Discovery of novel sulfonamides as potent and selective inhibitors against human and mouse 11β-hydroxysteroid dehydrogenase type 1

Molecular and Cellular Endocrinology 358 (2012) 46–52

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Discovery of novel sulfonamides as potent and selective inhibitors against human and mouse 11b-hydroxysteroid dehydrogenase type 1 Guangxin Xia a,b, Lin Liu a,b, Mengzhu Xue a, Haiyan Liu b, Jianxin Yu b, Ping Li b, Qian Chen b, Bing Xiong a,⇑, Xuejun Liu b, Jingkang Shen a,b,⇑ a b

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China Central Research Institute, Shanghai Pharmaceutical Holding Co., Ltd., 898 Ha Lei Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China

a r t i c l e

i n f o

Article history: Received 9 September 2011 Received in revised form 15 February 2012 Accepted 17 February 2012 Available online 3 March 2012 Keywords: 11b-HSD1 Inhibitor Sulfonamide Diabetes Docking

a b s t r a c t Several classes of non-steroid 11b-HSD1 inhibitors have been developed as promising treatments for Type 2 Diabetes (T2D). Using a human 11b-HSD1 selective inhibitor as a starting point, we designed and synthesized a new class of derivatives of 1-arylsulfonyl piperidine-3-carboxamides. It was found that the large lipophilic group on the amino moiety may lead to cross-species potency towards human and mouse, allowing drug development by evaluating compounds in rodent model. By exploring structure– activity-relationship, the (R)-(+)-bornylamine derivative is identified as the most potent inhibitor of mouse enzyme 11b-HSD1 with an IC50 of 18 nM. Docking studies revealed the different possible interaction modes of the S-enantiomer and R-enantiomer bound to h11b-HSD1, and explained why the S-enantiomer is more active than the R-enantiomer. Finally, two potent and isoform-selective compounds, (+)-isopinocampheylamine derivative 8m and (R)-(+)-bornylamine derivative 8l, with suitable in vitro properties, could be selected for future PK/PD evaluation in rodent models. Then, 8l was subjected a pharmacodynamics study in vivo with rodent model. It was shown that 8l have 71% and 63% inhibition in adipose and liver tissue at 1 h after administration, but it was a short-acting compound displaying a significant drop in potency in the subsequent 3 h. This study not only provides compounds as novel h11b-HSD1 inhibitors, but also presents structure–activity relationships for designing potent human/ mouse 11b-HSD1 inhibitors suitable for in vivo evaluation in rodent models. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Type 2 Diabetes (T2D) is becoming an epidemic world wide, especially in Asia and developing countries, with the World Health Organization (WHO) estimating that about 300 million people will be affected in 2025 (Zimmet et al., 2001). There has been a common understanding that ‘‘metabolic syndrome’’ is associated with type II diabetes. This syndrome is a collection of several metabolic and cardiovascular abnormalities, such as abdominal obesity, impaired glucose tolerance, impaired fasting glucose, insulin resistance, dyslipidemia, low levels of high density lipoprotein cholesterol (HDL-C) and hypertension (Kahn et al., 2005; Hu et al., 2005). Glucocorticoid receptor (GR) signaling plays a significant role in metabolic regulation, and defects in this signaling pathway

⇑ Corresponding authors. Address: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China (J. Shen). Tel.: +86 21 50806600x5407; fax: +86 21 50807088. E-mail addresses: [email protected] (B. Xiong), [email protected] (J. Shen). 0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2012.02.017

have been implicated in the development of several phenotypes of metabolic syndrome (Rose et al., 2010; Tomlinson and Stewart, 2007). GR signaling depends not only on the circulating cortisol levels but also on the intracellular production of cortisol through reduction of cortisone, the inactive glucocorticoid. The enzymes catalyzing the conversion between cortisone and cortisol are 11b-hydroxysteroid dehydrogenases (11b-HSDs). Among them, the type 1 isoform (11b-HSD1), highly expressed in liver and adipose tissue, are dominantly reduce cortisone to cortisol, and the type 2 isoform (11b-HSD2), primarily expressed in kidney, oxidizes cortisol to cortisone (Saiah, 2008). A potential role for 11b-HSD1 inhibitors in metabolic disease has been established using transgenic mice. Based on these findings, in recent years, 11b-HSD1 is recognized as a promising target in metabolic disease (Ge et al., 2010; Hale and Wang, 2008; Su et al., 2009). Many researchers have developed several classes of 11b-HSD1 inhibitors, including Incyte’s compound INCB-13739 (structure undisclosed), which significantly improves insulin sensitivity of T2D patients who failed on metformin treatment, and lowered triglyceride and cholesterol levels of patients with hyperlipidaemia and hypertriglyceridemia in a phase II trial (Rosenstock et al.,

