2 Inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1

2 Inhibitors of 11b-Hydroxysteroid Dehydrogenase Type 1 XIANGDONG SU, NIGEL VICKER and BARRY V.L. POTTER Medicinal Chemistry, Department of Pharmacy and Pharmacology and Sterix Ltd., University of Bath, Bath, BA2 7AY, UK INTRODUCTION

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THE SDR SUPERFAMILY AND 11b-HSD1 ENZYMOLOGY

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PROTEIN CRYSTAL STRUCTURES OF 11b-HSD1

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POTENTIAL THERAPEUTIC INDICATIONS Metabolic Disorders Inflammation CNS Disorders Other Diseases

38 38 40 41 42

BIOLOGICAL ASSAYS In Vitro Assays Cell-Based Assays In Vivo Assays

44 44 47 48

INHIBITORS OF 11b-HSD1 Natural Compounds and their Synthetic Analogues Arylsulphonamide Analogues Triazole Derivatives Inhibitors with a Ketone Linker Inhibitors with an Amide Linker Thiazolone and Isoxazole Derivatives Pyrazole, Pyrazolone, Pyridazine and Tetrazole Derivatives

49 49 52 63 72 76 108 110

IN VIVO STUDIES OF SELECTED 11b-HSD1 INHIBITORS

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CLINICAL STUDIES ON 11b-HSD1 INHIBITORS

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CONCLUSION

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REFERENCES

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Progress in Medicinal Chemistry – Vol. 46 Edited by G. Lawton and D.R. Witty DOI: 10.1016/S0079-6468(07)00002-1

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r 2008 Elsevier B.V. All rights reserved.

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

INTRODUCTION The 11b-hydroxysteroid dehydrogenase isozymes (11b-HSDs) are members of the short-chain dehydrogenase/reductase (SDR) family, a functionally heterogeneous protein family comprising the majority of known hydroxysteroid dehydrogenases [1]. The 11b-HSDs are microsomal enzymes catalysing the inter-conversion of active glucocorticoids (GCs) and their 11-keto counterparts in specific tissues [2, 3]. The type 1 enzyme (11b-HSD1) is a nicotinamide adenine dinucleotide phosphate NADP(H)-dependent enzyme, widely expressed in many glucocorticoid target tissues. Its predominant function is to reduce cortisone (E) to active cortisol (F) in vivo, thereby locally amplifying glucocorticoid action. The type 2 enzyme is an exclusive nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase of glucocorticoids, converting cortisol to inactive cortisone. Highly expressed in classical aldosterone target tissues, 11b-HSD2 has the very important function of protecting the mineralocorticoid receptor from activation by glucocorticoids, thus allowing regulation of the receptor by aldosterone [4]. Glucocorticoid hormones play essential roles in various vital physiological processes. These include regulation of carbohydrate, lipid and bone metabolism, maturation and differentiation of cells and modulation of inflammatory responses and stress. Glucocorticoid actions primarily depend on binding to glucocorticoid receptors (GRs), leading to altered target gene transcription and also to the pre-receptor metabolism of the ligand cortisol and its non-binding precursor cortisone; the latter process is mediated by 11b-HSD isozymes. Recent studies have indicated that excessive glucocorticoid action is often associated with insulin and leptin resistance, leading to the development of obesity, type 2 diabetes and metabolic syndrome [5–8]. Therefore, inhibition of tissue-specific glucocorticoid action by regulating 11b-HSD1 constitutes a promising treatment for metabolic and cardiovascular diseases. Pharmacological inhibition of 11b-HSD1 leads to lowered hepatic-glucose production and increased insulin sensitivity in animal models. Clinical studies with a non-selective 11b-HSD inhibitor suggested that inhibition leads to improvement of insulin sensitivity in healthy individuals [9]. More recently, animal studies with selective inhibitors indicate that modulation of 11b-HSD1 activity has beneficial effects on various conditions of the metabolic syndrome, including insulin resistance, dyslipidemia, obesity and arterial hypertension. Further studies also suggest other potential target areas, such as improvement of cognitive function and treatment of ocular hypertension, due to the role of glucocorticoids and cellular activation by 11b-HSD1 in the control of these systems. The development of specific 11b-HSD1 inhibitors that have the potential to be novel treatments for cardiovascular and other metabolic diseases has

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attracted intense interest for the last few years and some excellent reviews in this area have been published [10–14]. The fast growing development of potent selective inhibitors combined with the structural knowledge and regulation of the 11b-HSD1 target should provide novel treatment strategies against 11b-HSD1-related metabolic diseases.

THE SDR SUPERFAMILY AND 11b-HSD1 ENZYMOLOGY The SDRs are a large well-established superfamily of B3,000 functionally heterogeneous enzymes present in all forms of life. The 11b-HSD1 enzyme is a member of this family of oxidoreductases, which is distinct from the metallo-dehydrogenases containing zinc and iron [15–17]. The classical family enzymes typically have 250–300 amino acid residues and have also been called short-chain alcohol dehydrogenases [18] or secondary alcohol dehydrogenases [19] and differ from the aldoketo-reductases [20] and medium-chain dehydrogenases/reductases [21]. An extended family with more than 400 amino acid residues exists, but most enzymes fall into subfamilies related to the charged amino acids in the cofactor-binding region [15, 22]. The SDR enzyme structure contains an active site and a nucleotide cofactor-binding region with a characteristic coenzyme-binding fold, the Rossmann fold [23], with a highly conserved GXXXGXG segment which gives specificity for the cofactor NAD(P)(H) [15, 24]. The catalytic region of the SDRs always contains a tyrosine (Y) and lysine (K) residue with serine (S) residues also highly conserved, denoting the catalytic triad of these enzymes. A tetrad of catalytic residues has been implicated, as an asparagine (N) is conserved in many SDR enzymes [25]. In human 11b-HSD1, this asparagine is residue N119; indeed N119 is conserved in the 11b-HSD1 sequences of many species [7]. In the crystal structure of human 11b-HSD1, N119 is some distance from the catalytic triad and is positioned behind the backbone of the cofactor. It is therefore unlikely to be involved in catalysis, but may stabilise the cofactor in the binding pocket [26]. In human 11b-HSD2 there are asparagine residues at positions 166, 167 and 171 and in the cofactor-binding region the conserved GXXXGXG sequence is GCDSGFG between residues 89 and 95. The catalytic triad in 11b-HSD2 is positioned between residues 232 and 236 with the sequence YGTSK. These differences in the cofactor-binding region and catalytic regions between human 11b-HSD1 and 11b-HSD2 will not only determine cofactor preference and substrate affinity but also inhibitor selectivity (Figure 2.1). The a-helices and b-strands of SDR proteins can be overlaid showing a repeat a-b-a-b structure to form a Rossmann fold for cofactor binding [23],

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

11β -HSD1 -------------------------------------------MAFMKKYLLP---ILGL 14 11β -HSD2 MERWPWPSGGAWLLVAARALLQLLRSDLRLGRPLLAALALLAALDWLCQRLLPPPAALAV 60 11β -HSD1 FMAYYYYSANEEFRPEMLQGKK--VIVTGASKGIGREMAYHLAKMGAHVVVTARSKETLQ 72 11β -HSD2 LAAAGWIALSRLARPQRLPVATRAVLITGCDSGFGKETAKKLDSMGFTVLATVLELNSPG 120 11β -HSD1 KVVS-HCLELGAASAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNHITNTSLNLFHD 131 11β -HSD2 AIELRTCCSPRLRLLQMDLTKPGDISRVLEFTKAHTTSTGLWGLVNNAGHNEVVADAELS 180 11β -HSD1 DIHHVRKSMEVNFLSYVVLTVAALPMLKQSNGSIVVVSSLAGKVAYPMVAAYSASKFALD 191 11β -HSD2 PVATFRSCMEVNFFGALELTKGLLPLLRSSRGRIVTVGSPAGDMPYPCLGAYGTSKAAVA 240 11β -HSD1 GFFSSIRKEYSVSRVNVSIT---------------------LCVLGLIDTETAMKAVSGI 230 11β -HSD2 LLMDTFSCELLPWGVKVSIIQPGCFKTESVRNVGQWEKRKQLLLANLPQELLQAYGKDYI 300 11β -HSD1 VHMQAAPKEECALEIIKGG-----------ALRQEEVYYDSS-----LWTTLLIRNPCRK 274 11β -HSD2 EHLHGQFLHSLRLAMSDLTPVVDAITDALLAARPRRRYYPGQGLGLMYFIHYYLPEGLRR 360 11β -HSD1 ILEFLYSTSYNMDRFINK--------------------------- 292 11β -HSD2 RFLQAFFISHCLPRALQPGQPGTTPPQDAAQDPNLSPGPSPAVAR 405

Fig. 2.1 Sequence alignment of human 11b-HSD type 1 and 2. The cofactor-binding and active site regions are underlined and dotted underlined, respectively. Asparagine (N) amino acid residues near the cofactor-binding region are double underlined.

although this similarity is not present in the substrate-binding pocket [27]. Amino acid residues that stabilise the dimer interface on human 11b-HSD1 and 2 have been postulated on the a-F helix [28]. Pioneering work from the New York groups of Monder and White led to purification of 11b-HSD from rat liver, and an antiserum was raised against the protein to clone rat cDNA, the sequence of which was later updated [29–31]. The enzyme, subsequently named 11b-HSD1, is microsomal and its activity is NADP dependent [32]. In cell-free systems, 11b-HSD1 behaves mainly as a dehydrogenase with no reductase activity being detected in the purified preparation. Subsequently, cDNAs and protein sequences were published for the human [33], mouse [34], squirrel monkey [35], sheep [36], rabbit [32], pig, cow and guinea pig [37, 38] 11b-HSD1. Human liver 11b-HSD1 was eventually purified in an active form and was postulated to exist as a dimer [39]. Maser et al. discovered an unusual kinetic mechanism of action of the human liver 11b-HSD1 [39, 40]. They determined that this isoform exhibits Michaelis–Menten kinetics with respect to cortisol, but co-operative kinetics with cortisone. In this way, 11b-HSD1 could operate at both nanomolar and micromolar substrate concentrations. However, by using recombinant purified guinea pig and human proteins, no evidence for co-operative kinetics has been found [38]. Mouse liver 11b-HSD1 can use NAD as well as NADP as cofactor [41, 42]. Guinea pig liver 11b-HSD1 has been shown to

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have equal affinity for cortisone and cortisol with an apparent Km value of 3 mM in intact cells for both substrates [37] and the purified protein exhibiting a Km value of 0.8 mM for cortisone [38]. In the original purification studies, 11b-HSD1 in the liver was shown to be bidirectional although, in contrast with its dehydrogenase activity, the reductase activity was unstable in vitro [31]. A series of studies subsequently demonstrated that the enzyme acts as a reductase unless cells are disrupted [43, 44]. Importantly, when intact cell systems, including primary cultures of hepatocytes [45], fibroblasts [46], adipose stromal cells [47, 48], lung [49] and cultured hippocampal cells [50] were studied, 11b-HSD1 activity was reductive in nature. This is supported by kinetic analysis of the enzyme, as in vitro this enzyme has a higher affinity for E (Km=0.3 mM) than for F (Km=2.1 mM), suggesting that the enzyme acts predominantly as a reductase in vivo, thereby generating F [33, 51]. However, in a few studies, 11b-dehydrogenase activity has been reported in intact cell preparations, with the direction of 11b-HSD1 catalysis appearing to vary according to the physiological or developmental status of a particular cell type. In Leydig cells, both 11b-dehydrogenase and oxoreductase activities have been reported [52–54]. Freshly isolated cells display dehydrogenase activity that dramatically decreases after several days’ culture in vitro. However, others have found predominant 11b-reduction [55]. In human omental adipose stromal cells, 11b-HSD1 switches from a dehydrogenase to a reductase when these cells differentiate into adipocytes [56]. In neuronal cells, 11bHSD1 reductase and dehydrogenase activities have been reported [57, 58]. These findings indicate a possible important role for 11b-HSD1 dehydrogenase activity in normal physiology, with the relative contribution of the dehydrogenase and reductase activities being important in controlling the overall equilibrium of local glucocorticoid levels [59]. In every case, however, when cells are disrupted or the enzyme purified, reductase activity is lost. This striking change in directionality between intact cells and homogenates seems to reflect the specific intracellular localization of 11b-HSD1 within the lumen of the endoplasmic reticulum (ER), where neighbouring enzymes may be powerful generators of the reduced cosubstrate NADPH. Indeed, studies using purified human enzyme have shown that the equilibrium constant for the E to F direction (defined as the concentration of products divided by concentration of reactants) is 0.03. Given that a value of 1 would represent the exact equilibrium position, a value of 0.03 indicates a strong preference towards dehydrogenase (F to E) activity [60]. Reductase activity can be regained from tissue homogenates and purified enzyme, upon inclusion of an NADPH regeneration system employing the cytosolic enzyme glucose-6-phosphate dehydrogenase [60, 61]. This suggests that reductase activity predominates in intact cells

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

due to a high level of NADPH present within the ER lumen. Recently, it has been shown that hexose-6-phosphate dehydrogenase (H6PDH) has a key role in generating NADPH levels in the ER [62]. The 11b-HSDs are different from most members of the SDR family owing to the presence of one or more amino-terminal transmembrane domains. Other members of the SDR family having this secondary structural characteristic include some 17b-HSD isozymes and follicular variant translocation protein isozymes. There is a high level of sequence homology of 11b-HSD across species, particularly within the cofactorbinding region (GASKGIG) and the catalytic site (YSASK) (Figure 2.2). The 11b-HSD1 protein has a single hydrophobic N-terminal extension preceding the cofactor-binding domain, indicating that this region anchors the protein within microsomes. The precise topology of 11b-HSD1 was demonstrated using 11b-HSD1 constructs with FLAG epitopes attached at the N- and C-terminal regions [63]. The protein was shown to be intrinsic to the membrane of the ER, having a five amino acid region on the cytosolic side of the membrane, followed by a single transmembrane domain (Figure 2.3). Most of the enzyme is present in the lumen of the ER. Chimeric proteins, where the N-terminal regions from 11b-HSD1 and 11b-HSD2 were exchanged, led to inverted orientation within the ER. Both chimeric proteins were inactive [63]. In the single N-terminal transmembrane region, the charge distribution of two positively charged lysine residues on the cytoplasmic side, and two negatively charged glutamate residues, indicates that these are key residues for the orientation of 11b-HSD1 in the ER membrane. Mutation of the Lys5 residue suggests that it is critical in the determination of 11b-HSD1 topology and that its charge and specific side chain are both important [63]. The importance of the transmembrane domain upon 11b-HSD1 activity has been studied, but with conflicting results [64, 65]. The lumen of the ER promotes the formation of disulphide bonds, and studies have indicated that there are important intra-chain disulphide bonds within the 11b-HSD1 protein [32]. The importance of glycosylation upon 11b-HSD1 activity has been widely reported. Examination of the 11b-HSD1 peptide sequence revealed the presence of two potential N-linked glycosylation sites in the cloned rat enzyme (asparagine-X-serine, residues 162–164 and 207–209), consistent with the original description of the purified rat hepatic 11b-HSD1 as a glycoprotein [66]. Interestingly, studies in the vaccinia expression system showed that although partial inhibition of glycosylation decreased dehydrogenase activity by 50%, it did so without affecting reductase activity [61]. The relative importance of the two glycosylation sites was further studied by mutagenesis in Chinese hamster ovary (CHO) cells.

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Fig. 2.2 Alignment of 11b-HSD1 amino acid residues across species created using CLC Free Workbench. The white shading represents primary consensus sequence (identical amino acids), light grey shading indicates the secondary consensus sequences (groups amino acids with similar chemical structures), and dark grey shading represents amino acids that vary by chemical structure across species. Boxed residues indicate the cofactor-binding region (GXXXGXG) and the catalytic site (YXXXK). The double arrow highlights the residues proposed to form the dimer interface. The asterisk highlights putative N-linked glycosylation sites.

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

O

OH

O

OH

O

OH

HO

H H

OH

H H

H

O

H

O

NADPH

cortisol

11β-HSD1

cortisone

Cytosol

ER lumen

NADP

H6PDH

6PGL

G6P

Fig. 2.3 Schematic representation of the interaction between 11b-HSD1 and H6PDH, which provides NADPH as cofactor to permit reductase (cortisone to cortisol) activity. G6P, glucose-6phosphate; 6PGL, 6-phosphogluconolactonate.

Modification of the first site decreased dehydrogenase and reductase activities to 75% and 50% of the wild type, whereas mutation of the second site caused an almost complete abolition of both the activities [67]. These findings show that, in the rat, glycosylation of 11b-HSD1 at N207 plays a major role, and at N162 a minor role in catalysis. This is consistent with the incomplete conservation of the corresponding residues between species. In human 11b-HSD1, there are three putative glycosylation sites. The Asn-X-Ser sites are at positions 123–125, 162–164 and 207–209 of the protein (Figure 2.2). Human 11b-HSD1 has been expressed in Escherichia coli, where the biosynthesis of N-linked glycoproteins does not occur. Fully active non-glycosylated 11b-HSD1 enzyme activity generated in E. coli, with kinetic properties for both dehydrogenase and reductase activities similar to those reported in mammalian systems, has been reported [52]. Glycosylation is therefore not required for correct protein folding or enzyme activity of the human 11b-HSD1, but nevertheless may play a role in preventing protein aggregation, in addition to stabilizing the overall structure within the ER.

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PROTEIN CRYSTAL STRUCTURES OF 11b-HSD1 A number of crystal structures of 11b-HSD1 have been solved including murine [68], guinea pig [69], human [26], human in complex with NADP and carbenoxolone (CBX) and human in complex with a potent selective small molecule inhibitor of 11b-HSD1 [70, 71]. These have Protein Data Bank (PDB) [72] codes 1Y5M, 1XSE, 1XU9, 2BEL, 2ILT, 2IRW and the best resolution achieved is 1.55 A˚. Although the active site variability of 11b-HSD1 revealed by selective inhibitors and cross-species comparisons has recently been reported, the above crystal structures are useful tools in structure-based drug design [73]. Amgen crystallised a binary complex of murine 11b-HSD1 with NADP(H) to a resolution of 2.3 A˚ and a ternary complex with corticosterone and NADP(H) to a resolution of 3.0 A˚ [68]. The enzyme forms a homodimer in the crystal and has a fold similar to those of other members of the family of SDRs. The structure shows a novel folding feature at the C-terminus of the enzyme. The C-terminal helix insertions provide additional dimer interactions that exert an influence on the conformations of the substratebinding loops and present hydrophobic regions for potential membrane attachment. The structure also reveals how 11b-HSD1 achieves its selectivity for its substrate. Biovitrum reported the crystal structure of recombinant guinea pig 11b-HSD1 which was determined in complex with NADP to a resolution of 2.5 A˚ [69]. The overall structure of guinea pig 11b-HSD1 shows a clear relationship to other members of the superfamily of SDRs but contains a unique C-terminal helical segment that meets the three key functions involved in subunit interactions, contributes to the active site architecture, and is necessary for lipid–membrane interactions. The structure provides a model for enzyme–lipid bilayer interactions and suggests a funnelling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site. Hosfield et al. reported biophysical, kinetic, mutagenesis and structural data on two ternary complexes of human 11b-HSD1 [26]. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11b-HSD1 C-termini converge to form an as yet uncharacterised tetrameric motif. A C-terminal Pro-Cys unit is positioned at the centre of the tetramer and forms reversible enzyme disulphide bonds that modify the activity of the enzyme. Conformational flexibility at the tetramerisation interface is linked to structural changes at the enzyme active site, thus implying how the central Pro-Cys moiety may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding the activity of 11b-HSD1.

