Thiazolopyridines Improve Adipocyte Function by Inhibiting 11 Beta-HSD1 Oxoreductase Activity

Article abstract of DOI:10.1155/2017/3182129

Background. Glucocorticoid excess has been linked to clinical observations associated with the pathophysiology of metabolic syndrome. The intracellular glucocorticoid levels are primarily modulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme that is highly expressed in key metabolic tissues including fat, liver, and the central nervous system. Methods. In this study we synthesized a set of novel tetrahydrothiazolopyridine derivatives, TR-01-4, that specifically target 11β-HSD1 and studied their ability to interfere with the glucocorticoid and lipid metabolism in the 3T3-L1 adipocytes. Results. Based on the docking model and structure-activity relationships, tetrahydrothiazolopyridine derivatives TR-02 and TR-04 showed the highest potency against 11β-HSD1 by dose-dependently inhibiting conversion of cortisone to cortisol (IC₅₀ values of 1.8 μM and 0.095 μM, resp.). Incubation of fat cells with 0.1-10 μM TR-01-4 significantly decreased cortisone-induced lipid accumulation in adipocytes and suppressed 11β-HSD1 mRNA expression. Observed reduction in adipocyte fat stores could be partially explained by decreased expression levels of adipogenic markers (PPAR-γ, aP2) and key enzymes of lipid metabolism, including fatty acid synthase (FAS), hormone sensitive lipase (HSL), and lipoprotein lipase (LPL). Conclusions. The tetrahydrothiazolopyridine moiety served as an active pharmacophore for inhibiting 11β-HSD1 and offered a novel therapeutic strategy to ameliorate metabolic alterations found in obesity and diabetes.

Full text of DOI:10.1155/2017/3182129

Hindawi
Journal of Chemistry
Volume 2017, Article ID 3182129, 10 pages
https://doi.org/10.1155/2017/3182129
Research Article
Thiazolopyridines Improve Adipocyte Function by
Inhibiting 11 Beta-HSD1 Oxoreductase Activity
Thirumurugan Rathinasabapathy,1 Kimberly Marie Palatini Jackson,2,3
Yiwen Thor,3 Ayuba Sunday Buru,1 Debora Esposito,2,4 Xu Li,2,5 Mallikarjuna Rao Pichika,1
Ahmad Sazali Hamzah,6 and Slavko Komarnytsky2,3
1 Department of Pharmaceutical Chemistry, School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19,
Bukit Jalil, 57000 Kuala Lumpur, Malaysia
2Plants for Human Health Institute, North Carolina State University, North Carolina Research Campus, 600 Laureate Way,
Kannapolis, NC 28081, USA
3Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, 400 Dan Allen Drive,
Raleigh, NC 27695, USA
4Department of Animal Science, North Carolina State University, 120 Broughton Drive, Raleigh, NC 27695, USA
5Department of Plant and Microbial Biology, North Carolina State University, 100 Derieux Place, Raleigh, NC 27695, USA
6Center for Synthesis and Chemical Biology, Institute of Science, Universiti Teknologi MARA, 40450 Shah Alam,
Selangor Darul Ehsan, Malaysia
Correspondence should be addressed to Slavko Komarnytsky; komarnytsky@ncsu.edu
Received 25 December 2016; Revised 26 February 2017; Accepted 12 March 2017; Published 29 March 2017
Academic Editor: Shengmin Sang
Copyright © 2017 irumurugan Rathinasabapathy et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Background. Glucocorticoid excess has been linked to clinical observations associated with the pathophysiology of metabolic
syndrome. e intracellular glucocorticoid levels are primarily modulated by 11ꢀ-hydroxysteroid dehydrogenase type 1 (11ꢀ-HSD1)
enzyme that is highly expressed in key metabolic tissues including fat, liver, and the central nervous system. Methods. In this study
we synthesized a set of novel tetrahydrothiazolopyridine derivatives, TR-01–4, that specifically target 11ꢀ-HSD1 and studied their
ability to interfere with the glucocorticoid and lipid metabolism in the 3T3-L1 adipocytes. Results. Based on the docking model and
structure-activity relationships, tetrahydrothiazolopyridine derivatives TR-02 and TR-04 showed the highest potency against 11ꢀ-
HSD1 by dose-dependently inhibiting conversion of cortisone to cortisol (IC values of 1.8 ꢁM and 0.095 ꢁM, resp.). Incubation of
50
fat cells with 0.1–10 ꢁM TR-01–4 significantly decreased cortisone-induced lipid accumulation in adipocytes and suppressed 11ꢀ-
HSD1 mRNA expression. Observed reduction in adipocyte fat stores could be partially explained by decreased expression levels of
adipogenic markers (PPAR-ꢂ, aP2) and key enzymes of lipid metabolism, including fatty acid synthase (FAS), hormone sensitive
lipase (HSL), and lipoprotein lipase (LPL). Conclusions. e tetrahydrothiazolopyridine moiety served as an active pharmacophore
for inhibiting 11ꢀ-HSD1 and offered a novel therapeutic strategy to ameliorate metabolic alterations found in obesity and diabetes.