G. Xia et al. / Molecular and Cellular Endocrinology 358 (2012) 46–52

2010). Other examples (shown in Fig. 1) such as the triazole Merck 544 (1), the thiazolone AMG-221 (2), the urea derivative (3) and sulfonamides 4–6 displayed excellent potency on human 11bHSD1 (h11b-HSD1) and selectivity over human 11b-HSD2 (h11bHSD2) (Ge et al., 2010; Hale and Wang, 2008; Tice et al., 2010; Xia et al., 2011). While they have excellent potency in human, some inhibitors, such as INCB-13739 and PF-915275 (5), lack potency against mouse 11b-HSD1 (m11b-HSD1). So for in vivo PK/ PD studies of these compounds require primate models (Bhat et al., 2008), which hinder development of inhibitors of 11b-HSD1. To identify novel and proprietary 11b-HSD1 inhibitors we conducted a scaffold hopping study on BVT-14225 and found (Fig. 1) a 1-arylsulfonyl piperidine-3-carboxamide (7) displaying micromolar activity against h11b-HSD1 only. In order to facilitate optimal drug candidate selection by evaluating inhibitors in rodent models, we utilized 7 as a starting point and focused our work on finding new derivatives efficiently inhibiting both human and mouse enzymes. Here, we report the discovery, synthesis and structure– activity relationships of this series against human and mouse 11b-HSD1.

2. Methods 2.1. Synthesis of target compounds General synthetic procedures are described as follows, and detailed procedure plus analytical data of intermediates and target molecules are provided in Supporting information.

2.1.1. General method A (S) or (R)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (10a, b) was condensed with cyclohexanamine to provide corresponding (S) or (R)-amides (11a, b). After removing protecting group under acidic condition, the resulting product was then

O

N

N N

N S

N

OH

O

HN

O

HN Br

1 Merck 544 hIC50 = 7.5 nM

2 AMG 221 hK i = 13 nM

CF3

O

3 Vitae Compound hIC50 = 1.1 nM NC

HO NH2

N N S O O

S O

4 Amgen hIC50 = 0.7 nM mIC50 > 300 nM

6 BVT-14225 hIC50 = 52 nM mIC50 = 284 nM

N

O S N O N

2.1.2. General method B Sulfonylation of (S) or (R)-ester (12a, b) were prepared with 3chloro-2-methyl benzene-1-sulfonyl chloride by combined with (S) or (R)-benzenesulfonamide (13a, b). Target compounds with varied amino moieties (8h–m, 9l, and 9m) were obtained by two successive steps: hydrolysis of ester 13a or 13b and condensation of acid and primary amines (Scheme 2). 2.2. Scintillation proximity assay (SPA) for 11b-HSDs Inhibition by compounds of h11b-HSD1 and m11b-HSD1 and h11b-HSD2 enzymatic activities was determined by the scintillation proximity assay (SPA) using microsomes containing 11b-HSDs according to previous studies (Mundt et al., 2005; Yang et al., 2009). Briefly, the full-length cDNAs of human or mouse 11b-HSDs were isolated from cDNA libraries provided by NIH Mammalian Gene Collection and cloned into pcDNA3 expression vectors (Invitrogen, Carlsbad, CA, USA) by PCR. HEK-293 cells were transfected with the pcDNA3-derived expression plasmids and selected by cultivation at the presence of 700 lg/ml G418. The microsomal fraction overexpressing 11b-HSDs was prepared from the HEK-293 cells stably transfected with 11b-HSDs and used as the enzyme source for SPA. The assay was performed in a 96-well microtiter plate. Compounds with different concentrations were added, followed by adding 80 lL of 50 mM HEPES buffer, pH7.4 containing 25 nM cortisone[1,2-(n)3H] (Amersham, Buckinghamshire, UK) and 1.25 mM NADPH (for 11b-HSD1 assay) or 12.5 nM cortisol[1,2,6,7-(n)3H] (Amersham, Buckinghamshire, UK) and 0.625 mM NAD (for 11b-HSD2 assay). Reactions were initiated by the addition of enzyme preparations as microsome fractions from HEK-293 cells in a final concentration of 80 lg/ml for 11b-HSD1. Following 60 min incubation at 37 °C, the reaction was stopped by adding 35 lL of 10 mg/ml protein A-coated SPA beads (GE, Piscataway, NJ, USA) suspended in SuperblockÒ Blocking Buffer (Pierce, Rockford, IL) with 3 lg/ml of murine monoclonal cortisol antibody (East Coast Biologics, North Berwick, Maine, USA) and 314 lM glycyrrhetinic acid (Sigma–Aldrich, St. Louis, MO). The plates were incubated under plastic film on an orbital shaker for 120 min at room temperature before counting. The amount of [3H]cortisol generated by 11b-HSD1 was captured on the beads and measured in a microplate liquid scintillation counter. Percent inhibition was calculated relative to noninhibited control. Data were obtained from at least three independent experiments. IC50 values were calculated by using Prism Version 4 (GraphPad Software, San Diego, CA). 2.3. In vitro metabolic stability and CYP450 inhibition evaluation

