Discovery of Clinical Candidate 2-((2S,6S)-2-Phenyl-6

May 24, 2017 - BMS-816336 (6n-2), a hydroxy-substituted adamantyl acetamide, has been identified as a novel, potent inhibitor against human 11β-hydro...
1 downloads 18 Views 5MB Size
Article pubs.acs.org/jmc

Discovery of Clinical Candidate 2‑((2S,6S)‑2-Phenyl-6hydroxyadamantan-2-yl)-1-(3′-hydroxyazetidin-1-yl)ethanone [BMS816336], an Orally Active Novel Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor Xiang-Yang Ye,*,† Stephanie Y. Chen,† Shung Wu,† David S. Yoon,† Haixia Wang,† Zhenqiu Hong,† Stephen P. O’Connor,† Jun Li,† James J. Li,† Lawrence J. Kennedy,† Steven J. Walker,† Akbar Nayeem,§,◆ Steven Sheriff,§,◆ Daniel M. Camac,§,◆ Vidyhashankar Ramamurthy,§,◆ Paul E. Morin,¶ Rachel Zebo,∥ Joseph R. Taylor,∥ Nathan N. Morgan,∥ Randolph P. Ponticiello,∥ Thomas Harrity,∥ Atsu Apedo,‡ Rajasree Golla,⊥ Ramakrishna Seethala,⊥ Mengmeng Wang,‡ Timothy W. Harper,‡ Bogdan G. Sleczka,‡ Bin He,∥ Mark Kirby,∥ David K. Leahy,# Jianqing Li,∇ Ronald L. Hanson,# Zhiwei Guo,# Yi-Xin Li,‡ John D. DiMarco,+,■ Raymond Scaringe,+ Brad Maxwell,∇ Frederick Moulin,○ Joel C. Barrish,† David A. Gordon,∥ and Jeffrey A. Robl† †

Discovery Chemistry, ‡Pharmaceutical Candidate Optimization, §Computer-Assisted Drug Design, ∥Metabolic Diseases Biology, Lead Evaluation, #Process Chemistry, ∇Chemical Synthesis, ○Discovery Toxicology, Research and Development, Bristol-Myers Squibb, 350 Carter Road, Princeton, New Jersey 08540, United States ◆ Molecular Structure and Design, ¶Protein Science, +Solid State Chemistry, Research and Development, Bristol-Myers Squibb, P.O. Box 4000, Princeton, New Jersey 08543-4000, United States ⊥

S Supporting Information *

ABSTRACT: BMS-816336 (6n-2), a hydroxy-substituted adamantyl acetamide, has been identified as a novel, potent inhibitor against human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme (IC 50 3.0 nM) with >10000-fold selectivity over human 11βhydroxysteroid dehydrogenase type 2 (11β-HSD2). 6n-2 exhibits a robust acute pharmacodynamic effect in cynomolgus monkeys (ED50 0.12 mg/kg) and in DIO mice. It is orally bioavailable (%F ranges from 20 to 72% in preclinical species) and has a predicted pharmacokinetic profile of a high peak to trough ratio and short half-life in humans. This ADME profile met our selection criteria for once daily administration, targeting robust inhibition of 11β-HSD1 enzyme for the first 12 h period after dosing followed by an “inhibition holiday” so that the potential for hypothalamic−pituitary−adrenal (HPA) axis activation might be mitigated. 6n-2 was found to be well-tolerated in phase 1 clinical studies and represents a potential new treatment for type 2 diabetes, metabolic syndrome, and other human diseases modulated by glucocorticoid control.



INTRODUCTION According to the World Health Organization, the number of type 2 diabetes patients worldwide has reached 422 million and is predicted to become the seventh leading cause of death in the world by the year 2030.1,2 This chronic progressive disease is generally characterized by insulin resistance as a result of abdominal obesity and relative insulin deficiency due to progressive deterioration of pancreatic beta-cell function. Metabolic syndrome, a cluster of metabolic abnormalities (insulin resistance, obesity, dyslipidemia, hyperglycemia, and hypertension) is well recognized as a major cause of cardiovascular diseases and type 2 diabetes. Although the exact causes of metabolic syndrome are complex and not fully understood, it has been postulated that stress hormones such as glucocorticoids may be contributing to the disease state.3,4 Glucocorticoids are present in two forms in humans: the active © 2017 American Chemical Society

cortisol (corticosterone in rodents) and inactive cortisone (11dehydrocorticosterone in rodents). It is well-known that an excess of glucocorticoid tone via secretion from the adrenal gland, such as that observed in Cushing’s disease, leads to major perturbations in glucose and lipid metabolism including hyperglycemia, type 2 diabetes, hyperlipidemia, and accelerated cardiovascular diseases.5−7 A correlation of salivary cortisol levels with components of the metabolic syndrome has been reported.8 These findings support the strategy of modulating intracellular glucocorticoid levels (specifically intracellular concentration in the metabolically relevant tissues) as a potential therapeutic for metabolic syndrome. Received: February 24, 2017 Published: May 24, 2017 4932

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

candidates had relatively long pharmacokinetic half-lives and low clearances in both preclinical models as well as humans, resulting in prolonged inhibition of the enzyme with once daily dosing. For both compounds, we observed a mild HPA (hypothalamic−pituitary−adrenal) axis activation in both primates and humans, manifested by a small but definitive elevation of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS).36 Although the clinical manifestation of chronic but modest HPA axis activation in humans is unclear, we hypothesized if inhibition of 11β-HSD1 for a shorter time period during the 24 h circadian rhythm cycle, such as the 10−12 h when diurnal rhythm is maximal, could potentially inhibit the enzyme sufficiently to generate efficacy while ameliorating HPA axis activation (through relief of inhibition at the nadir of diurnal rhythm). The rationale for this approach is that, when the diurnal rhythm is in the peak phase, the availability of the substrate for 11β-HSD1 would also be at its peak, and high concentration of inhibitor at this period might be of importance.37 “Relief of inhibition” for the remainder of the 24 h cycle may circumvent HPA axis activation. With this goal in mind, we targeted discovery of a compound with good oral bioavailability but with higher clearance and shorter half-life pharmacokinetics (i.e., inhibiting 11β-HSD1 enzyme for ∼12 h period). Our efforts culminated in the discovery of clinical candidate 6n-2, the details of which will be discussed below.

11β-Hydroxysteroid dehydrogenase type 1 enzyme (11βHSD1) is an NADPH-dependent enzyme that is ubiquitously expressed in the human body, but primarily in the liver, the adipose tissue, and the central nervous system, where it catalyzes the conversion of inactive cortisone to the active glucocorticoid hormone cortisol.9 Transgenic mice that overexpress 11β-HSD1 in adipose have developed many of the features of metabolic syndrome, including glucose intolerance, insulin resistance, dyslipidemia, hypertension, and obesity.10,11 Conversely, whole-body genetic knockout of 11β-HSD1 expression in mice placed on a high-fat diet has been shown to improve glucose tolerance and reduce triglyceride levels relative to wild-type controls on a similar diet.12 These and other preclinical findings build a strong case that inhibition of 11β-HSD1 may be an efficacious approach for the treatment of metabolic syndrome. As a result, much attention has been focused on the discovery and development of inhibitors of this enzyme.13−27 In clinical trials, Incyte, Merck, and others have demonstrated that 11β-HSD1 inhibitors improved glycaemic control, lipid profiles, and blood pressure with modest weight loss.28 In addition to this indication, we recently explored and proposed atheroprotection as an emerging indication for this target. This was supported by our findings that 11β-HSD1 knockout mice cross bred with ApoE−/− mice showed an atheroprotective effect in the vessel wall.29 Supporting this observation, Merck found that cholesterol deposition was reduced by 3-fold in ApoE−/− mice treated with an 11β-HSD1 inhibitor for 8 weeks.30 Translationally, 11β-HSD1 expression levels increase 5.5-fold in aorta in metabolic syndrome patients.31 These findings highlight the potential of atheroprotection for 11β-HSD1 inhibitors. Through our drug discovery program in the 11β-HSD1 field, we identified two clinical candidates, 4-(8-(2-chlorophenoxy)[1,2,4]triazolo[4,3-a]pyridin-3-yl)bicyclo[2.2.1]heptan-1-ol (BMS-770767, 1a) 3 2 and 2-(3-(1-(4-chlorophenyl)cyclopropyl)-[1,2,4]triazolo[4,3-a]pyridin-8-yl)propan-2-ol (BMS-823778, 1b),33 generated from our lead triazolopyridine framework (Figure 1).34,35 These compounds demonstrated



RESULTS AND DISCUSSION We previously reported a series of adamantyl carboxylic acids (exemplified by 2a in Figure 2) that are potent, selective human 11β-HSD1 inhibitors. They have many good developmental attributes, including excellent metabolic stability, low CYP inhibition, and no pregnane X receptor (PXR) transactivation liability.38 However, rapid glucuronidation (as demonstrated in vitro in liver microsomes; UGT T1/2 ∼ 1 min) resulted in low oral bioavailability and short pharmacokinetics, precluding this class of inhibitors from further development. Through an acid isostere replacement strategy, we extended our work to tetrazoles exemplified by 2b (Figure 2), resulting in a series with an improved but not ideal glucuronidation potential and PK properties.39 Further research on other isostere replacements led to the identification of amide 3 as an early lead compound. Primary amide 3 was modestly active against human 11βHSD1 (IC50 175 nM) but was weakly active versus the mouse enzyme. Encouraged by the significant improvement in UGTT1/2 of 3 (>120 min vs 1 min for compound 1), we further explored the SAR of the amide portion. Initially, we examined analogues derived from small cyclic amines such as aziridine, azetidine, and pyrrolidine (Table 1), focusing only on in vitro

Figure 1. Triazolopyridine-based clinical compounds 1a and 1b.

excellent in vitro/in vivo profiles and pharmacokinetic properties suitable for dosing in humans. These two clinical

Figure 2. Glucuronidation profile of adamantyl carboxylic acid 2a and its isosteric tetrazole 2b and isosteric amide 3. 4933

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

the human enzyme. Analogues bearing a nonpolar substituent on the azetidine ring (such as 4d and 4k) often had good mouse activity (IC50 89 and 8 nM, respectively), while analogues bearing polar substituents showed variable effects. Thus, 4e, 4g, and 4j tended to be much less active against mouse enzyme, whereas 4f, 4h, and 4i, which also had a polar substituent (hydroxy), could be tolerated at certain positions and still maintain good mouse activity. These observations could be rationalized using the X-ray cocrystal structure of human 11β-HSD1 with 4k together with the docking experiment of 4k in mouse enzyme (Figure 3). As depicted below, cocrystal structures of human 11β-HSD1 show that the central interaction with the ligand involves

Table 1. Initial SAR of Amide Portion

a IC50 values refer to biochemical assay versus the human or mouse 11β-HSD1 enzyme; average of at least two replicates.