1. Introduction
in the developing world, including Malaysia, where 30% and
15% of adults are overweight and obese, respectively [3].
e worldwide surge in prevalence of obesity and associated
type 2 diabetes mellitus increases the need for novel preven-
tive and therapeutic strategies as stated by the World Health
Organization [1]. e metabolic disorders are no longer con-
fined to residents of affluent industrialized Western countries
(i.e., United States, where about 30% of adults are overweight
and another 30% are obese) [2] but become more widespread
High levels of circulating glucocorticoids, as seen in
Cushing syndrome, promote hyperphagia, central obesity,
and insulin resistance [4]. In the majority of obese subjects,
however, circulating glucocorticoids do not correlate with
obesity, blood glucose, or insulin levels due to increased
cortisol clearance [5] and tissue-specific metabolism by

2
Journal of Chemistry
11ꢀ-hydroxysteroid dehydrogenases (11ꢀ-HSDs) [6]. Intracel-
lular regeneration of active steroids cortisol from cortisone
(in humans) and corticosterone from 11-dehydrocortico-
2.2. Synthesis of 2-Amino-5-benzyl-4,5,6,7-tetrahydrothia-
zolo[5,4-C]pyridine (TR-01). 2-Amino-5-benzyl-4,5,6,7-tet-
rahydrothiazolo[5,4-c]pyridine was synthesized using a pre-
sterone (in rodents) is achieved through 11ꢀ-HSD type 1
(11ꢀ-HSD1), a lower-affinity NADP(H)-dependent enzyme
that acts as oxoreductase in tissues with high sensitivity
to glucocorticoids [7]. e opposing dehydrogenase activity
of 11ꢀ-HSD1 (conversion of cortisol to cortisone) becomes
evident only in cell culture and tissue homogenates or upon
purification due to decrease of ER luminal cofactor NADP(H)
generated by hexose-6-phosphate dehydrogenase [8].
viously reported procedure with some modifications [19]. 1-
Benzyl-4-piperidone (1.0 g, 5.3 mM) was dissolved in 30 ml
of cyclohexane. To this solution, pyrrolidine (0.4 g, 5.5 mM)
and p-toluenesulphonic acid (catalytic quantity) were added
and refluxed for 2.5 h using Dean-Stark trap. en, the
reaction mixture was cooled to room temperature and filtered
and the filtrate was concentrated to dryness in vacuo. e
formed residue was dissolved in 25 ml of dry methanol;
Several studies have highlighted 11ꢀ-HSD1 as a novel ther-
apeutic target in metabolic syndrome. 11ꢀ-HSD1 knockout
mice had low intracellular glucocorticoid levels and were
protected from obesity, diabetes, and dyslipidemia [9, 10].
Conversely, transgenic overexpression of 11ꢀ-HSD1 in white
adipose tissue resulted in elevated intracellular glucocorti-
coid levels, central obesity, insulin resistance, hyperglycemia,
and dyslipidemia in mice [11]. A number of structural classes
of 11ꢀ-HSD1 inhibitors have been described in the literature
[12], including the benzothiazole class of compounds which
are bioisostere to tetrahydrothiazolopyridine [13] and are
frequently reported as dipeptidyl peptidase IV inhibitors.
