nM (353)); selectivity was several thousand fold over MC3R and greater than 79 fold over MC5R. Compounds containing Tic amino acids, while potent MC4R agonists, suffered from low bioavailability (70).345 Incorporating isoquinuclidine preserved MC4R agonist potency and selectivity, despite lacking the R-amino group, and improved pharmacokinetic properties (RY764, 360, EC50 11 nM, selectivity index >75, and oral bioavailiability F 32%, rats; 50%, dogs; t1/2 3.2 h, rats; 4.9 h, dogs).346 In ViVo studies of selected MC4 agonists strongly support the therapeutic potential of such ligands in treatment of obesity and sexual dysfunction. When administrated orally, 354, 356, 358 and 360 were efficacious in reducing food intake and/or body weight.342-344,346 Furthermore, iv administration of 356 or 358 significantly increased erectile responses in rats over vehicle alone.343,344 The hydrophobicity and basicity of tetrahydroisoquinoline are not required for tight binding to MC4R. In 2003, Ruel and colleagues demonstrated that β-alanine and its analogues could replace Tic in agonist 340.347 Small alkyl groups, or even acetylation of the amine, had little influence on binding affinity for MC4R (e.g., 361-363, IC50 10-16 nM), and amines 361 (EC50 170 nM) and 362 (EC50 40 nM) (Chart 81) were still full agonists. Tic was also replaced by arylsubstituted alanines, quinoline, isoquinoline, naphthalene, azetidine, and piperidine in agonists 347 and 355,342,348 with nanomolar affinities for MC4R but reduced agonist potencies compared with Tic analogues (e.g., 364, Ki 4.8 nM, 17% agonism of R-MSH at 10 µM) such as the full agonist 347.348 Compound 365 (IC50 67 nM; EC50 1.8 nM) was 2-4-fold less active than 355.342 There was no information about the effect of Tic replacements on MC receptor selectivity. Alternatively, replacing the amide backbone of the dipeptide in agonist 366 (IC50 130 nM; EC50 < 0.1 nM) with a succinamide349 impacted on functional activity, with many compounds being potent antagonists. Compound 367 was the most active agonist reported (IC50 5.8 nM, EC50 15 nM) for this series. Ala-scanning, mutagenesis and computational modeling350 were used to probe the binding of 340 and 358 to MC4R receptor, the amine groups being thought to make electro-


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Chart 81

Chart 82

static contacts with acidic residues in TM2 and TM3 helices; the substituted piperidine mimicking Trp9 of melanocortin and making contacts with TM5 and TM6, while D-4fluoroPhe interacts with Leu133, Trp258, and Phe261. It was proposed that the important agonist components (positive charge, aromatic, and cyclohexane rings) reside in the same area as for corresponding groups of AGRP and NDP-MSH. Pyridazinone and dihydropyridazinone have been used as scaffolds in MC4R agonists.351,352 Compound 368 (Chart 82) is a partial agonist (IC50 144 nM) without the basic group present in other MC4R agonists, and the aromatic ring of the benzoazepinone region is important for binding. SAR studies established that replacing the benzoazepinone and related 4,5-dihydropyridazinone with piperidine- and pyrrolidine-caboxylic acids, especially when an R-methyl on the amine was in the (R) configuration, enhanced agonist affinity and potency. Compound 369 is such a pyridazone with trans stereochemistry of the piperidine ring (IC50 33 nM; partial

agonist EC50 177 nM).351 Further optimization gave a full MCR4 agonist 370 (IC50 18 nM, EC50 66 nM).352 High-throughput screening of large corporate compound collections and combinatorial libraries has successfully generated hits for MC4R. Compound 371 (Chart 83) , with an indole privileged structure for GPCRs, was identified from a large combinatorial library (IC50 612 nM, MC4R).353 Thiadiazolium salt 372 (IC50 174 nM) served as starting hit for an optimization program to potent agonist 373 (IC50 22 nM).354 Administration ip of 373 to rats caused significant reduction in food intake.

2.16.2. MC4R Antagonists The first MC4R antagonist was published in 2003 by Amgen researchers.335 Piperazine 374 (Chart 84) was identified from high-throughput screening (IC50 249 nM, MC4R); a small methyl N-substitution instead of phenyl of one piperazine ring increased affinity (375, IC50 of 52 nM



Chart 83

Chart 84

(AGRP) 217 nM (R-MSH); MC4R) and antagonist potency against R-MSH. The structurally related MCL0129 (376)355 is also a high-affinity (Ki 7.9 nM, radiolabeled ligand) and potent antagonist of MC4R (dose-dependently attenuating cAMP stimulation by R-MSH) with no appreciable affinity for MC1R and MC3R at 1 µM. Oral administration of 376 significantly improved stress-induced depression and anxiety in rats.355 Benzamidines are a different class of MC4R antagonists discovered by high-throughput screening at Millennium.356 A hit 377 (Ki 2.7 µM) (Chart 85) was modified by using a 5-bromo-2-methoxybenzyl group as a 2-substitutent on the benzamidine, and a methylene instead of the oxidizable thioether, to afford 378 with improved stability, MCR4 Chart 85

Chart 86

binding (Ki 160 nM), antagonism (IC50 103 nM), brain permeability, and the property of protecting mice from tumorinduced weight loss when administered sc. To increase oral bioavailability, the cyclic amidine was converted to the proposed imidazole metabolite and alkylated imidazoles357 to furnish compounds like 379 (Ki 180 nM, IC50 51 nM) that were 1-2 orders of magnitude more selective for MC4R than MC1R, MC3R, and MC5R. Just as modifications around Tic of MC4R agonists altered function, a derivative of the full agonist 347, namely 364, was an MC4R antagonist (IC50 300 nM), dose-dependently inhibiting R-MSH induced cAMP production.348 The pyridazinone derivative 380 (Chart 86), which differed from agonist 369 by addition of two chloro substituents on the phenyl ring in the piperidine region,352 also antagonized the activity of R-MSH (KB 3.1 nM) and had high affinity in a radioligand binding assay (IC50 3.2 nM). Likewise, variation of the Tic mimetic in agonists 366 and 367 altered function,349 replacing the dipeptide backbone with succinamide to give a ‘reverse amide’ increased affinity for MC4R; alkylation, amidation, or carbamylation of N4 atom of piperazine reduced agonist activity; with 381 (IC50 1.4 nM) that did not stimulate cAMP even at 10 µM, being typical of such compounds. Subtlely changing potent agonist 367 by replacing cyclobutylamine with N-Boc-piperazine (382) resulted in complete loss of agonism while affinity improved slightly (IC50 2 nM). Researchers from Neurocrine Biosciences used moderately active agonist 348 to investigate optimization of Tic substitutions.358 Small amino acids such as β-alanine (383) (Chart 87) and piperidine-3-carboxylic acid (384) increased binding affinity, but decreased functional response at 10 µM,


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Chart 87

Chart 88

exemplified by 386 (Ki 4.2 nM, MCR4) (Chart 88) and 387 (Ki 6.3 nM, MCR4).362,363 In functional assays, 387 competitively inhibited R-MSH-stimulated cAMP production (KB 47 nM). Alternatively, a set of small alkyls and alkoxyalkyls was investigated as potential surrogates for the flexible thienylethyl side chain of compound 385.360 2-Methoxypropyl was optimal and used in another series of compounds intended for optimizing the β-alanine. Small amide, carbamate, and urea groups were well-tolerated, and the basic β-amine was not required for potency.360,361 Similar findings were made for 385. The most promising compounds were 388-390, which had high affinity (Ki 6.9, 8.8, and 3.2 nM, respectively, MC4R). Compounds 386-390 all offered excellent selectivity against other MC receptors. Oral bioavailability of 387 (12%, rats) and 388 (8%, rats) also improved significantly compared to 385;360,362 their brain distribution was also favorable in line with logD of 1.8. In animal models, 387 po and 390 icv increased food intake in mice,361,362 suggesting that an MC4R antagonist might be a treatment for involuntary weight loss.

2.16.3. Other MCR ligands displaying dose-dependent inhibition of cAMP production stimulated by R-MSH (IC50 90 and 36 nM, respectively). Derivative 383 was a template in further work aimed at developing MC4R antagonists. Introducing an additional chloro substitutent at position 2 of D-4-chloro-phenylalanine increased receptor affinity 10-fold.359 Ligand 385 bound tightly to MC4R (Ki 1.8 nM) with >100-fold selectivity over MC1R, MC3R, and MC5R. It did not activate cAMP production at 10 µM, was a competitive antagonist by Schild analysis (pA2 7.9), was not orally bioavailable (F 1%), but did penetrate the blood-brain barrier when given iv despite its high hydrophilicity (logD 1.1, pH7.4). The key pharmacophoric groups in 385 (D-2,4-dichloroPhe aromatic ring, arylpiperazine, β-alanine) were extensively modified360-363 to show that 2,4-dichloro-Ph was the optimal aromatic ring.361 The arylpiperazine could be varied a great deal. As for agonists, replacing the phenyl ring with substituted cyclohexyl maintained tight receptor binding. The N or O containing substituents were in position 1 or 2 as Chart 89

With the knowledge that the bioactive conformation of melanocortins is a β-turn, a cyclic thioether β-turn mimetic was employed as a scaffold to appropriately project pharmacophore groups, generating libraries of putative MCR ligands.331,333 Several MC1R agonists were identified (337, 6.8 µM; 391, 2.7 µM; 392, 4.5 µM) (Chart 89) without much selectivity. Another library of urea compounds mimicking Phe-Trp-Lys gave nM MCR1 agonist (338), but no selectivity was observed over MC4R.364 The first potent and selective MC1R agonist 393 (IC50 120 nM, EC50 28 nM, MC1R; 900fold selectivity over MC3R, MC4R, MC5R)365 was developed through screening and extensive parallel synthesis. Interestingly, the amino group of His could be removed, acylated, or replaced by hydroxyl without significant effect on binding, but the chirality was essential for full activity and high potency. In ViVo 393 administered sc to mice (33 µmol/kg) significantly (82%) reduced TNF-R production stimulated by iv LPS. Recently, trans proline was reported to be a viable core for displaying the pharmacophore groups of melanocortin.


Compound 394 (Chart 90) was a potent MC3R ligand (Ki 66 nM), though it had comparable affinity for MC4R (Ki 109 nM).366 The phenylguanidine derivative 395 was a potent MC5R antagonist (Ki 2.1 nM), with over 400-fold selectivity over MC1R, MC3R, and MC4R,367 and it potently inhibited R-MSH stimulated cAMP production (IC50 72 nM).