Figure 3. (a) Co-crystal structure of 4k (green carbon atoms) with selected residues of human 11β-HSD1 (gray carbon atoms) with Fo− Fc density (cyan) contoured at 2.5 rmsd derived from the final model with 4k omitted. In this and all succeeding cocrystal structures, only the nicotinamide, ribose, two phosphates, and part of the adenine’s ribose of the NADP are shown, and thin sticks have been used to reduce overlap with other parts of the binding site. Hydrogen bonds are shown as dashed lines. (b) Superposition of the flipped orientation docking model of 4k (wheat carbon atoms, ligand carbon atoms in violet) in mouse 11β-HSD1 on the cocrystal structure of 4k (green carbon atoms) in human 11b-HSD1 enzyme (gray carbon atoms, ligand carbon atoms in green). Identical residues between human and mouse 11β-HSD1 are labeled in blue, whereas differing residues are labeled in gray (human) or wheat (mouse). Hydrogen bonds are shown as dashed lines. Superposition of 4k in mouse 11β-HSD1 the same orientation as in human is shown in Supporting Information, Figure S1.

potency against human and mouse enzymes. Aziridinyl amide 4a showed significant activity improvement against both species compared to 3 (9 nM vs 175 nM for human; 520 nM vs 10 μM for mouse). As the ring size expanded from three (4a) to four (4b) and then to five (4c), the human 11β-HSD1 activity increased, but the mouse activity peaked at the ring size of four (4b). Four-membered ring azetidinyl analogue 4b had the most favorable balance between human and mouse 11β-HSD1 activity (1 nM for human and 37 nM for mouse). This led us to further explore other azetidine analogues (4d−4k, Table 1). In general, both nonpolar and polar substituents were welltolerated at various positions on the azetidine ring for the human 11β-HSD1 enzyme, as indicated by their IC50 values (from 0.6 to 65 nM, 4d−4k). In contrast, substituent effects on the mouse 11β-HSD1 enzyme were more pronounced than on 4934

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

hydrogen bonding between the amide carbonyl group from the ligand and the hydroxy moieties of residues Tyr183 and Ser170 on the enzyme. Because of the symmetric nature of the CO group, however, this implies that the ligand can bind in two opposite orientations, which we refer to as “flipped orientations” (see Figure 3b). In both orientations, the interactions with Ser170 and Tyr 183 are preserved. Which of these two binding modes will prevail will depend on (i) the other surrounding residues in the substrate binding site as well as crystallographic waters that may be conserved, (ii) differences in ligand substituents on the adamantyl group and/or the azetidine ring, and, (iii) between human and mouse enzymes, the amino acid differences. Because our docking simulations show that preferences between the two binding modes are often small (less than 2 kJ/mol as measured by the docking scores), SAR differences between similar compounds are probably a consequence of binding in two different (flipped) orientations, a point to which we will return later. The crystal structure of 4k in human 11β-HSD1 (Figure 3a) shows that the side chains of residues around the methoxy group in 4k are mostly small and nonpolar, consisting of Ile121, Thr124, Ala223, Ala226, and Val227. The hydroxy group from Thr124, which points into the binding pocket, can act as a hydrogen bond donor for a suitable side chain substituent on the azetidine ring. This could explain why a diverse range of substituents, varying in size and polarity, can be tolerated in the human 11β-HSD1 binding pocket. A docking model of 4k in mouse 11β-HSD1, modeled using the X-ray structure of murine 11β-HSD1 complexed with compound 6n-2 (Figure 3b), shows some significant differences between the residues surrounding 4k in human versus the mouse enzyme. As shown in Figure 3b, Tyr177, Ala226, and Met233 in human 11β-HSD1 are replaced with Gln177, Glu226, and Ala233, respectively, in mouse 11βHSD1. The replacement of Ala226 in human 11β-HSD1 with Glu226 in the mouse enzyme explains why a carboxyl (4g) or nitrile (4j) side chain, on account of their negative polarity, are not as well tolerated in mouse 11β-HSD1 compared to human enzyme. The low potency seen in mouse 11β-HSD1 for compound 4e cannot easily be explained using the binding mode of 4k because the residues in human and mouse are identical in the vicinity of the azetidine moiety (Figure 3b). The mouse enzyme likely favors the “flipped” binding orientation because the adamantyl moiety would be relatively disfavored in the 4k binding mode due to Tyr177 and Met233 in human being replaced in mouse by a Gln and Ala, respectively, leading to a more hydrophilic environment in the mouse. Thus, the low potency may be a consequence of this flipped binding orientation, which favors placing the azetidine moiety rather than the hydrophobic adamantyl group in the vicinity of Gln177 as seen in Figure 3b. Among the substituted azetidinyl analogues having potent and balanced human and mouse activities (such as 4f, 4h, 4i, and 4k), we chose to profile 3-hydroxyazetidinyl amide 4i before further SAR optimization. While 4i was not subject to glucuronidation and was a weak PXR activator (EC50 6.7 μM, Ymax 37%), it was prone to the oxidative metabolism, presumably on the adamantyl ring, as suggested by liver microsomal biotransformation studies. This result was consistent with the literature40 as well as our earlier findings.38,39 A fluoro group was first used to block the C-5 and C-7 positions, two known metabolic soft spots on the adamantane ring (Table 2). Unexpectedly, the resulting analogues (5a and 5b, respectively) showed little improvement in liver microsomal

Table 2. Subsequent SAR of Adamantane Portion

a

IC50 values refer to biochemical assay versus the human or mouse 11β-HSD1 enzyme; average of at least two replicates. bRepresents percentage of compound remaining after incubation in human, mouse, or rat microsomes after a 10 min incubation period. cRacemic mixture. d NT = not tested under these conditions but demonstrated in other assays to be extremely low. 4935

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

molecule in the vicinity of the adamantyl group in the cocrystal structure of 5g, which has a methoxy at the C5-position, with 11β-HSD1 suggests a preference for a hydroxy substituent on the adamantyl ring at the C-6 position (vs C-5 or C-7). Distance measurements show that a hydroxy group at the C-6 position can form a hydrogen bond with both the water molecule and Thr124 carbonyl oxygen, whereas the latter direct hydrogen bond is unlikely for the hydroxy group at the C-5 or C-7 positions. Therefore, we synthesized analogue 6a (Scheme 1). To our delight, 6a not only maintained excellent metabolic stability similar to 5e and 5f but also demonstrated a 4−8-fold enhancement in human activity, and a 22−38-fold increase of mouse activity (Table 3). Introduction of an additional methyl group at the C-6 position of 6a gave analogue 6b, which showed comparable in vitro potency but slightly reduced metabolic stability. Primary amide 6c had excellent metabolic stability and good human 11β-HSD1 activity, but its mouse activity was poor (IC50 1733 nM). The analogue bearing a cyano group at the C-6 position (6d) had attenuated metabolic stability (27−53% remaining) and moderate mouse 11β-HSD1 activity (IC50 488 nM), despite good human 11β-HSD1 activity (IC50 12 nM). Thus, the C-6 hydroxy-substituted analogue 6a, which not only maintained good human and mouse 11β-HSD1 activity but also enhanced metabolic stability and reduced PXR transactivation (EC50 > 50 μM, Ymax = 4%), had the most optimal profile. Compound 6a was a racemic mixture bearing a “pseudo” chiral center through the adamantane ring system.41 To quickly establish SAR, we decided to optimize the lead 6a using the racemic mixture for initial in vitro activities screening and metabolic stability assessment. On the basis of the results, we would then choose compounds of interest for further investigation as their optically pure enantiomers. Thus, 3-hydroxyazetidinyl and 6-hydroxy-adamantyl groups were kept unchanged and the Ar group was varied (Table 4). Among the methyl-substituted phenyl at three different positions (o-, m-, and p-; 6e−6g), the mouse enzyme, and to a lesser extent human enzyme, had a clear preference for parasubstitution, i.e.p-methylphenyl (6g). This was also true when we compared p-Cl-phenyl analogue (6i) with m-Cl-phenyl analogue (6h). This led us to focus on the substituents at the para-position of the phenyl. Analogues bearing a p-methoxy group (6j) and a p-CF3 group (6k) had good human activity but were less active in the mouse enzyme. Polar functional groups such as methyl sulfone (6l) and cyano (6m) were poorly tolerated, although they demonstrated excellent metabolic stability across three different species. Unsubstituted phenyl analogue (6n) was equally as potent as p-F-phenyl analogue 6a. Incorporating a nitrogen atom into the phenyl ring (6o−6q) enhanced the metabolic stability significantly but came at the expense of in vitro potency. To our surprise, larger groups such as biphenyl (6r) or 4-pyridylphenyl (6s) were tolerated for in vitro activity potency but had poor metabolic stability. Binding can be rationalized by the X-ray cocrystal structure of 6r (Supporting Information, Figure S2). In addition, we examined benzyl (6t) and cyclohexyl group (6u) as phenyl replacements, both of which showed good human 11β-HSD1 activity but weak mouse activity and poor metabolic stability. Comparison of X-ray cocrystal structures of 6t (Supporting Information, Figure S2) and 6r (Supporting Information, Figure S3) with the cocrystal structure of 4k (Figure 3)