In preclinical studies with C57BL/6J mice fed high fat diet,
the beneficial effects of 11ꢀ-HSD1 inhibition were observed,
including reduced body weight, food intake, and fasting
glucose and insulin levels [14]. However, phase IIb clinical
trials with existing 11ꢀ-HSD1 inhibitors resulted in modest
improvements of glucose homeostasis in type 2 diabetic
subjects [15, 16]. For example, salicylates administered at
50 mg/kg/day for 2 weeks reduced 11ꢀ-HSD1 mRNA levels in
human subcutaneous fat, and a similar effect was observed in
fully differentiated human SGBS adipocytes in the dose range
of 10–100 ꢁM [17].
sulphur (S , 0.17 g, 0.66 mM) was added at once and the
8
reaction mixture was stirred for 10 min at room temperature.
en, the reaction mixture was cooled to 0∘C, cyanamide
(0.22 g, 5.3 mM) in 5 ml of dry methanol was added slowly
in dropwise manner, and the reaction was continued for 5 h
at the same temperature. Afer completion of the reaction,
the mixture was filtered and concentrated to obtain a crude
compound. TR-01 was chromatographed over silica gel with
dichloromethane/methanol (99 : 1) as eluent, yielding 51.2%.
e purity of the compound was confirmed by TLC using
CHCl /CH OH [(95 : 5); ꢃꢀ = 0.78], ESI-Q-TOF-MS: (ꢄꢅ/)
3
3
246.1 [M + H] and NMR spectra; 1H NMR (500 MHz,
DMSO-d ) ꢆ = 2.48 (t, 2H, J = 6.0 Hz), 2.70 (t, 2H, J = 6.0 Hz),
6
3.53 (s, 2H), 3.64 (s, 2H), 6.69 (s, 2H, NH ), 7.25 (dt, 1H, J
2
= 4.0 Hz), 7.34 (m, 4H); 13C NMR (125 MHz, DMSO-d ) ꢆ =
6
26.9, 50.0, 50.3, 61.2, 112.6, 127.4, 128.7, 129.2, 139.0, 143.9, 166.6.
2.3. Synthesis of N-(5-Benzyl-4,5,6,7-tetrahydrothiazolo[5,4-
c]pyridine-2-yl)-3-(4-chlorophenyl)-2-cyanoacrylamide (TR-
02). e intermediate cyanoester compound was synthesized
by Knoevenagel condensation method [20] in which 4-chlo-
robenzaldehyde (1.0 g, 7.1 mM) was dissolved in 25 ml of
methanol. tert-Butyl cyanoacetate (1.0 g, 7.1 mM) and pipe-
ridine (5 drops) were added and the mixture was refluxed
for 45 min. Afer the completion of the reaction, 100 ml of
ice-cold water was added, and the formed precipitate was
collected by filtration, recrystallized from ethanol-water to
give tert-butyl-3-(4-chlorophenyl)-2-cyanoacrylate. is es-
ter compound was dissolved in 10 ml of dichloromethane,
to which 10 ml of trifluoroacetic acid was added and stirred
at room temperature for 1 h. e reaction mixture was
evaporated in vacuo; the solid was triturated with water,
filtered, and dried to give 3-(4-chlorophenyl)-2-cyanoacrylic
acid. e purity of this compound was confirmed by TLC
using CHCl /CH OH [(90 : 10); ꢃꢀ = 0.4] and melting point
Given the public health significance of metabolic disor-
ders and the observation that past therapeutic approaches
have not met with success in addressing this issue, new strate-
gies are clearly needed. We therefore explored a novel class
of 11ꢀ-HSD1 inhibitors based on the tetrahydrothiazolopy-
ridine skeleton. Previously, several members of this class of
compounds were reported to inhibit phosphatidylinositol 3-
kinase [18]. Here, we report on the synthesis of four TR1-
4 derivatives, their inhibitory effects on 11ꢀ-HSD1 activity,
adipocyte differentiation, and expression of key adipose genes
associated with lipid metabolism and insulin resistance.