Cl O

reacted with aryl sulfonylchlorides to give target compounds (8a–g, 9a) (Scheme 1).

NH2

O

5 PF-915275 hK i = 1 nM mK i = 750 nM

Cl O S NH N O S

H N

47

N O 7 Initial compound hIC50 = 1602 nM

Fig. 1. Non-steroidal 11b-HSD1 inhibitors. All the enzyme binding affinities are reported in references. Compound 1 and 6 (Hale and Wang, 2008); compound 2 (Véniant et al., 2010); compound 3 (Tice et al., 2010); compound 4 (Tu et al., 2008); compound 5 (Bhat et al., 2008).

Microsomes (Human microsome: Xenotech, Lot No. H0610; Rat microsome: Xenotech, Lot No. R1000) (0.5 mg/ml) were preincubated with 1 lM test compound for 5 min at 37 °C in 0.1 M phosphate buffer (pH 7.4) with 1 mM EDTA, and 5 mM MgCl2. The reactions were initiated by adding prewarmed cofactors (1 mM NADPH). After 0, 5, 10, and 30 min incubations at 37 °C, the reactions were stopped by adding an equal volume of cold acetonitrile. The samples were vortexed for 10 min and then centrifuged at 10,000g for 10 min. Supernatants were analyzed by LC/MS/MS for the amount of parent compound remaining, and the corresponding loss of parent compound also determined by LC/MS/MS. The CYP enzymatic activities were characterized based on their probe reactions: CYP3A4 (midazolam 1-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), and CYP1A2 (phenacetin O-deethylation). Incubation mixtures were prepared in a total

48

G. Xia et al. / Molecular and Cellular Endocrinology 358 (2012) 46–52

Boc

N

OH

a

Boc

H N

N

b, c

O

O 11a 3S 11b 3R

10a 3S 10b 3R

H N

Ar N S O O

O

8a-g 3S 9a 3R Ar See Table 1

Scheme 1. Synthesis of 1-(arylsulfonyl)-N-cyclohexylpiperidine-3-carboxamides. Reagents and conditions: (a) cyclohexanamine, EDCI, HOBt, CH2Cl2, rt; (b) HCl(g)/1,4dioxane, CH2Cl2, rt; and (c) ArSO2Cl, Et3N, CH2Cl2, rt.

HN

O O

12a 3S 12b 3R

a Cl

O

S

N O

O

b,c Cl

O

O

S

8h-m 9l,m

13a 3S 13b 3R

H N

N O

A

O 3S 3R

A See Table 2 Scheme 2. Synthesis of 1-(3-chloro-2-methylphenylsulfonyl)piperidine-3-carboxamides. Reagents and conditions: (a) 3-chloro-2-methyl benzene-1-sulfonyl chloride, Et3N, CH2Cl2, rt; (b) MeOH, 50% aq NaOH, reflux; and (c) RNH2, EDCI, HOBt, CH2Cl2, rt.