stability (5−38% remaining in rodent microsomes after 10 min) and a 13-fold loss of mouse activity. We then examined a hydroxy group incorporation at various positions of the adamantyl ring (e.g., C-1, C-5, C-7, and C-8) based on the synthetic feasibility. The hydroxy groups at C-1 (5c) and C-8 (5d) not only decreased the metabolic stability but also attenuated 11β-HSD1 activity for both human and mouse enzymes (5c vs 4i and 5d vs 4i). Interestingly, the analogues bearing a hydroxy group at the C-5 (5e) and the C-7 (5f) positions significantly improved metabolic stability (greater than 80% remaining) while maintaining good human 11βHSD1 activity (IC50 45 and 22 nM respectively) but at the expense of inhibition versus the mouse enzyme. Encouraged by the good metabolic stability of 5e and 5f, we made their corresponding ether analogues 5g and 5h to probe the enzyme hydrophobic binding pocket occupied by the adamantane ring. Both ether analogues (5g and 5h) showed a near 20-fold increase in human 11β-HSD1 activity but were metabolically less stable compared to their hydroxy counterparts. 5g had much improved mouse activity (vs 5e), but 5h remained much less active in mouse enzyme (vs 5f). Furthermore, several analogues bearing polar functional groups such as carboxamide or cyano group at either the C-5 or the C-7 positions showed much improved metabolic stability and excellent in vitro potency against human enzyme, but all suffered from poor mouse 11β-HSD1 activity (5i−5k). Diminished activity vs the mouse enzyme was not desirable because the mouse was one of our more accessible in vivo animal models. We hypothesized that an optimal balance between polarity and lipophilicity might be required in order to obtain good in vitro human and mouse potency and good metabolic stability. It was worth noting that introduction of a substituent (such as F, OH, or OMe) into the adamantane ring (5a−5k) all resulted in a decrease of PXR transactivation compared to unsubstituted analogue 4i. To gain insight into the binding mode in the enzyme hydrophobic pocket and to aid future compound design, we obtained the cocrystal structure of 5g with human 11β-HSD1 enzyme (Figure 4). The presence of a crystallographic water

Figure 4. X-ray structure of 5g (green carbon atoms) with selected residues of human 11β-HSD1 (gray carbon atoms) with Fo−Fc density (cyan) contoured at 2.5 rmsd derived from the final model with 5g omitted. Hydrogen bonds are shown as dashed lines. In addition to the hydrogen bonds between the ligand and Ser 170 and Tyr 183, hydrogen bonds to two water molecules are shown. The one toward the upper left forms a hydrogen bond to Thr 124 N and is too far to form a hydrogen bond to the ligand methoxy, but the proximity is indicated by a narrow line and a distance label (3.4 Å). 4936

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of 6n-2a

a Conditions: (i) ethylene glycol, TsOH·H2O, dry CH2Cl2, RT, overnight, 72%; (ii) Meldrum’s acid, anhydrous pyridine, cat. piperidine, RT, 5 days, 58%; (iii) PhMgBr, CuBr, THF, 5 °C to RT, 6 h, 81%; (iv) DMF-H2O, 110 °C, 12 h, 93%; (v) TsOH, acetone−H2O, reflux, 70%; (vi) NaBH4, THF−H2O, RT, 83%; (vii) 3-hydroxyazetidine HCl salt, HOBt, EDCI, DIPEA, DCM, RT, 56%; (viii) chiral SFC separation, ∼90%; (ix) Br2/ pyridine, CCl4, 30−35 °C, 2.5 h, 81%; (x) enzymatic reduction, see ref 42.

while preserving the hydrogen bonds between the amide carbonyl group in 6t and Ser170/Tyr183, places the biphenyl group in an orientation that points outside the binding pocket and is free of steric clashes. While this explained the flipped orientation of 6t relative to 4k as seen in the cocrystal structures with human 11β-HSD1, this observation also made structure-based design more challenging. As the leading compounds for the program, racemates 6a and 6n were chosen for chiral supercritical fluid chromatography (SFC) separation to their respective enantiomers. The second eluting enantiomers 6a-2 and 6n-2 had better human in vitro potency (1.5−5-fold) and marginally superior metabolic stability compared to their first eluting enantiomers 6a-1 and 6n-1 (Figure 5). To determine their absolute stereochemistries, the four enantiomers were resynthesized from the corresponding optically pure carboxylic acids with known chirality35 via amide coupling conditions (HOBt/EDCI/DIPEA). Thus, the first eluting enantiomers 6a-1 and 6n-1 were assigned with (2R,6R)- chirality, while the second eluting enantiomers 6a-2 and 6n-2 were assigned with (2S,6S)- chirality according to the literature. 41 It is worth mentioning that in this 2,2disubstituted-6-substituted adamantane system, the imaginary chiral axis passes through the two substituted terminal carbon atoms (i.e., C-2 and C-6) and the geometrical center of the ring system, creating a chiral environment. Table 5 lists the activities of the resolved enantiomers of 6a and 6n. In addition to the human and mouse 11β-HSD1 enzymes, all the four isomers were tested against cynomolgus monkey enzyme. Potency versus primate 11β-HSD1 was critical because this species is ideal to assess HPA axis activation preclinically. As anticipated, the IC50 values correlated very well between cynomolgus monkey and human. On the basis of its overall more favorable in vitro characteristics, 6n-2 was chosen over others for additional

Table 3. Structure-Guided Substitution on the Adamantane Ring

11β-HSD1 IC50 (nM)b compda R1 = R2 = human 6a 6b 6c 6d

OH H OH CH3 CONH2 H CN H

mouse

metabolic stability % remaining (hu/m/r)c

5

43

95%/92%/88%

8

54

84%/66%/47%

30

1733

100%/99%/92%

12

488

53%/47%/27%

a

Racemic mixture. bIC50 values refer to biochemical assay versus the human or mouse 11β-HSD1 enzyme; average of at least two replicates. c Represents percentage of compound remaining after incubation in human, mouse, or rat microsomes after a 10 min incubation period.

indicated that the binding orientation of 6r and 6t was flipped relative to 4k. A molecular docking simulation shows that a binding orientation of 6t similar to 4k would have resulted in very unfavorable steric clashes between the biphenyl group in 6t and residue side chains in the binding site. The simulation also shows a preference for an alternative binding model which, 4937

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

Table 4. Optimization of Ar Group

a All compounds are racemic mixtures. bIC50 values refer to biochemical assay versus the human or mouse 11β-HSD1 enzyme; average of at least two replicates. cRepresents percentage of compound remaining after incubation in human, mouse or rat microsomes after a 10 min incubation period.

3.5 μM, respectively. While the selectivity ratio for mouse 11βHSD1 was still significant (∼176×), it was much lower (∼7×) versus the rat enzyme due to the relative weak activity against rat 11β-HSD1 (IC50 500 nM). This relatively poor rat selectivity did raise a concern for the toxicological assessment of 6n-2 in rats. Fortunately, no evidence for hypokalemia, plasma volume expansion, hypertension, or renal toxicity was seen in the 2 week rat exploratory toxicology study with the highest dose of 300 mg/kg and AUC0−24 exposure of up to 650 μM·h. The structure of 6n-2 has been determined in both the human (Figure 6a) and mouse (Figure 6b) enzymes and, as shown below (Figure 6c), binds very similarly in these two

characterization. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) is an isoform of 11β-HSD1 that catalyzes the irreversible oxidation of the 11-hydroxy group of cortisol to produce cortisone. Because inhibition of 11β-HSD2 would be expected to result in the highly undesirable side effects of hypertension and hypokalemia, all compounds of interest were counter-screened in an in vitro 11β-HSD2 assay. Testing of 6n2 in this assay showed no inhibition up to 30 μM versus the human 11β-HSD2 enzyme. Therefore, 6n-2 was at least 10000fold selective versus human 11β-HSD2. It was also highly selective (IC50 = 48 μM) versus the cynomolgus monkey 11βHSD2 enzyme. In contrast, it was less selective versus the mouse and rat 11β-HSD2 enzymes, with IC50 values of 7.9 and 4938

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

Figure 5. Chiral resolution of 6a and 6n and chirality assignment.

Table 5. In Vitro Characterization of Enantiomers 6n-1 and 6n-2

IC50 values refer to biochemical assay versus the human, mouse, or cynomolgus monkey 11β-HSD1 enzyme; average of at least four replicates ± SD. bRepresents percentage of compound remaining after incubation in human, mouse or rat microsomes after a 10 min incubation period.

a

CYP3A4), no transactivation of PXR (EC50 > 50 μM), and no cytotoxicity in a HEPG2 cell line at a concentration of 80 μM. The compound weakly inhibits the hERG channel in a patch clamp assay (10% at 30 μM). Compound 6n-2 has excellent permeability as seen in Caco-2 assays (Pc A-B 105 nm/s, Pc B-A 285 nm/s; efflux ratio 2.7) and excellent aqueous solubility (1.8 mg/mL at pH 6.5). In a human liver microsome stability assay, 6n-2 showed a half-life of 121 min. The compound has low protein binding in all species: human, 54%; mouse, 63%; rat, 65%; dog, 57%; cynomolgus monkey, 68%. In liver microsomal glucuronidation studies in the presence of UDPGA, the compound exhibited a T1/2 of >200 min in all species (human, cyno, dog, mouse) except rat (T1/2 47 min). No glutathione conjugates were detected when 6n-2 was incubated with liver microsomes from human, monkey, dog,

species, with the same orientation as 6r. The carbonyl group in 6n-2 serves as the critical hydrogen-bonding pharmacophore interacting with Tyr183 and Ser170. The differences in interaction of 6n-2 with human and mouse residues 177, 231, and 233 (substrate binding site residues which differ between the human and mouse enzymes: Tyr177, Val231, Met233 in human 11β-HSD1, and Gln177, Ile231, Ala233 in mouse 11βHSD1) may be responsible for the small potency differential of 6n-2 between these species (human IC50 3.0 nM, mouse IC50 43.5 nM). Compound 6n-2 was further evaluated in the in vitro safety and metabolism assays. It exhibits an excellent profile in most of the safety assays: minimal inhibition (IC50 > 40 μM) of nine cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and 4939