3
3
2. Materials and Methods
(m.p. 192∘C). e compound (0.5 g, 2.4 mM) was further dis-
solved in 30 ml of dichloromethane, EDCI (0.55 g, 2.9 mM),
HOBt (0.39 g, 2.9 mM), and DIPEA (0.69 g, 5.3 mM) and
stirred for 1 h at room temperature. en TR-01 (0.59 g,
2.4 mM) was added, and the stirring was continued at
same temperature for 24 h. e reaction mixture was
2.1. Chemicals and Analytical Methods. All chemicals and
solvents (anhydrous and ACS grade) were purchased from
Sigma-Aldrich (Saint Louis, MO) unless specified otherwise.
Structures and purity (>95%) of all compounds were con-
firmed by LC-MS using Bruker (Billerica, MA) Avance Elec-
trospray Ionization quadrupole time-of-flight mass detector
(ESI-Q-TOF-MS) with MS/MS (Triple quadrupole) interface,
using Agilent Technologies (Santa Clara, CA) Extend C18
reverse phase column (250 mm × 4.6 mm, 5.0 ꢁm). 1H NMR
and 13C NMR spectra were recorded on Bruker Avance 300,
500 MHz NMR stations.
washed with water, saturated NaHCO , and brine and con-
centrated by drying over anhydrous Na SO . e crude
3
2
4
compound was chromatographed over silica gel using dichlo-
romethane/methanol (95 : 5) as eluent, yielding 47.6% of
titled TR-02. e purity of the compound was confirmed by
TLC using CHCl /CH OH [(95 : 5); ꢃꢀ = 0.65] and NMR
3
3

Journal of Chemistry
3
spectra; 1H NMR (300 MHz, DMSO-d ) ꢆ = 2.62 (t, 2H, J =
filtered and concentrated to get the crude TR-04. TR-04
was further chromatographed over silica gel with dichlo-
romethane/methanol (98 : 2) as eluent, yielding 67.8%. e
purity of the compound was confirmed by TLC using CHCl /
6
5.1 Hz), 2.79 (t, 2H, J = 5.1 Hz), 3.50 (s, 2H), 3.70 (s, 2H), 7.28
(t, 1H, J = 3.9 Hz, ArH), 7.33 (m, 4H), 7.65 (d, 2H, J = 8.4 Hz),
8.00 (d, 2H, J = 8.4 Hz), 8.34 (s, 1H), 13.19 (s, 1H, NH).
3
1
CH OH [(95 : 5); ꢃꢀ = 0.72] and NMR spectra; H NMR
3
(300 MHz, DMSO-d ) ꢆ = 2.30 (s, 3H, N-CH ), 2.43 (t, 2H,
6
3
2.4. Synthesis of 2-Amino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyr-
J = 5.4 Hz), 2.59 (t, 2H, J = 5.4 Hz), 3.28 (s, 2H), 6.68 (s, 2H,
idine
(TR-03). 2-Amino-4,5,6,7-tetrahydrothia-zolo[5,4-
NH ).
2
c]pyridine was synthesized using a previously reported pro-
cedure [19]. 1-tert-Butoxycarbonyl-4-piperidone (1.0 g,
5.0 mM) was dissolved in 30 ml of cyclohexane. To this so-
lution, pyrrolidine (0.38 g, 5.3 mM) and p-toluenesulphonic
acid (catalytic quantity) were added and refluxed for 2.5 h
using Dean-Stark trap. en, the reaction was cooled to room
temperature and filtered and the filtrate was concentrated to
dryness in vacuo. e formed residue was dissolved in 25 ml
2.6. Molecular Docking Studies. e molecular docking
tool, GLIDE (Schrodinger Inc., USA; release 2014-2), was
used for 2-amino-5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-
c]pyridine (TR4) docking studies into 11ꢀ-HSD1 enzyme
binding pocket. e crystal structure of 11ꢀ-HSD1 was
obtained from the RCSB protein data bank (PDB ID: 4K1L)
[21]. e protein preparation was carried out using the default
parameters of “protein preparation wizard,” in Maestro 9.8.