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

Oppermann et al. solved the structure of human 11b-HSD1 in complex with NADP and CBX (PDB code 2BEL) [72]. The high-resolution structures of human, murine and guinea pig 11b-HSD1 show critical differences in active site architecture across species. Abbott reported X-ray crystal structures of human 11b-HSD1 with potent adamantyl amide 11b-HSD1 inhibitors (PDB code 2ILT, 2IRW) [70, 71]. X-ray data were collected to 3.1 A˚ resolution for 2IRW and 2.3 A˚ resolution for 2ILT, and molecular replacement with refinement revealed electron density for the cofactor NADP and compound. Comparison with previously determined structures of h-11b-HSD1 reveals that the inhibitor is bound in the steroid-binding site neighbouring the bound cofactor NADP [26]. The hydrophobic adamantyl group is located near the nicotinamide portion of the cofactor and the primary amide is in close proximity to the pyrophosphate moieties of the cofactor. The central amide is predicted to form interactions with active site residues and the carbonyl is 2.8 and 2.9 A˚ from the hydroxyl groups of Ser170 and Tyr183, respectively. The gem-dimethyl and ether-linked phenyl groups extend into the hydrophobic cavity of the steroid-binding site, where Tyr177 is positioned in the bottom of the pocket. The close packing around the phenyl moiety of the inhibitor suggests the protein may adopt different conformations to accommodate compounds with varied substitution at different positions of the aromatic ring.

POTENTIAL THERAPEUTIC INDICATIONS Selective inhibitors of 11b-HSD1 have considerable potential as treatments for a number of important diseases including type 2 diabetes, obesity and metabolic syndrome [12]. 11b-HSD1 has also been implicated in inflammation [74], atherosclerosis [75] and central nervous system (CNS) disorders [76, 77].

METABOLIC DISORDERS

Metabolic syndrome is a group of metabolic abnormalities associated with increased cardiovascular and mortality risks. Glucocorticoid excess has been linked to the development of metabolic syndrome, and intracellular glucocorticoid levels are regulated by 11b-HSD1. Inhibitors of the enzyme are being investigated as a potential therapy for insulin resistance and metabolic syndrome. Many structurally different classes of 11b-HSD1 inhibitors are currently under investigation throughout the pharmaceutical industry, highlighting the progress already made in this area [10].

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The role of glucocorticoid action in the pathophysiology of metabolic syndrome has been studied clinically and a positive correlation exists between adipose tissue 11b-HSD1 expression or activity and features of metabolic syndrome and body-mass index [78]. The first evidence that the correlation between 11b-HSD1 expression in adipose tissue and body-mass index is independent of genetic background was validated in a study of young adult monozygotic twins [79]. Direct evidence that inhibition of the adipose 11b-HSD1 is appropriate to treat aspects of metabolic syndrome has been provided by the observation that adipose-specific glucocorticoid inactivation protects against diet-induced obesity [80]. Although both impaired hepatic regeneration of cortisol by 11b-HSD1 and elevated adipose 11b-HSD1 activity were observed in obese humans [81, 82], the association of adipose 11b-HSD1 activity with obesity, insulin resistance and other features of metabolic syndrome has been consistently observed in different groups of obese subjects, including obese men and women [81, 83, 84]. As there was no detectable difference in 11b-HSD1 activity between obese type 2 diabetics and their obese controls, this suggests the dysregulation of 11b-HSD1 is better associated with obesity than with the diabetic phenotype [85]. There is a high correlation between visceral obesity with insulin resistance and metabolic syndrome, indicating that increased 11b-HSD1 activity in adipose tissue may have a role in metabolic syndrome [86]. Adipose tissue appears to be the primary target tissue for this multifactorial disease with the liver being the secondary target. When 11b-HSD2 is selectively over-expressed in adipose tissue, protection against dietinduced obesity is observed, concurring with this suggestion [75]. With the known effects of glucocorticoids on adipose tissue function and distribution, it has been postulated that the enhanced conversion of cortisone to cortisol within omental adipose tissue plays an important role in the pathogenesis of central obesity. Cortisol is essential for adipocyte differentiation [87] and the autocrine generation of cortisol through the action of 11b-HSD1 is able to regulate this process. Both cortisol and cortisone promote differentiation. Inhibition of 11b-HSD1 prevents cortisone-mediated adipocyte differentiation by blocking the activation of cortisone to cortisol [48]. In both rat and human preadipocytes, glucocorticoids exert an antiproliferative effect [88] and the inhibition of 11b-HSD1 was shown to overcome the antiproliferative action of cortisone [89]. The pharmacological inhibition of 11b-HSD1 has been reported to be therapeutic in mouse models of metabolic syndrome [75]. Administration of a selective potent 11b-HSD1 inhibitor lowered body weight, insulin, triglyceride and cholesterol levels in diet-induced obese mice. Fasting glucose, glucagon and free fatty acids levels were also lowered. Improved glucose tolerance was observed in this mouse model of type 2 diabetes.

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Most importantly, inhibition of 11b-HSD1 slowed plaque progression in a murine model of atherosclerosis, a key clinical outcome of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E (apoE) exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11b-HSD1 inhibitor [75]. These data provide the first evidence that pharmacological inhibition of intracellular glucocorticoid activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself. Recent reviews have implicated 11b-HSD1 as a target for the treatment of metabolic diseases [12, 13], and with proof of concept for the pharmacological inhibition of insulin resistance, obesity, dyslipidemia and hypertension obtained, positive clinical data are eagerly awaited.

INFLAMMATION

Glucocorticoids are widely used for their potent anti-inflammatory effects. Endogenous glucocorticoids are immunomodulatory and control both adaptive and innate immune responses. Recently, it has become apparent that an important level of control over endogenous glucocorticoid action is exerted by 11b-HSD1 and 11b-HSD2. Although 11b-HSD1 activity has been shown to play an important role in the metabolic actions of glucocorticoids, its role in the immune response has, until recently, remained unclear. Recent evidence has been reviewed as to the role of 11b-HSD1 in the inflammatory response [74]. Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. The differential expression, function and response to inflammatory stimuli of 11b-HSD1 in human fibroblasts have been reported as a possible mechanism for tissue-specific regulation of inflammation [90]. Expression, activity and function of 11b-HSD1 were measured in matched fibroblasts derived from the synovium, bone marrow and skin obtained from patients with rheumatoid arthritis or osteoarthritis. 11b-HSD1 was expressed in fibroblasts from all tissues, but mRNA levels and enzyme activity were higher in synovial fibroblasts. In the presence of 100 nmol/L cortisone, IL-6 production was significantly reduced in synovial, but not dermal or bone marrow, fibroblasts. This was prevented by co-treatment with an 11b-HSD1 inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data showed that differences in fibroblast-derived glucocorticoid production

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from 11b-HSD1 between cells from distinct anatomical locations may predispose certain tissues to develop an inflammatory response [90]. Glucocorticoids promote mechanisms important for the normal resolution of inflammation, notably macrophage phagocytosis of leucocytes undergoing apoptosis. In a recent study in a mouse model of acute inflammation, a single thioglycollate injection resulted in high expression of 11b-HSD1 oxoreductase but not 11b-HSD1 dehydrogenase activity in peritoneal cells. The 11b-HSD1 oxoreductase activity remained high in peritoneal cells until the inflammation resolved. In vitro, the 11b-HSD1 substrate, 11-dehydrocorticosterone, increased macrophage phagocytosis of apoptotic neutrophils to the same extent as corticosterone. This effect was dependent upon 11b-HSD1, as these cells solely expressed 11b-HSD1. CBX, a non-selective 11b-HSD1 inhibitor, prevented the increase in phagocytosis elicited by 11-dehydrocorticosterone. Macrophages from 11b-HSD1deficient mice failed to respond to 11-dehydrocorticosterone. In vivo, 11b-HSD1-deficient mice showed a delay in acquisition of macrophage phagocytic competence and had an increased number of free apoptotic neutrophils during sterile peritonitis. Importantly, in preliminary experiments, 11b-HSD1-deficient mice exhibited delayed resolution of inflammation in experimental arthritis. These findings suggest that 11b-HSD1 may be a component of mechanisms engaged early during the inflammatory response that promote its subsequent resolution [91].

CNS DISORDERS

Glucocorticoids influence a broad range of CNS processes, altering neurotransmission, electrophysiological activity, metabolism, cell division and survival. Recent results have highlighted the important and very different roles that the two isozymes play in the brain [76], where they modify learning, memory and fear behaviour, as well as regulate their own secretion by a negative feedback action. In the CNS, 11b-HSD1 is expressed principally in the cerebellum, cortex and hippocampus [92–94]. Lack of tissue glucocorticoid reactivation in 11b-HSD1 knockout mice ameliorates age-related learning impairments, as this results in apparent lower intrahippocampal corticosterone levels and reduces glucocorticoid-associated cognitive decline during aging [95]. This low corticosterone tissue environment is maintained even though there is a hyperactive hypothalamic–pituitary–adrenal (HPA) axis and elevated basal and stress-induced plasma corticosterone levels. Conversely, the major central effects of 11b-HSD1 are seen in development, as expression of 11b-HSD1 is high in foetal and certain parts of the neonate brain, but is confined to a few

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discrete regions of the adult brain. Loss of 11b-HSD2 from the foetus and tissues derived from the foetus results in altered development of the cerebellum in the neonatal period and a life-long phenotype of anxiety, consistent with early-life glucocorticoid programming. Inhibition of 11b-HSD by administration of CBX has been shown to improve cognitive function in healthy elderly men and in type 2 diabetics [96]. The glucocorticoid-related genetic susceptibility for Alzheimer’s disease (AD) has been investigated in a population of 814 AD patients and unrelated control subjects. This study revealed that a rare haplotype in the 5u regulatory region of the gene encoding 11b-HSD1 was associated with a six-fold increased risk for sporadic AD [97]. Merck has recently shown that an 11b-HSD1 inhibitor demonstrated positive effects in animals subjected to novel object recognition and passive avoidance tests. The role of the 11b-HSDs in the brain has been discussed [76], and transgenic mice with tissue-specific and temporal-regulation of enzyme expression in central regions of the brain are needed to further understand the effects of 11b-HSDs in the CNS and the potential of 11b-HSD1 inhibitors to treat neurodegenerative disorders.

OTHER DISEASES

It has been reported that there are partial defects in 11b-HSD1 in patients with polycystic ovarian syndrome (PCOS) and 11b-HSDs are dysregulated with hyperandrogenism in PCOS [98]. Adrenal secretion of cortisol and androgens is increased in women with PCOS and these increases may be explained by dysregulation of 11b-HSD causing increased oxidation of cortisol to cortisone, which cannot be accounted for by obesity [98]. There is a genetic component to PCOS together with other factors [99, 100]. Genetic analysis of HSD11B1 in cortisone reductase-deficient patients will give a wider perspective of the importance of 11b-HSD1 in PCOS. The human eye is an important target tissue for steroid hormones, and glucocorticoids have been implicated in the pathogenesis of ocular disease, including glaucoma. Systemic administration of glucocorticoids increases intraocular pressure (IOP) and patients with primary open angle glaucoma (POAG) show increased GC sensitivity [101, 102]. The expression and putative role of 11b-HSD enzymes within the human eye have been investigated and results suggest that the 11b-HSD1 modulates the steroidregulated sodium transport across the ciliary nonpigmented epithelial (NPE) cells and thus can influence IOP [101]. Data suggest that CBX lowers IOP in patients with ocular hypertension and this is mediated through 11b-HSD1 inhibition in the NPE [103]. Therefore, topical administration of

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selective inhibitors of 11b-HSD1 may provide a novel treatment for glaucoma. The identification of 11b-HSD1 in human bone has raised the question of whether 11b-HSD1 has a role in the pathogenesis of age-related osteoporosis, since chronic exposure to GCs has integral effects on bone structure and function [104]. The expression and functional consequences of 11b-HSD activity in human bone have been described [105]. The expression of 11b-HSD isozymes in human osteosarcoma cell lines, osteoblast cultures and foetal bone have been reported. The characterisation of 11b-HSD expression in adult human bone using specific anti-human 11b-HSD antibodies, riboprobes and enzyme activity has been studied [105]. In addition, the effect of 11b-HSDs on bone metabolism in vivo was assessed using the non-selective 11b-HSD inhibitor CBX in eight normal male volunteers. In fresh normal human bone tissue, both 11b-dehydrogenase and reductase activities were demonstrated. There was considerable interindividual variation in the dehydrogenase, but not reductase, activity. In bone homogenates, activity was NADP-dependent, suggesting the presence of 11b-HSD1. This was confirmed by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. Immunohistochemical and in situ hybridization studies demonstrated 11b-HSD1 expression in cells of the osteoblast lineage and in the osteoclasts. The 11b-HSD2 enzyme was expressed, but only in osteoblasts and at a low level. Ingestion of 300 mg of CBX by eight normal volunteers for 7 days resulted in a significant decrease in the bone resorption markers pyridinoline and deoxypyridinoline, with no overall change in the bone formation markers procollagen type I C-terminal peptide (PICP) and procollagen type I N-terminal peptide (PINP). These data suggest that local tissue metabolism of GCs is likely to be important in determining the sensitivity of both osteoblasts and osteoclasts to glucocorticoids. In particular, variation in 11b-HSD isozyme expression and activity may explain individual variation in susceptibility to glucocorticoid-induced osteoporosis. Many genes are important in the pathogenesis of osteoporosis [106]; however, to date the contribution of 11b-HSD1 has not been evaluated with large genetic studies. The H6PDH gene has the cytogenetic chromosome locus 1p36 which has been associated with hip and femoral neck bone mass densities. It is reported that 11b-HSD expression and GC synthesis are directed by a molecular switch during osteoclast differentiation in the human SV-HFO osteoclast cell line [107]. Dexamethasone (DMX) induces differentiation of SV-HFO cells, but in the absence of DMX, 11b-HSD1 mRNA activity strongly increased from 12 to 19 days. Promoter–reporter studies showed that specific regions of the 11b-HSD1 gene are involved in the

44

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

differentiation-controlled regulation of this enzyme. The functional implications of these changes in 11b-HSD1 are shown by the induction of osteoblast differentiation in the presence of cortisone. The study demonstrated the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11b-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism. At this early stage, the effect of 11b-HSD1 inhibition on osteoporosis requires further examination before the pharmacological potential of this approach can be predicted. Both the genetic and enzymatic characterization of 11b-HSD1 and its role in physiology and pathology in a tissue-specific manner have been reviewed [7]. The molecular basis of cortisone-reductase deficiency, the putative ‘‘11b-HSD1 knockout state’’ in humans, was defined and is caused by intronic mutations in HSD11B1 that decrease gene transcription, together with mutations in H6PDH, an endoluminal enzyme that provides reduced NADP as cofactor to 11b-HSD1 to permit reductase activity. The speculation that H6PDH activity, and therefore reduced NADP supply, may be crucial in determining the directionality of 11b-HSD1 activity, has been raised.

BIOLOGICAL ASSAYS The discovery of novel 11b-HSD type 1 inhibitors from concept to clinic requires rational, robust and convenient screening systems, encompassing relevant in vitro and in vivo methodologies. Initial assays are needed to confirm the mechanism of action of a compound and to show that the required target is being inhibited. In vitro assays using isolated enzymes or microsomal enzyme supernatants have demonstrated that compounds inhibit cortisol formation from cortisone by the inhibition of 11b-HSD1. To demonstrate efficacy in vivo, the inhibition of 11b-HSD1 in cell lines and in animal models has been established. Assay development to identify novel 11b-HSD1 inhibitors has been explosive over the last decade and the spectrum of effort is outlined.

IN VITRO ASSAYS

The initial attempts to identify inhibitors of 11b-HSD generally involved assays using microsomal fractions or homogenates from animal tissues, such as rat liver and kidney [108–114] or sheep liver and kidney [114, 115].

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The activities were usually measured using either a thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC) method to detect the conversion rate of radio-labelled cortisone to cortisol for the type 1 enzyme or cortisol to cortisone for the type 2 enzyme. It is known that in tissue homogenates and microsomal fractions 11b-HSD1 works bidirectionally, oxidizing and reducing physiological glucocorticoids depending on the cofactor [31]. The protein sequence of 11b-HSD1 differs between species, which leads to significant species variability in the potency of inhibitors [116]. Therefore, human enzyme-based assays were developed to provide a more accurate measurement of a given compound’s inhibitory property. Hult et al. [117] obtained human 11b-HSD type 1A cDNA by RT-PCR of a total RNA preparation from a liver transplantation sample and overexpressed the human 11b-HSD1 in yeast Pichia pastoris. The cell extract or microsomes from the transformed strains displayed both dehydrogenase and reductase activities, which were up to 10 times higher than in human liver microsomes. In the whole cell, recombinant human 11b-HSD1 showed reductive activity only, the same as was found in mammalian systems [44, 118]. Using this enzyme system combined with an HPLC methodology, some synthetic steroids and xenobiotics have been screened against human 11b-HSD1 [117]. The same methodology was also applied in the identification of perhydroquinolylbenzamide derivatives as novel inhibitors of 11b-HSD1; the inhibitor’s selectivity over human 11b-HSD2 was determined using lysates of an SW-620 human colon carcinoma cell line as the enzyme source [119]. The microsome preparations from human liver tissue taken from unaffected liver segments were also used to demonstrate the bi-directional activity of human 11b-HSD1 in the same system and were utilised in the assay of inhibitors of this enzyme by a TLC method [120]. Selectivity over 11b-HSD2 was measured with the same protocol using microsomes from human kidney cortex tissue, which possess exclusively 11b-HSD2 activity [121]. Schweizer et al. developed rapid screening assays for 11b-HSD1 and 11b-HSD2 using lysates from stably transfected cells [122, 123]. Human embryonic kidney 293 (HEK293) cells were transfected with the plasmid for expression of carboxy-terminally FLAG-epitope tagged 11b-HSD1 or 11b-HSD2, respectively [63]. The lysates from the 11b-HSD1-transfected HEK293 cell line were used for incubation with [3H]cortisone, unlabelled cortisone and NADPH for measuring reductase activity. Similarly, the lysates from the 11b-HSD2-transfected HEK293 cells were used for incubation with [3H]cortisol, unlabelled cortisol and NAD+ for measuring oxidative activity. The conversion rate of cortisone to cortisol or vice versa was determined by a TLC method using a chloroform/methanol (9:1) solvent system.

46

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

The TLC or HPLC methodology used in the assays to separate cortisol from cortisone is generally labour-intensive and time-consuming, and is therefore not suitable for high-throughput screening. The well-established scintillation proximity assay (SPA) has been widely used for the rapid and sensitive measurement of a range of biological processes and has been reviewed recently [124]. Initially, the SPA was applied in the determination of cortisol in plasma and this showed excellent agreement with a conventional radioimmunoassay [125]. This methodology was adapted to high-throughput screening of inhibitors of recombinant human and murine 11b-HSD1 enzyme produced by over-expression in the yeast P. pastoris [126]. In the assay, the enzyme was treated with a substrate/cofactor mixture of [3H]cortisone/NADPH and inhibitors. After quenching the enzymatic reaction by adding glycyrrhetinic acid, the [3H]cortisol product was captured by mouse monoclonal anti-cortisol antibodies and was then bound to SPA beads coated with anti-mouse antibodies. The amount of [3H]cortisol bound to bead could be measured by the scintillation count (Figure 2.4). An SPA was also further developed and optimised at Merck to facilitate the identification of novel 11b-HSD1 inhibitors in 96- or 384-well highthroughput formats [127, 128]. The [3H]cortisol produced by the enzymatic process was detected by a monoclonal antibody bound to protein A-coated SPA beads. The 11b-HSD2 screening was also performed by incubating 11b-HSD2 microsomes with [3H]cortisol/NAD+ and monitoring the disappearance of the substrate [127]. Moreover, the SPA for 11b-HSD1 was applied with an enzyme source from the crude lysates of E. coli expressed with truncated human or mouse 11b-HSD1 (lacking the first 24 amino acids) using the pET28 expression system [129–131]. Homogeneous time-resolved fluorescence (HTRF) offers a homogeneous, more robust method and is easier to automate than SPA. Unlike SPA, the

Fig. 2.4 Principle of SPA of 11b-HSD1. The generated [3H]cortisol binds to the SPA bead with monoclonal anti-cortisol antibodies and the complex emits light signal to be detected. (See colour plate section at the end of the book.)