volume of 100 lL as follows: 0.2 mg/ml microsome (Human microsome: Xenotech, Lot No. H0610), NADPH (1 mM), 100 mM phosphate buffer (pH 7.4), probe substrates cocktail (midazolam 5 lM, dextromethorphan 5 lM, phenacetin 100 lM) and 10 lM tested compound or positive control cocktail (ketoconazole 10 lM, quinidine 10 lM, a-naphthoflavone 10 lM or negative control (PBS). The final concentration of organic reagent in incubation mixtures was less than 1% v/v. There was a 5 min preincubation period at 37 °C before the reaction was initiated by adding a NADPH-generating system. Reactions were conducted for 20 min for CYP3A4, CYP2D6 and CYP1A2. For each probe drug, the percentage of metabolite conversion was less than 20% of substrate added. The inhibition rate was calculated as: (the formation of the metabolite of probe substrates with 10 lM tested compound)/(the formation of the metabolite of probe substrates with PBS)  100%. The samples for the time-dependent inactivation screening assay were preincubated for 0, 5, 10, 20 min at 37 °C with 0.2 mg/ ml Human microsome (Xenotech, Lot No. H0610) and 10 lM test compound or positive control (troleandomycin 10 lM, paroxetine 10 lM and furafylline 10 lM) with or without 1 mM NADPH. The percentage of remaining activity of CYP3A4, CYP2D6, and CYP1A2 was measured by the formation of the metabolite of their marker substrates: midazolam, dextromethorphan and phenacetin at single concentration approximating their Km values (midazolam 5 lM, dextromethorphan 5 lM, phenacetin 100 lM). The percentage of remaining activity of microsomes preincubated with NADPH was compared with that of microsomes without preincubation. 2.4. Pharmacodynamics evaluation Male BALB/c mice from SIPPR-BK Lab Animal Co., Ltd., fed ad libitum under standard regimen with free access to water and maintained on a 12/12 h light–dark cycle in a temperature-controlled room, were used in the study (18–22 g at the time of experiment). All animal experiments were conducted under principles of Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China on November 14, 1988 and followed the guidelines of the Animal Care and Use Committee of Shanghai Institute of Materia Medica. Compound 8l was dissolved in 0.5% methyl cellulose/ 0.1% Tween 80 in water and administered to the mice as a single oral dose at 50 mpk. At 1 and 4 h post-dose, animals were

sacrificed by cervical dislocation, and fresh tissues including liver and epididymal fat pad (EFP) were removed, immersed in PBS buffer, and weighed (three mice per data point were used). The total volume of PBS buffer added was equivalent to approximately five times the mass of tissue. Tissues were minced into 2–3 mm pieces and cortisone (LabPartner Shanghai Co., Ltd., 10 mg/kg in 0.5% methyl cellulose) was added to a final concentration of 10l M. The tissues were then incubated at 37 °C in a 5% CO2 atmosphere for 20 min for liver and 3 h for epididymal fat pad (EFP). The cortisol concentration in the media was determined by LC/MS/MS detection. The percent inhibition of 11b-HSD1 activity was calculated relative to a vehicle control (0.5% CMC/0.1% Tween 80 in water) treated group. 2.5. Docking method The Glide module in Maestro (Schrödinger Inc., Version 7.5) was used to perform the docking study (Friesner et al., 2004). The 3.0 Å X-ray structure of m11b-HSD1 complexed with corticosterone (PDB code: 1Y5R) and the 2.19 Å human enzyme complexed with a sulfonyl-piperazine inhibitor (PDB code: 3H6K) were chosen as the receptors in docking stimulations (Wan et al., 2009; Zhang et al., 2005). For each receptor, the grid-enclosing box was centered on the centroid of the corresponding ligand and defined to enclose residues located within 20 Å around the binding site, and a scaling factor of 1.0 was set to van der Waals (VDW) radii of those receptor atoms with partial atomic charge of less than 0.25. Compounds 8a, 9a and 8l were chosen as ligands to be docked, and prepared by the Ligprep module. Lastly, for each docking stimulation, the standardprecision (SP) mode was used to ensure it with default parameters, and top 20 poses of each compound were reserved for the binding mode analysis. 3. Results and discussion The activities of target compounds were determined in vitro for inhibiting h11b-HSD1 and m11b-HSD1, and the results listed in Tables 1 and 2. Initially, we compared the S-enantiomer 8a with R-enantiomer 9a. The S-enantiomer is about 10 times more potent than R-enantiomer, so more (S)-derivatives were then prepared to explore the SAR. As summarized in Table 1, the 5-bromothiophen-2-ylsulfonyl,

49

G. Xia et al. / Molecular and Cellular Endocrinology 358 (2012) 46–52 Table 1 Inhibitory activity of piperidine-3-carboxamides with varied 1-arylsulfonyl groups.