e active site of the receptor was defined using the default
settings of “receptor grid generation.” e chemical struc-
ture of TR-04 was drawn using ChemBiodraw Ultra 12.0,
imported into Maestro and its 3-D structure was prepared
by using the default parameters of “Ligprep.” e lowest
energy conformation of TR-04 was selected for docking
studies. e docking was performed using default parameters
of “Ligand Docking wizard” with a standard precision (SP)
docking mode. e final evaluation is done with the glide
score and the single best pose is generated as the output for
TR-04. e docking reliability was evaluated by calculating
the root mean square deviation (RMSD) between the pose
of cocrystallized ligand and the binding pose predicted by
of dry methanol; sulphur (S , 0.16 g, 0.63 mM) was added
8
at once and the reaction mixture was stirred for 10 min at
room temperature. en, the reaction mixture was cooled
to 0∘C, cyanamide (0.21 g, 5.0 mM) in 5 ml of dry methanol
was added slowly in dropwise manner, and the reaction was
continued for 5 h at the same temperature. Afer completion
of the reaction, the mixture was filtered and concentrated to
get the crude TR-03a. TR-03a was further chromatographed
over silica gel with dichloromethane/methanol (98 : 2)
as eluent, yielding 73.4%. e purity of the compound
was confirmed by TLC using CHCl /CH OH [(95 : 5);
3
3
ꢃꢀ = 0.72], ESI-Q-TOF-MS: (ꢄꢅ/) 256.3 [M + H], and NMR
1
spectra; H NMR (500 MHz, DMSO-d ) ꢆ = 1.41 (s, 9H),
6
2.43 (t, 2H, J = 6.0 Hz), 3.56 (t, 2H, J = 5.5 Hz), 4.29 (s, 2H),
6.80 (s, 2H, NH ).
2
˚
the docking protocol. If the RMSD is below 2 A [22, 23], the
e confirmed TR-03a was then treated with 1 : 1 ratio of
dichloromethane and trifluoroacetic acid and stirred for 1 h
at room temperature. e reaction mixture was concentrated
and washed with water and diethyl ether. en the reaction
mixture was dissolved in dichloromethane, washed with
water and brine, and concentrated by drying over anhydrous
Na SO to obtain TR-03. e purity of the compound was
docking protocol was considered reliable for predicting the
binding poses of ligands.
2.7. Adipocyte Cell Culture. e mouse embryonic fibroblast
cell line 3T3-L1 (CL-173) that undergoes a preadipose to
adipose conversion and expresses the major pathways of
glucose and lipid metabolism was obtained from ATCC
(Manassas, VA). Cells were routinely passaged every 3-4 days
and maintained in high glucose DMEM containing 10% fetal
bovine serum FBS (Life Technologies, Carlsbad, CA) and
1% penicillin-streptomycin (Fisher Scientific, Pittsburg, PA)
2
4
confirmed by NMR spectra; 1H NMR (300 MHz, DMSO-d )
6
ꢆ = 2.32 (t, 2H, J = 5.4 Hz), 2.86 (t, 2H, J = 5.4 Hz), 3.59 (s,
2H), 6.63 (s, 2H, NH ).
2
at 37∘C and 5% CO , following an established protocol [24]
2.5. Synthesis of 2-Amino-5-methyl-4,5,6,7-tetrahydrothia-
zolo[5,4-c]pyridine (TR-04). 2-Amino-5-methyl-4,5,6,7-tet-
rahydrothiazolo[5,4-c]pyridine was synthesized using a pre-
viously reported procedure [19]. 1-Methyl-4-piperidone
(1.0 g, 8.84 mM) was dissolved in 30 ml of cyclohexane. To
this solution, pyrrolidine (0.66 g, 9.3 mM) and p-toluenes-
ulphonic acid (catalytic quantity) were added and refluxed for
2.5 h using Dean-Stark trap. en, the reaction was cooled
to room temperature and filtered, and the filtrate was
concentrated to dryness in vacuo. e formed residue was
2
with some modifications. Cells were subcultured into 6-well
5
dishes at a density of 1 × 10 cells/well (day 0), changed
to fresh DMEM/FBS medium once confluent (day 2), and
induced to adipose conversion by subsequently changing
to D1 medium (DMEM/FBS, 1 ꢁg/ml insulin, 500 ꢁM 3-
isobutyl-1-methylxanthine, and 0.25 ꢁM dexamethasone) on
day 4 and to D2 medium (DMEM/FBS, 1 ꢁg/ml insulin) on
day 6. Mature adipocytes were changed to fresh DMEM/FBS
medium on day 8 and used for treatments on the same day.