2.17. Neurokinin Receptors There are over 40 tachykinin peptides known to bind to at least 3 neurokinin receptor subtypes (NK1, NK2, and NK3). The major endogenous ligands for the NK1 and NK2 receptors are substance P (SP) and neurokinin A (NKA), respectively, both peptides originating from the same gene (TAC1). Substance P and NKA are potent vasodilators and contractors of smooth muscle, cause mucus secretion, and increase vascular permeability. The TAC1 gene encodes two other tachykinins, neuropeptide K (NPK) and neuropeptide gamma (NPγ) that contain the same C-terminal amino acid sequence as NKA, but also additional N-terminal residues. Both NPA and NPγ elicit their effects by binding specifically to the NK2 receptor. The endogenous ligand for NK3 receptor is neurokinin B (NKB), which is expressed in the CNS and placenta. Tachykinins are ubiquitously expressed throughout the body, but sensory nerve fibers are typically the primary source of tachykinins, although several types of immune cells may synthesize and release substance P. The pursuit of nonpeptidic antagonists of the NK1 receptor proved elusive for more than half a century after the discovery of the endogenous peptidic ligand, substance P. A search for a nonpeptidic antagonist was undertaken by researchers at Pfizer using random screening of compound libraries for substances that could displace [3H]SP in bovine Chart 91


caudate membranes. The outcome was the discovery of a lead compound, 2-diphenylmethyl-3-benzylamino quinuclidine, that showed activity of 16 nM. A limited structure activity study of only 15 compounds lead to the orthomethoxybenzylamino derivative with enhanced potency. Subsequent resolution of the racemic mixture afforded CP 96345 396 (Chart 91) where the (2S, 3S)-enantiomer was shown to be responsible for the activity of 4 nM. The (2R, 3R)-enantiomer was inactive at the NK1 receptor.368 CP 96345 396 displayed complete specificity for the NK1 receptor over the other tachykinin receptors in functional assays and was devoid of any agonist properties. Interestingly, 396 displayed a species-dependent activity and bound to rat and mouse NK1 receptors approximately 100 times less potently than the human receptor and all other species studied. The presence of nonspecific effects on a number of ion channels were observed for 396 at high doses (e.g., L-type calcium channels IC50 27 nM) and prompted the search for more active and specific compounds. Further development at Pfizer showed that the diphenylmethyl group could be replaced by a single phenyl ring and the quinuclidine ring could be replaced by a piperidine. The resulting compound CP 99994 397 was equipotent with 396 at the NK1 receptor but displayed at least 100 times reduced binding to L-type calcium channels compared to 396. Again the (2S, 3S)-stereoisomer was responsible for all activity.369 Despite promising results in preclinical models of nociception and inflammation, 397 in clinical trials for analgesia following dental surgery was found to be less effective than the common over-the-counter NSAID, ibuprofen.370 Another quinuclidine analogue with an additional isopropyl group 398 (CJ11974, IC50 5 nM) was also evaluated in clinical trials for chemotherapy-induced emesis.371 Researchers at Glaxo also prepared analogues of the leading Pfizer compounds, the most promising of which were a series containing a tetrazole substituent exemplified by 399 (GR 205171, Ki 0.4 nM).372 Potent binding and selectivity for the NK1 receptor was accompanied by a 1000 times reduction in nonspecific binding to calcium channels. With good oral bioavailability, 399 was evaluated in clinical trials and found effective for reducing the rate of postoperative nausea but ineffective for motion-induced nausea. Researchers at Merck pursued a development program based on Pfizer’s lead compounds in an effort to further minimize nonspecific binding to calcium channels and to improve bioavailability. A significant discovery was the 3,5bis-trifluoromethylbenzyl ether as a replacement for the benzylamino substituent in 397 resulting in compound 400 (L 733060, IC50 1 nM) (Chart 92) . Common to other potent NK1 antagonists 400 was effective in animal models of neurogenic plasma protein extravasation and nociception and was the first compound used to demonstrate that NK1 antagonists possess antidepressant activity. A milestone was


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Chart 92

Chart 93

achieved with the development of the more advanced analogue 401 (L 754030, Aprepitant IC50 0.1 nM), that displayed up to 10 times greater efficacy than the earlier clinical candidate 399 due to enhanced bioavailability and duration of action. Compond 401 has demonstrated antidepressant and anxiolytic activity and is highly effective at preventing nausea associated with chemotherapy for which it has gained approval in the U.S.A. Another quinuclidine compound, 402 (SR140333, Ki 0.02 nM), displaying potent NK-1 antagonism and greater than 1000-fold selectivity over NK2 and NK3 receptors, was reported by Sanofi.373 In ViVo activity was also demonstrated for the [Sar9-Met(O2)11]substance P induced hypotension in dogs, bronchoconstriction in guinea pigs, and plasma extravasation in rats (ED50 3-42 µg/kg iv). Almost a decade later, the improved compound 403 (SSR240600, Ki 0.006 nM) was disclosed that was orally bioavailable with a long duration of action and was centrally active.374 Simple L-tryptophan derivatives including the N-acetylL-Trp-benzyl ester 404 (L 732138, IC50 40 nM)375 (Chart 93) and the dipeptide Cbz-Trp-Phe-NH2 (Ki 4 µM) served as leads for the development of more potent and metabolically more stable NK1 antagonists concurrently at Merck, Lilly and Park-Davis. Compounds 405 (L 737488, IC50 6 nM)376 and 406 (LY 303870, IC50 6 nM)377,378 both contain tertiary amines at the N-terminus and a modified C-terminus incorporating a short chain linking a phenyl substituent and are orally active. Surprisingly and in contrast to the proceed-

ing two examples, 406 and 407 (PD 154075, Ki 3 nM)379 were derived from the D-Trp series and the (S)-epimer was inactive. Compound 407 had good oral bioavailability (F ) 49%) and crossed the blood-brain barrier.380 A number of structurally similar NK1 antagonists based on a 4-aminopiperazine with an N-3,5-dimethylbenzoyl and 2-benzyl substituents have been reported independently by three pharmaceutical companies. The Ciba-Geigy compound 408 (CPG 49823 IC50 12 nM) (Chart 94) was discovered by screening the subset of Ciba-Geigy compounds that contained two aromatic groups at least five to nine atoms apart, in recognition that residues Phe7-Phe-8 of substance P were important for NK1 activity.381 Novartis reported 409 (NKP 608 IC50 3 nM) that was centrally active following oral administration and displayed long lasting antidepressant and anxiolytic effects in gerbils.382 Most recently, Johnson & Johnson reported 410 (R 116301 Ki 0.45 nM) that displayed superior pharmacology when compared directly with 10 other leading NK1 antagonists.383 Rhone-Poulenc described the perhydroisoindole 411 as a potent and selective NK1 antagonist that was an equally effective analgesic as morphine in two well-established rodent models.384 An improved compound 412 containing an extra aromatic ring and opposite stereochemistry at the fused rings was orally active and crossed the blood-brain barrier.385 A three point pharmacophoric model based on the early quinuclidines such as 396 proposed that a diphenylmethane unit, an appropriately spaced phenyl ring, and a basic



Chart 94

Chart 95

nitrogen were requirements for NK1 antagonists.386 Synthesis of 4-phenylisoquinolone derivatives and subsequently naphthyridones incorporating a basic nitrogen atom led to discovery of 413 (IC50 0.21 nM)387 (Chart 95) effective for reducing capsaicin-induced tracheal extravasation in guinea pigs (ED50 0.068 mg/kg). Interestingly, 413 exists in a 7:1 equilibrium mixture of stable, and separable cis and trans amide bond rotamers. NK1 activity was largely confined to the more abundant trans isomer with the cis isomer 20 times less potent. Additionally, each rotamer exists as a racemic mixture of non-interconverting atropisomers separable by chiral HPLC, in which the (aR)-trans is at least 5 times more active than the enantiomer (aS)-trans. This implies that less than half of the molecules are actually present in the most bioactive shape, and the inhomogeneous composition was expected to cause problems for drug approval. These observations led to the design of the cyclic analogue 414 (TAK 638, Ki 0.5 nM)388 where the ring dictates a trans amide and the asymmetric (9R)-methyl group directed an atropoisomerically specific ring closure giving the most active (aR,9R)-isomer. The X-ray structure of TAK 638 potentially offers a detailed description of the geometry at the receptor binding site. Further recent development has resulted in 415 as a possible clinical candidate for urinary incontinence.389 The first potent and selective non-peptide antagonist of the NK2 receptor 416 (SR 48968, Saredutant) (Chart 96) was described in 1992 following optimization of a random screening hit at Sanofi pharmaceuticals.390,391 The methyl group on the amide nitrogen was essential for activity, whereas exchange of the acetamido group with various polar substituents such as hydroxyl or acetoxy was tolerated. The benzoyl group could be replaced by 2- or 3-thienyl, or the larger 1-naphthoyl group with similar receptor binding

potency but reduced in ViVo activity. In receptor binding assays, 416 inhibited [123I]NKA binding to NK2 receptors in rat duodenum membranes and hamster urinary bladder membranes (Ki 0.5 and 1.2 nM, respectively) but did not bind to NK1 or NK3 receptors (Ki > 5 µM). The (R)enantiomer was considerably less active (Ki 1 µM). Compound 416 was also orally active and brain-permeable (displaying antidepressant/anxiolytic effects) and has been used extensively to investigate the pharmacology of NK2 receptors in ViVo. More recently, the improved compound 417 was reported by Sanofi as having greater NK2 activity (Ki 0.04 nM) than 416. The known important N-methyl group in 416 was extended in the form of a morpholine ring that also incorporated the asymmetric center and rigidified the molecule. Other modifications included the difluoro and dimethylurea substituents. Selectivity over NK1 and NK3 receptors (Ki >1 µM) was maintained also with long lasting oral activity and brain permeability. Additionally, 417 did not affect a large range of other receptors or ion-channels in binding assays up to 1 µM.392 Numerous analogues of 416 have been reported that retain the highly favorable dichlorophenyl, N-methyl benzamide, and piperidine ring features together with other minor modifications including 418393,394 and 419. Other cyclized analogues somewhat reminiscent of 417 are represented by structures 420395 and 421396. Researchers at Glaxo had optimized peptidic antagonists selective for the NK2 receptor and realized that the most potent compounds all contained a D-Trp residue and another aromatic group.397 Screening libraries of compounds that presented these features, and subsequent optimization of hits resulted in the discovery of 422 (GR159897) (Chart 97).398 In CHO cells expressing the hNK2 receptor, 422 was a potent antagonist (Ki 0.3 nM) and was highly selective over both NK1 and NK3 receptors (Ki < 1 µM). In ViVo activity for


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Chart 96

Chart 97

422 was also confirmed in a guinea-pig model of bronchoconstriction together with good oral bioavailability of 87% in beagle dogs. The (R)-sulfoxide contributed significantly to the overall potency of 422 as the enantiomeric sulfoxide was 30-fold less active, and the corresponding sulfone was even less active. Utilizing L- and D-phenylalanine as a template for a “fragment approach” to ligand identification, numerous hydrophobic groups were positioned at the N-terminus together with a water solubilizing morpholinopropyl C-terminal cap, ultimately resulting in 423 (MEN 14933).399 Compound 423 bound to the hNK2 receptor expressed in CHO cells (Ki 16 nM) showing 40-fold selectivity over hNK1 receptors (Ki 620 nM), and the ability to inhibit NKB induced contractions in human urinary bladder strips (Kb130 nM). Compound 424 (Ki 1 nM, hNK2R) was representative of a series of 2-phenylquinolines developed by SmithKline Beecham.