Ar N S O O

Compound

Configuration

8a

3S

Ar

Cl

9a

3S

O

IC50a (nM) h11b-HSD1

Percent inhibitionb m11b-HSD1

0.8

24

8.1

12

0.9

26

5.7

29

3.3

33

11

32

46

ND

3.5

8

*

3R Cl

8b

H N

*

Br S *

8c

3S

8d

3S

8e

3S

8f

3S

8g

3S

*

*

* O O

NC a b

*

*

The IC50 values are reported as the mean of three measurements with variance less than 15%. Percent inhibition measured at a concentration of 100 nM and reported as the mean value of two measurements. ND: not determined.

mesitylenesulfonyl, 4-biphenylsufonyl and 3-cyano phenylsulfonyl derivatives (8b, 8c, 8d and 8g) have nanomolar or subnanomolar inhibitory activity against h11b-HSD1, while substitution with 3,4-dimethoxyphenylsufonyl and 4-t-Bu-phenylsufonyl groups lead to reduced activities (8f and 8e). However, all of these compounds exhibit low activity against m11b-HSD1 (percent inhibition <35% at a concentration of 100 nM). Next, we investigated the effect on activity of amino moiety (Table 2). Cyclopropyl amine derivative 8h appears to have only 24% inhibition at 100 nM concentration against the human enzyme, whereas its cyclopentyl, cyclohexyl and cycloheptyl (8i, 8a and 8j) derivatives have dramatically increased the activities when a large alicyclic hydrocarbon (substituent A) was introduced. If one introduces a hydroxyl group on the cyclohexylamine of 8a, the activity decreases 3.6-fold (8k, IC50 = 2.9 nM). However, except for the cycloheptyl derivative 8j, with IC50 of 139 nM, all of cycloalkyl derivative (8a–k in Tables 1 and 2) did not show significant inhibition against m11b-HSD1. Many known 11b-HSD1 inhibitors have large lipophilic groups, such as 2-norbornyl in 2 and adamantyl in 1 and 3. Therefore, further modification of this series was conducted by introducing bridged hydrocarbon with high steric hindrance into the amino moiety. The (+)-isopinocampheylamine derivative 8m and (R)(+)-bornylamine derivative 8l were then found to be the most potent compounds (IC50 of 0.03 and 0.4 nM) against h11b-HSD1. Interestingly, these two compounds also inhibit m11b-HSD1 effectively in vitro. Although the inhibitory activity against m11b-HSD1 of 8m decreases by 4100-fold (IC50 = 123 vs. 0.03 nM) compared with h11b-HSD1, the most potent inhibiting the mouse enzyme is 8l with an IC50 of 18 nM, which is only 45 times lower than

the activity against h11b-HSD1 (0.4 nM), and is suitable for preclinical PD studies in rodent models. For comparison, the (R)-form piperidine-3-carboxylic amide derivatives with high steric bridged hydrocarbon (9l and 9m) were also synthesized and inhibitory assays against both h11b-HSD1 and m11b-HSD1 were performed. Although these compounds showed nanomolar activities against human enzyme, they are all less potent compared with the corresponding (S)-enantiomers (9l, 9m vs. 8l, 8m in Table 2). Inhibition of 11b-HSD2 might cause sodium retention, hypokalemia, and hypertension, so we also measured inhibitory activities against h11b-HSD2 of this series of compounds. As shown in Table 2, all these tested derivatives have high selectivity ratios for h11bHSD1 over h11b-HSD2 (>1000-fold). Compound 8l and 8m show moderate metabolic stability in the human liver microsome assay. Moreover, 8l neither inhibits 3A4, 2D6, 2C9 directly nor shows time-dependent inhibition (TDI) of these three key CYPs. Compound 8m exhibits a slight inhibition of 3A4 with IC50 of 3.2 lM, but no direct inhibition of 2D6 and 2C9 or TDI on the three CYPs were observed. Consequently, we tested 8l in a pharmacodynamics (PD) rodent model and the 11b-HSD1 inhibition was measured in target tissues. Mice were sacrificed 1 or 4 h after oral dosing. The liver and epididymal fat pad (EFP) were collected and incubated in the presence of cortisone in the media. Cortisol levels were then measured to determine the level of compound inhibition from cortisone to cortisol. As summarized result in Table 3, 8l showed 71% and 63% inhibition in adipose and liver tissue at 1 h after administration, but it was a short-acting compound displaying a significant drop in potency in the subsequent 3 h.