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

rat, and mouse in the presence of NADPH and GSH. These results suggest that under the in vitro experimental conditions tested, 6n-2 does not form detectable levels of reactive metabolites. Because 11β-HSD1 is located in the lumen of the endoplasmic reticulum, the effect of the various membrane barriers, and perhaps intracellular protein binding, on the intrinsic potency of 6n-2 was assessed in a cell-based assay (Table 6). Two versions of this assay were utilized. The first Table 6. Cell-Based Inhibitory Activities of 6n-2 against 11βHSD1 Enzymes 11β-HSD1 IC50 (nM) 6n-2

HEK cells

3T3L1 cells

37.3 ± 11.6 (n = 25)

28.6 ± 9.4 (n = 27)

version employs human embryonic kidney (HEK) cells that are stably transfected with an expression vector encoding human 11β-HSD1. The average IC50 from 25 data sets was 37.3 ± 11.6 nM (SEM), indicating an approximately 10× decrease in potency from the Ki (3.8 ± 1.3 nM, 37 °C) measured under cell-free conditions. A similar reduction in cellular potency vs the IC50 in the cell-free assay was observed with our two other clinical candidates. The second version of the assay makes use of the mouse adipocyte-like 3T3-L1 cell line. In this case, no genetic manipulation was applied to the cells. Instead, native mouse 11β-HSD1 activity expressed by these cells was used to determine inhibitor potency. The IC50 value was 28.6 ± 9.4 nM, which is similar to the value measured in the cell-free biochemical assay for the mouse enzyme. This suggests that unlike the HEK cells, the 3T3-L1 cells provide no significant barrier to the compound reaching the target in the lumen of the endoplasmic reticulum. It is unclear whether or not this predicts an advantage in gaining access to 11β-HSD1 in intact adipose tissue in vivo. Regardless, the most important point from these cell culture models is that 6n-2 clearly enters the cells, reaches the target, and inhibits 11β-HSD1. Table 7 lists the pharmacokinetic profiles of compound 6n-2 in four animal models. Following an IV dose, the total plasma clearance (CLTp) of 6n-2 was high in rats, moderate in mice and monkeys, and low in dogs. Apparent elimination half-life estimates were 2 h (mouse), 3 h (rat), 7 h (dog), and 6 h (monkey). 6n-2 distributes extravascularly in all animal species tested, with Vss of 2.0, 0.5, 3.0, and 4.2 L/kg in mouse, rat, dog, and monkey, respectively. In mice, the tissue-to-plasma concentration ratio averaged ∼0.15 in the adipose but showed concentration-dependency in the liver (1 to 56). Compound 6n-2 was deemed not to penetrate the blood−brain barrier in mice as brain concentrations were below the limit of quantification and brain/plasma ratios were found to be 90% yield. Racemate 6n was later separated into two enantiomers 6n-1 and 6n-2 using chiral SFC. Alternatively, 12 was separated by chiral SFC to afford

two enantiomers 12-1 and 12-2. The desired carboxylic acid 12-2 (also second eluent in chiral SFC) was then converted to optically pure 6n-2 via HOBt/EDCI/DIPEA coupling reaction. The undesired carboxylic acid 12-1 was recycled back to ketone A in 81% yield upon treating with Br2/pyridine. This synthetic route has been proven to be reliable for generating compound 6n-2 to support multiple in vivo studies. However, the approach using chiral SFC to resolve two enantiomers in either acid stage or in the final product stage was not efficient and practical when the program advanced to full development. Our process team discovered that a ketoreductase cloned and expressed from Escherichia coli was able to convert ketone A to optically pure 12-2 in excellent yield (>99%) and enantioselectivity (>99% ee).42 This improved protocol significantly accelerated the large scale synthesis of the API.



CONCLUSION In summary, 6n-2 is a potent and highly selective human 11βHSD1 inhibitor, with excellent aqueous solubility and in vitro and in vivo safety profiles and acceptable pharmacokinetic properties. Structure-based drug design contributed to the incorporation of the 6-hydroxy group in the adamantane ring as the optimal substitution for activity and metabolic stability. Despite structural similarities, we observed flipped binding orientations of 4k and 6n-2 in their cocrystal structures with human 11β-HSD1 enzyme. The PK profile of 6n-2 was selected to enable partial (e.g., 12 h) enzyme inhibition in vivo upon once daily dosing, allowing for a “holiday” from inhibition, potentially resulting in amelioration of HPA axis activation 4943

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

2.45−2.19 (d, J = 13.2 Hz, 1 H), 2.37−2.28 (m, 1 H), 2.12−2.02 (m, 1 H), 1.99−1.83 (m, 2 H), 1.80−1.68 (m, 1 H), 1.77 (d, J = 1.9 Hz, 1 H), 1.60 (d, J = 13.2 Hz, 1 H), 1.52 (bs, 1 H), 1.47−1.32 (m, 2 H). 13 C NMR (100 MHz, DMSO-d6) δ 169.9, 145.4, 127.9, 125.6, 72.4, 58.7, 56.2, 45.8, 40.3, 33.1, 32.0, 31.9, 31.9, 31.1, 30.4, 30.2, 26.8, 26.6, 25.8. HRMS (ESI) calcd for C21H27NO3 341.44, found [M + H] 342.20. Alternatively, 6n-2 was obtained from chiral resolution of (±)-6n using chiral SFC condition. (±)-6n was synthesized from acid 12 and 3-azetidinol hydrochloride using standard amide coupling condition described above. The chiral SFC condition used to separate (±)-6n into two enantiomers 6n-2 and 6n-1 is identical to the condition described for 12. 6-Dioxalane-adamantan-2-one (8). A solution of 2,6-adamantanedione (9.5 g, 57.85 mmol), ethylene glycol (3.22 mL), and TsOH monohydrate (1.099 g, 5.785 mmol) in dry CH2Cl2 (870 mL) was stirred at room temperature overnight. Solvent was removed under reduced pressure, and the residue was purified via column chromatography (SiO2, 300 g ISCO cartridge, 25% EtOAc in nhexane) to provide compound 8 as a white solid (8.69 g, 72% yield); mp 106 °C. 1H NMR (400 MHz, CDCl3) δ ppm 3.72 (s, 4 H), 2.12 (br s, 2 H), 1.95−2.08 (m, 4 H), 1.54−1.68 (m, 6 H). 13C NMR (100 MHz, CDCl3) δ 215.7, 108.9, 64.7, 45.5, 36.3, 36.2. HRMS (ESI) calcd for C12H16O3 208.11, found [M + H] 209.18. 2,2-Dimethyl-5-{spiro[adamantane-2,2′-[1,3]dioxolane]-6-ylidene}-1,3-dioxane-4,6-dione (9). Compound 8 (1.94 g, 9.32 mmol) and Meldrum’s acid (1.611 g, 11.18 mmol) were placed in a flask under argon, and pyridine (9.32 mL) was added, followed by piperidine (0.046 mL, 0.466 mmol). The mixture was stirred at room temperature for 5 days then was kept for another 2 days without stirring. Ice-cooled water (15 mL) was added. The mixture was stirred at room temperature for 15 min. The precipitate was collected via filtering and rinsed with small amount of cold water. The solid was dried in vacuum to afford 9 as a white solid (1.82 g, 58% yield). 1H NMR (400 MHz, CDCl3) δ ppm 4.00 (s, 4 H), 3.97 (br s, 2 H), 2.32 (d, J = 12.1 Hz, 4 H), 1.91 (br s, 2 H), 1.84 (d, J = 12.6 Hz, 4 H), 1.76 (s, 6 H). 13C NMR (101 MHz, CDCl3) δ 187.9, 161.2, 112.1, 109.7, 103.8, 64.8, 37.3, 35.9, 34.6, 27.0. 2,2-Dimethyl-5-(6-phenylspiro[adamantane-2,2′-[1,3]dioxolan]6-yl)-1,3-dioxane-4,6-dione (10). To a suspension of copper(I) bromide (0.619 mL, 21.5 mmol) in dry THF (100 mL) at −5 °C (ice−acetone bath) was added phenylmagnesium bromide (1 M in THF, 86 mL, 86 mmol) slowly. After completion, the resulting mixture was stirred at this temperature for another 10 min to give a light-brown solution. A solution of 9 (3.594 g, 10.75 mmol) in dry THF (100 mL) was cannulated into the above mixture. The resulting mixture was stirred and gradually warmed to room temperature overnight. NH4Cl (saturated, aqueous, 100 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with NH4Cl (saturated, aqueous, 50 mL), H2O (100 mL), and brine (100 mL). It was dried and concentrated to afford crude product. It was partitioned between Et2O (7.5 mL) and pentane (15 mL). The solid was collected via filtering to afford first batch of product as a white solid (3.48 g). The mother liquor was concentrated and the residue was purified via column chromatography (SiO2, 10% EtOAc/hexane) to afford second batch of product as a white solid (0.09 g). The combination of two batches yields 3.57 g (81% yield, 95% purity). 1H NMR (400 MHz, CDCl3) δ 7.35 (m, 2 H), 7.26 (m, 3 H), 4.29 (s, 1 H), 4.01−3.88 (m, 4 H), 2.96 (s, 2H), 2.30 (d, J = 14.2 Hz, 2 H), 2.20 (d, J = 14.2 Hz, 2 H), 1.90 (m, 4 H), 1.70 (d, J = 13.3 Hz, 2 H), 1.47 (s, 3 H), 0.64 (s, 3 H). LC-MS: m/z = 413.5 [M + H]. 2-(6-Phenylspiro[adamantane-2,2′-[1,3]dioxolan]-6-yl)acetic Acid (11). The solution of 10 (7.02 g, 17.02 mmol) in DMF (55 mL) and water (5.5 mL) was heated in 110 °C oil bath for 12 h. HPLC showed the reaction completed. Solvent was removed under reduced pressure, and the residue was freeze-dried in lyophilizer to afford 12 as a white solid (5.2 g, 93% yield). Crude product was used in next reaction without further purification. 1H NMR (400 MHz, CDCl3) δ 7.34−7.20 (m, 5 H), 3.98−3.92 (m, 4 H), 2.72 (s, 2 H), 2.56 (m, 2 H),

typically observed with other inhibitors of this enzyme. While we suspect the pharmacokinetic profile of 6n-2 may alter its ability to activate the HPA axis, it is also noted that 6n-2 does not reach the brain in significant concentrations (brain:plasma ratio ∼0.05 in preclinical species), and this may also contribute to the lack of apparent HPA axis activation in the cyno. On the basis of an acceptable pre-IND safety and toxicity profile, 6n-2 has been advanced to phase 1 clinical studies and was found to be well-tolerated up to the maximally tested dose of 900 mg. The data from this study will be reported in due course. In addition to the treatment of type II diabetes and metabolic syndrome, 11β-HSD1 inhibition may find other potential clinical utilities such as atheroprotection and cognitive protection. These areas await further exploration.