Compound 425 reported by Zenica was the most potent member of a series of pyrrolopyrimidines that competitively inhibited [3H]NKA binding to hamster urinary bladder NK2 receptors (Ki 2 nM) and showed good selectivity over NK1 receptors (Ki 2 µM inhibition of [3H]SP binding to NK1 receptors in guinea pig lung membranes). Because of a substantial species selectivity difference, 425 was 48-fold weaker for inhibition of NKA binding in cloned human NK1 receptors and was 90-fold less potent as a competitive antagonist of NKA-induced contractile responses in human bronchus.400,401

2.18. Neuropeptide Y Receptors 2.18.1. Neuropeptide Y1 Neuropeptide Y (NPY) is a 36 residue C-terminally amidated peptide found ubiquitously throughout the peripheral and central nervous systems to activate a family of 6



Chart 98

receptor subtypes (Y1-Y6). Additionally, there are two closely related peptides that share a common C-terminal amino acid sequence, termed peptide YY (PYY) and pancreatic peptide (PP) that are also ligands for these receptors. Numerous physiological functions have been ascribed to NPY, but interest from pharmaceutical companies has focused mainly on anti-obesity and anti-hypertensive agents. An alanine scan of NPY revealed that the C-terminal pentapeptide sequence Thr-Arg-Gln-Arg-Tyr-NH2 was most important for recognition by both Y1 and Y2 receptors. Molecular modeling suggested that distinct turn and helical conformations were recognized by Y1 and Y2, respectively, and that these structures needed to be stabilized since the pentapeptide alone was inactive.402 On the basis of this pharmacophore, a series of compounds were designed, culminating in the discovery of the first potent and selective Y1 antagonist 426 (BIBP3226, IC50 5 nM) (Chart 98) .403 The (R)-configuration was essential for activity ((S)-enantiomer, IC50 >10 000 nM), consistent with results from a D-amino acid scan of NPY that showed [D-Arg35]NPY was tolerated.404 Replacement of the Arg within BIBP3226 with Lys resulted in complete loss of activity. Interestingly, [Lys35]NPY also does not bind to the Y2 receptor, but retains some Y1 activity.405 The peptidomimetic 427 (SR 120819A) was reported by Sanofi to bind selectively to human Y1 receptors (Ki 15 nM) and was the first orally active antagonist described.406 Researchers at Lilly screened their compound collection and identified a simple 1,2,3-trisubstituted indole 428 (Chart 99) that bound to the human Y1 receptor (Ki 2 µM). Exploration of a series of analogues, in particular, those incorporating two basic piperidine groups appropriately tethered to the indole scaffold, culminated in the highly potent and selective Y1 antagonist 429 (LY 357897, Ki 0.75 nM).407 Not surprisingly, similarly potent compounds were derived from isosteric benzimidazole408,409 and benzothiophene410 templates, as in 430 (Ki 0.05 nM) and 431 (Ki 11 nM). High-throughput compound screening at Parke-Davis identified the phenylsulfonyl quinoline compound 432 (PD 9262) (Chart 100), with reasonably good receptor binding activity at Y1 (Ki 282 nM) and selectivity over Y2 (Ki > 10 000 nM).411 This discovery was interesting because, unlike the previously mentioned compounds that mimicked the two C-terminal arginine residues of NPY, 432 was only weakly basic owing to the delocalised nature of aniline and quinoline nitrogens, together with two electron withdrawing groups. SAR revealed that both the amine and nitro groups on the quinoline ring system were essential for good activity as were both the phenyl ring and sulfone linkage. A variety of substituents were allowable in the phenyl ring with improved activity observed for the ortho fluoro analogue 433 (Ki 58 nM) and ortho isopropyl compound 434 (PD 160170, Ki 48 nM). Interestingly analogues with larger halogen atoms than

fluorine resulted in less potent compounds (F > Cl > Br), whereas alkyl groups showed the opposite trend (iPr > Me > Et) with the bulky isopropyl being preferred. Screening of a Shionogi compound library identified the trisubstituted benzoazepinone 435 (Chart 101) that bound to the Y1 receptor (IC50 1.6 µM).412 The methoxy group was not important for activity, and replacement of the carbamate Chart 99


Chart 101

Chart 102

(Boc group) with the corresponding urea improved activity 20-fold. Exploration of a series of aryl and benzo-fused heterocycles attached to the 3-ureido group afforded several potent Y1 antagonists, for example, 436 (Ki 5 nM), that were also selective over Y2, Y4, Y5, and 17 other receptors assayed.413 Because of limited aqueous solubility and bioavailability, however, the series was not developed further. Morpholinopyridine derivatives exemplified by 437 (Chart 102) were reported by Banyu pharmaceuticals as subnanomolar Y1 antagonists, with excellent subtype selectivity (Y2, Y4 > 10 µM, Y5 6 µM) and also no significant binding to 50 other unspecified receptors.414,415 Binding of [125I]PYY to human and rat receptor membrane preparations was inhibited by 437 (Ki 0.29 and 0.54 nM, respectively); also intracellular Ca2+ mobilization induced by NPY in CHO cells expressing the human receptor was inhibited dose-dependently (IC30 3.2 nM). No increase in intracellular Ca2+ was induced by 437 alone (up to 1 µM), thus, confirming selective Y1 antagonism. Compound 437 was also the first orally bioavailable and brain-permeable Y1 antagonist described, Chart 103

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with levels of 0.5 µM detectable in brain tissue 24 h after oral administration (100 mg/kg) in rats. Intracerebroventricular injection of NPY (5 µg) induced robust feeding in satiated rats; however, when co-administered with 437 (200 µg), there was a 74% reduction in spontaneous feeding. Administration of 437 either intracerebroventricularly or orally to Zucker fatty rats, that are known to have high levels of hypothalamic NPY, also led to a significant reduction in spontaneous feeding (25 and 18%, respectively) for 24 h. These results clearly demonstrate that the Y1 receptor is involved in pathophysiological feeding and may represent a viable target for obesity management. High-throughput screening at Bristol-Meyers Squibb identified a novel dihydropyridine compound 438 (BMY-20429) (Chart 103) that was a competitive Y1 antagonist (Ki 109 nM).416 Structure-activity studies were conducted that culminated in several potent analogues, in particular, the interesting cyanoguanidine 439 with subnanomolar Y1 antagonism. Replacement of the original ethyl ester from the racemic 438 with a methyl ester removed the chiral center and gave almost 10 times improvement in potency. The dihydropyridine ring (a vinylogous urethane) was optimal, as both dihydropyrazines417 and pyridines were much less active. Replacement of the urea with thiourea made little difference, and cyanoguanidines (e.g., 439) were highly active. A variety of aryl piperazines and piperidines were tolerated for the terminal amine fragment, including the spiroindane piperidine 440 (H 394/84) which was found to be active in ViVo. The vasoconstrictor response to both exogenous and endogenous (neuronally released) NPY was significantly antagonized in rats and pigs following iv infusion of 440, when plasma levels reached 29 nM and 70% of the antagonism remained 90 min after the drug was given.418

2.18.2. Neuropeptide Y2 Selective, nonpeptidic Y2 antagonists had not been described in the literature until 1999 when the peptidomimetic 441 (BIIE0246) (Chart 104), based on the C-terminal sequence of NPY was disclosed.419 Radioligand displacement studies using [125I]NPY confirmed that 441 was a potent (IC50 3.3 nM hY2 receptor binding) antagonist for human, rat, and rabbit Y2 receptors, with good selectivity as no displacement was observed at Y1, Y4, and Y5 receptors up to 1 µM, nor did it cross-react with 60 other unspecified receptors or enzymes. The compound was rather large (MW 895) and peptidic, suggesting that oral bioavailability may be low. High-throughput screening at Johnson and Johnson identified the first small molecule Y2 antagonist 442 (IC50 4 µM, receptor binding).420 SAR studies probing the major features of the molecule resulted in considerably improved analogues, in particular, 443 (JNJ-5207787, IC50 100 nM). All modifica-



Chart 104

Chart 105

tions of the trans cinnamide were deleterious to activity, except small groups at the 3-postion of the phenyl ring that afforded a modest improvement to potency. Replacement of the N-acetyl-6-indoline fragment by a small series of heterocyclic analogues was not beneficial. The basic tertiary amine present in the benzylpiperidine fragment was important for activity, and replacement of the benzyl group with the longer, saturated cyclopropylethyl group led to a 30-fold improvement in activity. JNJ-5207787 443 inhibited PYY binding similarly to human and rat Y2 receptors (pIC50 7.0 and 7.1, respectively) and was >100-fold selective over Y1, Y4, and Y5 receptors and a wide range of other unspecified receptors and enzymes. Antagonism was confirmed by inhibition of PYY-stimulated binding of [35S]GTPγS in KAN-Ts cells expressing the human Y2 receptor and in rat brain sections using autoradiography to detect binding of [125I]PYY that was inhibited by 443. Brain permeability was also demonstrated following ip administration of 443 (30 mg/kg, Cmax 1351 ng/mL after 30 min), suggesting that 443 may be a useful pharmacological tool for the evaluation of the Y2 receptor in ViVo.421

Researchers at Bristol-Meyers Squibb disclosed a series of low molecular weight Y2 antagonists based on an initial screening hit, the most potent analogue being 444 (IC50 450 nM).422 No biological data has been reported for 444 which appears to share common pharmacophore with 442 and 443.