50

G. Xia et al. / Molecular and Cellular Endocrinology 358 (2012) 46–52

Table 2 Inhibitory activity and selectivity of piperidine-3-carboxamides with varied amino groups.

Compound

Configuration

8h

3S

8i

3S

8a

3S

8j

3S

8k

3S

8l

3S

IC50a (nM)

A

h11b-HSD1

m11b-HSD1

h11b-HSD2

24% @ 100 nM

ND

ND

3.9

21% @ 100 nM

ND

0.8

24% @ 100 nM

>100,000 (26% @ 100 lM)

1.1

139

>100,000 (37% @ 100 lM)

2.9

15% @ 100 nM

>10,000 (21% @ 10 lM)

0.4

18

>10,000 (32% @ 10 lM)

0.03

123

7800

8.1

20% @ 100 nM

>100,000 (16% @ 100 lM)

5.5

189

>10,000 (21% @ 10 lM)

5.6

1263

6604

*

*

*

* OH

* *

H

H 8m

3S

9a

3R

9l

3R

*

* *

H

H 9m

a

3R

*

The IC50 values are reported as the mean of three measurements with variance less than 15%. ND: not determined.

Table 3 Ex vivo pharmacodynamic data. Compound

8l

% Inhibition in fat

% Inhibition in liver

1h

4h

1h

4h

71

32

63

26

To investigate the activity difference against h11b-HSD1 between compound 8a and its enantiomer 9a, we scrutinized their binding poses generated by molecular docking (Fig. 2). Compared with that observed in the complex of 3H6K (data not shown), 8a binds to the enzyme in a similar manner; in particular, one of the sulfone oxygens makes a hydrogen bond with the backbone nitrogen of the residue Ala 147 (distance 3.34 Å). In addition, the chlorine substituent phenyl ring participates in a p–p stack interaction with the residue Tyr 152. Moreover, 8a makes additional interactions with the protein, which may also contribute to its superior activity. In detail, the oxygen of the amide linker forms a hydrogen bond with the hydroxyl of Tyr 158 (distance 3.27 Å), while the terminal cyclohexyl contacts the enzyme by a van der Waals interaction with Ala 201. As for 9a, it retains the hydrogen bond with Ala 147 while the distance increases to 3.78 Å. Besides, the amide linker turns to the opposite orientation, so that the

hydrogen interaction with Tyr 158 is abolished and the van der Waals interaction with Ala 201 is attenuated. Consequently, these changes combined together may result in the 10-time decrease in inhibitory potency of 9a. To explain the inhibitory potency difference between h11bHSD1 and m11b-HSD1, compound 8l which shows the better inhibitory potency against m11b-HSD1 and 8a with good activity against the human enzyme was docked into corresponding binding sites of the two proteins. The m11b-HSD1 structure 1Y5R with the docked compounds was chosen as a template, and aligned by that of h11b-HSD1 (Fig. 3). Similar to 8a in the binding site of the h11bHSD1, 8l can make critical interactions with the same residues; and the compound makes a slight shift to the entrance of the pocket, which may due to the larger terminal hydrophobic group. However, the compound binds to the m11b-HSD1 in the same location as 8a to the h11b-HSD1. So it is surmised that the murine binding site prefers a larger terminal hydrophobic group. Similarly, 8m with a large terminal hydrophobic group shows moderate activities against the murine enzyme, compared with the inactive 8a. In addition, since the Tyr 152 in the human enzyme is changed into the Gln 177, the original p–p stack interaction induced by the chlorine substituent phenyl ring is eliminated, which may also contribute to the selectivity of these compounds.