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HTRF assay is not radiometric, and provides a more attractive alternative. Based on patented technology [132–134], Cisbio has developed an HTRF assay allowing rapid and accurate cortisol measurement even in complex samples such as liver microsomes, whole cells and animal serum with low cross-reactivity with cortisone and other steroid hormones. This assay is based on fluorescence resonance energy transfer (FRET) between a Eu3+ cryptate donor and allophycocyanin, a second fluorescent label (acceptor). Using this HTRF methodology, novel 11b-HSD1 inhibitors have been identified with human microsomal enzyme systems [135–137].

CELL-BASED ASSAYS

Cell-based assays have the advantage of simultaneously evaluating a ligand’s ability to penetrate the cell membrane and its binding properties, therefore, cell-based assays generally provide more accurate measurements of a given ligand’s efficacy. It was demonstrated that 11b-HSD1 functions primarily as a reductase within the intact whole cell with both the yeast [117] and mammalian systems [44, 118]. A cellular 11b-HSD1 assay was developed in a primary rat hepatocyte system. Compounds were evaluated for their ability to inhibit the conversion of 11-dehydrocorticosterone to corticosterone using a radioTLC method [119]. Similar assays were also performed with endogenous 11b-HSD1 in intact mouse C2C12 myotubes and 3T3-L1 adipocytes [138]. Untransfected HEK293 cells lack endogenous 11b-HSD activity and this cell line has been shown to be a suitable system for evaluating 11b-HSD activity after being transfected with the plasmid for expression of 11b-HSD1 or 11b-HSD2 [63, 122, 139]. The high-throughput cell-based assays were conducted on the human 11b-HSD1 transfected HEK293 cell line using either an SPA [135, 140, 141], an enzyme-linked immunosorbent assay (ELISA) [142–144] or a fluorescence polarisation immunoassay (FPIA) [131, 145]. The expression of H6PDH within the cell is essential for the 11b-HSD1 oxoreductase activity as it determines the reaction direction by reproducing the cofactor NADPH in situ [146–148]. Therefore, the HEK293 cell line transfected with both 11b-HSD1 and H6PDH showed much higher oxoreductase activity and was utilised for evaluating enzyme inhibitors by a TLC method [138]. Since endogenous CHO cells do not have detectable 11b-HSD1 or 11b-HSD2 activity, they could also be transfected with human 11b-HSD1 or 11b-HSD2 and these have been used in cell-based assays to determine the potency and selectivity of enzyme inhibitors [149].

48

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

A non-radioactive cell-based assay was performed on human cervical carcinoma derived (HeLa) cells co-transfected with full-length human 11b-HSD1 with the glucocorticoid response element (GRE) linked to a b-galactosidase reporter gene [150–154]. The principle of the assay is that cortisol, converted from cortisone by 11b-HSD1 within the HeLa cell, binds to and activates the glucocorticoid receptor. The activated glucocorticoid receptor then binds to the GRE and initiates transcription and translation of b-galactosidase. The 11b-HSD enzyme activity can then be evaluated with high sensitivity by a colourimetric method. Another high-throughput non-radioactive cell-based assay is based on a liquid chromatography-tandem mass spectrometry technique; it was used on a human 11b-HSD1 transfected HeLa cell line or a hepatic human (WRL) cell line [155]. This assay could monitor cortisol and cortisone levels simultaneously. The cell-based assays were also conducted with human Fa2N-4 immortalised cells, which are derived from human hepatocytes and exhibit many characteristics of normal human hepatocytes. Using an immunoassay method, inhibition of 11b-HSD1 was assessed with this cell model by measuring the decrease of enzyme-produced cortisol accumulation in cultures co-treated with cortisone and potential inhibitors [156].

IN VIVO ASSAYS

Many groups are now placed to take 11b-HSD1 inhibitors from concept to man. The in vivo model used initially to evaluate an inhibitor’s glucoselowering effect was the KKAy mouse, a rodent model of type 2 diabetes [126]. A compound’s effect on corticosterone production through 11bHSD1 inhibition could be evaluated in adrenalectomised (ADX) mice to avoid the confusing influence of adrenal-derived steroids [119]. Other rodent models for profiling the 11b-HSD1 inhibitor’s pharmacodynamic (PD) activities include a diet-induced obesity (DIO) mouse model, an HF/ streptozotocin (STZ) type 2 diabetes mouse model and an apoE knockout mouse model [75, 145]. The adipose tissue, which is regarded as one main target for 11b-HSD1 inhibition, was used in an ex vivo assay for evaluating an inhibitor’s pharmacodynamic effect [75]. With a novel method provided by scientists from Merck, the measurement of 11b-HSD activity could be performed in intact whole animal tissues in the presence of systemically or ex vivo administered inhibitors [157]. The data from these in vivo studies would aid the investigation of 11b-HSD1 inhibitor effects on multiple

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49

aspects of metabolic syndrome. A preliminary study showed a method of in vivo quantification of 11b-HSD1 activity in human patients with cirrhosis of the liver [158].

INHIBITORS OF 11b-HSD1 NATURAL COMPOUNDS AND THEIR SYNTHETIC ANALOGUES

Many 11b-HSD inhibitors have been identified from various natural resources including glycyrrhizin from liquorice root and polyphenols from tea and herb extracts. A recent report also revealed that a heat-stable component from coffee extract inhibits endogenous and recombinant 11bHSD1 activity with a selectivity of 7–10-fold over 11b-HSD2 and 17b-HSD1, which contributes at least part of the anti-diabetic effect of coffee consumption [159]. Glycyrrhizin (1) and its derivatives (2, 3) exhibit pseudoaldosteroidism, hypoleukaemic activity and hypertensive effects [160–162]. It was later revealed that these effects were induced by inhibition of 11b-HSD2 rather than a direct mineralocorticoid effect [108, 163]; although some evidence suggested that liquorice-induced hypertension involved more than simply 11b-HSD inhibition [164]. 18b-Glycyrrhetinic acid (18b-GA; 2) and its hemisuccinate derivative CBX (3) showed non-selective inhibition of 11b-HSDs with IC50 values in the nanomolar range [108, 165]. Attempts to identify selective 11b-HSD1 inhibitors by optimisation of 18-GA derivatives at the 3- or 30-positions of the skeleton generated mostly nonselective inhibitors or the potent type 2 selective inhibitor (4) [111–113, 166]. The 18b-GA derivatives (5–7), with apparent selectivity for rat 11b-HSD1, showed IC50 values from 400 nM to low micromolar range, but these compounds exhibited very weak inhibition of 11b-HSD1 in a HEK293 cell line [167]. CBX (3), originally developed for the treatment of peptic ulcers, has been used to investigate the consequences of 11b-HSD1 inhibition on healthy men and men with type 2 diabetes [9, 168, 169]. When administered orally at a dose of 100 mg/8 h for 7 days, CBX could increase hepatic insulin sensitivity and decrease glucose production through the inhibition of hepatic 11b-HSD1. It was also reported that the topical application of 18b-GA in healthy women could reduce the thickness of subcutaneous thigh fat possibly through the blocking of 11b-HSD1 [170]. Nevertheless, the non-selectivity of these GA derivatives limits their potential clinical application as 11b-HSD1 inhibitors. Chenodeoxycholic acid (CDCA, 8) was reported as a selective 11b-HSD1 inhibitor with IC50 values of 2.8 mM for oxoreductase activity and 4.4 mM

50

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

for dehydrogenase activity on the human liver microsomal enzyme, whereas its analogue lithocholic acid (LCA, 9) is equally potent for 11b-HSD1 with an IC50 value of 2.4 mM, but lacks selectivity over 11b-HSD2 [120]. Studies with lysates from 11b-HSD2-transfected HEK293 cells indicated that CDCA, LCA and deoxycholic acid (DCA) (10) inhibited 11b-HSD2 with IC50 values of 22, 7 and 38 mM, respectively [171]. The controversy regarding the inhibitory activity of CDCA lies in the isoform specificity and its directional effects on 11b-HSD1. It was demonstrated that CDCA preferentially affects 11b-HSD1 dehydrogenase and only inhibits 11b-HSD1 oxoreductase and 11b-HSD2 dehydrogenase at high concentrations exceeding 37 and 70 mM, respectively [172].

R2

COOH O

H

R1

R2

H

COOH

H RO

HO

HO

H

R1

(1) R = -(glucuronide)2 (4) R1 = O, R2 = CONHCH2CH2OH (8) R1 = OH, R2 = H (2) R = H (5) R1 = O, R2 = CH2OCH2CH2OH (9) R1 = H, R2 = H (3) R = -OCOCH2CH2COOH (6) R1 = α-CH , β-OH, R2 = COOH (10) R1 = H, R2 = OH 3 (7) R1 = CH2, R2 = COOH

Endogenous steroids (metabolites of progesterone) have been identified as 11b-HSD inhibitors. 11b-Hydroxy-progesterone (11) selectively inhibits 11b-HSD from the homogenates of rat vascular smooth muscle cells in the direction of dehydrogenase, whereas 11-keto-progesterone (12) inhibits 11b-HSD only in the direction of oxoreductase [173]. Further studies indicated that 3a, 5a-tetrahydro-cortisone (13), 5a-dihydro-corticosterone (14) and 3a,5a-tetrahydro-11-dehydrocorticosterone (15) inhibit 11b-HSD1 from the homogenates of rat Leydig cells in the oxoreductase direction, with IC50 values at 4.3, 6.3 and 0.7 mM, respectively [114]. It has been shown that the steroid precursor dehydroepiandrosterone (DHEA, 16) causes downregulation of 11b-HSD1 and dose-dependent reduction of its oxoreductase activity in both liver and adipose tissue. DHEA also reduces the expression of H6PDH, thereby further contributing to the inhibition of 11b-HSD1 oxidoreductase activity through limiting the availability of NADPH [174].

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OH O

O

O

R

R2

H

R3

H

H HO

O

R1

H (13) R1 = α-OH, β-H, R2 = O, R3 = OH (14) R1 = O, R2 = α-H, β-OH, R3 = H (15) R1 = α-OH, β-H, R2 = O, R3 = H

(11) R = α-H, β-OH (12) R = O

(16) DHEA

Screening with recombinant human 11b-HSD1 expressed in yeast P. pastoris revealed that some synthetic steroids such as DMX (17) and stanozolol (18) inhibited the oxoreductase activity of 11b-HSD1 with Ki values in the micromolar range and were also equally potent in their dehydrogenase activity [117]. Compound (19), a potent 11b-HSD1 inhibitor, was discovered through common feature pharmacophore modelling and virtual screening [138]. Compound (19) showed significant inhibitory activity for 11b-HSD1 with an IC50 value of 144 nM and a 27-fold selectivity over 11b-HSD2 when tested on lysates of HEK293 cell expressed with recombinant human 11b-HSD1 or 11b-HSD2. It also showed high potency and selectivity on cell-based assays with IC50 values of 0.41, 0.33 and 0.65 mM in transfected HEK293, endogenous mouse C2C12 myotubes and 3T3-L1 adipocytes, respectively [138]. OH OH

O OH

HO

O

HN N H

(17)

COOH

H

H F

O

(18)

AcO

O

H (19)

Other natural products exhibiting 11b-HSD oxoreductase inhibition activity include some flavonoids: 2u-hydroxy-flavanone (20), flavanone (21) and 4u-hydroxy-flavanone (22). These selectively inhibited 11b-HSD1 from lysates of transfected HEK293 with IC50 values of 10, 18 and 34 mM, respectively [122]. Another flavanone analogue, naringenin (23), was reported to be a very weak inhibitor of 11b-HSD1 in both oxoreductase

52

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

and dehydrogenase directions [117]. The same report also revealed that furosemide, a known loop diuretic, strongly inhibits 11b-HSD1 bi-directionally with IC50 values in the sub-micromolar range [117]. Abietic acid (24) also shows inhibitory activity for 11b-HSDs in either direction [122]. O

OH

O iPr

R1

Me

O

HO

O

R2 R1

R2

(20) = OH, =H (21) R1 = H, R2 = H (22) R1 = H, R2 = OH

OH (23)

Me

COOH (24)

In general, these natural compounds or synthetic derivatives can be characterised as having either low potency or low selectivity or being devoid of favourable physicochemical and pharmacokinetic (PK) profiles, and are therefore not regarded as suitable lead compounds for selective 11b-HSD1 inhibition. However, an interesting strategy based on a structural classification of natural products, followed by combinatorial chemistry, has been applied to identify selective 11b-HSD1 inhibitors and resulted in the discovery of several potent compounds with IC50 values ranging from 310 to 740 nM [175].

ARYLSULPHONAMIDE ANALOGUES

Biovitrum pioneered the area of selective 11b-HSD1 inhibitors with a series of patent publications [176–181]. The core structures claimed in these patents belong to the arylsulphonamidothiazole family. Based on the SPA, the initial hit (25) was identified from a high-throughput screen as a potent 11b-HSD1 inhibitor with a Ki value of 0.82 mM. Optimisation of (25) indicated that in area A, a combination of small hydrophobic substituents was preferred; the sulphonamide linker in area B was unique, as N-methylation or using an amide linker instead usually led to loss of activity; and in area C, the substituents with H-bond donor/acceptor capacities on the thiazole ring were preferred [182]. Compounds (26, 27) were highlighted for their potency and selectivity [126]. BVT14226 (26) is a highly potent 11b-HSD1 inhibitor with IC50 value of 52 nM on the human

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enzyme and 284 nM on the mouse enzyme, whereas BVT2733 (27) showed inhibition of mouse 11b-HSD1 with an IC50 value of 96 nM and relatively weak inhibition of the human enzyme (IC50=3.3 mM). Furthermore, they are both more than 200-fold selective over human 11b-HSD2. The discovery highlighted the structural differences of 11b-HSD1 in different species, which was in agreement with data from the resolved crystal structures [26, 68]. With a favourable pharmacokinetic profile, BVT2733 (27) was evaluated in vivo in the hyperglycemic KKAy mouse model, and the results indicated a significant blood glucose level reduction in a dose-dependent manner after twice daily oral administration of (27) for 3–11 days [126].

A

B

C

Cl

Me

S SO2 NH

S SO2NH

Cl

N

CO2Et

N

CONEt2

Cl Me (25)

(26)

Me

S

O

SO2NH

Cl

N

N N .HCl Me

(27)

Other arylsulphonamidothiazole analogues claimed in Biovitrum’s patents as 11b-HSD1 inhibitors include compounds (28) (Ki=14 nM), (29) Ki=14 nM), (30) (Ki=21 nM), (31) (Ki=28 nM), (32) (Ki=18 nM) and (33) (Ki=91 nM) [176–181].

Me Cl

S SO2NH N

S

O

SO2NH Et Pr

(28)

(29)

N OMe

54

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

S SO2NH

O O

N

Cl

S SO2NH

N Cl

(30)

Me

Me

(31)

S

S SO2NH

Cl

Me Me

N

O

Ph

SO2NH

CH2OH

N

Cl

N

Pr (32)

(33)

N-thiadiazolyl arylsulphonamides and N-thienyl arylsulphonamides, as exemplified by (34–36) were also reported to be 11b-HSD1 inhibitors with Ki values in the range of 10 nM–10 mM when screened on recombinant human 11b-HSD1 using an SPA. The representative compound (36) showed a Ki value of 219 nM, but its selectivity is unknown [183, 184].

Me Cl

SO2NH

Me

N N S

Cl CO2Et

S

N

SO2NH

SO2NH

iPr

S N

CO2Et PhO

(34)

(35)

(36)

The arylsulphonamidooxazole compound (37) was also reported to be a potent inhibitor in a cell-based assay. Using CHO cells transfected with full-length human 11b-HSD1, (37) showed selective inhibition of human 11b-HSD1 with an IC50 value of 2.3 mM. It showed reasonable levels of exposure after oral dosing (50 mg/kg) in mice with an AUC of 6.23 mg h/mL [149]. A series of bicyclic arylsulphonamide compounds showed up to 100% inhibition of human 11b-HSD1 at 10 mM [185, 186]. The representative compounds (38, 39) exhibited IC50 values of 3.2 and 3.7 mM, respectively. The introduction of a 4-chloro substituent on the benzothiazole ring led to enhanced activity partly due to the alteration of the geometry of the two aromatic moieties in the molecule [187].

X. SU, N. VICKER AND B.V.L. POTTER Cl

O PhSO2NH

ArSO2NH

N

55

Me SO2NH

Cl

N

N

Me Ph

(37)

CO2Et

S

(38) Ar = 2,5-di-Cl-Ph (39) Ar = 4-Pr-Ph

(40)

Arylsulphonamidopyridyl derivatives were discovered as potent 11bHSD1 inhibitors by scientists from Agouron/Pfizer [188]. Inhibitory activities for exemplified compounds were evaluated with K iapp values which were shown in the 10 nM–10 mM range. Compound (40) showed a K iapp value of 42 nM and 72% inhibition at 100 nM. Modifications were performed with varied arylsulphonyl groups and substituted pyridyl groups at either end of the molecule, which revealed that a 6-methyl or 6-amino substituent on the pyridin-2-yl group is highly preferred. Compounds with varied aryl moieties selected from substituted phenyl, substituted biphenyl, substituted benzothiophenyl were made. Potent inhibitors (41–46) with virtually maximum inhibition at 100 nM are listed in Table 2.1.

Me

Me

Cl

Cl SO2NH S

SO2NH S

N

N

CO2Et

Me

(41)

(42)

Table 2.1 IN VITRO INHIBITION OF 11b-HSD1 BY ARYLSULPHONAMIDOPYRIDYL DERIVATIVES [188] Compound (40) (41) (42) (43) (44) (45) (46)

K iapp (nM) 42 2.8 3.2 6.4 4.6 9 17

56

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

SO2NH

SO2NH N

N Me

NC

NH2 Cl

(43)

(44)

Me SO2NH

Cl N

SO2NH N

NH2

F3C

NH2

(45)

(46)

Subsequent development of arylsulphonamidopyridyl derivatives indicated that the introduction of a hydroxyl group onto the side chain of the pyridyl moiety retained potent inhibition of 11b-HSD1 [156]. In HEK293 cell-based assays, compounds (47–49) exhibited IC50 values of 23, 51 and 59 nM, respectively [156]. The absolute configurations of compounds (47) and (48) are not clear, but they are identified as enantiomers with dextro-rotation.

SO2NH

SO2NH N

Me

N Me

R

HO

HO NC

NC (47)

(48) R = Et (49) R = H

Expansion of the linker with a nitrogen-containing aliphatic ring attached to the sulphonamide group generated new structural categories of 11b-HSD1 inhibitors [189]. The representative molecules (50–55) showed high potency against 11b-HSD1 with maximum inhibition at 100 nM in high-throughput screening.