2.18.3. Neuropeptide Y5 A search for selective Y5 antagonists by Synaptic pharmaceuticals began with the weak and poorly selective lead, benextramine 445 (Ki 5 µM, Y5; 2 µM,Y1) (Chart 105) that also bound to other GPCRs. Investigation of a variety of symmetrical polyamine analogues of 445 failed to improve activity; however, replacement of one amino group with the non-basic sulfonamide led to substantial improvements in both potency and Y5 selectivity, for example, 446 (Ki 0.1 µM, Y5; 7 µM, Y1).423 The optimal length of the aliphatic linker between nitrogen atoms was found to be six methylene units, but further gains in potency were achieved by introducing a rigidifing cyclohexane ring constraint. Naphthyl and various 2-substituted phenyl groups were preferred sulfonamides, and the other hydrophobic region tolerated


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Chart 106

Chart 107

both 1- and 2-naphthyl groups, tetrahydronaphthyl, and quinolinyl groups. The most potent compound from the series was 447 (Ki 8 nM, Y5; 16 µM, Y1) and was shown to be a pure antagonist in functional assays. Good selectivity was achieved over both human and rat Y1, Y2, and Y4 receptors as well as other GPCRs (usually >100-fold), although significant binding was observed for cloned human R2c receptors (Ki 100 nM) and human D2 receptors (Ki 63 nM). The origin of the Y5 receptor selectivity was rationalized using a homology model that showed a hydrogen bond between the sulfonamide NH and His398 in TM6, as this residue is not present in the other NPY receptors. Additionally, the model showed a salt bridge between the basic tertiary amine and Glu211 and hydrophobic contacts with the aromatic groups. Selective screening of the Novartis compound library using a pharmacophoric model derived from weak Y5 hits identified from a previous Y1 program revealed several aminoquinazolines with low micromolar affinity for the Y5 receptor,424 for example, 448 (IC50 1.4 µM, hY5; 60 µM, hY1). Further optimization through combinatorial chemistry generated compounds with subnanomolar affinity like 449 (IC50 0.6 nM); however, the preferred compound for in ViVo studies based on affinity and selectivity was 450 (IC50 3 nM, hY5; 8 µM, hY1; 2 µM, hY2; 6 µM, hY4) that incorporated the known naphthalenesulfonamide and trans 1,4-bis-(aminomethyl)cyclohexane, components also present in the Synaptic pharma compound series. Numerous reports of other potent Y5 antagonists containing the arylsulfonamide and trans 1,4-bis-(aminomethyl)cyclohexane fragments appended to a variety of different hydrophobic units have subsequently appeared in the literature. The benzocycloheptene 451 (IC50 16 nM, hY5; 10 µM, hY1) (Chart 106) is a 7-membered ring homologue of the tetralin 447 also with piperidine replacing the cyclohexane ring and shows similar potency.425 The 1-hydroxy-1substituted tetralin 452 (IC50 0.5 nM) was however considerably more potent, and the sulfonamide group, previously considered essential for activity, could be replaced by the benzothiazolinone 453 (IC50 0.7 nM) without loss of activ-

ity.426 Although possessing good Y5 receptor affinity, the brain permeability determined ex ViVo after oral administration (100 mg/kg) to rats was found to be poor. Because of the poor oral bioavailability and low brain permeability of 450, Novartis continued to search for improved Y5 antagonists. Screening revealed that certain aryl thiazoles could effectively replace the aminoquinazoline ring system present in 448-452, and the arylsulfonamide fragment, that was suspected of causing the poor absorption, could be replaced by various aliphatic amides. Among several promising candidates, the tricyclic thiazole 454 was selected for further pharmacological evaluation.427 Excellent affinity and selectivity were observed for 454 (IC50 5 nM, hY5; >30 µM, hY1, hY2, hY4) together with high levels in plasma and the hypothalamus following oral administration (30 mg/ kg) to rats. Screening at Banyu for Y5 antagonists with pharmacologically acceptable properties focused on compounds with molecular weight ∼350 and identified a novel aryl pyrazole 455 (Ki 59 nM) (Chart 107).428 Modification of the chlorophenyl group did not lead to major gains in potency, although a factor of 2 was accomplished with a 3,4dimethoxyphenyl analogue. SAR of a series of aromatic acids revealed that the indane-2-carboxyl fragment was critical for high activity as most other derivatives had affinities >1 µM. An exception was the naphthalene-2-acetic acid derivative 456 (Ki 8.3 nM) with good binding affinity, but 456 may have raised ADME concerns as it was not discussed further. Modification of the initial screening lead to incorporate the novel chiral benzoindane carboxamide gave a series of very potent compounds, but with low oral bioavailability. The (-)enantiomer was seven times more potent than the (+)enantiomer; however, the absolute configuration remains to be determined. Reinvestigation of the 5-aryl substituent finally led to the discovery of the potent and selective ethylpyridine 457 (Ki 3.5 nM, hY5; > 10 µM, Y1, Y2, Y4) with good oral bioavailability after 10 mg/kg dosing (F 70%, t1/2 2 h, Tmax 2 h, Cmax 5 µM) in SD rats; however, the brain concentration after 2 h was only 0.45 µM, suggesting that brain distribution was inefficient. Functional antagonism


Chart 109

(IC50 14 nM) was demonstrated with the dose-dependent inhibition of NPY-induced [Ca2+] increase in LMtk- cells expressing the human Y5 receptor. The structurally distinct xanthene 458 (Chart 108) has been reported by Banyu and Merck to inhibit [125I]-PYY binding to the human Y5 receptor (Ki 26 nM) but not human Y1, Y2, or Y4 receptors at 10 µM. Additionally, 458 inhibited NPY-induced [Ca2+] increase in LMtk- cells expressing the human Y5 receptor, dose-dependently, but did not itself cause [Ca2+] increase even at 10 µM, thus, confirming pure antagonism.429 Oral administration of 458 (10 mg/kg) to rats led to brain concentrations 2.9 µM after 2 h, which was 100fold higher than the IC50 value obtained from the functional assay. Furthermore, 458 did not cross-react significantly with a variety of 120 other unspecified receptors or 7 enzymes. High-throughput screening at Amgen identified the potent pyrrolo[3,2-d]pyrimidine 459 that inhibited [125I]PYY binding to hY5 receptor expressing HEK293 cells (IC50 1 nM).430 Subsequent investigation of a large series of analogues revealed even more potent compounds, in particular, with various halogen atoms on the phenyl ring with IC50 < 0.1 nM; however, ADME results were still being examined. AstraZeneca developed a series of carbazole derivatives with potent Y5 binding affinities with the intention of treating obesity.431 The initial lead compound 460 (IC50 2 nM, hY5; >10 µM, Y1, Y2, Y4) (Chart 109) suffered from poor oral bioavailability, and there were toxicity concerns surrounding the N-ethyl-3-aminocarbazole unit that was a known animal carcinogen. Improving the ADMET properties of 460 proved relatively straightforward, with 461 emerging as a promising candidate for pre-clinical evaluation. A large variety of amides and ureas still conferred potent Y5 binding affinity, Chart 110


and replacement of the pyridine-propionamide unit with the morpholine-urea fragment in particular led to greatly improved adsorption and metabolic stability. The N-isopropyl4-methyl-3-aminocarbazole unit was completely devoid of mutagenic activity as determined by the Ames test but retained highly favorable Y5 affinity. Compound 461 bound with high affinity to the hY5 receptor (IC50 3 nM) and was a functional antagonist inhibiting Ca2+ flux (IC50 6 nM) but did not bind to Y1, Y2, Y4, and 80 other receptors at >10 µM. Compound 461 also showed excellent oral bioavailability of 76% and serum half-life of 3.7 h, and levels in the CNS of 153 nM at 1 h after oral dosing (10 mg/kg) exceeded the Y5 binding IC50 by at least 15-fold. Compounds 454, 457, 458, and 461 represent a structurally diverse range of potent and selective Y5 antagonists, and have been used to investigate the role of the Y5 receptor in feeding and the possibility that antagonists may be useful as anti-obesity agents. Intracerebroventricular administration of NPY or a Y5 selective agonist such as bovine pancreatic polypeptide (bPP) leads to robust feeding in satiated rats. The Y5 antagonists were effective only at reducing bPPinduced feeding, but not that induced by NPY which also antagonizes other NPY receptors. Of more clinical relevance, fasting-induced feeding and spontaneous free feeding in rats was not reduced by these Y5 antagonists, suggesting that the Y5 receptor is not alone involved in regulation of food intake. No Y5 antagonist has entered human clinical trials.

2.19. Opioid Receptors Opioid receptors are expressed throughout the central nervous system (CNS) including the spinal cord. They are classified according to their ligand binding profile and comprise three main types (µ, κ, δ) which are divided into further subtypes (µ1-µ3, κ1-κ3, δ1-δ2).432-434 For centuries, opioid agonists have been utilized therapeutically to relieve severe pain. Today, morphine (462) (Chart 110) and its derivatives still continue to be the most commonly prescribed analgesics. In addition, opioid agonists are also been used clinically to induce anaesthesia, to treat diarrhea, while opioid antagonists are important therapeutics to combat drug (e.g., morphine 462, heroin 463) addiction and overdose. Selective and specific endogenous opioid peptides were discovered as early as 1970s. The endogenous opioid ligands were produced from larger pro-opioid proteins by the action of proteases, and most of them consisted of a conserved N-terminal YGGF sequence.433,434 The enkephalin-derivatives, Met-enkephalin (YGGGM) and Leu-enkephalin (YGGFL), were discovered in 1975 as δ and µ selective agents. β-Endorphin is 31-residue long produced from proopiomelanocorin and also has high affinity toward both δ- and