EXPERIMENTAL SECTION

General Methods. For anhydrous reactions, DRISOLV solvents from EMD were used. For other reactions, reagent or HPLC grade solvents were used. All commercially available solvents and reagents were used as received. All reactions were carried out under an argon atmosphere unless noted otherwise. Reaction mixtures were concentrated under reduced pressure at 40−65 °C on a rotary evaporator. Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 plates from Merck. Flash column chromatography separations were performed on RediSep normal phase silica flash columns (230−400 mesh, 60 Å, Teledyne ISCO). LC/MS measurements were obtained using either a Shimadzu HPLC system with a Phenomenex Luna C18 column (5 μm, 100 Å, 4.6 mm × 30 mm), operated at 40 °C with 0.1% TFA modified ACN/water mobile phases or a Waters Acquity UPLC system with a Acquity UPLC BEH C18 (1.7 μm, 130 Å, 2.1 mm × 50 mm) column operated at 50 °C with 0.05% TFA modified ACN/water mobile phases. UV detection was carried out with a Shimadzu SPD-10AV at 220 nm, and mass detection was carried out with waters ZQ single quadrupole mass spectrometer hybrid system using positive electrospray ionization. Final HPLC purity determination was made with a Shimadzu HPLC system with a Waters Sunfire C18 (3.5 μm, 100 Å, 3.0 mm × 150 mm) column and a Waters Xbridge Phenyl (3.5 μm, 130 Å, 3.0 mm × 150 mm) column with 0.05% TFA modified ACN/water mobile phases with gradient run from 10% to 90% ACN. UV detection was carried out with a Shimadzu SPD-20AV at 220 and 254 nm. All final compounds achieved a minimum of 95% purity. All chromatography was carried out with HPLC grade organic solvents. 1 H NMR spectra were obtained on a Bruker 400 MHz spectrometer using the indicated solvent. Chemical (δ) shifts are reported in ppm from tetramethylsilane with the residual solvent signal as the internal standard, signals are expressed as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad, coupling constants (J) are in hertz (Hz). All animals were treated according to the standards of the BMS Animal Care and Use Committee (ACUC). Animals were maintained at the Bristol-Myers Squibb animal facilities (Hopewell, NJ) with 12 h light/dark cycle and free access to food and water. 2-((2S,6S)-6-Hydroxy-2-phenyladamantan-2-yl)-1-(3-hydroxyazetidin-1-yl)ethanone (6n-2). To a solution of 12-2 (0.15 g, 0.524 mmol), 3-azetidinol hydrochloride (68.9 mg, 0.629 mmol), and 1hydroxybenzotriazole monohydrate (88.2 mg, 0.576 mmol) in dichloromethane (2 mL) was added diisopropylethylamine (0.2 mL, 1.15 mmol), followed by a slow addition of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (110 mg, 0.576 mmol). The resulting mixture was stirred at room temperature for 16 h. The solvent was removed under reduced pressure. The residue was purified in preparative HPLC to afford 6n-2 as a white solid (100 mg, 56% yield); [α]25D = +15.3 (c 2.5, MeOH). 1H NMR (400 MHz, DMSOd6) δ 7.40−7.24 (m, 5 H), 5.34 (t, J = 5.8 Hz, 1 H), 4.51 (t, J = 2.9 Hz, 1 H), 3.90−3.78 (m, 1 H), 3.63 (dd, J = 9.6, 7.2 Hz, 1 H), 3.56 (br s, 1 H), 3.13 (ddd, J = 10.1, 5.4, 5.2 Hz, 1 H), 3.04−2.76 (m, 2 H), 2.60− 2.45 (bs, 1 H), 2.51−2.42 (bs, 1 H), 2.51−2.26 (d, J = 12.8 Hz, 1 H), 4944

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

2.40−1.61 (m, 10 H). 13C NMR (100 MHz, CDCl3) δ 177.4, 144.0, 128.2, 126.5, 126.0, 110.6, 64.0, 45.1, 44.8, 36.0, 35.3, 31.6, 30.5, 29.7. HRMS (ESI) calcd for C20H24O4 328.40, found [M + H] 329.33. 2-(6-Oxo-2-phenyladamantan-2-yl)acetic Acid (Ketone A). A solution of 11 (5.2 g, 15.83 mmol) and TsOH (0.358 g, 1.88 mmol) in acetone (111 mL) and water (47.5 mL) was heated in a 56 °C oil bath for 12 h. The solvent was removed under reduced pressure. The residue was purified in a reversed phase chromatography (120 g of C18 column). Solvent A, water; solvent B, CH3CN; method, 10% B for 5 min then 10% B to 100% B in 15 min gradient. The desired product was eluted in ca. 80% B. The fractions containing desired product were concentrated under reduced pressure to afford ketone A as a white solid (3.15 g, 69.9% yield). 1H NMR (400 MHz, CDCl3) δ 7.37−7.24 (m, 5 H), 2.84 (s, 2 H), 2.79 (s, 2 H), 2.60−2.56 (m, 3 H), 2.37−1.81 (m, 7 H). 13C NMR (100 MHz, CDCl3) δ 217.2, 177.0, 143.1, 128.5, 126.4, 126.2, 45.8, 45.1, 44.9, 44.9, 34.8, 34.0, 32.1. HRMS (ESI) calcd for C18H20O3 284.35, found [M + H] 285.26. (±)-2-(6-Hydroxy-2-phenyladamantan-2-yl)acetic Acid (12). To a solution of ketone A (257 mg, 0.904 mmol) in THF (15 mL) was added NaBH4 (85 mg, 2.26 mmol) in small portions. After completion, the mixture was stirred at room temperature for 30 min. LCMS showed reaction complete. It was quenched with HOAc (0.2 mL). The solvent was removed under reduced pressure, and the residue was purified in a reversed phase chromatography (120 g C18 column). Solvent A, water; solvent B, CH3CN; method, 10% B for 5 min, then 10% B to 100% B in 16 min gradient. The desired fractions were collected and concentrated to afford 12 as a white solid (227 mg, 83% yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.27−7.39 (m, 4 H), 7.15−7.24 (m, 1 H), 3.81 (br s, 1 H), 2.64−2.80 (m, 2 H), 2.47−2.62 (m, 2 H), 2.30 (dq, J = 13.7, 3.3 Hz, 1 H), 2.14−2.24 (m, 1 H), 1.87− 2.02 (m, 4 H), 1.68−1.79 (m, 2 H), 1.65 (dd, J = 13.0, 2.7 Hz, 1 H), 1.47−1.57 (m, 1 H). 13C NMR (101 MHz, CDCl3) δ ppm 176.1, 144.2, 128.3, 126.4, 126.0, 74.3, 45.9, 44.9, 34.0, 33.4, 32.3, 32.3, 31.6, 31.4, 26.9, 26.3. LC/MS (m/z) = 285.5 (M − H)−. 2-((2S,6S)-6-Hydroxy-2-phenyladamantan-2-yl)acetic Acid (122). Racemic 12 (540 mg) was subjected to chiral SFC purification (Chiralpak AD-H, 250 mm × 30 mm ID; 5 μm, mobile phase, CO2/ MeOH (80/20); flow rate, 60 mL/min; UV detection, 220 nm; injection volume, 2 mL of 10 mg/mL in methanol) to provide compound 12-1 (269.1 mg, white solid; retention time = 5.7 min) and compound 12-2 (243.9 mg, white solid; retention time = 14 min). NMR spectra of 12-1 and 12-2 are identical to that of 12. Compound 12-1: [α]25D = −10.17° (c = 0.83 mg/mL, methanol). Chiral analytic HPLC: Chiralpak AD, 250 mm × 4.6 mm ID; 10 μm, room temperature; mobile phase, CO2/MeOH (80/20); flow rate, 2 mL/ min; UV detection, 220 nm; retention time, 3.4 min ee > 99.9%. Compound 12-2: [α]25D = +12.37° (c = 1.4 mg/mL, methanol). Chiral analytic HPLC: Chiralpak AD, 250 mm × 4.6 mm ID; 10 μm; room temperature; mobile phase, CO2/MeOH (80/20); flow rate, 2 mL/min; UV detection, 220 nm; retention time, 7.7 min ee > 99.9%. Alternatively, optical pure 12-2 was obtained from the enzymatic reduction of ketone A in excellent yield and excellent enantioselectivity. See ref 42 for the detailed procedure. Synthesis of Ketone A from 2-((2R,6R)-6-Hydroxy-2-phenyladamantan-2-yl)acetic Acid (12−1). Undesired enantiomer 12-1 was converted to ketone A using the following procedure. Compound 12-1 (475 mg, 1.659 mmol) and pyridine (0.40 mL, 4.98 mmol) in CCl4 (19 mL) and CH2Cl2 (19 mL) was heated until it became clear solution. To the above solution at 0 °C was added Br2 (0.268 mL, 5.22 mmol). The mixture was heated at 35 °C for 2.5 h with exclusion of light. During the reaction, precipitate was formed in the orange solution. The reaction was cooled to 0 °C and quenched with a solution of sodium metasulfite (0.3 g in 5 mL water) until the orange color disappeared. The solvent was evaporated to dryness. The residue was purified in a reversed phase chromatography (120 g C18 column). Solvent A, water; solvent B, CH3CN; method, 10% B for 5 min then 10% B to 100% B in 16 min gradient; flow rate, 40 mL/min; monitor, 220 nm. The desired fractions were collected and concentrated to afford ketone A as a white solid (387 mg, 81% yield). The