X. SU, N. VICKER AND B.V.L. POTTER

57

Me

N

SO2NH

N F3 C

N

N

SO2NH N Me

Me

N (50)

N

(51)

SO2NH

N

SO2NH N

N

Et

CONEt2 (52)

N

N

F

(53)

SO2NH N

N

SO2NH N

Me

Et

Pr

Ph (54)

(55)

With the same assay method, sulphonylpyrrolidine analogues were also identified as potent 11b-HSD1 inhibitors. All the exemplified compounds (56–58) exhibited maximum inhibition at 100 nM and K iapp values less than 10 nM (Table 2.2) [190]. No selectivity data were released for individual compounds. Table 2.2 IN VITRO INHIBITION OF 11b-HSD1 BY ARYLSULPHONAMIDOPYRROLIDYL DERIVATIVES [189, 190] Compound

K iapp (nM)

(50) (51) (52) (53) (54) (55) (56) (57) (58)

4.1 2.8 5.7 5.4 8.2 1 6.7 o1 o1

58

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

Me

F

F

SO2 N

SO2 iPr

S

N CH2OH

NC

Me

Me

(56)

(57)

Me F SO2 N S

N O

(58)

After acquiring Biovitrum’s 11b-HSD1 portfolio, scientists from Amgen expanded the sulphonamide inhibitors to include sulphonylpiperazine, sulphonylpiperidine and sulphonylpyrrolidine analogues, as exemplified by compounds (59–64) [140, 141]. The compounds were tested on both microsomal 11b-HSD1 and intact HEK293 cells transfected with human recombinant 11b-HSD1. It was claimed that the exemplified compounds had IC50 values from 200 nM to less than 1 nM, but no selectivity data were disclosed. A more recent patent from Amgen specifically claimed aniline sulphonamide derivatives as 11b-HSD1 inhibitors [191]. Arylsulphone derivatives were also claimed in a patent publication from Amgen as potent 11b-HSD1 inhibitors, but no specific activity data were released for individual compounds [192]. Similarly, more sulphonylpiperazine and sulphonylpyrrolidine derivatives were claimed as human 11b-HSD1 inhibitors in a patent from Takeda [193]. However, biological data for individual compounds were not released.

SO2 N Me HO F3C

SO2 N

N N

Me (59)

tBu

Cl (60)

X. SU, N. VICKER AND B.V.L. POTTER

SO2 N

59

SO2 N

N CONH2

Me HO F 3C

N

Me (61)

(62)

SO2 N

N

SO2 N CONH2

Me HO F3 C

N

tBu

Me

Me HO F 3C

N

(63)

(64)

It was also disclosed by Hoffmann-La Roche that arylsulphonylpiperidine analogues with varied amides substituted at the 3-position of the piperidine group were potent human 11b-HSD1 inhibitors [194]. The exemplified compounds (65–70) were screened against purified human 11b-HSD1 using an SPA (Table 2.3). Some compounds were also tested in vivo for the inhibition of cortisone conversion on male C57BL/6J mice with an i.p. dose of 100 mg/kg.

Me

SO2 N

SO2 N NH

Cl

NH

Cl

O

O (65)

(66)

Table 2.3 IN VITRO INHIBITION OF HUMAN 11b-HSD1 BY ARYLSULPHONAMIDOPIPERIDINYL DERIVATIVES [194] Compound

IC50 (nM)

(65) (66) (67) (68) (69) (70)

25 31 47 190 290 390

Ph

60

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

SO2 N SO2 N NH S

O

NH O

(67)

(68)

SO2 N

SO2 N NH

Cl

NH

iPr

O (69)

O (70)

Some adamantamine or adamantyl methylamine arylsulphonamides, exemplified by compounds (71–73), were disclosed by Sterix as showing W60% inhibition of human microsomal 11b-HSD1 at 10 mM concentration when tested with an HTRF assay [135].

SO2NH

SO2NH S

Pr

(71)

SO2NH S

(72)

(73)

Further discoveries of 11b-HSD1 inhibitors in the arylsulphonamidothiazole family were demonstrated by patents from Taisho and Takeda [136, 137, 195]. Modification of the substituent at the 5-position of the thiazole ring gave some very potent 11b-HSD1 inhibitors, as shown by representative compounds (74, IC50=16 nM) and (75, IC50=42 nM) [136]. The adamantyl group at the 5-position of the thiazole ring was well tolerated indicating possible hydrophobic interactions with the enzyme at that site. Other compounds (76–81) with substituents possessing hydrogenbonding capacity at the 5-position retained the enhanced 11b-HSD1 inhibition with IC50 values below 10 nM when tested with an HTRF method (Table 2.4) [137].

X. SU, N. VICKER AND B.V.L. POTTER

61

Table 2.4 IN VITRO INHIBITION OF 11b-HSD1 BY ARYLSULPHONAMIDOTHIAZOLE DERIVATIVES [137] Compound

IC50 (nM)

(76) (77) (78) (79) (80) (81)

2.0 8.8 3.2 3.1 6.6 2.1

N

ArSO2NH

N

ArSO2NH

O

N

ArSO2NH

SO2tBu

S S

S Ar

Ar Me

(74) Cl

(76)

∗

(75)

(78)

∗ S

S

Ar

Me

Cl

∗ S

∗

(77)

∗

Me

Cl

∗

(79)

S NC

Br

Me

Cl

S

S

Me

N

SO2NH

SO2 N Cl

(80)

N

SO2NH

Me

O O

S

S

(81)

Scientists from Hoffmann-La Roche explored the possibility to expand the scope of arylsulphonamidopyridyl derivatives as 11b-HSD1 inhibitors. With aryl or aryloxyl groups attached to the 5- or 6-position of the pyridyl ring through a bond or an extra methylene, these exemplified compounds inhibit human 11b-HSD1 with IC50 values below 1 mM on microsomes from transfected HEK293 cells using an ELISA method [142, 144].

62

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

Me SO2NH N

F

SO2NH

Cl

N

Me

Me

Cl Cl

(82)

(83)

F

O

SO2NH N

SO2NH N F

O

Cl

(84)

(85)

F

With the same assay, some arylsulphamidopyrimidyl derivatives exemplified by compounds (86, 87) were also identified as 11b-HSD1 inhibitors [196]. The most potent inhibitor identified by Hoffmann-La Roche in the sulphonamide series was an indazolone analogue. Compound (88) shows extremely high activity with an IC50 value of 7 pM [143]. The activity for representative compounds (82–89) is listed in Table 2.5. Evotec also disclosed that arylsulphonamidopyridyl analogues with aryl or acetamido substituents at the 5- or 6-position of pyridyl ring inhibit 11b-HSD1 in both the HEK293 and human adipocyte cell lines [197–199]; but no specific data were released for individual compounds.

Table 2.5 IN VITRO INHIBITION OF HUMAN 11b-HSD1 BY ARYLSULPHONAMIDOPYRIDYL, PYRIMIDINYL AND INDAZOLYL DERIVATIVES [142–144, 196] Compound

IC50 (nM)

(82) (83) (84) (85) (86) (87) (88) (89)

3 12 16 5 20 167 0.01 0.1

X. SU, N. VICKER AND B.V.L. POTTER

N

SO2NH

63

N

SO2NH

N

N

Ph

iPr (87)

(86)

O

Cl

SO2NH

O NH

NH

N

N

Bz

NC

SO2NH

iPr

F (88)

(89)

TRIAZOLE DERIVATIVES

Novel classes of compounds with a triazole ring as the core structure are disclosed as 11b-HSD1 inhibitors in a series of patents from Merck [200–204]. The initial set of compounds with the generic structure (90) show that the most preferred R1 is an optionally substituted adamantyl group attached through a bond or methylene to the 5-position. The substituents at the 3- or 4-positions are either independently alkyl or aryl groups or can together form an aliphatic ring. The preferred compounds inhibit 11bHSD1 with IC50 values of o100 nM and possess 10–100-fold selectivity over 11b-HSD2 in an SPA [200]. Compound (93) (MK544) was highlighted from the SAR study as a very promising human 11b-HSD1 inhibitor with an IC50 value of 7.8 nM (98 nM for mouse) [205]. Its selectivity over 11bHSD2 was demonstrated with IC50 values of W3,000 nM for human and W10,000 nM for mouse. In the pharmacodynamic mouse model assay, (93) inhibited 59% of [3H]cortisone conversion 1 h after oral dosing (10 mg/ kg), and 17% of conversion after 4 h. The SAR study revealed that the number of methylene units in the aliphatic ring of compounds (91–98) correlated with the in vitro activity. Compound (97) was the most potent, which suggested that the active site could accommodate large hydrophobic substituents. On the other hand, ring expansion from 7-to 11-membered rings resulted in an increase of inhibition of 11b-HSD2 (Table 2.6). The hydrophobic substituents at the 3- and 4-positions varied from alkyl to aryl groups; and these analogues generally retained potent activity in vitro as shown in compounds (99–104). It was also found that additional substituents on the adamantyl ring generally led to poorer activity [205].

64

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

Table 2.6 IN VITRO INHIBITION OF 11b-HSD AND MOUSE PHARMACODYNAMIC ASSAY RESULTS OF TRIAZOLE DERIVATIVES [205] Compound

IC50 (nM), human

(91) (92) (93) (94) (95) (96) (97) (98) (99) (100) (101) (102) (103) (104)

PD assay (% inhibition)

11b-HSD1

11b-HSD2

1h

4h

2,180 278 7.8 2.2 4 2.5 1.4 3.6 11 52 11 72 3 37

2,000 W3,000 W3,000 787 30 25 8.7 358 2,840 W4,000 W4,000 W4,000 92 W4,000

0 NR 59 83 43 69 45 17 31 24 55 NR 48 85

0 NR 47 24 44 31 29 3 21 4 37 NR 23 47

NR=Not reported.

1 2 N N 1

R X

5 N

3

ZR

4 WR2 (90)

N N

N N

N

N

3

(CH2)n (91) (92) (93) (94) (95) (96) (97) (98)

n= n= n= n= n= n= n= n=

1 2 3 4 5 6 7 8

R2

1

R (99) (100) (101) (102) (103) (104)

R1 = Me; R2 = Bu R1 = Et; R2 = Pr R1 = Pr; R2 = Et R1 = Bu; R2 = Me R1 = Me; R2 = Bz R1 = Me; R2 = Ph

Replacement of the adamantyl group on the triazole template with phenyl-substituted spirocycles generated another cluster of 11b-HSD1 inhibitors [201]. At the 5-position of the generic structure (90), the preferred compounds usually contain a p-chloro- or p-fluoro-benzyl group with a spirocyclopropyl or spirocyclobutyl group attached to the benzylic position. The cyclopropyl groups as substituents at both the 3- and 4-positions of the

X. SU, N. VICKER AND B.V.L. POTTER

65

triazole ring also characterise this series of inhibitors. Compounds with a methyl group at the 4-position generally have a substituted aryl group at the 3-position. Compounds (105–107) are typical examples with IC50 values below 100 nM, but no specific biological data were released. Compound (106) was also claimed in its crystalline anhydrate and crystalline monohydrate forms as a potent 11b-HSD1 inhibitor in a later patent [206]. A novel process for preparing this compound was also disclosed by Merck [207]. Cl

Cl

Cl

N N

N N

N

N

Cl

N N N

Cl

Me F

(105)

(106)

(107)

Another category of triazole compounds characterised by the attachment of a bicyclic[2.2.2] octyl group to the 5-position of the triazole ring as shown in the generic structure (108) was also claimed to provide 11b-HSD1 inhibitors [202, 203]. The substituents R2 at the 4-position were selected from hydrogen, alkyl, alkenyl and cycloalkyl groups, whereas R3 at the 3-position was usually limited to aryl or heteroaryl that was linked through a bond, a carbonyl group or an aliphatic chain. The linking group X at the bridgehead of bicyclo[2.2.2]octane consisted of sulphone, ketone, aromatic, aliphatic or heteroaromatic groups. It was claimed that the preferred compounds in the patents have IC50 values less than 100 nM for 11b-HSD1 and more than 10-fold selectivity over 11b-HSD2. N N N R1 X

N N R3

N

R2

(108)

N N

F3C

N Me

(109)

(110)

A comprehensive SAR study with this group of compounds revealed that the replacement of the alkyl tail at the bridgehead of the bicyclo[2.2.2] octane with a heteroaryl group led to more potent and selective 11b-HSD1 inhibitors with a promising PK profile [208]. Compound (109) showed high potency against 11b-HSD1 with IC50 values of 5 nM for the human enzyme

66

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

and 3 nM for the mouse enzyme. In a mouse PD study, (109) inhibited 74% and 52% of [3H]cortisone conversion at 1 and 4 h after oral dosing, respectively. Optimisation at the 3- and 4-positions led to compound (110) with an IC50 value of 2 nM for human 11b-HSD1 and 3 nM for mouse 11b-HSD1. Compound (110) also exhibited nearly maximal inhibition of [3H]cortisone conversion after 1 and 4 h in PD study. But both (109) and (110) suffered from poor PK properties with high clearance and low bioavailability. The replacement of the n-pentyl chain with 3-methyloxadiazole gave a compound (111) with modest potency and good selectivity on both human and mouse enzymes. Increasing the hydrophobicity of the substituent by the introduction of 4-chlorophenyl onto the oxadiazole ring gave (112) with potency and selectivity matching the lead compound (109). Furthermore, variation of the phenyl ring substituents, in combination with the replacement of the heptyl ring, led to the discovery of some highly potent 11b-HSD1 inhibitors (113–117) with good selectivity (IC50 W1 mM for 11b-HSD2) (Table 2.7). Compound (114) was highlighted as being particularly potent and highly selective (B1,800-fold over 11b-HSD2). In the mouse PD assay, (114) showed 86% inhibition of [3H]cortisone conversion at 4 h and 74% inhibition at 16 h. In addition, (114) had a very good PK profile with low clearance, a long half-life and high bioavailability in mouse, rat and dog. (Table 2.8).

Table 2.7 IN VITRO INHIBITION OF 11b-HSD BY TRIAZOLE DERIVATIVES [208] Compound

(111) (112) (113) (114) (115) (116) (117)

IC50 (nM), human

IC50 (nM), mouse

11b-HSD1

11b-HSD2

11b-HSD1

11b-HSD2

289 13 4 2.2 2.6 9.3 4.1

W4,000 228 W1,000 W1,000 W1,000 W1,000 W1,000

136 17 2 1.9 2.6 5.2 3.2

W4,000 4,000 W1,000 W1,000 W1,000 W1,000 W1,000

Table 2.8 PK PROPERTIES OF COMPOUND (114)[208] Species

nAUC (mM h)

Clp (mL/min/kg)

t1/2 (h)

F (%)

Mouse Rat Dog

5.7 6.2 9.55

5.88 5.39 3.51

17.7 5.1 9.87

58 83 100

X. SU, N. VICKER AND B.V.L. POTTER

N N

N N

N

N

67

R

N

N

1

Me

R

N O

R2

N O (113) R1 = 4-Cl-Ph; R2 = 2-CF3-Ph (114) R1 = 4-F-Ph; R2 = 2-CF3-Ph (115) R1 = 2,4-di-F-Ph; R2 = 2-CF3-Ph (116) R1 = 4-F-Ph; R2 = 2-CHF2O-Ph (117) R1 = 4-F-Ph; R2 = 2-Cl-Ph

(111) R = Me (112) R = 4-Cl-Ph

The effect of replacing the 1,2,4-oxadiazole with other 5-membered heterocycles was also investigated [208]. The results indicated that the compounds (118–121) retain almost the same level of activity as (114) in vitro. However, the imidazole and triazole analogues (120, 121) show only weak inhibition of [3H]cortisone conversion after 16 h in the PD assay. In order to improve the water solubility of (114), other polar groups were introduced onto the phenyl ring to give compounds (122–125) which although exhibiting high-level in vitro inhibitory activity, show only weak to modest inhibition of [3H]cortisone conversion after 16 h oral dosing in the PD assay (Table 2.9).

N N

F

N N

F3C

X ∗

O

∗

N N N

(118) X = (119) X =

O N

∗

Me

R

Me ∗

N

N

N ∗

(120) X = (121) X = ∗

N

N N

OMe

(122) R =

NH H N

N O N

∗ ∗ ∗

(123) R = ∗

F3C

∗

(124) R = O S Me O

(125) R =

O S Me O O S CF3 O

∗

It was also claimed by Merck that some 2,5-diaryl-1,2,4-triazole derivatives represented by the generic structure (126) are potent 11b-HSD1 inhibitors [204]. The aromatic group attached to the 5-position was selected from aryl or heteroaryl group, such as pyridyl, thienyl, furyl, pyrazolyl, thiazolyl, oxazolyl, imidazolyl, indolyl; the benzene ring at the 2-position was substituted with various groups with 2-CF3 being preferred, as exemplified by compounds (127,128). No specific biological data for individual compounds were released.

68

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

Table 2.9 IN VITRO INHIBITION OF 11b-HSD1 AND PHARMACODYNAMIC ASSAY RESULTS OF TRIAZOLE DERIVATIVES [208] Compound

IC50 (nM)

(118) (119) (120) (121) (122) (123) (124) (125)

Human 11b-HSD1

Mouse 11b-HSD1

4h

16 h

7.2 2 5.3 4.7 4.1 4.9 4.2 1.9

5 o1 2.4 1.6 4.5 1.8 9.5 o0.98

85 93 83 93 81 95 71 76

79 91 32 28 0 40 11 71

N N Ar

PD assay (% inhibition)

N N

Me N R

N

F3C

Cl

OMe N N

N

N

Me

Me

Me

(126)

(127)

F3C

(128)

The patent publications for pharmaceutical use of 11b-HSD1 inhibitors from Novo Nordisk are also based on the 1,2,4-triazole core structure (129) [209, 210]. The linker at the 3-position is sulphur or oxygen in the first patent as exemplified by compounds (130, IC50=160 nM for 11b-HSD1) and (131, IC50=190 nM for 11b-HSD1) [209]. The main difference to the patents from Merck is that the substituent at the 5-position is selected, for example, from aryl, cycloalkyl, heteroaryl and heterocycloalkyl rather than an adamantyl group. Another patent claims compounds with a fused triazole ring substituted with an aryl ring at the 5-position as 11b-HSD1 inhibitors [210]. The biological data were released for compounds (132, IC50=230 nM for 11b-HSD1) and (133 IC50=110 nM for 11b-HSD1). The assay was performed on recombinant human 11b-HSD1 using an SPA.

N N R3

N R2

(129)

R1 X

Br

N N N Et

(130)

N N N

S

N O

Me

(131)

Ph

S O

X. SU, N. VICKER AND B.V.L. POTTER

PhO

Br

69

PhO

N N

N N

N N

N

N

N

(132)

(133)

(134)

The triazole series was expanded by introducing an optionally substituted benzyl group with a spirocyclopropyl or spirocycloheptyl group attached to the benzylic position onto the triazole ring at 5-position and a varied aryl, cycloalkyl or heterocycloalkyl group at the 3-position. These proved to be highly potent 11b-HSD1 inhibitors, as exemplified by compounds (135–143) [211]. Using an SPA, these compounds were shown to inhibit recombinant human 11b-HSD1 with IC50 values less than 10 nM. The preferred compounds generally contain a phenylcyclopropyl-4-methyl-4H-1,2,4-triazole core structure. The variations at the 3-position of the triazole ring indicate the diversity of potential interactions with the enzyme at this site. F N N

Cl

Ph N

N N Ph N

N

Me

Cl

N N

N

N

N

N

(136)

(137)

Cl

N N

Cl

N N

Ph

Cl

Me

Cl

OH

(135)

Cl

Ph

N

N

NHSO2Me

Me

N

Me

(138)

(139)

N N

N N

Ph

Ph

OH

N Me

(140)

NAc

N

N CONH2

Me

(141)

70

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

N N Ph

N

N

Cl

N N Ph

N

O

Me

Cl N

(142)

N

O

Me

(143)

The development of a triazole series based on the triazolo[4,3-a]azepine or triazolo[4,3-a]azocine core structures demonstrated that the compounds with a thiophenylcyclopentyl or thiophenylisopropyl motif attached to the 5-position retain high inhibitory activity for 11b-HSD1 with very good selectivity over type 2 enzyme, as shown by compounds (144–147) [212]. Similarly, compounds (148, 149) with 2-chlorophenyl group at the 3-position of the triazole ring also show high activity (Table 2.10).