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Chart 111

µ-receptors. The third precursor of endogenous-peptides is prodynorphin; its protease-cleaved products are dynorphin A, dynorphin B, R-neoendorphina, and β-endorphin and are κ opioid receptor selective peptides. In 1997, selective and highly potent µ-receptor ligands, endomorphin-1 (YPWFNH2) and endomorphin-2 (YPFF-NH2), which were analogues of the common YGGF sequence, were isolated from mamalian membrane.433,434 Systematic substitution of key amino acids and introduction of constraints into endogenous peptides provided important pharmacophore information that is now being utilized in the design of active peptidomimetic and nonpeptidic opioids. Modifications to morphine have also produced potent ligands that target opioid receptors. Numerous SAR studies of morphine and peptidic ligands have provided a better understanding of ligand selectivity and of the biological roles of opioid receptors. These and site-directed mutagenesis studies have revealed that three components, a protonated amino group, an aromatic ring, and a hydrophobic moiety, contribute significantly to the tight binding and potency of morphine. It has been proposed that an ionic contact of the amine group with an Asp residue belonging to the TM3 helix, π-π stacking of the aromatic portion, and hydrophobichydrophobic interactions of the ligand with aromatic and aliphatic side chains of the transmembrane helices are the key contributors to high receptor affinity.434-436 Molecular modeling of opioid receptors and putative interactions with ligands enabled refinement of a 3D pharmacophore model with four motifs.434-437 Opioid ligands are considered to have “message” and “address” components, the message binds to the receptor and causes signal transduction, while the address binds elsewhere on the receptor and confers high affinity and selectivity to ligands.438 In morphine derivatives, the A ring and the amino group are proposed to mimick the Tyr ‘message’ domain of endogenous peptides. Variation on this part is minimal; the R1O constituent is often hydroxyl or methoxy group, and larger groups were found to be detrimental for activity.437 Other regions of the morphine (462) ring system and N-substituents have been extensively modified to produce many potent and selective opioid receptor modulators.434,437 Buprenorphine (464) (Chart 111) is an oripavin derivative, and was recently approved by the FDA for opioid detoxification and maintenance.298,439 It is a partial µ-agonist and a weak κ-antagonist, with 25-40-fold higher potency than

morphine. It also has a higher safety margin, as at higher doses the agonist activity of buprenorphine plateaus and its pharmacological effects become those of a µ-antagonist.439 Sufentanil (465), originating from the 4-anilinopiperidine scaffold, is 600-800-fold more active than morphine and has 100-fold higher selectivity for the µ-receptor.434 The clinically available naloxone (466) and naltrexone (467) are opioid antagonists having several fold higher selectivity against µ-receptor. These compounds become inverse agonists in the morphine-dependent state, probably by suppressing the basal activity of µ-receptor.440 Derivatization of naltrexone 467 through the amino group of the C ring with large receptor-binding hydrophobic groups and removal of the phenolic hydroxy moiety led to identification of numerous potent µ-selective antagonists, including DOC-CAM (469).434 The methylated derivative (468) of naltrexone is a peripherally acting antagonist; its co-administration with other opioid agonists is a promising approach to overcome adverse side effects of morphine derivatives such as constipation.434,440,441 Alvimopan (470) is a potent µ-antagonist based on a phenyl piperidine skeleton.442 The compound exists as a zwitterion due to the presence of the carboxylate group. Its high polarity restricts gastrointestinal absorption and CNS penetration, rendering it able to manage opioidinduced peripheral adverse side effects. The compound has recently completed a Phase III clinical trial for treating postoperative ileus, and accelerated gastrointestinal recovery in patients undergoing major abdominal surgery. Peripherally restricted agonists are also valuable therapeutics. The potent µ-selective agonist loperamide (471) (Chart 112) is marketed as an antidiarrhoeal and is currently under investigation for treating local dermal and ophthalmic pain and itch.441 Extending the proposed δ-address site, the C ring of the morphinan scaffold with aromatic groups afforded δ receptor selective ligands. In agonist 472, the spiroindane group is perpendicular to the C ring, a position postulated to be favorable for δ receptor binding.434 Fusing the C ring with heterocycles, such as pyrrole, indole, and benzofuran, afforded modified morphinan scaffolds, from which many highly active and selective δ antagonists were obtained.434,437,438 Recently, the δ receptor selective naltrindole-derived antagonists (473-475) and ligands with similar ring systems were utilized for construction of three-dimensional models by comparative molecular field analysis (CoMFA).437 Three



Chart 112

Chart 113

contour maps were built for µ, δ, and κ receptors. It was found that the steric interactions are dominant factors in binding; the ratio of steric to electrostatic contributions was 75%/25%, 65%/35%, and 67%/33% for δ, µ, and κ models, respectively. Furthermore, the models were consistent with both a “message” and “address” and known SAR data. It was evident from the contour maps that large hydrophobic substitution is tolerated on the amino group, while bulky alkylation of the phenolic 3-OH group is unfavorable for binding to all three receptor types. In agreement with the proposal that the indole ring corresponds to the ‘address’ region, bulky substituents in this part (5′, 6′, and 7′) were well-tolerated by the δ-receptor. Selective δ-agonists show therapeutic promise, producing analgesia in animal models of pain, while lacking the undesirable side effects of morphine derivatives. TAN-67 agonist (476) (Chart 113) is an isoquinoline derivative with low nanomolar potency against δ-receptor and 3 orders of magnitude selectivity over other opioid receptors.434,443 The N-benzylpiperazine is a suitable molecular skeleton to appropriately present amino and aromatic pharmacophoric groups for tight binding. Decoration of this scaffold with substituted aromatic groups has produced numerous potent, δ-selective agonists such as SNC 80 (477) and BW373U86 (478).434,444 N-Substituents (cyclopropylmethyl) that tend to confer antagonism to morphinan derivatives failed to deliver

benzylpiperazine antagonists. The only ligand with reported antagonist activity from this series is DPI2503 (479), of which the cis configuration of the dimethyl groups is thought to switch the biological effects.434,444 Simplification of the SNC 80 (477) structure led to a new class of δ-selective agonists. In this series, one nitrogen atom of the piperazine ring was replaced by a methylene group that is connected to the aromatic region via a rigid double bond. The resultant planar structures (480) are the most selective and potent δ-agonists described to date, with < nanomolar affinity and >103-fold selectivity over µ-receptor.444 Selective morphine-derived κ-agonists have been hard to find. Early efforts identified benzomorphan derivatives like ethylketocyclazocine (481) as marginal κ-agonists.434,441 Arylacetamide derivatives (482-486) (Chart 114) were effective κ ligands, with low nanomolar affinities for the κ-receptor and exceptional selectivies over µ- and δ-receptors. The amide moiety contains an extra protonatable amino group, most often in the form of pyrolidinyl in β position. The disappointing clinical results of centrally acting κagonists (e.g., enadoline, 482) prompted the search for peripherally limited κ-agonists a decade ago.441 As for alvimopan (470), introduction of a free carboxylic group helps restrict absorption and systemic availability of ADL 01-0344 (485). The piperazine derivative GR 94839 (486) was also a peripherally selective κ-agonist, antinociceptive


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Chart 114

Chart 115

activity being demonstrated in a writhing and pruritis assay in mice.434,441 Recently, other morphinan derivatives (e.g., TRK-820, 487) were found to exhibit high potency and κ selectivity, with >85-fold potency and analgesic activity over morphine.434,445 Derivatization of the ‘address’ region of natrindole also resulted in κ selective ligands. An additional guanidino group in position 6′ of naltrindole confers κ selectivity, giving the agonist 6′-GNTI (488). Interestingly, the same guanidino substituent in position 5′ (489) converts naltrindole into a κ-selective antagonist; similarly, natrindole containing another positively charge group, such as amidine (490), in this position or in position 4′ also displays antagonistic activity.434 There are also other potent opioid ligands; many of them are in clinical use. The structures of representative compounds are displayed in Charts 114 and 115 (cyprodime 491,

meperidine 492, fentanyl 493, methadone 494, levorphanol 495, and SB219825 496).434 Dimerization of a ligand through a linker often produces a compound with increased potency; the success of such a strategy is well-documented for opioid receptors (497).434 The presence of a protonatable amino group is not an absolute requirement for high activity of opioid receptor ligands. Salvinorin A (498) is a natural product isolated from the plant SalVia diVinorum and was reported to be a highly selective κ-agonist. Derivatization efforts of this neoclerodane diterpene scaffold have also produced a potent µ-selective agonist (499) (Table 5).446 Dimerization and oligomerizations of GPCRs have been shown to exist and may be of biological relevance in ViVo. Recently, Whistler and colleagues have utilized the agonist 6′-GNTI (488) to demonstrate the existence of a δ and κ heterodimers in ViVo.447 The supposed κ-selective agonist 488



Table 5. Receptor Binding Affinities of Selected Opioid Agonists and Antagonists µ compounds Morphine (462) Buprenorphine (464) Sufetanil (465) Naloxone (466) Naltrexone (467) Alvimopan (470) Loperamide (471) Naltrindole (473) TAN-67 (476) SNC 80 (477) BW373U86 (478) SNC analogue (480) U50488(483) ADL01-0344 (485) TRK-820 (487) Salvinorin A (498) 499

IC50 (nM)

δ Ki (nM)

IC50 (nM)

1.2 0.08 0.025 0.4 0.1 0.77 3.3 27 2320 2467 46 3570

κ Ki (nM) 120 0.42 10 31.6 7.9 4.4 48 0.22 1.1

IC50 (nM)

Ki (nM)

ref

120 0.11 31.6 6.3 1 40 1156 30 1790

432 298 432 432 432 442 441 437 443 444 444 444 432 441

2.9 0.9 0.74 200 1300 53 >1000 12

7940 700 1200 5790 1170

1 4 3.5 1.9 90

446 446

Chart 116

was far more potent and efficacious in a Ca2+ mobilization assay using δ- and κ-receptors coexpressing HEK-293 cells. Importantly, intrathecal administration of this compound to mice induces antinociceptive effects with the ED50 value of 0.45 nmol per mouse. However, analgesic activity was not observed when 6′-GNTI (488) was administered to brain, suggesting tissue-specific function of the δ and κ receptor heterodimers. The results strongly indicate that heteromer of opioid receptors is a biologically functional entity, and this could provide a unique opportunity to develop tissuespecific opioid ligands that might result in therapeutics with better toxicological profiles.