dissolved in 25 ml of dry methanol; sulphur (S , 0.28 g,
8
1.1 mM) was added at once and the reaction mixture was
stirred for 10 min at room temperature. en, the reaction
was cooled to 0∘C, cyanamide (0.37 g, 8.84 mM) in 5 ml
of dry methanol was added slowly in dropwise manner,
and the reaction was continued for 5 h at the same
temperature. Afer completion, the reaction mixture was
2.8. Measurement of 11ꢀ-HSD1 Activity in Adipocytes. 11ꢀ-
HSD1 bidirectional oxoreductase (conversion of cortisone to
active cortisol) and dehydrogenase (conversion of cortisol to
inactive cortisone) activities were measured by incubating
3T3-L1 preadipocytes (days 0 and 4) or mature adipocytes
(day 8) in medium with either 1 ꢁM cortisone or cortisol,

4
Journal of Chemistry
respectively, for 20 min at 37∘C. Afer extraction with ethyl
acetate and evaporation in vacuo, glucocorticoid concen-
trations were measured in triplicate by LC-MS in posi-
tive mode (Agilent G1312B-1200 series Infinity Quaternary
HPLC system coupled with Agilent 6530A Accurate-Mass
Quadrupole Time-of-Flight MS with Agilent Jet Stream
Technologies), following the manufacturer’s protocol on an
ABI GeneAMP 9700 (Life Technologies).
e resulting cDNA was amplified in duplicate by real-
time quantitative PCR (qPCR) using SYBR green PCR
Master Mix (Life Technologies). To avoid interference due
to genomic DNA contamination, only intron-overlapping
source). Dry samples were reconstituted with 500 ꢁl 80%
primers were selected using the Primer Express version 2.0
sofware (Applied Biosystems, Foster City, CA) as follows:
methanol, centrifuged, and filtered, and 5ꢁl was injected onto
a Agilent ZORBAX Eclipse Plus C18 column (3 × 100 mm,
1.8 ꢁm). A gradient from 30% acetonitrile in 0.1% formic
acid to 90% acetonitrile in 0.1% formic acid was used for
HPLC separation. Mass data were acquired with the following
parameters: drying gas temperature 300∘C, drying gas flow
7 L/min, nebulizer pressure 40 psi, sheath gas temperature
350∘C, sheath gas flow 10 L/min, capillary voltage 3500 V,
nozzle voltage 500 V, fragmentor voltage 150 V, skimmer
voltage 65 V, and octopole RF peak voltage 750 V. Standard
curves for compounds of interest were obtained in a linear
dynamic range of 10–50,000 pg and used for quantitative
analysis. Peak area was determined by Agilent MassHunter
Qualitative Analysis (version B.05.00) sofware. e percent
ꢀ-actin (housekeeping gene), forward primer: 5ꢁ-AAC CGT
GAA AAG ATG ACC CAG AT-3ꢁ, reverse primer: 5ꢁ-CAC
AGC CTG GAT GGC TAC GT-3ꢁ; adiponectin (AdiQ),
forward primer: 5ꢁ-TGT TCC TCT TAA TCC TGC CCA-3ꢁ,
reverse primer: 5ꢁ-CCA ACC TGC ACA AGT TCC CTT-
3ꢁ; leptin (Lep), forward primer: 5ꢁ-GAG ACC CCT GTG
TCG GTT C-3ꢁ, reverse primer: 5ꢁ-CTG CGT GTG TGA
AAT GTC ATT-3ꢁ; glucose transporter type 4 (GLUT4),
forward primer: 5ꢁ-CAG CTC TCA GGC ATC AAT-3ꢁ,
reverse primer: 5ꢁ-TCT ACT AAG AGC ACC GAG-3ꢁ; fatty
acid synthase (FAS), forward primer: 5ꢁ-GGC ATC ATT
GGG CAC TCC TT-3ꢁ, reverse primer: 5ꢁ-GCT GCA AGC
ACA GCC TCT CT-3ꢁ; hormone sensitive lipase (HSL),
of conversion ratio was calculated as 100 − [(initial concen-
tration − final concentration)/(initial concentration)] ∗ 100%
ratio.