characterization data of ketone A obtained here are consistent to those obtained previously. 11β-HSD1 SPA Enzyme Assay. 11β-HSD1 was assayed by scintillation proximity assay (SPA) in a 384-well PerkinElmer white plate. The dose response of the compounds was determined using 11 half-log dilutions of compound in DMSO in duplicate. To each well, 0.5 μL of compound dilution in DMSO were added. Then 15 μL of assay buffer (for blanks) or 15 μL of human microsomes in assay buffer were added next and the plates were incubated for 10 min at room temperature. The final microsomal protein concentration was 1.1 μg/assay. Duplicates were in the same plate one row below the other. Then 10 μL of 3H-cortisone (final concentration 40 nM) was added to each well and the plate was spun down to mix and bring down the contents to the bottom of the wells. The plates were incubated at room temperature with gentle shaking for 4 h. The reaction was stopped with addition of 10 μL of 10 mM carbenoxolone. Then 0.5 mg of yttrium silicate SPA beads coupled to anticortisol antibody in 20 μL were added to all the wells of plate, which were spun down once more and incubated at room temperature overnight. The plate was read in a TopCount (1 min/well). Data were uploaded automatically to Tool Set, a Lead Evaluation informatics program for data capture and calculation. Graphs were generated with the Curve Master program. In Vivo Pharmacodynamic Assessment in Mice. Nonfasting diet-induced obese male mice were weighed and separated into groups (n = 6) such that body weights were not statistically different from each other. Animals were bled via the tail for a −60 min time sample and then were dosed orally with vehicle or drug. The vehicle was composed of 0.5% Methocel, 0.1% Tween 80 in water. At 60 min after dosing, mice were bled again via the tail and dosed orally with DHC @ 10 mg/kg. All animals were subsequently bled at 30, 60, and 120 min post DHC dosing. Plasma was isolated for analysis of corticosterone using a commercially available enzyme immunosorbent assay. Drug levels were also measured in the terminal bleed samples. In Vivo Pharmacodynamic Assessment in Cynomolgus Monkey. The experimental design for the cynomolgus monkey pharmacodynamics study is very similar to the mouse model described above. The test compound was administered at a predetermined time point prior to initiation of the experiment based upon the pharmacokinetics of the compound. The monkeys were then given a dose of substrate, and blood samples were removed at various time points thereafter. A notable difference from the mouse protocol was that even though the natural substrate for 11β-HSD-1 in primates was cortisone, the natural rodent substrate 11-dehydrocorticosterone (DHC) was used instead. Human 11β-HSD1 Purification and Crystallization. A pET28 T7 plasmid encoding residues 24−293 of human 11B-HSD1 was constructed that included two site-specific mutations to improve solubility (L262R, F278E) and a thrombin-cleavable N-terminal Histag to simplify purification. The plasmid was used to transform BL21(DE3) E. coli cells (Novagen) and expressed protein at 37 °C in Terrific Broth induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown overnight. Harvested cell paste was suspended in 50 mm Tris pH 8.0, 300 mM NaCl, 20% glycerol, 5 mM βmercaptoethanol (buffer A) supplemented with 0.4% β-dodecyl maltopyranoside (DDM, Anatrace) and complete EDTA-free protease inhibitor cocktail (Roche). DNaseI and 10 mm MgCl2 were added to help remove DNA. The cell suspension was lysed with an Emulsiflex homogenizer and the lysate clarified via centrifugation. The supernatant was decanted into a vessel containing Ni-NTA Superflow resin (Qiagen) equilibrated with buffer A supplemented with 20 mM imidazole and 0.1% DDM and the slurry gently stirred for 40 min. This slurry was poured into a column, and the resin bed washed with buffer A supplemented with 0.1% DDM until the baseline UV absorbance was re-established. Protein of interest was eluted using a linear gradient of 20−300 mM imidazole. Fractions containing a major band at ∼34 kDa were pooled, concentrated, and dialyzed against buffer A supplemented with 0.1% DDM, The sample was treated with thrombin (Boehringer Ingelheim) to cleave the His-tag, and the mixture was reapplied to a Ni-NTA column equilibrated with 50 mm 4945

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

Tris pH 8.0, 300 mM NaCl, 20% glycerol, 0.1% DDM, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) to separate the desired His-tag free 11β-HSD1 product. Cleaved product was verified by mass spectroscopy. Final polish of the material was performed on a Superdex 200 column (GE Healthcare) equilibrated with 50 mm Tris pH 8.0, 100 mM NaCl, 20% glycerol, and 5 mM TCEP. The NaCl concentration increased to 200 mM, and the protein concentrated to 4.6 mg/mL using centrifugal filter concentrators (Vivaspin, 30 K MWCO). The final product was flash-frozen in liquid N2 and stored at −80 °C. Crystal growth was accomplished by adding Zwittergent 3-12 (Calbiochem) to a final concentration of 1.5 mM followed by concentration to 9.2 mg/mL using a Vivaspin concentrator. Compound 2b of ref 32 was added to 5 mM, and the solution was allowed to incubate at 4 °C for 5 h. The mixture was further concentrated to 15−20 mg/mL and set up in 1 + 1 μL drops in Qiagen hanging drop trays using 22% PEG 3350 and 0.2 M potassium formate pH 7.3 as precipitant at 4 °C. Crystals grew within 48−72 h. Replacement of compound 2b of ref 32 in the parent crystals was accomplished by soaking the fully formed crystal in a drop of matched precipitant containing 5 mM compound of interest stored and then at 4 °C for 16 h to 32 days. Crystals were flash-cooled in liquid nitrogen using a cryoprotectant solution of 20% (v/v) ethylene glycol, 22% (w/ v) PEG 3350, and 0.2 M potassium formate pH 7.3. Mouse 11β-HSD1 Purification and Crystallization. Purification and crystallization of murine 11β-HSD1 followed the method of Zhang et al.43 Data Collection and Processing. Data were collected either in the laboratory using a Rigaku FR-E generator, Rigaku MicroMax confocal mirrors, and a Rigaku Saturn92 CCD detector or at the Advanced Photon Source 17-BM beamline with a MAR CCD 165 detector. In all cases, the crystals were flash-cooled in liquid nitrogen and maintained at 100 K by a nitrogen gas stream. The images were processed with HKL2000.44 Refinement and Model Building. The structures were refined starting with models derived from 1XU745 for human or 1Y5M43 for mouse using Buster from GlobalPhasing, Ltd. Stereochemical restraints were derived by MAKETNT from GlobalPhasing, Ltd. Model building used either Quanta (Accelrys) or Coot.46 Figures for publication were made with PyMol (Schrodinger). Omit Fo−Fc electron density maps were calculated from the final model without the ligand. Prior to deposition in the PDB, further refinement was carried to take advantage of tools that were not available at the time of the original work: (1) Grade (GlobalPhasing, Ltd.) for assignment of stereochemical restraints and (2) implementation of local structure similarity restraints (LSSR) in Buster. Modeling Methods. Compound 4k was prepared for Glide-SP docking47−49 using LigPrep in Maestro version 10.0 (Schrodinger, LLC, NY). The A chain of the mouse 11β-HSD1 crystal structure with compound 6n-2 was used because 6n-2 was the nearest analogue to 4k with a crystal structure in mouse 11β-HSD1. The protein structure was optimized using the Protein Preparation Wizard (Schrodinger, LLC, NY) by removing water molecules and bound ligand (6n-2) and adding hydrogen atoms followed by energy minimization with the OPLS 2005 force field,50 not allowing deviations beyond 0.3 Å RMSD in heavy atoms with respect to the original crystal structure. The docking grid was generated around the original ligand (6n-2) with a box size of 20 × 20 × 20 Å3. Glide-SP docking was carried out using the constraint that the amide oxygen atom on the ligand lie within hydrogen bonding distance of the hydroxy groups from Ser170 and Tyr183. The two favored docking poses, consistent with hydrogen bonding with Tyr183 and Ser170 as required by the constraints, were merged with the protein for energy minimization using Prime (Schrodinger, LLC, NY).51,52



Crystallographic statistics for complexes of 11β-HSD1 with various inhibitors, figures showing the X-ray structures of 6t and 6r (PDF) Molecular formula strings (CSV) Docked model of 4k in mouse in the same orientation as human and computational models of the docked poses of compound 4k in mouse 11β-HSD1 shown in Figure S1 (PDB) Docked model of 4k in mouse in the same orientation as human and computational models of the docked poses of compound 4k in mouse 11β-HSD1 shown in Figure 3b (PDB) Accession Codes

Structures of complexes of human 11β-HSD1 with compounds 4k (5PGU), 5g (5PGV), 6r (5PGW), 6t (5PGX), and 6n-2 (5PGY) and the structure of mouse 11β-HSD1 with compound 6n-2 (5PGZ) have been deposited in the Protein Data Bank.



AUTHOR INFORMATION

Corresponding Author

*Phone: 609-466-5101. E-mail: [email protected]. ORCID

Xiang-Yang Ye: 0000-0003-3739-0930 Brad Maxwell: 0000-0001-8507-2078 Notes

The authors declare no competing financial interest. ■ Deceased.



ACKNOWLEDGMENTS Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the IMCA-CAT beamline 17-BM at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the University of Chicago.