2.20. Opioid-Like Receptor The opioid-like receptor (ORL-1) and its endogenous ligand, nociceptin, are widely expressed in the CNS but typically not at the same place as opioid receptors.448,449 Although sharing close homology with conventional opioid receptors, most opioid peptidic and small molecule ligands do not have appreciable affinity for ORL-1, and likewise, nociceptin does not bind appreciably to µ-, δ-, or κ-receptors. Pharmacological and biological studies have indicated that ORL-1 agonists may have therapeutic potential for treating anxiety, stress, and drug and alcohol addiction, while its antagonists could be valuable in managing and treating locomotor diseases such as Parkinsonism. Currently, the natural ligand nociceptin is in development (Phase II) for treating urinary incontinence. Another peptide (ZP-120), a peripherally restricted ORL-1 agonist, is also in Phase II clinical trials to treat acute decompensated heart failure. Given the homology with classical opioid receptors, potent nonpeptidic ORL-1 agonists have been sought after from among morphine derivatives.448 Buprenorphine (464) was shown to be a modest ORL-1 agonist (Ki 285 nM).298 TRK-

820 (487) was described as a κ-selective agonist and is a ORL-1 antagonist (Ki 380 nM).445 Recently, compounds based on the naltrindole scaffold (e.g., 500) (Chart 116) were reported to be potent ORL ligands (IC50 g 25 nM). More potent and more selective ORL agonists and antagonists were obtained by modification of the piperidine scaffold.448 Substitution of the nitrogen by a large hydrophobic group, often aromatic, and decoration of position 4 of the piperidine by aromatic substitutents increased potency. Both ORL agonists and antagonists (501-505) (Charts 116 and 117) were identified with this general structure; their affinities being single digit or subnanomolar.448,450 Compound 503 was reported to be an ORL-specific agonist displaying subnanomolar affinity and 10-100-fold selectivity over opioid receptors. This compound was shown to have notable anxiolytic activity in several animal models after ip administration. Substituted quinoline derivatives are also claimed to be ORL ligands; antagonist 506 exhibiting good affinity toward ORL (Ki 45 nM) with some selectivity over opioid receptors.448,450 Compound 506 was taken to Phase II clinical trial by Japan Tobacco for analgesic activity, but was discontinued for undisclosed reasons. The structurally relative quinazoline scaffold was also utilized to develop potent ORL ligands. Compound 507 is a specific ORL antagonist with subnanomolar binding affinity and exceptional selectivity over µ opioid receptor. This quinazoline derivative was shown to have excellent efficacy in prevention of spontaneous scratching in mice with a destroyed dermal horny layer barrier. Recently, aminocyclohexanes and cyclohexanols have emerged as new, promising ORL modulators.448 The diaminocyclohexane 508 displays good affinity for ORL (Ki 3 nM) and antinociceptive activity in the mouse tail flick model at 1 mg/(kg/iv). A related compound (509), with one amino


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Chart 117

Chart 118

group replaced by hydroxyl, was also quite a potent ORL modulator (Ki 9 nM) and extremely effective in ViVo (ED50 0.001 mg/kg) eliciting a maximal protective effect of 96% at 0.01 mg/kg in a mouse tail flick model.

2.21. Orexin Receptors Orexin-A and B (hypocretin-1 and -2) are 33- and 28residue neuropeptides, both derived from a 130-residue precursor, prepro-orexin.451 These peptides activate two GPCRs, OX1R and OX2R, leading to increased intracellular calcium concentrations. OX1R couples exclusively to Gq heterotrimeric proteins with 10-fold greater affinity for orexin-A over orexin-B, while OX2R couples to both Gq and Gi/o heterotrimeric proteins with equipotent affinity with both orexin-A and -B.451 Orexins were discovered in 1998 and have important roles in the sleep/wake cycle, feeding, energy homeostasis, and arousal. Deficiency of orexins correlates with narcolepsy (excessive daytime sleepiness) and cataplexy (sudden muscle weakness), and is found in 90% of narcolepsy sufferers.451 Several small molcule nonpeptidic antagonists of the oxerin receptors have been reported. The first selective OX1R antagonist, SB334867 (510) (Chart 118), was developed at GlaxoSmithKline from a high-throughput lead 1-aryl-3(quinolin-4-yl)urea (511).452,453 The lead compound (pKb 7.5, OX1R) also had affinity at serotonin receptors (pKi 8.6, 5-HT2b; pKi 7.6, 5-HT2c). Since it had been previously shown that changes in the indoline structure affect serotonin

receptor affinity, researchers substituted 1-methylindol-5-yl for 2-methylbenzoxazol-6-yl and reduced affinity at serotonin receptors 10-100-fold.453 Changes were also made to the quinoline ring to increase solubility, with 1,5-naphthyridine in SB334867 increasing selectivity for OX1R (pKb 7.2) over 5-HT by 100-fold.453 SB334867 reduces food intake, decreases body weight, and increases behavioral satiety (suppresses active behavior) in rats. Banyu developed the first OX2R antagonist from a nonselective orexin antagonist identified by high-throughput screening (512, IC50 7 µM, hOX1R; IC50 2 µM, hOX2R).454 Modification of the aryl portion to 4-pyridinylmethyl (S)-tert-leucyl tetraisoquinoline (513) increased receptor discrimination (IC50 > 10 µM, OX1R; 40 nM, hOX2R) with 200-fold selectivity over other SSTRs. Compound 541 also inhibited growth hormone secretion by rat pituitary cells and rats. Urea-backbone cyclization of L-054,222, introduced by Pasternak and colleagues, resulted in significant improvement of pharmacokinetic characteristics486 (e.g., 542, Ki 8.5 nM, oral bioavailability 64%). In a landmark report, Rohrer et al. discovered highly active, subtype-selective, small molecule agonists for each of 5 SSTR.492 A potent SSTR2 cyclic hexapeptide agonist was used to identify potential hits from Merck’s in-house chemical library. A set of 75 compounds was screened and led to identification of 543 (IC50 100 nM, SSTR2) (Chart 126). From this lead, an iterative mix and split combinatorial library of several hundred thousand compounds were prepared with diverse variations of the pharmacophoric aromatic, tryptophan, and amine moieties. Subsequent screening and deconvolution resulted in potent SSTR agonists. L-779,976 (544) contained a constrained amino group and had >6000fold selectivity for SSTR2 (Ki 0.05 nM, SSTR2) over other SSTRs. It inhibited release of growth hormones from rat pituitary cells with comparable potency (IC50 0.025 nM) to SIRF-14. L-796,778 (545) was a partial agonist for SSTR3 (IC50 18 nM) for inhibition of forskolin-induced cAMP production. SSTR1 selective agonist L-797,591 (546) had IC50 of 3 nM against SSTR1 in cAMP response element (CRE)-β-galactosidase reporter assay. L-803,087 (547) and L-817,818 (548) were, respectively, selective SSTR4 (IC50 0.2 nM) and SSTR5 (IC50 1.3 nM) agonists in a cAMP accumulation assay. Recently, the 1,2,4-triazole core was exploited to project aromatic, indole, and amino moieties from the β-turn pharmacophore.493 Compound 549 was discovered from a library of more than 700 members, having single digit Ki binding affinities against SST2 and 5 receptors. In a functional assay, compound 549 displayed EC50 values of 4.0 and 2.3 nM on cells expressing SSTR2 and SSTR5, respectively.

To date, nonpeptidic antagonists have only been reported for SSTR1 and SSTR3. SRA-880 (550) (Chart 127) is a selective SST1 antagonist derived from the octahydro-benzo[g]quinoline backbone.486,488,494 SRA-880 displayed high binding affinity (pKd 8.05) and greater than 100-fold selectivity for SSTR1, behaving as a surmountable and competitive antagonist (pKB 7.5) of SRIF-14 on forskolinstimulated cAMP accumulation. In mice, oral administration of SRA-880 at 0.01-30 mg/kg resulted in reduction of aggressive behavior and was active in depression and bipolar diseases.486,488 The SST3 receptor antagonist BN-81674 (551) was obtained from a library of tetrahydro-β-carboline congeners, and competitively blocked the action of SIRF14 in a cell based assay (KB 2.8 nM).486,488,495 Compound 551 bound to the SST3 receptor (Ki 0.92 nM) and was >10 000-fold more selective over other SSTRs.

2.25. Urotensin Receptors Urotensin II (U-II) is an 11 residue disulfide-linked cyclic peptide, cyclo-(5,10)-[ETPDCFWKYCV],11 the most potent known vasoconstrictor in mammals with CNS and cardiovascular properties.496 It binds to a somatostatin-like GPCR (hUT) of the rhodopsin family that was previously known as orphan receptor GPR14.497 While some hUT-binding ligands have dual actions on urotensin and somatostatin receptors, a number of hUT agonists and antagonists have selective actions on just this receptor, and ligand docking in a homology model of the transmembrane region of hUT has been studied.498 Typically antagonists inhibit vasoconstriction and thus have potential for the treatment of cardiovascular and other disorders,497,498 but no hUT ligands have yet progressed into clinical trials. Receptor activation requires the Trp-Lys-Tyr segment of a β-turn,11 and thus, nonpeptidic compounds that contain a basic amine (for interaction with Asp130 in TM3 of hUT) and two aromatic groups to mimic these side chains have been identified as ligands for hUT. Among such compounds are nonpeptidic agonists (e.g., 552, EC50 300 nM) (Chart 128),499 and antagonists like indole 553 (S6716, IC50 400 nM, Ca2+ release)500 and biphenylamide 554 (IC50 10 nM, hUT; 5 nM, SST5).501 Indoles like 553 were discovered


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Chart 126

through screening in silico libraries followed by synthetic modifications. Analogues of 553, although nonselective antagonists of urotensin and somatostatin receptors, are reportedly valuable agents for treating diabetes and hypertension. Among other hUT antagonists reviewed elsewhere502 that are active at nanomolar concentrations are piperidines, with Chart 127

tetrahydrobenzazepine (e.g., 555 (Chart 129), IC50 2 nM, hUT) or sulfonamide (e.g., 556) substitutents, 4-aminoquinolines (e.g., 557; R ) H, Me, Br; IC50 33, 4, 2 nM), 2-aminoquinolines (558, R ) COCHPh2, Ki 90 nM, hUT) and related 2-aminoquinolones, 4-ureidoquinolines (e.g., 559) bearing tetrahydroisoquinoline, piperidine, pyrrolidine, or piperazine substitutents. One piperidine derivative of 559 (palosuran, ACT058362)503 with high hUT affinity (IC50 4 nM) is an effective UT antagonist reducing glomerular and Chart 128



Chart 129

Chart 130

tubular dysfunction and renal tissue injury induced by renal ischemia/reperfusion in male Wistar rats. A wide variety of sulfonamides like diarylsulfonamides 560 and 561 have also been described with UT antagonist potency. Compounds 560 (IC50 10 µM, Ca2+ release) and 561 (SB706375, Ki (affinity) 5 nM, SJRH30 cells; pKb 7.5 (rat aorta contractions) and 7.3 (Ca2+ release, HEK293 cells))140 appear to represent a selective pharmacophore for UT antagonist activity.