Cl

N N

N N

S

Cl

S

N

N N S

N

N Me Me

(144)

(145)

N N

Me

S N Me Me

PhSO2

N N

N N Me

(147)

(148)

(146)

N N

Cl

Cl

N Me

N Me Me

Me

(149)

Patents from BMS disclosed compounds based on the triazolo[4,3-a] pyridine structure, as exemplified by compounds (150–153), that also inhibit human recombinant 11b-HSD1 with IC50 values of less than 10 mM [213, 214]; no specific data were released.

X. SU, N. VICKER AND B.V.L. POTTER

71

Table 2.10 IN VITRO INHIBITION OF HUMAN 11b-HSD BY TRIAZOLE DERIVATIVES [212] Compound

IC50 (nM)

(144) (145) (146) (147) (148) (149)

11b-HSD1

11b-HSD2

5.3 4.4 5.2 6.6 15 18

W3,000 W1,000 W1,000 W1,000 ND W3,000

ND=Not determined.

Cl

N N

Me

N N Cl

O

OMe

N

N

O Cl (150)

(151)

Cl O

N N N

N N

O N

N

Cl (152)

(153)

Mochida Pharmaceuticals has identified potent 11b-HSD1 inhibitors from 3-oxy-1,2,4-triazole derivatives, 3-amino-1,2,4-triazole derivatives and 3,5-diamino-1,2,4-triazole derivatives [215–217]. Characterised by the aromatic amino groups attached to both the 3- and 5-position of the triazole ring, the exemplified compounds (154, 155) show potent inhibition of 11bHSD1 with IC50 values of 50 and 2 nM, respectively, when tested on HepG2 cells with an HTRF protocol. It was also revealed that (154) inhibits the 11b-HSD1 activity on human hepatocytes with an IC50 value of 3 nM. In an ex vivo assay, (154 and 155) suppress the 11b-HSD1 activity by 64 and 54% respectively, when administered 30 mg/kg orally [216].

72

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

F

F

F

N N N Me

N N N

N

N HO

Me

Me

N

Me

(154)

Me

N Me

(155)

INHIBITORS WITH A KETONE LINKER

Compounds with a ketone linker connecting varied groups have appeared in a few patents and publications as potent 11b-HSD1 inhibitors. The ketone functionality in these series may be mimicking the role of the ketone moiety in the natural substrate cortisone. Scientists from AstraZeneca have discovered various sets of aryl ketone derivatives as potent 11b-HSD1 inhibitors based on the assay results performed on HeLa cells which were stably transfected with a construct containing the GRE linked to a b-galactosidase reporter gene and a construct containing full-length human 11b-HSD1 [152–154]. For the generic structure (156), ring-A was defined as aryl or heteroaryl and ring-B was defined as carbocyclic or heterocyclic; R1 was selected from alkyl, halogen, nitro and cyano groups and R2–R5 could be selected from H, OH, NH2, CN, substituted alkyl and heterocyclic groups; X and Z were generally selected from C, SO2, S, CO, CONR, NRCO, CO2, SO2NR and NRSO2; p, q and s are 0 or 1 and r is 1 or 2. Biological data were reported for exemplified compounds (157–162) (Table 2.11) [152, 154]. O R1

X

A

p

Z r q R2 R3 R4 R5

s

R6 B

(156)

S

Me

Cl

NHAc

O

(157)

O O

(158)

F SO2 N O

(159)

X. SU, N. VICKER AND B.V.L. POTTER

73

Table 2.11 INHIBITION OF HUMAN 11b-HSD1 IN HELA CELLS BY ARYL KETONE DERIVATIVES [150–154] Compound

IC50 (nM)

(157) (158) (159) (160) (161) (162) (164) (165) (166) (167) (168) (169) (170) (171) (172)

77 153 94 72 60 47 254 97 50 10 83 206 75 70 447

O

O

O

SO2NiPr2

SO2NMe2

SO2NMe2

N

N

N

iPrO

(160)

(161)

(162)

The aryl ketone series was further expanded to include a piperidine moiety as part of the linker system as depicted by the generic structure (163), for which ring-A could be either a carbocycle or a heterocycle, and R1 may be selected from alkyl, halogen, nitro and cyano groups; the optional R2 may be halogen or OCF3; X is a bond, CO, SO2, CONR or CH2 and Y is H, alkyl, carbocyclic or heterocyclic groups; q is 0 or 1. Biological data were reported for exemplified compounds (164–166) (Table 2.11) [153].

O R1

O R2

A

F

q N (163)

X

Y

N

MeO (164)

O

74

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

O

O F N

MeO

N

F3CO O

(165)

OCF3

O (166)

Similarly, aryl ketone derivatives with a carbonyl group attached to the 3-position of the piperidine ring, as exemplified by compounds (167–169), were also disclosed by AstraZeneca as 11b-HSD1 inhibitors [151]. In addition, the piperidine ring could be replaced by a pyrrolidine ring as shown in typical compounds (170–172) (Table 2.11) [150]. O

O N

SO2iPr

O N

F

F (167)

F (168)

O

O

O S

N

N

Cl

F

O

F (169)

Cl (170)

O

O

Cl

O

N F

N F

(171)

O S O F

(172)

It was claimed that some aryl ketone derivatives with the carbonyl group connected to a sulphonamide moiety through a methylene unit could also inhibit human 11b-HSD1 [218]. The results were based on a cell-based assay using an 11b-HSD1 stably transfected CHO cell line. The representative compounds (173–175) were claimed to have IC50 values less than 500 nM.

X. SU, N. VICKER AND B.V.L. POTTER

O

O Ph

N

NHSO2

NHSO2 Cl

O NHSO2

Ph

N Me

Me

75

Me

MeO

(173)

(174)

Cl

Me

(175)

Sterix discovered that the adamantyl ketone derivatives, as represented by generic structure (176) are also potent human 11b-HSD1 inhibitors [135]. The exemplified compounds (177–180) show W60% inhibition at 10 mM on both the human microsomal enzyme and the HEK293 cell line transfected with human 11b-HSD1. For the generic structure, the optional R1 could be alkyl, halo or hydroxyl groups; X was selected from O, S, NH, SO, SO2, or CH2; ring-A was normally an optionally substituted aromatic ring; and n is 0 or 1.

Ph R1

Cl

n X

O

A

O

S

O

(177)

(176)

Cl

O

(178)

Me S

S O

O O (179)

O

O

Cl

(180)

In the process of synthesising sulphonamidooxazoles as 11b-HSD1 inhibitors, scientists from Wyeth have identified a minor by-product b-keto sulphone (181) as a very potent 11b-HSD1 inhibitor with an IC50 value of 190 nM and over 1,000-fold selectivity over type 2 enzyme on cell-based assays [149]. Further optimisation of (182) indicated that the b-keto sulphone linker is unique, as a replacement of either the keto or sulphone functionality leads to reduced activity or inactive compounds. The introduction of a 3-OMe group at the meta position results in enhanced activity as shown by (182) (IC50=60 nM), indicating both electron donating and electron-withdrawing groups are tolerated in that area [219].

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

O

O SO2Ph

SO2Ph

MeO

F3C (181)

(182)

INHIBITORS WITH AN AMIDE LINKER

Scientists from Janssen discovered that a new class of adamantylacetamide derivatives could inhibit 11b-HSD1 in both a non-cellular recombinant protein assay and a cell-based assay with differentiated mouse 3T3-L1 cells and rat hepatocytes [220]. The compound’s effect on 11b-HSD2 was studied in HepG2 and LCC-PK1 cells. Selected examples (183–187) were disclosed as potent inhibitors with pIC50 values greater than 6 for type 1 enzyme and pIC50 values less than 5 for type 2 enzyme. It was revealed that (183) suppresses the formation of corticosterone in the liver and in fat tissue dosedependently, when administered orally to normal mice [221]. In low-fat diet db/db mice and high-fat diet KKAy mice treated with 0.3% compound in food for 4 weeks, the blood glucose levels are attenuated. Me Me R

Me Me

H N

H N

O N (183) R = 4-F (184) R = 4-OMe (185) R = 3-Me

O

(186)

N

H N

O

(187)

The scope of adamantylacetamide derivatives was further expanded by Janssen to include compounds with a 1,4-disubstituted adamantine group [222]. The substituent at the bridgehead of the adamantane moiety was selected from a hydroxyl, fluoro, phenyl, amino, N,N-dimethylamino, acetamido or tolylsulphonamido group. The exemplified compounds were tested in cellular assays for both 11b-HSD1 inhibition (3T3-L1 cell line) and 11b-HSD2 inhibition (HepG2 cell line). The representative compounds (188–196) showed high inhibitory activity (pIC50 W6) and good selectivity over type 2 (pIC50 o5) [222].

X. SU, N. VICKER AND B.V.L. POTTER

MeO

Me

Me H N

Me N H

F3C R

O

(188) R = OH (189) R = F (190) R = NHAc

Me

Me H N

O

Me

NMe2

O

Ph

O

(193)

(194)

Me F3C

R

O

Me

Me H N

O

Me H N

(191) R = F (192) R = NHAc

Cl Me

N H

77

Me H N

O

O NHTs

O

Me

(195)

H N OH

O (196)

It was also found that compounds with pyrrolidin-2-one as a linker system, as shown by typical examples (197–199), are highly active against 11b-HSD1 with IC50 values less than 1,000 nM and selectivity greater than 10-fold over 11b-HSD2 [223]. Cl

Ph N

N Me

O

Cl (197)

Cl

Ph

N Me

O

Cl (198)

Cl

O

Ph

(199)

On a similar template, with the 2-adamantyl group attached to the nitrogen of the pyrrolidine ring, some representative compounds (200–204) were shown to inhibit 11b-HSD1 on both a microsomal enzyme assay and a cellular assay (pIC50 W6); their selectivity over 11b-HSD2 was greater than 10-fold [224]. Further optimisation by Janssen revealed that compounds

78

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

with a pyrrolidine ring forming a tricyclic system, as typified by compounds (205–207), also exhibit inhibitory activity for 11b-HSD1 with IC50 values less than 1 mM [225]. Compounds (205, 206) are 10-fold selective over type 2 enzyme, whereas (207) shows the same level of inhibition for both 11bHSD1 and 11b-HSD2. F R

R

N

N

Me O

F

O

(200) R = H (201) R = Me

N

(202) R = H (203) R = Me

O

H

MeO

N

MeO O (204)

H N

O

H (205)

H

O N

N H (206)

(207)

Novartis claimed a wide range of amide compounds as 11b-HSD1 inhibitors and their use in the treatment of type 2 diabetes and other conditions such as syndrome X, dyslipdaemia, hypertension and central obesity [226]. The core structure, as represented by (208), indicates that the attachment on the nitrogen of the amide group is usually a large saturated carbocycle, which implied that hydrophobic interactions with the enzyme were highly preferred. The in vitro assays are performed with recombinant human 11b-HSD1 from transfected P. pastoris and with rat hepatocytes at the cellular level. The selectivity was established by quantifying the inhibition of the [3H]cortisol conversion to [3H]cortisone using lysates of SW-620 human colon carcinoma cells as the enzyme source. The in vivo

X. SU, N. VICKER AND B.V.L. POTTER

79

activity was determined by evaluating the inhibition of corticosterone production in adrenalectomised (ADX) mice after oral administration. It was disclosed that the representative compounds (209, 210) are highly active in vitro with IC50 values of 6.5 and 7.7 nM, respectively; their inhibition for 11b-HSD2 is below 34% at 10 mM indicating good selectivity. In addition, (209, 210) inhibit corticosterone production in ADX mice by 57 and 67%, respectively, at an oral dose of 25 mg/kg. R2 R

W

R

N Y

Me Me N

O

1

X

3

O H

O

N O

R4

(208)

H

NO2

(209)

(210)

Coppola and coworkers performed a comprehensive optimisation and SAR study in a perhydroquinolylbenzamide series starting with the initial hit compound (211), (IC50=128 mM for human 11b-HSD1) [119]. Modification by varying the combination of substituents on the left-hand phenyl ring and the right-hand side tertiary amide revealed compounds (212–214) with greatly enhanced activity with IC50 values of 370, 220 and 730 nM, respectively. It was discovered that the highly preferred tertiary amide consists of trans-decahydroquinoline moiety as in (213). The corresponding decahydroisoquinoline analogue (214) loses 3 to 4-fold activity relative to (213) and is not as selective over 11b-HSD2 [119]. OH

O N

O MeO

O Cl

N H

R

O N H

MeO

Cl OMe (212)

(211)

Me N R=

∗

(213) ∗ H N H

(214) H N H

∗

80

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

Based on the new template (215), further modifications with substituents on the benzamide phenyl ring at the left-hand side indicated that the substituent effect of R1 is quite similar regardless of the electronic features of the substituent as shown by (216–220) (Table 2.12) [119]. Compound (220), with an IC50 value of 0.16 mM, is 3.75-fold more active than unsubstituted (215) and marginally more active than (213). It appears that the position of the substituents on the phenyl ring has little effect on inhibitory activity. The combination of 2,4-dichloro substituents proved to be the most preferred and increases the activity by 5-fold relative to the 2- or 4-monochloro analogues. The modifications on the central phenyl ring give mixed results as shown by (225–229); the R4 substitution with Cl, OMe or OPr generates compounds roughly twice as active as (213). The methoxysubstituted analogue (227) is significantly less-selective over 11b-HSD2. Replacing the methoxyl group with the n-propoxyl group (228) retain 11b-HSD1 inhibition and increases the selectivity. The naphthalene analogue (229) was identified as equally potent with an IC50 value of 100 nM and W100-fold selectivity over 11b-HSD2. Replacing the phenyl ring with a heterocycle (230) retains the activity, with an IC50 value of 320 nM. (Table 2.12) [119].

R4 R2

O

R1

O

R3 H

N

N H

H

R1 O O

H N H

N Me

N H

(230)

(215) (216) (217) (218) (219) (220) (221) (222) (223) (224) (225) (226) (227) (228) (229)

R2

R3

R4

H H H H F H H H OMe H H H Me H H H COOH H H H SO2NPr2 H H H H F H H H OMe H H F F H H H H Cl OMe Cl Cl OMe H Cl Cl H Cl OMe Cl Cl H OPr Cl Cl H Cl Cl -CH=CH-CH=CH-

Compound (231) with a meta-substituted benzamide was shown to be very potent with an IC50 value of 14 nM and W700-fold selectivity over 11b-HSD2. Unfortunately, this compound shows only weak inhibition at the cellular level (Table 2.12). Modification of the secondary amide results in some very potent inhibitors (232–234) with IC50 values from 50 to 60 nM, indicating the amide linker could be replaced (Table 2.12) [119].

X. SU, N. VICKER AND B.V.L. POTTER

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Table 2.12 INHIBITION OF 11b-HSD BY DECAHYDROQUINOLINE DERIVATIVES [119] Compound

11b-HSD1 IC50 (mM)

11b-HSD2 (%inhibition) (10 mM)

(215) (216) (217) (218) (219) (220) (221) (222) (223) (224) (225) (226) (227) (228) (229) (230) (231) (232) (233) (234)

0.60 0.56 0.41 0.49 0.53 0.16 0.55 0.15 0.48 0.36 0.28 0.10 0.12 0.12 0.10 0.32 0.014 0.06 0.05 0.05

0 2 0 23 4 33 47 51 2 71 6 37 81 17 22 26 12 8 64 17

Ph

O

O

H N

H

O

N

H

N

SO2NH

H

H F

(231)

(232)

O H N H F

O N

O N

H F

(233)

H

N H

Me (234)

The active compounds identified from the above in vitro studies were further evaluated for inhibition with cellular assays on rat hepatocytes [119]. In addition, the compounds that show W50% inhibition at 1 mM at the cellular level were studied in vivo on the ADX mouse model. Compounds

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

were administered orally at 25 mg/kg at 4 and 2 h before sacrifice (total dose 50 mg/kg). Homogenised liver samples were used to measure the corticosterone concentration, which was determined by an RIA. Compounds (213, 216, 229) were shown to cause a W70% decrease of liver corticosterone concentration in mice (Table 2.13) [119]. A diverse set of amide compounds is claimed by Novo Nordisk in patent publications as 11b-HSD1 inhibitors and is useful for the treatment and prevention of diseases in which a decreased intracellular concentration of active glucocorticoid is desirable [227–229]. Generally, the tertiary amide unit in the typified compounds features a substituted nitrogen attached to, or forming part of, a saturated or partially saturated cyclic, bicyclic or tricyclic ring system, while the carbonyl group is attached to an aryl ring. A more specific patent was based on a pyrazolo[1,5-a]pyrimidine core structure, as typified by compounds (239, 240) [228]. Compounds (235–240) were tested in an SPA using recombinant human 11b-HSD1 from transfected yeast as enzyme source and their IC50 values were reported [227, 228] (Table 2.14). O Pr Cl

O

N

N H

Me

N

N N

Me

O

N

Me

Me Me

(235)

(237)

(236)

F3C

O H N

O

N

Ph N

O

N

N

O

N

N Me

(238)

Me

N

N Me

(239)

Me

N

N Me

(240)

More recently, another patent from Novo Nordisk disclosed amide derivatives with a spirocyclic core structure, as exemplified by compounds (241, 242), as potent 11b-HSD1 inhibitors useful for the treatment of metabolic syndrome [230]. The compounds were tested with the same SPA protocol, but no specific data were released. Novo Nordisk also claimed that the combination therapy of 11b-HSD1 inhibitors with anti-hypertensive agents can be used for the treatment or prevention of disorders involving metabolic syndrome, insulin resistance, dyslipidaemia, obesity or

X. SU, N. VICKER AND B.V.L. POTTER

83

Table 2.13 CELLULAR AND IN VIVO INHIBITION OF 11b-HSD1 BY DECAHYDROQUINOLINE DERIVATIVES [119] Compound

% inhibition (1 mM)a

In vivob

(213) (215) (216) (219) (220) (225) (226) (228) (229) (230) (231) (232) (234)

81 84 86 23 76 74 50 17 59 100 19 47 45

70 57 73 NR 37 NR 15 NR 73 67 NR NR NR

NR=Not reported. a Assayed in rat hepatocytes. b % change of mouse liver corticosterone.

hypertension [231]. Furthermore, the combination therapy of an 11b-HSD1 inhibitor with glucocorticoid receptor agonists was claimed to be useful in treating cancer and inflammation-associated diseases and to minimise the side effects associated with glucocorticoid receptor agonist therapy [232]. O

O Ph

N

N

N N H O (241)

NH

N H (242)

Apart from 11b-HSD1 inhibitors based on the 1,2,4-triazole core structure, researchers from Merck also identified a series of amide compounds as selective 11b-HSD1 inhibitors, which can be potentially used in the treatment of diabetes and other symptoms associated with metabolic syndrome [233, 234]. Pyrazole carboxamides can be depicted by the general structural formula (243). R1 was defined as hydrogen or C1–C4 alkyl, R2 as substituted aryl ring and R3 was selected from hydrogen, chlorine or methyl group; R4 was defined as hydrogen or C1–C4 alkyl and R5 was claimed to be selected from aryl, cycloalkyl, bicycloalkyl or tricycloalkyl group. The representative compounds (244, 245) showed that

84

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1 Table 2.14 INHIBITION OF 11b-HSD1 BY AMIDE DERIVATIVES Compound

IC50 (nM)

Ref.