ABBREVIATIONS USED 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; 11βHSD2, 11β-hydroxysteroid dehydrogenase type 2; CL, plasma clearance; CYP, cytochrome P450; EDCI, 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide; HOBT, N-hydroxybenzotriazole; HEK cells, human embryonic kidney cells; SFC, supercritical fluid chromatography; NAD, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; PPB, plasma protein binding; SAR, structure−activity relationship



REFERENCES

(1) Breton, M.-C.; Guénette, L.; Amiche, M. A.; Kayibanda, J.-F.; Gregoire, J.-P.; Moisan, J. Burden of Diabetes on the Ability to Work: A systematic review. Diabetes Care 2013, 36, 740−749. (2) Global Report on Diabetes; World Health Organization (WHO): Geneva, 2016; http://www.who.int/diabetes/global-report/en/ (accessed April 2, 2017). (3) Björntorp, P.; Rosmond, R. Obesity and cortisol. Nutrition 2000, 16, 924−936. (4) Wamil, M.; Seckl, J. R. Inhibition of 11β-hydroxysteroid dehydrogenase type 1 as a promising therapeutic target. Drug Discovery Today 2007, 12, 504−520. (5) Tomlinson, J. W.; Stewart, P. M. Mechanisms of disease: selective inhibition of 11β-hydroxysteroid dehydrogenase type 1 as a novel

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00211. 4946

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

treatment for the metabolic syndrome. Nat. Clin. Pract. Endocrinol. Metab. 2005, 1, 92−99. (6) Gathercole, L. L.; Lavery, G. G.; Morgan, S. A.; Cooper, M. S.; Sinclair, A. J.; Tomlinson, J. W.; Stewart, P. M. 11β-Hydroxysteroid dehydrogenase 1: translational and therapeutic aspects. Endocr. Rev. 2013, 34 (4), 525−555. (7) Morton, N. M. Obesity and corticosteroids: 11β-hydroxysteroid type 1 as a cause and therapeutic target in metabolic disease. Mol. Cell. Endocrinol. 2010, 316, 154−164. (8) Oltmanns, K. M.; Dodt, B.; Schultes, B.; Raspe, H. H.; Schweiger, U.; Born, J.; Fehm, H. L.; Peters, A. Cortisol correlates with metabolic disturbances in a population study of type 2 diabetic patients. Eur. J. Endocrinol. 2006, 154, 325−331. (9) Jamieson, P. M.; Walker, B. R.; Chapman, K. E.; Rossiter, S.; Seckl, J. R. 11β-Hydroxysteroid dehydrogenase type 1 is a predominant 11β reductase in the intact perfused rat liver. J. Endocrinol. 2000, 165, 685−692. (10) Masuzaki, H.; Paterson, J.; Shinyama, H.; Morton, N. M.; Mullins, J. J.; Seckl, J. R.; Flier, J. S. A transgenic model of visceral obesity and the metabolic syndrome. Science 2001, 294, 2166−2170. (11) Masuzaki, H.; Yamamoto, H.; Kenyon, C. J.; Elmquist, J. K.; Morton, N. M.; Paterson, J. M.; Shinyama, H.; Sharp, M. G. F.; Fleming, S.; Mullins, J. J.; Seckl, J. R.; Flier, J. S. Transgenic amplification of glucocorticoid action in adipose tissue causes high blood pressure in mice. J. Clin. Invest. 2003, 112, 83−90. (12) Morton, N. M.; Holmes, M. C.; Fievet, C.; Staels, B.; Tailleux, A.; Mullins, J. J.; Seckl, J. R. Improved lipid and lipoprotein profile, hepatic insulin sensitivity, and glucose tolerance in 11β-hydroxysteroid dehydrogenase type 1 null mice. J. Biol. Chem. 2001, 276, 41293− 41300. (13) For the recent overview of medicinal chemistry in 11β-HSD1 field, see: Scott, J. S.; Goldberg, F. W.; Turnbull, A. V. Medicinal Chemistry of Inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). J. Med. Chem. 2014, 57, 4466−4486 and the references cited therein. (14) Ge, R.; Huang, Y.; Liang, G.; Li, X. 11β-Hydroxysteroid dehydrogenase type 1 inhibitors as promising therapeutic drugs for diabetes: status and development. Curr. Med. Chem. 2010, 17, 412− 422. (15) Veniant, M. M.; Hale, C.; Hungate, R. W.; Gahm, K.; Emery, M. G.; Jona, J.; Joseph, S.; Adams, J.; Hague, A.; Moniz, G.; Zhang, J.; Bartberger, M. D.; Li, V.; Syed, R.; Jordan, S.; Komorowski, R.; Chen, M. M.; Cupples, R.; Kim, K. W.; St. Jean, D. J., Jr.; Johansson, L.; Henriksson, M. A.; Williams, M.; Vallgarda, J.; Fotsch, C.; Wang, M. Discovery of a potent, orally active 11β-hydroxysteroid dehydrogenase type 1 inhibitor for clinical study: Identification of (S)-2-((1S,2S,4R)bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)one (AMG 221). J. Med. Chem. 2010, 53, 4481−4487. (16) Siu, M.; Johnson, T. O.; Wang, Y.; Nair, S. K.; Taylor, W. D.; Cripps, S. J.; Matthews, J. J.; Edwards, M. P.; Pauly, T. A.; Ermolieff, J.; Castro, A.; Hosea, N. A.; LaPaglia, A.; Fanjul, A. N.; Vogel, J. E. N(Pyridin-2-yl) arylsulfonamide inhibitors of 11β-hydroxysteroid dehydrogenase type 1: Discovery of PF-915275. Bioorg. Med. Chem. Lett. 2009, 19, 3493−3497. (17) Rosenstock, J.; Banarer, S.; Fonseca, V. A.; Inzucchi, S. E.; Sun, W.; Yao, W.; Hollis, G.; Flores, R.; Levy, R.; Williams, W.; Seckl, J. R.; Huber, R. The 11β-hydroxysteroid dehydrogenase type 1 inhibitor INCB13739 improves hyperglycemia in patients with type 2 diabetes inadequately controlled by metformin monotherapy. Diabetes Care 2010, 33, 1516−1522. (18) Shah, S.; Hermanowski-Vosatka, A.; Gibson, K.; Ruck, R. A.; Jia, G.; Zhang, J.; Hwang, P. M. T.; Ryan, N. W.; Langdon, R. B.; Feig, P. U. Efficacy and safety of the selective 11β-HSD-1 inhibitors MK-0736 and MK-0916 in overweight and obese patients with hypertension. J. Am. Soc. Hypertens. 2011, 5, 166−176. (19) Xu, Z.; Tice, C. M.; Zhao, W.; Cacatian, S.; Ye, Y.; Singh, S. B.; Lindblom, P.; McKeever, B. M.; Krosky, P. M.; Kruk, B. A.; Berbaum, J.; Harrison, R. K.; Johnson, J. A.; Bukhtiyarov, Y.; Panemangalore, R.; Scott, B. B.; Zhao, Y.; Bruno, J. G.; Togias, J.; Guo, J.; Guo, R.; Carroll,

P. J.; McGeehan, G. M.; Zhuang, L.; He, W.; Claremon, D. A. Structure-based design and synthesis of 1,3-oxazinan-2-one inhibitors of 11β-hydroxysteroid dehydrogenase type 1. J. Med. Chem. 2011, 54, 6050−6062. (20) Scott, J. S.; Bowker, S. S.; deSchoolmeester, J.; Gerhardt, S.; Hargreaves, D.; Kilgour, E.; Lloyd, A.; Mayers, R. M.; McCoull, W.; Newcombe, N. J.; Ogg, D.; Packer, M. J.; Rees, A.; Revill, J.; Schofield, P.; Selmi, N.; Swales, J. G.; Whittamore, P. R. O. Discovery of a potent, selective, and orally bioavailable acidic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor: Discovery of 2-[(3S)-1[5-(cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-3-piperidyl]acetic acid (AZD4017). J. Med. Chem. 2012, 55, 5951−5964. (21) Scott, J. S.; deSchoolmeester, J.; Kilgour, E.; Mayers, R. M.; Packer, M. J.; Hargreaves, D.; Gerhardt, S.; Ogg, D.; Rees, A.; Selmi, N.; Stocker, A.; Swales, J. G.; Whittamore, P. R. O. Novel acidic 11βhydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor with reduced acyl glucuronide liability: The discovery of 4-[4-(2adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]benzoic acid (AZD8329). J. Med. Chem. 2012, 55, 10136−10147. (22) Yu, J.; Liu, H.; Xia, G.; Liu, L.; Xu, Z.; Chen, Q.; Ma, C.; Sun, X.; Xu, J.; Li, H.; Li, P.; Shi, Y.; Xiong, B.; Liu, X.; Shen, J. Discovery of 2-Alkyl-1-arylsulfonylprolinamides as 11β-hydroxysteroid dehydrogenase type 1 Inhibitors. ACS Med. Chem. Lett. 2012, 3, 793−798. (23) Wan, Z.-K.; Chenail, E.; Li, H.; Ipek, M.; Xiang, J.; Suri, V.; Hahm, S.; Bard, J.; Svenson, K.; Xu, X.; Tian, X.; Wang, M.; Li, X.; Johnson, C. E.; Qadri, A.; Panza, D.; Perreault, M.; Mansour, T. S.; Tobin, J. F.; Saiah, E. Discovery of HSD-621 as a potential agent for the treatment of type 2 diabetes. ACS Med. Chem. Lett. 2013, 4, 118− 123. (24) Goldberg, F. W.; Dossetter, A. G.; Scott, J. S.; Robb, G. R.; Boyd, S.; Groombridge, S. D.; Kemmitt, P. D.; Sjogren, T.; Gutierrez, P. M.; deSchoolmeester, J.; Swales, J. G.; Turnbull, A. V.; Wild, M. J. Optimization of brain penetrant 11β-hydroxysteroid dehydrogenase type I inhibitors and in vivo testing in diet-induced obese mice. J. Med. Chem. 2014, 57, 970−986. (25) Byun, S. Y.; Hong, S. P.; Shin, Y. J.; Nam, K. Y.; Ahn, S. K. A novel highly potent and selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, UI-1499. Life Sci. 2015, 120, 1−7. (26) Robb, G. R.; Boyd, S.; Davies, C. D.; Dossetter, A. G.; Goldberg, F. W.; Kemmitt, P. D.; Scott, J. S.; Swales, J. G. Design of pyrozolopyrimidines as 11beta-HSD1 inhibitors through optimization of molecular electrostatic potential. MedChemComm 2015, 6, 926−934. (27) Ryu, J. H.; Lee, J. A.; Kim, S.; Shin, Y. A.; Yang, J.; Han, H. Y.; Son, H. J.; Kim, Y. H.; Sa, J. H.; Kim, J.-S.; Lee, J.; Lee, J.; Park, H.-g. Discovery of 2-((R)-4-(2-Fluoro-4-(methylsulfonyl)phenyl)-2-methylpiperazin-1-yl)-N-((1R,2S,3S,5S,7S)-5-hydroxyadamantan-2-yl)pyrimidine-4-carboxamide (SKI2852): A Highly Potent, Selective, and Orally Bioavailable Inhibitor of 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1). J. Med. Chem. 2016, 59, 10176−10189. (28) Anderson, A.; Walker, B. R. Drugs 2013, 73, 1385−1393. (29) Garcia, R. A.; Search, D. J.; Lupisella, J. A.; Ostrowski, J.; Guan, B.; Chen, J.; Yang, W.; Truong, A.; He, A.; Zhang, R.; Yan, M.; Hellings, S. E.; Gargalovic, P. S.; Ryan, C. S.; Watson, L. M.; Langish, R. A.; Shipkova, P. A.; Carson, N. L.; Taylor, J. R.; Yang, R.; Psaltis, G. C.; Harrity, T. W.; Robl, J. A.; Gordon, D. A. 11beta-hydroxysteroid dehydrogenase type 1 gene knockout attenuates atherosclerosis and in vivo foam cell formation in hyperlipidemic apoE−/− mice. PLoS One 2013, 8, e53192. (30) Hermanowski-Vosatka, A.; Balkovec, J. M.; Cheng, K.; Chen, H. Y.; Hernandez, M.; Koo, G. C.; Le Grand, C. B.; Li, Z.; Metzger, J. M.; Mundt, S. S.; Noonan, H.; Nunes, C. N.; Olson, S. H.; Pikounis, B.; Ren, N.; Robertson, N.; Schaeffer, J. M.; Shah, K.; Springer, M. S.; Strack, A. M.; Strowski, M.; Wu, K.; Wu, T.; Xiao, J.; Zhang, B. B.; Wright, S. D.; Thieringer, R. 11β-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice. J. Exp. Med. 2005, 202, 517−527. (31) Atalar, F.; Vural, B.; Ciftci, C.; Demirkan, A.; Akan, G.; Susleyici-Duman, B.; Gunay, D.; Akpinar, B.; Sagbas, E.; Ozbek, U.; Buyukdevrim, A. S. 11β-hydroxysteroid dehydrogenase type 1 gene 4947