2.26. Vasopressin Receptors The cyclic nonapeptide, cyclo-1,6-[C1YFQNC6PRG]-NH2, or [Arg8]-vasopressin (AVP) is an antidiuretic hormone with a wide range of physiological activities, regulating water resorption, body fluid, osmolarity, blood volume, blood pressure, cell contraction, cell proliferation, and adrenocorticotropin secretion. There are three vasopressin receptors (V1-vascular, V1R or V1a; V2-renal, V2R; V3-pituitary, V3R or V1b). Each subtype is expressed on a different chromosome (12, X, and 1, respectively), has a different function, intracellular signaling, different receptor inactivation requirements, and is in a different physical location. The oxytocin receptor, modulated by the similar hormone oxytocin, is related to the vasopressin receptor family. Many peptide antagonists of vasopressin receptors have been developed through modifications to AVP, but none have successfully completed clinical trials. Several promising clinical trials are underway with more orally bioavailable nonpeptidic antagonists. AVP mediates vasoconstriction through V1R, as well as platelet aggregation, coagulation factor release, and cellular proliferation. Consequently, V1R has been implicated in heart failure, arterial hypertension, atherosclerosis, and dysmen-

orrhea. V1R is expressed in the liver, vascular smooth muscle, blood platelets, adrenal cortex, kidney, reproductive organs, spleen, adipocytes, and brain.504 Upon activation, V1R couples with Gq/11 and activates phospholipases C, D, and A; produces inositol 1,4,5-triphosphate and diacylglycerol; activates protein kinase C, p42/p44 MAP kinase, PI3kinase, and calcium/calmodulin-dependent kinase II; and mobilizes intracellular calcium,505 but no stimulation of cAMP accumulation is observed.504 Activation of V1R leads to a mitogenic response in a number of cell lines.504 Internalization of the receptor occurs shortly after agonist binding. Three selective V1R antagonists have been reported, OPC21268 (562) (Chart 130), SR49059 (563 Relcovaptan), and YM218 (564). Compounds 562 and 563 were developed by chemical optimization of a hit identified through random screening. Compound 562 is orally active with high affinity (IC50 5 nM) for rat smooth muscle cells,504 but poor affinity for hV1R (human liver Ki 14 µM). Thus, while effective in a rat deoxycorticosterone acetate-salt model of hypertension, it was ineffective in altering human AVP mediated effects in Vitro or in ViVo.504 Compound 563 was efficacious at both animal and human V1R receptors, reducing smooth muscle contraction and blood pressure. In the clinic, it did not alter blood pressure or heart rate, but it did increase the rate at which blood pressure returns to baseline, blocked AVP platelet aggregation, and antagonized vasopressin-induced uterine contraction, possibly effective in dysmenorrhea.504 Compound 564 was an orally bioavailable, potent selective antagonist (V1R affinity Ki 0.5 nM; selective for V1R over 28 other receptors/enzymes) which dose-dependently inhibited the pressor response caused by AVP.506


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Chart 131

V2R mediates AVP effects in the kidney, located on the basolateral membrane of the collecting duct in the medullary kidney for regulation of antidiuretic effects of AVP. Internal signaling of this receptor involves coupling with Gs heterotrimer G proteins, activation of adenylyl cyclase, production of cAMP, and activation of protein kinase A, resulting in increased synthesis and insertion of aquaporin 2 water channels on the luminal surface of the renal collecting tubule cells.504,505 It has been experimentally shown that agonist activation of V2R alters the conformation of the receptor, thereby exposing a region of extracellular loop 1 for cleavage and inactivation by a metalloprotease.504 The main aim of developing antagonists of V2R (‘aquaretic’ drugs) is to eliminate water without modifying electrolyte urine excretion. The potent V2R antagonists OPC-31260 (565 Ki 9.42 nM, V2R) (Chart 131) and OPC-41061 (566 tolvaptan, Ki 0.43 nM, V2R) were developed by modifying the V1R antagonist OPC-21268 (562). They both reduce free water excretion without effecting electrolytes. OPC-31260 (565) blocks cAMP and AQP-2 production, as well as being effective in experimental models of liver cirrhosis, congestive heart failure, SIADH (syndrome of inappropriate antidiuretic hormone secretion), nephrotic syndrome, and acute hyponatremia.504,505 OPC-31260 (565) has also been effective in experimental models of cerebral oedema, dose-dependently preventing water retention and alterations of brain water and sodium content. In clinical trials, OPC-31260 effectively increased urine volume and decreased urine osmolarity in 11 SIADH patients, and 8 patients with biopsy-proven liver cirrhosis, oedema, and ascites.504 VPA-985 (567 lixivaptan) is another potent V2R antagonist (affinity rat V2R Ki 0.48 nM) that is 100-fold selective for V2R over V1R and oxytocin receptor, with efficacy in a human heart failure trial (increased urine volume, and decreased urine osmolarity) and human liver cirrhosis.504 SR-121463A (568) another potent and selective V2R antagonist produces effective aquaresis in hydrated rats.504 Modification of compound 569 by differing methylation changes the in ViVo properties (CH3 ) agonist; H ) antagonist, at V2R). The change in intrinsic agonist and antagonist properties by a methylene substituent was also observed in compound 570 (NH(n-Pr) affinity IC50 18 nM, 79% agonist activity 571 (pyrrolidine affinity IC50

Chart 132

Chart 133

9 nM, 64% agonist activity; piperidine affinity IC50 120 nM, 8% agonist activity).507 AVP modifies ACTH secretion by corticotrophic cells through V3R (V1bR); therefore, this receptor is thought to be an important target in ACTH-secreting tumors. One V3R-selective nonpeptide antagonist has been developed, SSR149415 (572 Ki 1.26 nM hV3R CHO cells) (Chart 132).508 This 2-oxoindoline antagonist has uncovered roles for V3R in anxiety and depression, as well as roles in social and aggressive behavior identified by receptor knockout models.508 SSR149415 appears to be based on modification of SR49059 (563). Dual nonselective V1R/V2R antagonists have been designed to combat hypertension and congestive heart failure where they potentially can modify vasoconstriction and platelet aggregation, as well as fluid retention. YM087 (573 conivaptan) (Chart 133), is a tetrahydroimidazo[4,5-d]1benzazepine antagonist with affinity at ratV1R and ratV2R with Ki values of 0.48 and 3.04 nM, respectively, in a radiolabeled competition assay.504 Experimently, YM087 antagonizes the


pressor response and produces an aquaretic effect in rats. Clinical studies have established that YM087 is welltolerated, with more pronounced blockage of V2R over V1R, with the compound’s aquaretic effect lasting 6 h, and only skin vasoconstriction was inhibited; no significant change in blood pressure or heart rate was observed in single dose studies. YM087 (573 conivaptan) has recently been approved by FDA for euvolemic hyponatremia.509,510 Compound 574 developed by Johnson & Johnson was designed from a bridged bicycle attached to a benzodiazepine unit.511 The template design was based on the benzodiazepine scaffold previously represented in VPA-985 (567) and OPC-31260 (565). Compound 574 had high affinity at human V1R and V2R (IC50 24 and 5 nM, respectively), and functionally inhibited calcium mobilization by V1R (Ki 23 nM) and cAMP production by V2R (Ki 13 nM).511 Rat pharmacokinetics of 574 showed an oral half-life of 2.5 h and 7% oral bioavailability. It was also observed that 574 had affinity for CYP2C9 (IC50 7.4 µM), effectively producing an aquaretic effect and increasing the rate at which blood pressure returns to baseline.511 Other researchers have sought to replace the benzodiazepine scaffold with 2,3-dihydrothieno[2,3-b][1,4]-thiazine (575) (Chart 134), however, only moderate (low micromolar) antagonists were obtained with this scaffold.512 Spirobenzazepine scaffolds have also been explored, with compounds 576 being potent dual V1R/V2R antagonists (576 LogP 3.6; rat F 22%; affinity IC50 2 nM (V1R) and 8 nM (V2R); functional IC50 45 nM (V1R) and 36 nM (V2R)) and 577 a potent V1R antagonist (577 LogP 2.5; rat F 42%; affinity IC50 4 nM (V1R) and 341 nM (V2R); functional IC50 78 nM (V1R) and 646 nM (V2R)).513

3. Summary G protein-coupled receptors (GPCRs) are the largest family of proteins considered to be viable targets for pharmaceuticals, but less than 5% of these membrane-spanning cellsurface proteins are currently regulated by drugs in man. A major challenge in the design and development of druggable lead compounds for this receptor class is the creation of new nonpeptidic chemical entities with structural diversity and receptor selectivity. Native peptide ligands for GPCRs provide important clues to the design of nonpeptidic ligands


but they tend to bind in different ways to, or at subtly different sites on, any given receptor. Since many different GPCRs appear to recognize recurring structural fragments in their potent nonpeptidic ligands, the recombination of complementary fragments from compounds already known to be bioavailable drugs might be a successful approach to new drug leads, though with associated risks of losing receptor selectivity. To provide a more comprehensive basis for assessing the status and future prospects of GPCRdirected drug discovery, we have assembled a diverse portfolio of 577 nonpeptidic chemical structures known to interact with 76 peptide-activated GPCRs, and reported on their chemical composition, selective affinities for GPCRs, agonist/antagonist potencies, and pharmacological properties. We emphasized their advantages as drugs (over peptide ligands), described some of their origins, and identified some more promising chemical fragments that provide important clues for developing new nonpeptidic ligands for GPCRs through fragment assembly, privileged structure elaboration, and natural product modification. This portfolio of compounds provides a valuable guide to druggable fragments for lead compound discovery, and not only for GPCR target proteins. It may enable researchers to identify similarities in scaffolds, substituents, and other fragments across different compound and receptor classes. Peptide-activated GPCRs have historically been considered difficult pharmaceutical targets because of their large interacting protein-protein surfaces which are difficult to attack with small organic compounds. While this might have once been a reasonable consideration for agonist development, antagonists usually only need to interfere either with small ‘hot spots’ to block receptor activation by endogenous agonists, or with key interactions that define high-affinity binding. There are also now numerous examples of small molecule synthetic agonists, inverse agonists, and allosteric regulators that exert their functions through binding to only very small regions of a GPCR. It seems likely that the often huge receptor-binding domains of endogenous protein and peptide agonists that target GPCRs have evolved to confer high affinity and especially receptor selectivity for signal transduction via GPCRs. The actual activating or effector domain of GPCR ligands is however frequently a very small segment, often of low affinity alone, that is anchored in proximity of its ligand-binding site on a GPCR by a higher affinity GPCR-binding domain of the agonist that has no direct receptor-activating role in signal transduction. Clearly, based on the content of this review alone, peptide-activated GPCRs are certainly tractable and very promising targets for small nonpeptidic therapeutic candidates. Of the several hundred known peptide-activated GPCRs, most academic and industry researchers have necessarily focused on investigating the regulation of just one or a few GPCRs and usually for a single disease area, for which they painstakingly glean insights on structure-function relationships toward the development of a potent and selective drug. We decided to review the field laterally in a generic way, partly because we do not think this has been done previously to anything like the extent of this review, and partly because we wished to examine numerous targets to see if there were common approaches used successfully (and unsuccessfully) to develop drugs, as well as less common approaches that have not been used widely. In the present perspective survey, there are indeed a number of strikingly common structural features of ligands for GPCRs, as well as some promising