(235) (236) (237) (238) (239) (240)

40 45 6 118 4700 340

[227] [227] [227] [227] [228] [228]

2-adamantyl was the most common group linked to the amide [233]. A series of bicyclo[2.2.2]octane carboxamides, as represented by the general formula (246) and exemplified by selected compounds (247, 248), was also claimed as 11b-HSD1 inhibitors [234]. The compounds were screened with an SPA using microsomal 11b-HSD1 from CHO transfectants as the enzyme source. The compounds were claimed to possess IC50 values less than 500 nM and over 2-fold selectivity. O R3 R2

N

R4 N R5

O F

N

O

Cl

NH

N

Cl

N

Me

NH

N

N

R1

Me

Me

(243)

(244)

(245)

O O

O NH

NH

L R S O O

(246)

Q MeSO2

EtSO2

(247)

(248)

A series of substituted benzamides featuring a bicyclic aromatic system attached to the amide nitrogen was reported by Sterix to inhibit human microsomal 11b-HSD1 in a radioimmunoassay [235]. The representative compounds (249, 250) exhibit 54 and 71% inhibition of the enzyme at 10 mM, respectively.

X. SU, N. VICKER AND B.V.L. POTTER

S

O

S

O

Me N

N H

85

Me N

N

Pr

Me

Pr (249)

(250)

Further development revealed that the amide derivatives depicted by general structure (251) encompass another novel cluster of 11b-HSD1 inhibitors [135]. The preferred ring-A is usually an optionally substituted adamantyl group, branched alkyl or substituted carbocycle; ring-B is selected from diverse set of aromatic systems; X can be a bond or methylene unit; the linker length can be varied by changing n from 0 to 3; R2 is defined as H, alkyl or is taken together with the nitrogen and ring-B to form a cycle. The exemplified compounds (252–262) show W60% inhibition at 10 mM on both human microsomal enzyme and in a HEK293 cell line transfected with human 11b-HSD1 [135]. R2

O

n N

n

A

O

B

N

X

S

(252) (253) (254) (255)

(251)

N

(260)

N Me

n = 0, R =H n = 1, R =H n = 0, R =Me n = 1, R =Me

(256) (257) (258) (259)

Cl N

N

n = 0, R =H n = 1, R =H n = 0, R =Me n = 1, R =Me

O N

S

(261)

N

R

O

O

Me

n N

R

O

S

Me

(262)

Scientists from Pfizer have found a group of proline and morpholine derivatives to be potent 11b-HSD1 inhibitors and claimed methods of using these compounds to treat a condition mediated by the modulation of 11b-HSD1 [236]. The common structural feature is that the carbonyl group of the amide is normally attached to a 5- or 6-membered nitrogen-containing heterocarbocycle, whereas the nitrogen of the amide is attached to the group selected from carbocyclic groups, such as adamantane or 2,3-dihydro-1H-indene.

86

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

The core structure is generally d-prolinamide, 3-morpholine carboxamide or 2-piperazine carboxamide. The inhibition constant K iapp value was measured for human 11b-HSD1 using a high-throughput HPLC method and the percentage of inhibition at 100 nM was also reported. The representative compounds (263–276) selected from 373 examples all show about 1–3 nM K iapp values and give maximum inhibition at 100 nM (Table 2.15) [236]. R

R

O N

O N

N H

O

H N

N H

N H

O Cl

(263) (264) (265) (266) (267)

R = Me R = 4-Cl-Ph R = CF3 R = CH2OH R = 2-Pyridyl

(268) R = Me (269) R = Ph (270) R = 4-CN-Ph

R

N

O N

O N

(271) F

R

(273) R = 4-Cl-Ph (274) R = CF3

O N

N H

N H

(272)

O

HO

N H

(275) R = Me (276) R = Cyclopentyl

Recently, another patent from Pfizer claimed a set of adamantyl acetamide compounds as 11b-HSD1 inhibitors and their inhibitory activities were determined using the same HPLC method mentioned above [237]. The typified compounds (277–279) possess K iapp values of 1, 3 and 3 nM, respectively. Me N

O

O

O N H

(277)

N

N

N

N H

(278)

N H

(279)

Patent publications from Incyte claiming a broad range of amide derivatives as 11b-HSD1 inhibitors feature a core structural formula (280), for which Cy represents a phenyl ring substituted with a combination of groups such as halogens and aromatic rings. Both R1 and R2 at the benzylic position were

X. SU, N. VICKER AND B.V.L. POTTER

87

Table 2.15 IN VITRO INHIBITION OF 11b-HSD1 BY PROLINE OR MORPHOLINE DERIVATIVES [236] Compound

K iapp (nM)

(263) (264) (265) (266) (267) (268) (269) (270) (271) (272) (273) (274) (275) (276)

1.3 2.0 1 3.2 o1 2.1 2.4 1.1 1 o1 0.85 0.85 1.4 1

taken together with the carbon they attached to form a 3–6-membered ring, with the preference for spirocyclopropane. The nitrogen of the amide group is usually part of a 5- or 6-membered heterocycle, which was further substituted or fused to form a bicyclic or tricyclic system [238, 239]. The compounds were tested in vitro with an enzymatic assay using lysates from transfected HEK293 cells as the enzyme source. The activity at the cellular level was determined on peripheral blood mononuclear cells (PBMCs) with an ELISA method. Compounds with IC50 values less than 20 mM were regarded as active inhibitors; but no specific biological data were reported. The typical compounds (281–292) were selected from 655 examples [238]. R1 Cy

3 R2 R N

Cl

Cl

O

R4

N

O (280)

O Ph

N

OH

(281)

(282)

N OPh

Ph N

N

N Cl

O

Cl

(283)

Cl

O

O

(284)

(285)

Ph

88

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1 S tBu N

N N O

Cl

O

Cl

(286)

(287)

OH Cl N N

O

O

F3CS

O

O

Cl

(288)

N

(289)

H N N Cl

(290)

N

H

O

Cl

O

N Cl

F

(291)

O

O

O

(292)

Incyte claimed that amide compounds based on a similar template as above but with the nitrogen attached to a carbocycle or substituted alkyl, as exemplified by compounds (293–295), are 11b-HSD1 inhibitors in both enzymatic and cellular assays [240]. In addition, the combination of N-sulphonylpiperidine with the similar amide template also generates another cluster of inhibitors (296–299) [241, 242].

Cl

Cl

O N

(293)

Cl

O N H

(294)

Me

O N H

(295)

Ph OH

X. SU, N. VICKER AND B.V.L. POTTER

89

O N

O PhSO2

N

N

N

N Ph

PhSO2 N

(296)

(297) O O

O

N

O N PhSO2 N PhSO2 N (298)

(299)

Another patent from Incyte is more specifically based on the core structure of aryl amide as depicted by general structure (300), whereas Ar represents a phenyl ring substituted with a combination of substituents at the 2-, 3- and 4-positions (301–303). The compounds were tested in both enzymatic and cellular assays [243]. It was also claimed by Incyte that the replacement of the cyclopropyl group with a dimethyl group at the benzylic position of inhibitors based on the template mentioned above would retain the 11b-HSD1 inhibitory activity [242].

Cl N

Ar

O

O

N

O

O

O

O (300)

O

(301)

MeOCO PhO

N N

N O

O

Me N

O O

(302)

(303)

O

O

90

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

More recently, researchers from Incyte have found that compounds based on the core structure of 2,7-diazaspiro[4.5]decan-1-one, as represented by compounds (304–307), also inhibit human 11b-HSD1 with IC50 values less than 20 mM at both enzymatic and cellular levels [244]. However, no specific biological data were reported for individual compounds in patent publications. O

O

OH N

N

OH N

N

F N NC

(304)

Cl

O

OH

Me N SO2

(305)

Cl

O Me

N

N

N SO2

(306)

(307)

It was discovered by scientists from Eli Lilly that cycloalkyl lactam derivatives as depicted by general formula (308) are 11b-HSD1 inhibitors. Their use as medicaments to treat diabetes, hyperglycemia, obesity, hypertension, hyperlipidemia, syndrome X and other conditions associated with hyperglycaemia was claimed in a series of patent publications [245–247]. For the general structure (308), G is a methylene or ethylene unit; L is a bivalent linking group selected from C1–C4 alkylene, CH(OH), S and O; the R2 group attached to the nitrogen was normally a saturated carbocycle or heterocycle; R1, R3 could be selected from H, OH, alkyl groups and R0 is a substituted phenyl ring or substituted bicyclic aromatic ring. The exemplified compounds were screened using recombinant human 11b-HSD1 in a fluorescence assay. The activities were also evaluated with an acute in vivo cortisone conversion assay on C57BL/6 mice after oral dosing. While active compounds are defined as those with IC50 values less than 20 mM, representative compounds (309–312) show IC50 values less than 500 nM (Table 2.16) [245].

X. SU, N. VICKER AND B.V.L. POTTER

91

Table 2.16 IN VITRO INHIBITION OF HUMAN 11b-HSD1 BY LACTAM DERIVATIVES Compound

IC50 (nM)

Ref.

(309) (310) (311) (312) (313) (314) (315) (316) (317) (318) (319) (320) (321) (322) (323) (324) (325) (326)

230 378 384 273 190 682 257 427 162 508 276 354 134 213 344 247 353 423

[245] [245] [245] [245] [246] [246] [246] [246] [246] [246] [246] [247] [247] [247] [247] [247] [247] [247]

Cl R 0

Cl

O

O

R

O

3

R

G

OH

N

2 N R

L

R

Cl

N Ar

R1

(308)

(309) R = Cl (310) R = H

(311) R = H, Ar = 4-Pyridyl (312) R = Me, Ar = 3-Pyridyl

Further development based on the similar template led to the discovery of inhibitors with a bicyclic aromatic system linked to the lactam through a methylene unit. The most common bicyclic rings are benzothiophene, benzofuran or naphthalene, as exemplified by compounds (313–326), and their activity is reported as IC50 values for human 11b-HSD1 inhibition (Table 2.16) [246, 247]. O

R

X

Cl

O

N

(313) R = H, X = S (314) R = H, X = O (315) R = 2-F-4-Pyridyl, X = S (316) R = 4-CO2H-Ph, X = S

S

R1 R2

N

(317) R1R2 = OCH2O (318) R1 = H, R2 = OMe

92

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

R2

Cl

O

O

N

S N

R1

OH

(320) R1 = H, R2 = Br (321) R1 = OMe, R2 = H (322) R1 = OMe, R2 = Cl (323) R1 = 2,6-di-Cl-Ph, R2 = H

(319) O

OMe OH

N MeO

N

n O

(324) n =1 (325) n =2

(326)

Recently, the adamantyl amide core structure illustrated by the general structure (327) forms the basis of a series of patent publications from Abbott [248–250]. R1 and R2 are independently selected from varied groups such as optionally substituted alkyl, cycloalkyl, aryl, heterocycle or are taken together with the nitrogen to form a heterocycle; R3 and R4 are independently selected from hydrogen, alkyl, cycloalkyl, aryl, heterocycle or are taken together with the intervening atoms to form a cycloalkyl or heterocycle; R5 is a member selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heterocycle. The compounds were tested for their activity on truncated human 11b-HSD1 (residues 24–287) with an SPA protocol, and (328–331) were reported to have IC50 values of 35, 46, 34 and 48 nM, respectively [248]. Compound (328) is a selective inhibitor with IC50 value W10 mM for 11b-HSD2 and its in vivo activity was evaluated with a mouse dehydrocorticosterone challenged model. Male CD-1 mice were dosed with vehicle or compound at various times before being challenged with 11-dehydrocorticosterone (11-DHC). After 0.5 h, blood samples from the mice were analysed for corticosterone levels with an ELISA or HPLC/ MS method. Compound (328) exhibits significant inhibition of the conversion of 11-DHC to corticosterone [249, 250]. R5 R4 R3 R2 N N O

(327)

R1

Me

H N

H N

N O

(328)

OH

N O

(329)

N

X. SU, N. VICKER AND B.V.L. POTTER

H N

N O

Me

H N

OBz N

93

N O

O N O

(330)

(331)

Further development of adamantyl amide inhibitors by Abbott involved the introduction of a combination of substituents onto the adamantyl ring at the 1- and/or 3-position. The preferred substituents normally consist of groups selected from halo, hydroxyl, alkyl, aryl, carbonyl, sulphonyl, heterocyclic or amino groups [130, 251, 252]. The exemplified compounds generally have IC50 values less than 600 nM, preferably less than 50 nM. The selectivity over 11b-HSD2 is generally over 10-fold, and preferably over 100-fold. Compound (332) inhibits the conversion of 11-DHC to corticosterone significantly in vivo [130]. Compounds (333–337) were also shown to be potent selective inhibitors of human 11b-HSD1 (Table 2.17) [252]. A method of increasing the metabolic stability of a pharmaceutically active adamantyl compound by incorporating a combination of substituents onto the adamantyl ring has been disclosed [253]. The preferred groups attached to the 1- or/and 3-position of the adamantyl group include members such as carboxyl, amino, tetrazolyl, carboxyalkyl, SO2NHCO, CONH or NHCO. In an in vitro metabolic half-life study, (338) showed greatly enhanced microsomal stability in microsomes from human, mouse and rat (Table 2.18) [253].

Table 2.17 IN VITRO INHIBITION OF 11b-HSD BY ADAMANTYL AMIDE DERIVATIVES Compound

(333) (334) (335) (336) (337) (338) NR=Not reported.

IC50 (nM), human 11b-HSD1

11b-HSD2

110 92 150 140 82 50

W10,000 W10,000 W10,000 W10,000 W10,000 NR

Ref.

[252] [252] [252] [252] [252] [253]

94

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1 Table 2.18 PK PROPERTIES OF COMPOUND (338) [253]

Assay

Microsomes Hepatocyte

Liver microsomal and hepatocyte intrinsic clearance (CLint in L/h/kg) Human

Mouse

Rat

Monkey

Dog

0.29 0.09

7.61 0.82

2.4 0.31

1.89 0.28

0.45 0.22

Me

H N

N

HO

O N

CF3

(332)

Me

N

HO N

O

Me

H N

O Me

(333)

∗ RNH

N O

Me

H N

N

R= N

N

HO

∗ F

HO2CNH

(335)

(336) ∗

CF3

∗

HO2C

O F (334)

(337)

(338)

Rohde and coworkers investigated the SAR of 2-amino-N-(adamant-2-yl) acetamide derivatives aiming for the discovery of potent, selective 11bHSD1 inhibitors with a suitable PK profile [145]. Compound (339) was identified from a high-throughput screen as a potent 11b-HSD1 inhibitor on both human and mouse enzymes with good selectivity over 11b-HSD2 (IC50 W100 mM). Its cellular potency was also confirmed on the HEK293 cell line (Table 2.19) [145]. However, (339) suffers from rapid metabolism in both human liver microsomes (HLM) and mouse liver microsomes (MLM) as indicated by hepatic intrinsic clearance (CLint) values [254]. Based on mass spectral fragmentation patterns, further metabolism studies revealed three major metabolites from aryl ring oxidation, adamantane oxidation and N-piperazine dealkylation. Optimisation on the aryl moiety generated (340, 341), which demonstrates enhanced metabolic stability in both human and mouse microsomes (Table 2.19) [145].

X. SU, N. VICKER AND B.V.L. POTTER

95

Table 2.19 INHIBITION OF 11b-HSD1 AND PK PROPERTIES OF ADAMANTYL AMIDE DERIVATIVES [145] Compound

11b-HSD1 Ki (nM)

(339) (340) (341) (342) (343) (344) (345) (346) (347) (348) (349) (350) (351)

Human

Mouse

14 5 24 8 67 32 26 5 8 3 5 12 4

53 21 51 34 300 120 110 15 8 2 7 9 5

HEK293 IC50 (nM)

HLM CLint L/h/kg

MLM CLint L/h/kg

260 210 190 130 W15,000 550 450 29 46 35 71 180 21

110 35 10 1 11 2 14 3 6 o1 o1 o1 2

380 63 142 41 65 136 o6 7 130 60 23 o6 20

The predominant metabolites from (341) were identified as hydroxylated analogues; the hydroxylation is thought to occur at the bridgehead of the adamantane ring. H N

CN

N O

H N

N

N O

N

N Cl

(339)

(340)

H N

N O

N N

(341)

CF3

The introduction of a hydroxyl or fluoro group at the bridgehead of the adamantane was pursued and optimisation gave compounds (342–345), among which (342) was identified as the major metabolite of (341) and exhibited the best PK profile. Compound (342) also retained the potency

96

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

and selectivity across species for 11b-HSD1 and showed no significant cross-activity when assessed in W50 receptor binding assays [145]. The Z-isomer (343) is a very weak inhibitor at the cellular level. Although showing an improved PK profile, the fluorine-substituted analogues (344, 345) were less potent than (342) on a HEK293 cell line. Compounds (346, 347) with mono- or dimethyl substituents at the acetamido carbon linker maintained high potency on the microsomal enzyme and demonstrated enhanced cellular potency (Table 2.19) [145]. H N

H N

N

R1 O

(342) (343) (344) (345)

N

HO

N

R2

R1 R2

O N

N N

CF3

R1 = OH, R2 = H R1 = H, R2 = OH R1 = F, R2 = H R1 = H, R2 = F

CF3

(346) R1 = Me, R2 = H (347) R1 = Me, R2 =Me

The E-5-hydroxy-2-aminoadamantane moiety was held constant and the acetamido substitution was varied. Compounds (348–351) were reported to have a very good PK profile and highly potent activity towards 11b-HSD1 in microsomal and cellular assays (Table 2.19) [145]. The inhibitory activity of (346) following oral dosing (30 mg/kg) was further evaluated in an ex vivo mouse study using liver, adipose and brain tissue at 1 and 7 h post-dose. A modest inhibition (W3674%) was observed at 1 h in all three tissues, which then declined or disappeared by 7 h. In general both (342) and (346) proved to be potent selective 11b-HSD1 inhibitors with robust PK profiles. H N

N

HO

F N

HO

O

O CF3

(348)

Me

H N

N

HO

O (349)

Me

H N

Me

H N

N

HO

O

O

O CF3 (350)

O

(351)

X. SU, N. VICKER AND B.V.L. POTTER

97

Further modifications were performed on the acetamido carbon linker based on the COOH or CONH2 substituted adamantane template [255]. With a gemdimethyl attached to the linker carbon, the carboxamide substituted (353) is more potent on mouse enzyme and less-metabolically stable than the carboxylic acid substituted analogue (352). With a cyclopropane forming part of the linkage, compounds (354, 355) with E-COOH or E-CONH2 at the bridgehead of adamantane, were more potent than their Z-isomers (356–357) (Table 2.20). The carboxylic acid derivative (354) is more stable in MLM but relatively weaker against mouse 11b-HSD1 than (355). The ethyl or cyclopropylsubstituted compounds (358, 359) show high potency and selectivity on both human and mouse microsomes, as well as on a HEK293 cell assay. Compounds (358–360) also exhibit good metabolic stability on MLM (Table 2.20). Compound (354) was further evaluated for its mouse PK profile, which showed maximal bioavailability, a high oral AUC (197 mL/h/mL), with a moderate i.v. half-life (0.1 L/h/kg) and a low volume of distribution [255]. H N

Me Me

H N

N

R O

N

R1 N

O N

N

2

R

N

CF3

(352) R = CO2H (353) R = CONH2

(354) (355) (356) (357)

CF3

R1 = CO2H, R2 = H R1 = CONH2, R2 =H R1 = H, R2 = CO2H R1 = H, R2 = CONH2

Table 2.20 INHIBITION OF 11b-HSD1 AND METABOLIC STABILITY OF ADAMANTYL AMIDE DERIVATIVES [255] Compound

(352) (353) (354) (355) (356) (357) (358) (359) (360) (361)

11b-HSD1 Ki (nM) Human

Mouse

13 9 7 8 250 13 6 6 7 42

180 5 500 15 1,700 1,500 15 5 3 26

ND=Not determined.