G. Xia et al. / Molecular and Cellular Endocrinology 358 (2012) 46–52

51

Fig. 2. The binding modes of compound 8a and 9a to the h11b-HSD1. (A) Docking result of the compound 8a. The NAP and protein residues are colored gold, with the inhibitor in green. The residues making critical contacts are labeled and hydrogen bonds highlighted by red dashed lines. (B) Docking result of the compound 9a, compared with its 3S isomer 8a. The compound 9a is shown in green, with 8a in gray. Other legends are stated as in (A).

Fig. 3. The docking results of the compound 8l to human and murine 11b-HSD1. (A) Docking result of the compound 8l to the human enzyme. The NAP and residues making critical interactions are shown in gold, with the compound 8l in green; residues making critical interactions in the human protein binding site are labeled. (B) Docking result of the compound 8l to the murine enzyme. The NAP and residues of the murine protein are shown in gold, and residues of the human protein in magenta. Residues making critical interactions in the human protein binding site are labeled, compared with corresponding mutated residues in the murine enzyme.

4. Conclusions

Appendix A. Supplementary data

In the present work, we have reported the discovery of 1-arylsulfonyl piperidine-3-carboxamides as potent and selective inhibitors of 11b-HSD1. Based on the initial human 11b-HSD1 selective inhibitor, a class of new derivatives was synthesized and inhibition of human and mouse 11b-HSD1 evaluated. SAR studies indicate that a large lipophilic group with high steric hindrance on the amino moiety will lead to cross-species potency. Docking studies revealed the possible different interaction modes of S-enantiomer 8a and R-enantiomer 9a with h11b-HSD1, and show why S-enantiomer is more active than its enantiomer. Finally, potent and selective compound, the (R)-(+)-bornylamine derivative 8l, with suitable candidate-like properties, was tested on an ex vivo PD rodent model, and displayed inhibition of 11b-HSD1 in target tissues.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2012.02.017.

Acknowledgments We are indebted to Prof. Ying Leng, SIMM for the 11b-HSD1 assay. We are grateful for financial support from New Drug Creation Project of the National Science and Technology Major Foundation of China (Project 2010ZX09401-404), to Shanghai Pharmaceutical Holding, Shanghai Postdoctoral Sustentation Fund, China (Grant No. 07R214213 to G.X.) and Program of Excellent Young Scientist of Chinese Academy of Sciences (Grant to B.X.: KSCX2-EW-Q-3-01).

References Bhat, B.G., Hosea, N., Fanjul, A., Herrera, J., Chapman, J., Thalacker, F., Stewart, P.M., Rejto, P.A., 2008. Demonstration of proof of mechanism and pharmacokinetics and pharmacodynamic relationship with 40 -cyano-biphenyl-4-sulfonic acid (6amino-pyridin-2-yl)-amide (PF-915275), an inhibitor of 11b-hydroxysteroid dehydrogenase type 1, in cynomolgus monkeys. J. Pharmacol. Exp. Ther. 324 (1), 299–305. Friesner, R.A., Banks, J.L., Murphy, R.B., Halgren, T.A., Klicic, J.J., Mainz, D.T., Repasky, M.P., Knoll, E.H., Shaw, D.E., Shelley, M., Perry, J.K., Francis, P., Shenkin, P.S., 2004. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 47, 1739–1749. Ge, R., Huang, Y., Liang, G., Li, X., 2010. 11Beta-hydroxysteroid dehydrogenase type 1 inhibitors as promising therapeutic drugs for diabetes: status and development. Curr. Med. Chem. 17 (5), 412–422. Hale, C., Wang, M., 2008. Development of 11beta-HSD1 inhibitors for the treatment of type 2 diabetes. Mini Rev. Med. Chem. 8 (7), 702–710. Hu, G., Qiao, Q., Tuomilehto, J., 2005. The metabolic syndrome and cardiovascular risk. Curr. Diabetes Rev. 1 (2), 137–143. Kahn, R., Buse, J., Ferrannini, E., Stern, M., 2005. The metabolic syndrome: time for a critical appraisal. Joint statement from the American Diabetes Association and the European Association for the Study of Diabetes. Diabetologia 48 (9), 1684– 1699. Mundt, S., Solly, K., Thieringer, R., Hermanowski-Vosatka, A., 2005. Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1. Assay Drug Dev. Technol. 3 (4), 367–375.