directions in drug discovery that have not been fully exploited for many GPCRs. For peptide-activated GPCRs, the most obvious historical approach to drug discovery has been to examine the peptides that activate the receptor. This has often been the starting point for developing nonpeptidic drugs, with the peptide backbone structure and side chain composition affording valuable clues for the generation of constrained peptides, cyclic peptides, peptide fragments fused to nonpeptidic units, and peptidomimetics designed and iteratively optimized to make ligands more potent, more selective, less peptidic, and more druggable. Such peptidic compounds have been valuable mechanistic probes of biological processes, helped to validate therapeutic targets, and sometimes have proven to be valuable drugs in their own right. We have previously overviewed such ligands for over 100 peptide-activated GPCRs and thus do not mention them herein.11,514,515 Generally, such peptidomimetics have ultimately had to be converted to more nonpeptidic compounds to generate cheaper drugs with improved bioavailability, stability, favorable pharmacokinetic and pharmacodynamic profiles. In the present review, we have instead focused on over 500 nonpeptidic ligands for over 70 peptide-activated GPCRs for the potential treatment of a large and diverse range of diseases and briefly commented on their pharmacological properties. Unlike peptide ligands, which can bind to the extracellular N-terminus and/or the three extracellular loops of GPCRs, the vast majority of nonpeptidic ligands for GPCRs primarily target the cell membrane, localizing to the hydrophobic transmembrane helices that are fairly conserved throughout the GPCR families. The most common approach used to identify active compounds has been high-throughput assaying of natural product and synthetic compound libraries resulting in ‘hits’ that are usually of micromolar affinity for a receptor. Mostly, this effort has involved random compound screening, but more recent efforts have concentrated on more focused structural libraries either based on known active compound classes, drug-like structures, or smaller units that could conceivably be linked through fragment-based assembly. Combinatorial chemistry (or parallel synthesis) has probably been of best value for generating drug fragments or optimizing hits into more potent/selective ‘leads’, and of less value in directly finding high value hits or leads unless extensive structural diversity can be readily incorporated into the chemistry. More recently, homology models of receptors have become more useful for ligand docking, with ligand binding sites being assigned through combinations of docking studies and effects of site-directed receptor mutagenesis on structure-activity relationships for affinities/activities of tested ligands. This work, like the evaluation of ligand affinity/potency, can be compromised if the receptor is transfected into a particular cell type, because this process artificially increases the number and density of receptors, and thus, ligand activities rarely correspond in magnitude to their effects on whole cells with native expression levels of GPCRs. It is also not yet clear whether transfection alters downstream activity profiles of agonists. Very recently, it has become more widely appreciated that ligand binding to a GPCR is frequently species-dependent and that residues vary in a GPCR between species. Such variations, by influencing ligand affinity/potency, can provide valuable information for identifying the ligand binding site on a GPCR. Polymorphisms even within human populations can

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complicate the situation even further. Virtual screening of in silico compound libraries has recently also become more viable, and a cheaper alternative to finding hits, as homegrown computer software programs have improved to some extent allowing more accurate ligand fitting and scoring, though there are still no really effective commercial software programs available for this endeavor. In any case, ligand docking is inherently compromised by the lack of precise structural information on GPCRs and so modelling necessarily lacks the degree of accuracy required for confident predictions of appropriate drug composition. Another approach to hit finding is based on the detection of common ‘scaffolds’, termed “privileged structures”,8,14,25,48,49,51-57,64,70,193,337,516-521 in ligands that bind to different GPCRs. Even a cursory inspection of the nonpeptidic compounds assembled in this review reveals many components that are shared by ligands for different GPCRs. Just a few of these are benzimidazoles (10, 17, 147, 212, 319, 430), benzimidazolepyridines (2-6, 14, 18, 19), benzodiazepines (56, 57, 202-206, 565, 566), benzofurans (475, 482, 523), benzothiophenes (431), biphenylimidazoles (214, 17, 18), biphenyltetrazoles (7-14), diaminopyridines (58-60), dihydropyridines (48, 438-440), dihydroquinoxalinone (53-55), imidazoles (1, 8, 12, 15, 16, 100, 101, 213, 289, 379), imidazopyridines (2-6, 14, 21, 146, 236, 237), imidazolopyrimidinone (270), indanes (98, 230-232, 278, 279, 283, 284, 440, 457, 472, 525), indazoles (320, 321, 530), indoles (77, 93-96, 207, 209, 211, 212, 252, 253, 297299, 317, 318, 428-430, 442, 473, 488, 500, 511, 528, 529, 535, 553), lactams (128, 263, 276, 277, 303, 368), quinazolin(on)es (184, 185, 207, 208, 247, 331, 332, 448-450, 507), quinolines (33-35, 37-39, 322-328, 506), piperazines (38, 57, 80, 83, 84, 152-155, 343-350, 358, 359, 374-376, 381-390, 410, 438, 477-479, 486, 520, 524, 525, 555, 556) pyridazin(on)es (368-370, 380), pyrazol(in/inon)es (287, 288, 455), pyrazolopyrimidin(on)e (224, 269), pyrroles (31, 37, 86), pyrroloquinoline (49), pyridine (63, 150-152, 193, 229, 437, 457, 460), pyrimidines (129-132, 175,176, 227229, 264, 265), spiropiperidines (85, 87, 88, 515-517), and tetrazoles (12-14, 60, 276, 399). An even greater number of common substituents have been used to decorate such scaffolds in the compounds exhibited herein. The scaffolds themselves have also been used as substituents to decorate other scaffolds. Thus, one can view this set of compounds as a useful library of druggable fragments which could be combined in innumerable ways to achieve therapeutic agents that target one or more GPCRs. The scaffolds in particular tend to be small, rigid, constrained, hydrophobic templates with few rotatable bonds, and they are usually decorated with the same types of substituents. This approach has allowed receptor ‘hopping’,86,192,233,463,522 using the same scaffolds with subtly different decoration with substituents to increase affinity and selectivity for a particular GPCR. Of course, it is precisely this advantage of being ‘privileged’ that can also compromise selectivity. Have GPCR ligands really been adequately tested for specificity among large numbers of GPCRs? Unfortunately, there just is not enough data in the literature at this time to know whether privileged structures can be appropriately decorated to generate truely selective effects on a single GPCR in a single isoform. Most studies to date have tended to focus narrowly on a specific receptor or receptors, reporting selectivity between isoforms of a receptor and sometimes between a handful of like receptors, but very few


studies have reported comprehensive screening of compounds against many different GPCRs. Without such broad selectivity screening, it is not yet possible to know whether the advantages conferred by privileged scaffolds will ultimately outweigh the potential disadvantages of promiscuous receptor interactions. On this point, there is emerging evidence in support of common binding sites in GPCRs for small organic compounds, especially those recognizing privileged structural fragments.64,523 If indeed ligand binding sites in the transmembrane domains of GPCRs prove to be highly homologous, it may be quite difficult to obtain very highly selective ligands for a single GPCR out of the currently reported 850 GPCRs. As pharmaceutical development increasingly moves toward even higher target selectivity, to avoid undesirable side effects, it may become necessary to instead (or as well) target the extracellular domains of GPCRs, much as nature does, in order to obtain much higher target selectivity. The quest for safer therapeutic agents through significantly enhanced target selectivity has been a major driver in the dramatic recent shift by pharma toward protein and antibodybased therapeutic agents. Exposed on the exterior surfaces of cells, GPCRs are attractive targets for protein-based therapeutics, since the latter do not need to passively diffuse into cells to exert their effects. This review has assembled what we believe to be by far the most comprehensive single compilation of nonpeptidic ligands for peptide-activated GPCRs. In view of the comparative under-exploitation of such receptors for disease regulation, the promising stages described here in the development of pharmacologically effective new drug leads, the problems associated with target selectivity, and the clues provided which distinguish effective from fruitless drug development, we urge readers to carefully analyze this data in detail (as we have been doing) toward more effective design and development of druggable compounds that can selectively target specific peptide-activated GPCRs.

4. Abbreviations Aib AKH Cit Cpa DCIN EC50 GPCRs HTS IC50

Ki Lys(Nic) Ilys Dpr Nal Nme Nle Orn Pal pE Pen pHPPA Tfa Tho Sar

alpha-aminoisobutyric acid Adipokinetic hormone Citrulline p-Chlorophenylalanine Dark-colour-inducing neurohormon Molar concentration of an agonist that produces 50% of the maximum effect of the agonist G protein-coupled receptors High-throughput screening Molar concentration of an unlabeled agonist/antagonist that inhibits 50% radioligand binding or molar concentration of antagonist that inhibits 50% of a known concentration of agonist activity Equilibrium dissociation constant of a ligand determined in inhibition studies Ne-nicotinoyllysine Ne-isopropyllysine diaminopropionic acid b-(2-naphthyl)alanine N-methyl Norleucine Ornithine b-[pyridyl]alanine pyro glutamic acid Penicillamine p-hydroxy-3-phenyl-proprionyl Trifluoroacetyl reduced threonine sarcosine


5. Acknowledgments We thank the Australian Research Council (ARC) and the Australian National Health and Medical Research Council (NHMRC) for partial funding of the researchers who conducted this work.

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