HEK293 IC50 (nM)

MLM % remaining

39 45 45 22 1,900 490 24 38 19 29

89 65 98 70 ND ND 88 93 91 ND

98

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1 n R

H N

H N

N

H2NOC

O

H2NOC

N N

(358) R = Et (359) R = Cyclopropyl

N O

N N

CF3

(360) n = 1 (361) n = 2

CF3

The scope of the adamantyl amide inhibitors with enhanced metabolic stability was further expanded to include compounds with varied substituents on the acetamido carbon [131, 256], especially extended ether analogues as represented by compounds (362–365). Potent inhibitors of 11b-HSD1 were identified with IC50 values less than 600 nM, with the most active ones showing an IC50 less than 50 nM. Me H N H2NOC

Me Me

H N

O

HO2C

O

HO2C

(363)

Me Me O O

(364)

O O

(362)

H N

Me Me

CF3

Cl H N H2NOC

Me Me O O

(365)

Compound (365) was highlighted for its excellent in vitro activity and selectivity against the target enzyme, with IC50 values of 32 and 71 nM in human and mouse 11b-HSD1, respectively, and an IC50 value of 22 nM on the HEK293 cell line. However, its poor PK profile in mice, due to the metabolism of phenoxy side chain, needs to be addressed by further modification [129]. A series of compounds with the carboxamide at the bridgehead replaced by heterocyclic bioisosteres was synthesised and evaluated. Polar heterocyclic derivatives (366, 367) were found to be potent only in a microsomal enzyme assay and especially labile to mouse liver

X. SU, N. VICKER AND B.V.L. POTTER

99

Table 2.21 INHIBITION OF 11b-HSD1 AND METABOLIC STABILITY OF ADAMANTYL AMIDE DERIVATIVES [129] Compound

11b-HSD1 IC50 (nM)

(366) (367) (368) (369) (370) (371) (372) (373) (374) (375)

Human

Mouse

35 10 39 45 41 89 6 5 14 47

53 11 26 92 87 74 4 11 65 59

HEK293 IC50 (nM)

MLM % remaining

476 110 124 82 119 455 18 38 165 169

41 3 64 94 79 78 87 83 100 87

microsomal metabolism (Table 2.21). The hydroxyamidine derivative (368) showed improved stability, whereas (369–371) exhibited a robust PK profile. Further examination in mouse ex vivo pharmacodynamic and PK studies revealed that (368), although suffering from rapid clearance (4.6 L/h/kg) and a short half-life (0.4 h), exhibited potent inhibition of 11bHSD1 at 7 h and 16 h in liver (89/84%), fat (92/67%) and brain (85/79%) after oral dosing 30 mg/kg on DIO mice. This observation might be attributed to active metabolites of (368) [129].

∗

Cl H N

Me Me

R=

H N

H N

∗

NH2

∗

N N

N

N OH

(366)

(367)

(368)

O

R O

HN N R=

∗

NH2

CO2H

(369)

∗

N

(370)

N

∗

CO2H

(371)

Furthermore, the alterations to the aromatic side chain generated compounds (372–375), which all showed excellent metabolic stability and high potency in a microsomal enzyme assay (Table 2.21) [129].

100

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

R H N H2NOC

(372)

Me Me O

N

O

H N H2NOC

R = Br

(373)

R=

∗

N O

R

(374)

R = CN

(375)

R=

N N

Me Me

∗

N O

To obstruct the plasma hydrolysis of the primary carboxamide at the bridgehead of the adamantyl group in (365), further extensions at this position were pursued with a series of secondary amides [71]. Compound (376) shows robust in vitro activity and metabolic stability, but is subject to phase-II metabolism. Compounds (377, 378) as carboxylic acid mimics suffer from both low cellular activities and lower metabolic stabilities, whereas heterocycle-substituted compounds (379, 380) are metabolised very quickly in MLM (Table 2.22). Other attempts to find a suitable replacement for the primary carboxamide yielded no significant improvement. In ex vivo PD studies, (376) was found to be a quite potent inhibitor in liver at 1, 7 and 16 h with inhibition at 99, 93 and 77%, respectively; whereas in fat it only shows moderate activity with about 37% inhibition. The sulphonamide (377) exhibits moderate inhibition in liver, but weak activity in fat and brain tissue [71].

Table 2.22 INHIBITION OF 11b-HSD1 AND METABOLIC STABILITY OF ADAMANTYL AMIDE DERIVATIVES [71] Compound

(376) (377) (378) (379) (380) (381)

11b-HSD1 IC50 (nM) Human

Mouse

15 51 16 27 25 12

12 24 18 11 14 4

HEK293 IC50 (nM)

MLM % remaining

59 300 690 92 91 120

94 35 51 5 16 78

X. SU, N. VICKER AND B.V.L. POTTER CO2H H N

O R

Me Me

R=

O

SO2NH2

HN N N

∗

N

∗ ∗

O

N H

101

(376) Cl

(377)

S

R=

(378)

N N

∗

∗

∗

(379)

(380)

CONH2

(381)

Other modifications involved introducing a sulphone or sulphonamide group at the bridgehead of the adamantyl group [70]. The methyl sulphone (382) exhibited robust in vitro activity and moderated cellular potency and metabolic stability in MLM. Further extension to the ethyl sulphone gave poorer cellular activity, as in (383). Compounds (384, 385), although showing good in vitro activity, are less active at the cellular level. The sulphonamide series displayed similar SAR (Table 2.23). Small electronwithdrawing groups at the phenyl ring improve potency. The 4-substituted compound (387) is more stable in MLM than the 2-substituted analogues (386, 388). The methyl-substituted sulphonamide (390) is a weak inhibitor at the cellular level and is metabolised very quickly in MLM (Table 2.23) [70]. In mouse PK studies, (382) exhibits a good volume of distribution, excellent bioavailability, moderate half-life (t½ i.v. 1.0 h) and moderate Table 2.23 INHIBITION OF 11b-HSD1 AND METABOLIC STABILITY OF ADAMANTYL AMIDE DERIVATIVES [70] Compound

(382) (383) (384) (385) (386) (387) (388) (389) (390)

11b-HSD1 Ki (nM) Human

Mouse

7 26 5 7 4 5 7 6 8

4 7 2 8 2 5 3 3 8

ND=Not determined.

HEK293 IC50 (nM)

MLM % remaining

98 450 120 410 83 230 54 125 620

57 ND ND 70 36 96 38 61 o1

102

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

clearance (Clp i.v. 1.1 L/h/kg) at a 10 mg/kg dose. Further studies with (382) in the monkey at a dose of 2.5 mg/kg also show a longer half-life (t½ i.v. 5.1 h), excellent bioavailability, low clearance (Clp i.v. 0.4 L/h/kg) and a volume of distribution consistent with tissue penetration. The longer halflife in the monkey is possibly attributed to higher metabolic stability as revealed by the difference of intrinsic clearance (1.5 L/h/kg in monkey vs. 6.0 L/h/kg in mouse). Human and monkey liver microsomes give similar metabolic stability results (1.6 L/h/kg in human) with compound (382). In the ex vivo PD studies with DIO mice at an oral dose of 30 mg/kg, (382) was discovered to inhibit 11b-HSD1 in liver at 1, 7 and 16 h with inhibitions of 95, 95 and 89%, respectively; whereas in fat and brain it also showed robust activity at the same time interval, with inhibitions of 87, 93, 86% in fat and 90, 90, 77% in brain [70].

R

H N

Me Me O

O S O

(382) R = Me, (383) R = Et, (384) R = Me, (385) R = Me,

Ar

O

Ar = 2-Cl-4-F-Ph Ar = 2-Cl-4-F-Ph Ar = 2-Cl-Ph Ar = 4-Cl-Ph

R NH

H N

Me Me O

O S O

Ar

O

(386) R = H, Ar = 2-Cl-Ph (387) R = H, Ar = 4-Cl-Ph (388) R = H, Ar = 2-OCF3 (389) R = H, Ar = 2-Cl-4-F-Ph (390) R = Me, Ar = 4-Cl-Ph

The replacements of the adamantyl group with other carbocycles or heterocycles to overcome the metabolism problem associated with an unsubstituted adamantyl group were also pursued by scientists from Abbott [257]. In the aryl piperazine series, compounds (391–393) were shown to be relatively potent and selective for a human microsomal enzyme, as well as metabolically stable in MLM; meanwhile, their potency for the mouse enzyme varied from 62 to W10,000 nM, indicating the mouse enzyme may prefer a primary amide over an acid group at the bridgehead (Table 2.24). Compounds (391, 393) also show strong inhibition at the cellular level. In the aryl ether series, all the compounds (394–396) exhibit excellent potency and selectivity in human and mouse enzymes, as well as robust metabolic stability in MLM (Table 2.24). In the ex vivo PD studies, (396) shows strong inhibition of 11b-HSD1 in liver and brain at 7 and 16 h post-dose (80/70% in liver; 60/72% in brain) and weaker activity in fat (40/20%) [257].

X. SU, N. VICKER AND B.V.L. POTTER

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Table 2.24 INHIBITION OF 11b-HSD1 AND METABOLIC STABILITY OF AMIDE DERIVATIVES [257] Compound

11b-HSD1 IC50 (nM)

(391) (392) (393) (394) (395) (396)

Human

Mouse

97 44 62 22 11 13

W10,000 1,150 62 32 28 28

Me Me R

HN

N N

O

R=

N

MLM % remaining

75 606 65 80 22 450

97 91 85 90 89 85

∗ H

H

HEK293 IC50 (nM)

H

CO2H CF3

R

O O

R= HO2C

(393)

H

∗ N H

H

H

(392)

H

Cl Me Me

∗ N H

H2NO2C

H2NO2C

H

(394)

∗ N H

H2NO2C

H

(391)

H

∗ N H

HO2C

N H

(395)

∗

(396)

A novel series of butyrolactam 11b-HD1 inhibitors has been discovered and a highly efficient synthesis of these compounds reported [258, 259]. Compounds (397, 398, 400) all show excellent in vitro activity with IC50 values in the low nanomolar range in both human and mouse enzymes. Moreover, they are all metabolically stable in MLM and possess potent cellular activity (Table 2.25). With a bicyclo[2.2.2]octane moiety as the replacement for adamantane, (399) shows similar properties to its analogue (396), but also suffers from weaker cellular potency. Compounds (401, 402) with polar aromatic substituents exhibit improved water solubility while retaining the same relative in vitro potency in both species (Table 2.25). At a dose of 5 mg/kg i.v. or 10 mg/kg oral, (400) displays a good PK profile in mice; furthermore, the ex vivo PD studies reveal the inhibition for mouse liver 11b-HSD1 is 99, 94 and 67% at 1, 7 and 16 h, respectively [258].

104

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1 Table 2.25 INHIBITION OF 11b-HSD1 AND METABOLIC STABILITY OF BUTYROLACTAM DERIVATIVES [258]

Compound

11b-HSD1 IC50 (nM)

(397) (398) (399) (400) (401) (402)

Human

Mouse

7 5 15 3 41 19

3 9 10 2 32 14

HEK293 IC50 (nM)

MLM % remaining

39 46 500 45 43 260

88 92 91 97 ND 67

ND=Not determined.

Me O R

Me

H

N O

CN

R=

N

H ∗ HO2C

H H2NOC

Me

N

Me

∗

O R

H2NOC ∗

H

(397)

O

∗

(399)

(398)

∗

N

∗

R= CN

H2NOC

N

N

N

N

(400)

(401)

N

(402)

Another class of 11b-HSD1 inhibitor was discovered from a dichloroaniline amide family by Abbott after the initial hit compound (403) was identified from high-throughput screening [260]. While (403) shows potent inhibition in both human and rat enzymes, it suffers from quick metabolism in rat liver microsomes mainly because of the hydroxylation of the cyclopentyl ring. The cyclohexyl amide analogue (404) retains the same level of potency as (403), while the polycyclic amides (405, 407) show robust inhibition in a HEK293 cell line (Table 2.26). To introduce polar functionality into the molecule, substituted piperazine and amino piperidine dichloroaniline amides were evaluated; most show weak cellular activity. The trans-piperidine amides (408, 409) are well tolerated by human 11b-HSD1 and retain high activity in a HEK293 cell-based assay (Table 2.26) [260].

X. SU, N. VICKER AND B.V.L. POTTER

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Table 2.26 INHIBITION OF 11b-HSD1 BY DICHLOROANILINE DERIVATIVES [260] Compound

11b-HSD1 Ki (nM)

(403) (404) (405) (406) (407) (408) (409)

Human

Rat

8 14 4 3 4 12 22

6 5.3 8 6 4 160 39

O Cl

HEK293 IC50 (nM)

140 130 17 100 41 72 76

O N

H2N

Cl

O Cl

N

H2N Cl

H2N

(405)

O

O N Me

Cl

Me

Cl

Me

Cl

(404)

O

H2N

N

Cl

(403)

Cl

Me

N

Cl

N

H2N

H2N

Me

Me

N

Cl

Cl

O

(406)

(407)

X

(408) X = O (409) X = NH

Coincidently, high-throughput screening in Biovitrum/Amgen identified the hit compound (410) in a piperidinylbenzimidazolone family similar to that of (409) [261]. Based on the result of an SPA, (410) was found to be potent for human 11b-HSD1 inhibition with Ki value of 300 nM. The attempts to change the piperidine-amide linker to a sulphonamide, urea or carbamate resulted in decreased activity. Alterations to the substituents in the phenyl ring generated compounds (411, 412) with Ki values of 110 and 180 nM, respectively. Based on the 3-methoxy-4-methylbenzamide template, the replacement of the benzimidazolone moiety with oxindole or benzothiazolone gave relatively potent analogues (413) (Ki=240 nM) or (414) (Ki=170 nM). Furthermore, introduction of a methyl group at either

106

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

the 3- or 4-position of benzimidazolone ring maintained the same level of activity as shown by (415) (Ki=180 nM) and (416) (Ki=140 nM). O MeO

O MeO

N

R

N

N

Me

N

NH

O

X

O

(410) R = H (411) R = NH2 (412) R = Me

(413) (414) (415) (416)

R

R = H, X = CH2 R = H, X = S R = H, X = NMe R = Me, X = NH

The benzamide derivatives with general formula (417) form the basis of a recent patent from Amgen which claims these 11b-HSD1 inhibitors and their use in the treatment of diabetes, obesity and other related conditions [262]. The exemplified compounds feature an (S)-1,1,1-trifluoro2-phenylpropan-2-ol moiety attached to the carbonyl of an amide linker. The inhibitory activities were evaluated with an SPA method on both microsomal enzymes and the HEK293 cell line and were reported to have IC50 values between 1,000 and o1 nM. R4

O N

2

R1

O

R

R6

R5

HO

R3 (417)

N

F3C

F3C HO

Me (418)

F

O N

Me (419)

In addition to 11b-HSD1 inhibitors based on the triazolo[4,3-a]pyridine core structure, BMS also disclosed series of pyridyl amide or pyridyl sulphonamide compounds as selective 11b-HSD1 inhibitors which can be used in the treatment of diabetes and other symptoms associated with metabolic syndrome. It was claimed that the exemplified compounds have IC50 values less than 10 mM when screened on recombinant human 11b-HSD1; no specific data were released [213]. Recently Webster et al. have identified two adamantyl amides (420, 421) as 11b-HSD1 inhibitors from a focused library [263]. Both exhibit sub-micromolar potency in cellular assays and (421) is selective over 11b-HSD2 with no inhibition at 10 mM (Table 2.27). Further optimisation

X. SU, N. VICKER AND B.V.L. POTTER

107

Table 2.27 INHIBITION OF 11b-HSD BY ADAMANTYL AMIDE DERIVATIVES [263] Compound

11b-HSD1 IC50 (nM)

11b-HSD2 (% inhibition) at 10 mM

(420) (421) (422) (423) (424) (425) (426) (427) (428) (429) (430) (431) (432) (433)

600 82 53,000 800 600 1,000 7,200 12,000 15,000 200 700 1,500 1,500 500

NR 0 ND 11 51 0 11 0 0 0 13 31 80 40

NR=Not reported; ND=not determined.

revealed that the p-tolyl substituted compound (421) is about 10-fold more potent than unsubstituted analogue (423). Hydroxylation at the bridgehead position led to (422) with very weak cellular potency, which suggested that protection from microsomal oxidation at that position is necessary to retain in vivo activity. Compounds (424, 425) show reduced activity compared with the initial hit (421). Attempts to replace the adamantyl group with other hydrophobic groups were unsuccessful, as shown by compounds (426–428) (Table 27). O

N

N

Et

N R

N

Et

N

R O

(420)

O

O

(421) (422) (423) (424) (425)

R = Tolyl R = OH R=H R = 4-OMe-Ph R = Bz

Me R=

Bz

∗

∗ F

(426)

(427)

(428)

Modification of the nitrogen substituents in the amide group gave mixed results. The piperidine analogue (429) shows enhanced activity with an IC50 value of 200 nM. In the adamantyl series, replacing the ethyl with a methyl group retains the activity at the same level, while the analogue with ethanol

108

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

side chain (431) is less potent than (423) and (430) (Table 2.27). In the p-tolyl series, the best N-substituent is still the original ethyl group. Compound (421) was also tested on murine 11b-HSD1 and shows an IC50 value of 81 nM. In ex vivo PD studies, (421) exhibits robust inhibition of mouse 11b-HSD1 in liver (63%), fat (54%) and brain (39%) tissues at 1 h post-dosing (10 mg/kg i.p.) [263]. N N

Me

R

N

O

N

R

N

O O

(429)

(430) R = Me (431) R = CH2CH2OH

(432) R = Bz (433) R = CH2CH2OH

THIAZOLONE AND ISOXAZOLE DERIVATIVES

Patent publications from Biovitrum/Amgen disclosed a novel class of 11b-HSD1 inhibitors with a thiazolone core structure as depicted by representative compounds (434–436) [264, 265]. Generally, active compounds favour a carbonyl group at the 3-position of the thiazolone ring, hydrophobic substituents at the 4-position and a secondary amine attached at the 2-position. The exemplified compounds were screened with an SPA on recombinant human 11b-HSD1. Compounds (434–436) were shown to be active with Ki values of 384, 107 and 174 nM, respectively. O

N

N N H

O

O

S

(434)

Me

N N H

(435)

S

O

N

iPr

S

NH

Me

Cl

(436)

A comprehensive SAR study was performed based on the thiazolone template to find potent 11b-HSD1 inhibitors with suitable PK profile [266]. The initial hit (437) with a Ki value of 503 nM was disclosed in a patent publication [265]; but compounds in this series suffered from rapid clearance in the rat (Z2,000 mL/h/kg) [266]. Modifications to the 2-benzylamino thiazolone template gave (438) which shows a 10-fold increase of potency in vitro compared with (437) and exhibits excellent cellular activity (Table 2.28). With an (S)-methyl group introduced at the

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Table 2.28 INHIBITION OF 11b-HSD1 BY THIAZOLONE DERIVATIVES [263] Compound

11b-HSD1 inhibition

(437) (438) (439) (440) (441) (442) (443) (444)

SPA Ki (nM)

Whole cell IC50 (nM)

503 65 50 18 3 9 18 20

ND 53 20 41 18 10 18 56

ND=Not determined.

benzylic position, the potency of (439) at the cellular level is increased, whereas its (R)-epimer is much weaker (Ki=130 nM, IC50=268 nM). Introduction of a trifluoromethyl, fluoro or chloro group at the ortho position of the phenyl ring results in increased potency in vitro and maintains the cellular activity (Table 2.28). The C-5 (S)-enantiomer of (441) was shown to be very potent (Ki=4 nM, cellular IC50=4 nM). Although being highly potent, (441) also suffered from rapid clearance (2,570 mL/h/kg) in the rat with a 2 mg/kg i.v. dose. Replacing the isopropyl group at the 5-position with a fluorinated short alkyl group could possibly block the microsomal oxidation of the side chain and therefore slow down clearance. Compound (443) retains the activity and shows a dramatically improved PK profile (Clp=890 mL/h/kg, F=47%). Similarly, (444) also shows good potency and PK profile (Clp=501 mL/h/kg, F=55%). The C-5 (S)-enantiomer of (444) displays both high potency (Ki=22 nM, cellular IC50=33 nM) and robust PK properties (Clp=188 mL/h/kg, F=75%). In the ex vivo study in a mouse model, the C-5 (S)-enantiomer of (444) exhibits inhibition of 11b-HSD1 at 2 h (88%) and 6 h (91%) post-dose (30 mg/kg oral). O iPr

N

CF3

N H

(437)

O

S

2

R

Me

1

N H

(438) (439) (440) (441) (442)

iPr

N

R

O

S

Me

R1 = H, R2 = H R1 = Me, R2 = H R1 = Me, R2 = CF3 R1 = Me, R2 = F R1 = Me, R2 = Cl

F

R

N

Me N H

S

(443) R = CFMe2 (444) R = CF3

Me

110

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1 Table 2.29 IN VITRO INHIBITION OF 11b-HSD BY METHYLISOXAZOLE DERIVATIVES [267] Compound

IC50 (nM)

(445) (446) (447) (448) (449)

11b-HSD1

11b-HSD2

84 80 16 93 15

21,000 6,000 11,600 5,900 21,000

Shionogi has identified a series of amide or ketone compounds with a nitrogen-containing 5-membered heterocycle, usually 3-methylisoxazole, attached to the carbonyl group as 11b-HSD1 inhibitors [267]. With an HTRF protocol, the representative compounds (445–449) were reported with IC50 values in the nanomolar range (Table 2.29). Compounds with a hydrophobic carbocycle attached to the 4-position of the isoxazole ring through a bond, a sulphide or a sulphone linker provided an optimal IC50 value of 15 nM and possessed over 700-fold selectivity against 11b-HSD2 [267].