52

G. Xia et al. / Molecular and Cellular Endocrinology 358 (2012) 46–52

Rose, A.J., Vegiopoulos, A., Herzig, S., 2010. Role of glucocorticoids and the glucocorticoid receptor in metabolism: insights from genetic manipulations. J. Steroid Biochem. Mol. Biol. 122 (1–3), 10–20. Rosenstock, J., Banarer, S., Fonseca, V.A., Inzucchi, S.E., Sun, W., Yao, W., Hollis, G., Flores, R., Levy, R., Williams, W.V., Seckl, J.R., Huber, R., 2010. The 11-betahydroxysteroid dehydrogenase type 1 inhibitor INCB13739 improves hyperglycemia in patients with type 2 diabetes inadequately controlled by metformin monotherapy. Diabetes Care 33 (7), 1516–1522. Saiah, E., 2008. The role of 11beta-hydroxysteroid dehydrogenase in metabolic disease and therapeutic potential of 11beta-hsd1 inhibitors. Curr. Med. Chem. 15 (7), 642–649. Su, X., Vicker, N., Trusselle, M., Halem, H., Culler, M.D., Potter, B.V., 2009. Discovery of novel inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1. Mol. Cell. Endocrinol. 301 (1–2), 169–173. Tice, C.M., Zhao, W., Xu, Z., Cacatian, S.T., Simpson, R.D., Ye, Y.J., Singh, S.B., McKeever, B.M., Lindblom, P., Guo, J., Krosky, P.M., Kruk, B.A., Berbaum, J., Harrison, R.K., Johnson, J.J., Bukhtiyarov, Y., Panemangalore, R., Scott, B.B., Zhao, Y., Bruno, J.G., Zhuang, L., McGeehan, G.M., He, W., Claremon, D.A., 2010. Spirocyclic ureas: orally bioavailable 11 beta-HSD1 inhibitors identified by computer-aided drug design. Bioorg. Med. Chem. Lett. 20 (3), 881–886. Tomlinson, J.W., Stewart, P.M., 2007. Modulation of glucocorticoid action and the treatment of type-2 diabetes. Best Pract. Res. Clin. Endocrinol. Metab. 21 (4), 607–619. Tu, H., Powers, J.P., Liu, J., Ursu, S., Sudom, A., Yan, X., Xu, H., Meininger, D., Degraffenreid, M., He, X., Jaen, J.C., Sun, D., Labelle, M., Yamamoto, H., Shan, B., Walker, N.P., Wang, Z., 2008. Distinctive molecular inhibition mechanisms for

selective inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1. Bioorg. Med. Chem. 16 (19), 8922–8931. Véniant, 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 Jr., D.J., Johansson, L., Henriksson, M.A., Williams, M., Vallgårda, J., Fotsch, C., Wang, M., 2010. Discovery of a potent, orally active 11beta-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-methylthiazol4(5H)-one (AMG 221). J. Med. Chem. 53 (11), 4481–4487. Wan, Z.K., Chenail, E., Xiang, J., Li, H.Q., Ipek, M., Bard, J., Svenson, K., Mansour, T.S., Xu, X., Tian, X., Suri, V., Hahm, S., Xing, Y., Johnson, C.E., Li, X., Qadri, A., Panza, D., Perreault, M., Tobin, J.F., Saiah, E., 2009. Efficacious 11beta-hydroxysteroid dehydrogenase type I inhibitors in the diet-induced obesity mouse model. J. Med. Chem. 52 (17), 5449–5461. Xia, G., Xue, M., Liu, L., Yu, J., Liu, H., Li, P., Wang, J., Li, Y., Xiong, B., Shen, J., 2011. Potent and novel 11b-HSD1 inhibitors identified from shape and docking based virtual screening. Bioorg. Med. Chem. Lett. 21 (19), 5739–5744. Yang, H., Shen, Y., Chen, J., Jiang, Q., Leng, Y., Shen, J., 2009. Structure-based virtual screening for identification of novel 11beta-HSD1 inhibitors. Eur. J. Med. Chem. 44 (3), 1167–1171. Zhang, J., Osslund, T.D., Plant, M.H., Clogston, C.L., Nybo, R.E., Xiong, F., Delaney, J.M., Jordan, S.R., 2005. Crystal structure of murine 11beta-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes. Biochemistry 44 (18), 6948–6957. Zimmet, P., Alberti, K.G., Shaw, J., 2001. Global and societal implications of the diabetes epidemic. Nature 414 (6865), 782–787.