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948

Journal of Medicinal Chemistry

Article

of 11-β-hydroxysteroid dehydrogenase inhibitors. Org. Process Res. Dev. 2014, 18, 960−968. (43) Zhang, J.; Osslund, T. D.; Plant, M. H.; Clogston, C. L.; Nybo, R. E.; Xiong, F.; Delaney, J. M.; Jordan, S. R. Crystal structure of murine 11-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes. Biochemistry 2005, 44, 6948−6957. (44) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307−326. (45) Hosfield, D. J.; Wu, Y.; Skene, R. J.; Hilgers, M.; Jennings, A.; Snell, G. P.; Aertgeerts, K. Conformational flexibility in crystal structures of human 11beta-hydroxysteroid dehydrogenase type I provide insights into glucocorticoid interconversion and enzyme regulation. J. Biol. Chem. 2005, 280, 4639−4648. (46) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486−501. (47) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739−1749. (48) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 2004, 47, 1750−1759. (49) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra precision Glide: docking and scoring incorporating a model of hydrophobic enclosure for protein−ligand complexes. J. Med. Chem. 2006, 49, 6177−6196. (50) Banks, J. L.; Beard, H. S.; Cao, Y.; Cho, A. E.; Damm, W.; Farid, R.; Felts, A. K.; Halgren, T. A.; Mainz, D. T.; Maple, J. R.; Murphy, R.; Philipp, D. M.; Repasky, M. P.; Zhang, L. Y.; Berne, B. J.; Friesner, R. A.; Gallicchio, E.; Levy, R. M. Integrated modeling program, applied chemical theory (IMPACT). J. Comput. Chem. 2005, 26, 1752−1780. (51) Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J. F.; Honig, B.; Shaw, D. E.; Friesner, R. A. A hierarchical approach to all-atom protein loop prediction. Proteins: Struct., Funct., Genet. 2004, 55, 351− 367. (52) Jacobson, M. P.; Friesner, R. A.; Xiang, Z.; Honig, B. On the role of crystal packing Forces in determining protein sidechain conformations. J. Mol. Biol. 2002, 320, 597−608.

expression is increased in ascending aorta tissue of metabolic syndrome patients with coronary artery disease. GMR, Genet. Mol. Res. 2012, 11, 3122−3132. (32) Robl, J. A.; Wang, H.; Li, J. J.; Hamann, L.; Simpkins, L.; Golla, R.; Li, Y.-X.; Seethala, R.; Zvyaga, T.; Harper, T.; Hsueh, M.; Wang, M.; Hansen, L.; Patel, C.; Azzara, A.; Rooney, S.; Sheriff, S.; Morin, P.; Camac, D.; Harrity, T.; Zebo, R.; Ponticiello, R.; Morgan, N.; Taylor, J.; Gordon, D. Optimization of triazolopyridine based 11βhydroxysteroid dehydrogenase type-1 (11βHSD-1) inhibitors leading to the discovery of the clinical candidate 2a. Abstracts of Papers, 244th ACS National Meeting & Exposition, Philadelphia, PA, United States, August 19−23, 2012; American Chemical Society; Washington, DC, 2012; MEDI-217. (33) Li, J.; Kennedy, L. J.; Wang, H.; Li, J. J.; Walker, S. J.; Hong, Z.; O’Connor, S. P.; Ye, X.-Y.; Chen, S.; Wu, S.; Yoon, D.; Nayeem, A.; Camac, D. M.; Morin, P. E.; Sheriff, S.; Wang, M.; Harper, T.; Golla, R.; Seethala, R.; Harrity, T.; Ponticiello, R. P., Morgan, N. M.; Taylor, J. R.; Zebo, R.; Gordon, D. A.; Robl, J. A. Optimization of 1,2,4triazolopyridines as inhibitors of human 11β-hydroxysteroid dehydrogenase type 1: discovery of clinical candidate BMS-823778. Abstracts of Papers, 250th ACS National Meeting & Exposition, Boston, MA, USA, August 16−20, 2015; American Chemical Society: Washington, DC, 2015; MEDI-28. (34) Wang, H.; Robl, J. A.; Hamann, L. G.; Simpkins, L. M.; Golla, R.; Li, Y.-X.; Seethala, R.; Zvyaga, T.; Gordon, D. A.; Li, J. J. Generation of 3,8-substituted 1,2,4-triazolopyridines as potent inhibitors of human 11β-hydroxysteroid dehydrogenase type-1. Bioorg. Med. Chem. Lett. 2011, 21, 4146−4149. (35) Li, J.; Kennedy, L. J.; Wang, H.; Li, J. J.; Walker, S. J.; Hong, Z.; O’Connor, S. P.; Nayeem, A.; Camac, D. M.; Morin, P. E.; Sheriff, S.; Wang, M.; Harper, T.; Golla, R.; Seethala, R.; Harrity, T.; Ponticiello, R. P.; Morgan, N. N.; Taylor, J. R.; Zebo, R.; Gordon, D. A.; Robl, J. A. Optimization of 1,2,4-triazolopyridines as inhibitors of human 11βhydroxysteroid dehydrogenase type 1 (11β-HSD-1). ACS Med. Chem. Lett. 2014, 5, 803−808. (36) Li, J.; Li, J. J.; Kennedy, L. J.; Wang, H.; Walker, S. J.; Harrity, T.; Ponticiello, R. P.; Morgan, N. N.; Taylor, J. R.; Zebo, R.; Gordon, D. A.; Robl, J. A. Unpublished results. (37) Véniant, M. M.; Hale, C.; Komorowski, R.; Chen, M. M.; St. Jean, D. J.; Fotsch, C.; Wang, C. Time of the day for 11β-HSD1 inhibition plays a role in improving glucose homeostasis in DIO mice. Diabetes, Obes. Metab. 2009, 11, 109−117. (38) Ye, X.-Y.; Chen, S. Y.; Nayeem, A.; Golla, R.; Seethala, R.; Wang, M.; Harper, T.; Sleczka, B. G.; Li, Y.-X.; He, B.; Kirby, M.; Gordon, D. A.; Robl, J. A. Design, synthesis, and SAR studies of novel polycyclic acids as potent and selective inhibitors of human 11βhydroxysteroid dehydrogenase type 1 (11β-HSD1). Bioorg. Med. Chem. Lett. 2011, 21, 6699−6704. (39) Ye, X.-Y.; Yoon, D.; Chen, S. Y.; Nayeem, A.; Golla, R.; Seethala, R.; Wang, M.; Harper, T.; Sleczka, B. G.; Apedo, A.; Li, Y.-X.; He, B.; Kirby, M.; Gordon, D. A.; Robl, J. A. Synthesis and structure-activity relationship of 2-adamantylmethyl tetrazoles as potent and selective inhibitors of human 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). Bioorg. Med. Chem. Lett. 2014, 24, 654−660. (40) Rohde, J. J.; Pliushchev, M. A.; Sorensen, B. K.; Wodka, D.; Shuai, Q.; Wang, J.; Fung, S.; Monzon, K. M.; Chiou, W. J.; Pan, L.; Deng, X.; Chovan, L. E.; Ramaiya, A.; Mullally, M.; Henry, R. F.; Stolarik, D. F.; Imade, H. M.; Marsh, K. C.; Beno, D. W. A.; Fey, T. A.; Droz, B. A.; Brune, M. E.; Camp, H. S.; Sham, H. L.; Frevert, E. U.; Jacobson, P. B.; Link, J. T. Discovery and metabolic stabilization of potent and selective 2-amino-N-(adamant-2-yl) acetamide 11βhydroxysteroid dehydrogenase type 1 inhibitors. J. Med. Chem. 2007, 50, 149−164. (41) Gerlach, H. Bestimmung des Chiralitätssinns der enantiomeren 2,6-Adamantandiole. Helv. Chim. Acta 1985, 68, 1815−1821. (42) Hanson, R. L.; Goldberg, S. L.; Guo, Z.; Tully, T. P.; Goswami, A.; Ye, X.-Y.; Robl, J. A.; Patel, R. N. Enzymatic reduction of adamantanones to chiral adamantanol intermediates for the synthesis 4948

DOI: 10.1021/acs.jmedchem.7b00211 J. Med. Chem. 2017, 60, 4932−4948