O

S

R

O Me

N H

O N

(445) R = Ph (446) R = Cyclohexyl (447) R = Cycloheptyl

R Me

Ph

O N

(448) R = -SO2Ph (449) R = -SCyclohexyl

PYRAZOLE, PYRAZOLONE, PYRIDAZINE AND TETRAZOLE DERIVATIVES

Amgen claimed compounds with a pyrazole moiety as the core structure as 11b-HSD1 inhibitors and their use in the treatment of diabetes, obesity and metabolic syndrome [268]. Presumably, the pyrazole ring, as a linker system for the molecule, functions as a hydrogen-bonding acceptor/donor to interact with the enzyme. The compounds were screened against recombinant human 11b-HSD1 with an SPA protocol. Representative compounds (450–454) were reported to have IC50 values less than 10 nM.

X. SU, N. VICKER AND B.V.L. POTTER

F

HN N

HN N

R

Ph

111

R

Me

Me

Cl

∗

N ∗

∗

N

R=

N ∗

R = Ph Cl

Cl (450)

F

(451)

(452)

(453)

(454)

A novel class of 11b-HSD1 inhibitors based on the core structure of (1H-pyrazol-3-yl)(pyrrolidin-1-yl)methanone outline the basis of a patent publication from Japan Tobacco [269]. Compounds (455–462) were screened with an SPA and are reported to have IC50 values of o30 nM. For the active compounds, the substituent on the nitrogen of the piperidine ring is most likely selected from groups consisting of alkyl, carbocyclic and heterocyclic with suitable hydrogen-bonding properties. O CF3

(455) (456) (457) (458)

O

N N

N N

R

F F ∗

R=

N H

CH2OH

∗ N

(460)

Me Me ∗

OH ∗

(459)

R = NH2 R = NHCH2CH2OH R = CH2CH2NH2 R = CH2NHSO2Me

CONH2

N (461)

(462)

Hoffmann-La Roche also disclosed that compounds with a 1H-pyrazole4-carboxamide core structure were identified as potent 11b-HSD1 inhibitors [270]. While the substituent at the 1-position of pyrazole is usually kept consistent as a methyl group, the variations at the 5-position with substituted phenyl rings give highly potent compounds (463–468) with IC50 values less than 100 nM as identified by an HTRF screening method. O

R

N

N Me N

(463) R = 3-iPr-Ph (464) R = 4-MeS-Ph (465) R = 3-Cl-2-Me-Ph

(466) R = 3-CF3-Ph (467) R = 4-CH2OH-Ph (468) R = 3-Cl-4-EtO-Ph

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

The scope of inhibitors with a pyrazole ring system was further expanded to include compounds with pyrazolone core structure [271, 272]. The compounds were tested for their activity on a microsomal enzyme from transfected HEK293 cells with an ELISA kit. A pyrazolone fused with a polycyclic system at the 4- and 5-positions gave a good template for further optimisation. Compounds (472–474) with varied substituents at the 2-position of this template achieve high potency against human 11b-HSD1 with IC50 values from 10 to 23 nM (Table 2.30). Hoffmann-La Roche also claimed compounds with a pyridazine ring system as 11b-HSD1 inhibitors as shown by representative (477) with IC50 value of 3 nM on the recombinant human enzyme [273]. Me O Me Ph N

N N

N

O

R Me

Me (469)

(470) Me

Bz Ph

Me

Me N N

CF3

Me

Me N N

H

O (471) R = Ph (472) R = 2-Cl-Ph (473) R = 2,3-di-Me-Ph (474) R = 2-F-Bz (475) R = 3-Biphenyl

H O

(476)

N

N

(477)

Table 2.30 IN VITRO INHIBITION OF HUMAN 11b-HSD1 BY PYRAZOLONE DERIVATIVES Compound

IC50 (nM)

Ref.

(469) (470) (471) (472) (473) (474) (475) (476)

21 133 63 14 10 23 59 36

[271] [271] [272] [272] [272] [272] [272] [272]

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113

Recently, a group from Edinburgh University discovered that compounds with a 1,5-disubstituted-1H-tetrazole template are human 11b-HSD1 inhibitors [274]. Use of the compounds in the treatment of metabolic syndrome was also claimed in the patent. Exemplified compounds were all tested in a cellular assay (HEK293) using an SPA. Compounds (478–480) inhibit 11bHSD1 selectively at the cellular level with IC50 values of 173, 93 and 150 nM, respectively.

Me

N N N N

OMe

N N S

Me

tBu O

F

N N

N

N N

Ph

S

OMe O

Me

N

S O

Me

(478)

(479)

(480)

IN VIVO STUDIES OF SELECTED 11b-HSD1 INHIBITORS In searching for selective 11b-HSD1 inhibitors, scientists from Biovitrum discovered BVT2733 (27), which showed a high potency with an IC50 value of 96 nM for the mouse enzyme [126]. When administered twice daily at 25, 50 or 100 mg/kg orally to the hyperglycaemic KKAy mouse, the compound lowers blood-glucose level significantly in a dose-dependent manner. The maximal reduction of glucose was 53% of the control after 11 days of treatment at the highest dose. Further studies indicate that the maximal inhibition of hepatic 11b-HSD1 activity occurs at about 0.5 h after acute administration of BVT2733 with a single dose of 100 mg/kg, p.o. To achieve a steady-state blood concentration, BVT2733 was administrated to KKAy mice as a continuous subcutaneous infusion by osmotic mini-pumps (167 mg/kg/day). The results show that blood glucose, serum insulin levels and the hepatic concentration of mRNA encoding phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), the ratelimiting enzymes for gluconeogenesis are all reduced [275]. In an extended study, BVT2733 (200 mg/kg b.i.d. p.o.) exhibits a reduction of circulating glucose and insulin levels in ob/ob and db/db mice. BVT2733 treatment also improves whole-body glucose tolerance and increases insulin sensitivity in ob/ob and KKAy mice. Cholesterol, triglyceride and free fatty acids levels are reduced following a 4 h fast in the KKAy mouse [276]. When administrated to female C57BL/6 mice twice daily at a dose of

114

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

100 mg/kg, p.o., BVT2733 reduces food intake and weight gain, but increases water intake. Energy expenditure is 3878% higher in BVT2733 treated obese mice than in the pair-fed mice. BVT2733 prevents a concomitant reduction in lean body mass and energy expenditure, which may contribute to improved glucose tolerance [277]. Another 11b-HSD1 inhibitor that underwent extensive in vivo studies is MK544 (93), which exhibits high potency with an IC50 value of 7.8 nM for the human enzyme (98 nM for mouse) as well as excellent selectivity for 11bHSD2 (IC50 W3,300 nM for human, W10,000 nM for mouse) [205]. 11bHSD1 inhibition with MK544 in DIO mice at 20 mg/kg twice daily p.o. for 11 days lowers body weight gain by 7% and food intake by 12.1%. Furthermore, the central fat pad weight in animals is also reduced. In the same study, MK544 also reduces fasting serum glucose by 15% compared with vehicle-treated animals. Insulin levels are also lowered compared to lean controls. In an HF/STZ mouse diabetes model, MK544 (30 mg/kg twice daily p.o.) significantly reduces fasting and postprandial glucose levels and improves insulin sensitivity after a 9-day treatment [75]. MK544 also shows an improved lipid profile in murine models. Triglyceride levels are reduced by 18% and serum cholesterol level by 24% in treated DIO mice compared with controls. In HF/STZ mice, MK544 significantly lowers free fatty acid concentration. In apoE KO mice, inhibition of 11b-HSD1 with MK544 (10 mg/kg in feed for 8 weeks) lowered circulating cholesterol levels by 28% and serum triglyceride levels by 61%. Most intriguingly, this compound dramatically slows plaque progression in apoE KO mice, a murine model of atherosclerosis. This discovery provides the first evidence that 11b-HSD1 inhibition can effectively treat atherosclerosis, a major characteristic of metabolic syndrome [75]. Yeh et al. investigated the in vivo efficacy of a potent inhibitor, compound (400) in DIO mice as a metabolic syndrome animal model [258]. After dosing orally at 30 mg/kg b.i.d. for 2 weeks, several metabolic parameters were measured, including body weight, plasma insulin, plasma glucose and plasma triglyceride levels. In the study, RU-486, a glucocorticoid receptor antagonist, and rosiglitazone, a PPARg agonist were used as positive controls. Compound (400) induces significant efficacy in weight loss (body weight 40.7 g vs. 41.9 g in high-fat fed control) and lowering of plasma insulin levels (1.31 ng/mL vs. 1.93 ng/mL in high-fat fed control). Blood glucose levels are also decreased from 174.0 mg/dL in the high-fat diet control group to 164.7 mg/dL, but not to the same level as the other positive controls. Plasma triglyceride levels are remarkably normalised after the treatment (33.3 mg/dL vs. 64.9 mg/dL in high-fat fed control) [258]. PF-915275 (481) is a potent selective 11b-HSD1 inhibitor extensively examined in vitro and in vivo by Pfizer [278]. PF-915275 shows a Ki value of

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115

2.3 nM on the purified recombinant human 11b-HSD1 enzyme and a Ki value of 750 nM on the mouse enzyme. The species-dependent activity is further confirmed in cell-based assays with an EC50 value of 15 nM on HEK293 cells stably transfected with human 11b-HSD1 gene and an EC50 value of 14500 nM on rat Fao hepatoma cells. PF-915275 demonstrates the potency by inhibiting the cellular conversion of cortisone to cortisol in human, monkey and dog hepatocytes with EC50 values of 20 nM, 100 nM and 120 nM, respectively. The proof of mechanism is demonstrated with PF-915275 in cynomologous monkeys [278]. In vivo studies were performed in normal cynomologous monkeys using prednisone to prednisolone conversion, as a biomarker of 11b-HSD1 inhibition, to avoid the interference of normal feedback from endogenous glucocorticoids on the HPA axis. The results indicate the dose-dependent inhibitory activity of the prednisone to prednisolone conversion with a maximum 87% inhibition at the highest dose of 3 mg/kg. The relationship of exposure of PF-915275 to the response as indicated by the ratio of prednisolone to prednisone in plasma is demonstrated. The study also shows that insulin levels are decreased in a dose dependent manner [278].

SO2NH N NH2 NC (481)

CLINICAL STUDIES ON 11b-HSD1 INHIBITORS Initial clinical studies used the non-selective inhibitor CBX and examined its effects upon insulin sensitivity and glucose metabolism [9, 169]. CBX inhibits both 11b-HSD1 and 11b-HSD2 and as such causes hypertension and hypokalaemia, probably due to inhibition of 11b-HSD2, giving it little use in the clinical setting for the treatment of metabolic syndrome. Furthermore, when examining its effects upon insulin sensitivity and glucose metabolism, the impact of CBX on potassium is a concern. With the above observations taken into account, in healthy individuals CBX improves whole-body insulin sensitivity [9]. In similar studies in patients with type 2 diabetes, CBX decreased glucose production rate and, interestingly, caused a small but significant decrease in total circulating cholesterol [169].

116

INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

In these studies the hypothesis is that hepatic 11b-reductase enhances glucocorticoid receptor activation in the liver by inhibiting this enzyme with CBX and observing effects on insulin sensitivity. Seven healthy males took part in a double-blind randomised cross-over study in which oral CBX given at 100 mg every 8 h or placebo was administered for 7 days. Euglycemic hyperinsulinemic clamp studies were then performed, including measurement of forearm glucose uptake. CBX increased whole-body insulin sensitivity (M values for dextrose infusion rates, 41.172.4 for placebo vs. 44.672.3 mmol/kg/min for CBX; Po0.03), but had no effect on forearm insulin sensitivity. This implied that CBX, by inhibiting hepatic 11b-reductase and reducing intrahepatic cortisol concentration, increases hepatic insulin sensitivity and decreases glucose production. Thus, plasma cortisone provides an inactive pool that can be converted to active glucocorticoids at sites where 11b-reductase is expressed; abnormal hepatic 11b-reductase activity might be important in syndromes of insulin resistance and manipulation of hepatic 11b-reductase may be useful in treating insulin resistance. The non-selective 11b-HSD inhibitor CBX was evaluated in healthy men and lean male patients with type 2 diabetes. Six diet-controlled non-obese diabetic patients with haemoglobin A1c (Hb A1c), the most abundant glycosylated haemoglobin in human blood, being less than 8% and six matched controls participated in a double-blind cross-over comparison of placebo and CBX dosed orally at 100 mg every 8 h for 7 days. They were admitted overnight for infusions of insulin which are required to maintain arterialised plasma glucose of 5.0 mM and [13C6] glucose. Glucose kinetics were measured in the fasted state from 7.00 to 7.30 am, during a 3-h euglycemic hyperinsulinemic clamp, which included somatostatin infusion and replacement of physiological GH and glucagon levels, and during a 2-h euglycemic hyperinsulinemic clamp with a fourfold increase in glucagon levels. CBX had the expected effects of raising blood pressure and lowering plasma potassium. CBX reduced total cholesterol in healthy subjects by 5.2570.34 mM vs. 4.7870.40 mM, Po0.01, but had no effect on other serum lipids or on cholesterol in diabetic patients. CBX did not affect the rate of glucose disposal or the suppression of free fatty acids during hyperinsulinemia. However, CBX reduced the glucose production rate during hyperglucagonemia in diabetic patients by 1.9070.2 vs. 1.5370.3 mg/kg/min, Po0.05. This was due to reduced glycogenolysis by 1.3170.2 vs. 1.0170.2 mg/kg/min, Po0.005, rather than altered gluconeogenesis. These observations reinforce the potential metabolic benefits of inhibiting 11b-HSD1 in the liver of patients with type 2 diabetes. Clinical investigations of selective 11b-HSD1 inhibitors are in their infancy. Few data have been released and no compound data has been reported from phase II studies. The first company to enter clinical trials was

X. SU, N. VICKER AND B.V.L. POTTER

117

Biovitrum, with BVT3498. A clinical phase-I study with BVT3498 including 66 healthy volunteers was successfully concluded [279]. This trial aimed to demonstrate improved insulin sensitivity and glycemic control, and the development programme was designed to establish positive effects on body composition, lipid profile and other metabolic aberrations linked to insulin resistance, either as monotherapy or as an add-on treatment to other therapies. A phase-IIa clinical study was initiated in 2003. The placebocontrolled, double-blind study involved over 100 type 2 diabetes patients at centres in Finland and Sweden [280]. The results of this trial were not published, but the compound is thought to have been abandoned due to lack of efficacy. Amgen acquired the rights to the Biovitrum 11b-HSD1 inhibitor portfolio after BVT3498 entered the phase-IIa trial [281]. The structure of BVT3498 has not been reported. Incyte initiated a phase I trial with INCB13739 in June 2006 [282] which was completed and this compound entered phase II in 2007 [283]. In the phase-I trial, the endocrine safety and pharmacodynamic activity of INCB13739 were reported. After oral dosing INCB13739 is well tolerated and an MTD is not achieved. Multiple indices of cortisol homoeostasis are within normal limits after 9 days of treatment at pharmacodynamically active doses. Complete inhibition of adipose tissue and hepatic cortisone reductase activity are achieved after oral dosing of INCB13739. A 28-day phase IIa study in type 2 diabetic patients has started and Incyte reported positive interim results from the ongoing 28-day phase IIa placebocontrolled clinical trial in type 2 diabetes. In the 20 patients included in this interim analysis, positive effects on fasting plasma glucose and on dyslipidemia, including reduction of LDL, total cholesterol and triglycerides, as well as modest increases in HDL are demonstrated. A three-month phase IIb trial in type 2 diabetes is scheduled to begin in the first half of 2008, provided full results from the ongoing trial are comparable to the interim data. Full results from the phase IIa trial are expected in the first half of 2008. INCB20817. Their follow on 11b-HSD1 compound, for which the Investigational New Drug Application (IND) has been accepted, is expected to enter phase I trials in the first quarter of 2008 [284]. The structures of INCB13739 and INCB20817 have not been reported. Pfizer has compounds identified to be in clinical development for the treatment of type 2 diabetes whose mode of action has not yet been fully disclosed, but which is expected to be inhibition of 11b-HSD1. In July 2007, Pfizer listed PF-734200 as in phase II studies and PF-915275 in phase I clinical development. The phase I study on PF-915275 has now been discontinued due to formulation issues [285]. The structure of PF-91525 (481) has recently been published [278]. NCT00427401 is listed as a doubleblind, placebo-controlled, parallel group study to evaluate the safety,

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INHIBITORS OF 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 1

tolerability, pharmacokinetics, and pharmacodynamics of PF-915275 after oral administration to subjects with type 2 diabetes mellitus for 4 weeks. The study was due to start in February 2007 with an expected enrolment of 40 patients. The primary end point being glucose lowering over a 24 h mean glucose concentration and the secondary outcome being fasted blood glucose [286]. Merck has a number of compounds in clinical trial for diabetes with no disclosed mechanism of action [287]. The first set of data from a phase-II clinical trial of a selective 11b-HSD1 inhibitor to be reported is eagerly awaited.

CONCLUSION Recent efforts in the design of selective inhibitors of 11b-HSD1 have been intense. The plethora of new patent filings by most of the major pharmaceutical companies is evidence of this explosive field. There is now a vast array of different structural types of selective inhibitors of 11b-HSD1 as potential preclinical candidates. Such a raft of varied compounds is important as the physicochemical properties of the compounds will determine tissue distribution, HPA effects and ultimately clinical utility. Few compounds have entered the clinic and the results from clinical studies are sparse. The outcome of current trials on a number of selective inhibitors of 11b-HSD1 is eagerly awaited to assess the potential of this new field to treat disease areas of unmet medical needs.

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