forward primer: 5ꢁ-CCA AGT GTG TGA GCG CCT ATT-
3ꢁ, reverse primer: 5ꢁ-CAC GCC CAA TGC CTT CTG-
3ꢁ; lipoprotein lipase (LPL), forward primer: 5ꢁ-CTG AAA
GTG AGA ACA TTC CCT TCA-3ꢁ, reverse primer: 5ꢁ-CCG
TR-01–4 for cell culture studies was dissolved in DMSO as
1000x stocks and stored at −20∘C. When indicated, 11ꢀ-HSD1
activity was inhibited by exposure of cells for 2 h to vehicle
control (0.1% DMSO) or the appropriate TR-01–4 in the dose
range of 0.1–100 ꢁM. Cell viability was estimated using the
MTT assay [25] by absorbance read at 570 nm on a Synergy
H1 microplate spectrophotometer (BioTek, Sunnyvale, CA).
TGT AAA TCA AGA AGG AGT AGG TT-3ꢁ; peroxisome
proliferator activated receptor (PPAR-ꢂ), forward primer: 5ꢁ-
GCC CTT TGG TGA CTT TAT GGA-3ꢁ, reverse primer:
5ꢁ-GCA GCA GGT TGT CTT GGA TG-3ꢁ; and fatty acid
binding protein 4 (aP2), forward primer: 5ꢁ- TCA CCT GGA
AGA CAG CTC CT-3ꢁ, reverse primer: 5ꢁ- AAT CCC CAT
TTA CGC TGA TGꢁ. qPCR amplifications were performed
on an ABI 7500 Fast real-time PCR (Life Technologies). Cycle
conditions were 50∘C for 2 min, 95∘C for 10 min, and then 40
cycles at 95∘C for 15 s and 60∘C for 1 min. mRNA expression
was analyzed using the ΔΔCT method [26] and normalized
with respect to the expression of the ꢀ-actin using ABI
7500 Fast System SDS Sofware v1.3.0 (Life Technologies).
Amplification of specific transcripts was further confirmed by
obtaining dissociation (melting) curve profiles with 1 cycle of
1 min at 95∘C, 30 s at 55∘C, and 30 s at 95∘C.
2.9. Adipocyte Differentiation and Lipid Accumulation. e
3T3-L1 preadipocytes were induced to adipose conversion as
described above (dexamethasone was omitted) and treated
with 1 ꢁM cortisone and 0–10 ꢁM TR-01–4 throughout dif-
ferentiation. Cells were stained with Oil red O on day 8 to
visualize differentiated adipocytes and quantify lipid accu-
mulation following an established protocol [24] with some
modifications. Afer medium removal, cells were washed
twice with ice-cold PBS and fixed in 10% neutral buffered
formalin at room temperature for 10 min. Cells were then
washed twice with ice-cold PBS and stained with 1% Oil
red O in isopropanol, diluted 3 : 2 in PBS, for 1 h at room
temperature. Following two additional PBS washes, fresh PBS
was added to cover the cell surface and prevent dehydration.
Cells were photographed at 40x magnification using EVOS
FL Cell Imaging System (ermo Fisher Scientific, Waltham,
MA). Lipid-bound Oil red O stain was eluted by 30 min
incubation with isopropanol and quantified by absorbance
read at 520 nm on a Synergy H1 spectrophotometer (BioTek).
2.11. Statistical Analysis. Statistical analyses were performed
using Prism 6.0 (GraphPad Sofware, San Diego, CA). Data
were reported as means
SEM and analyzed by one-
way ANOVA or two-tailed Student’s ꢇ-test, as appropriate.
ANOVA post hoc analyses of differences between individual
experimental groups were made using Dunnett’s multiple
comparison tests. Significance was set at ꢈ < 0.05.
3. Results
3.1. Synthesis of Tetrahydrothiazolopyridine Derivatives and
Molecular Docking Simulations. e molecular docking stud-
ies were performed to understand the molecular interactions
between TR-04 and 11ꢀ-HSD1 enzyme. First, the reliability of
the docking protocol was determined by RMSD calculation
between the poses of cocrystallized ligand and its docked
conformation. e superimposition of cocrystallized ligand
2.10. RNA Extraction and Gene Expression by qPCR. e total
RNA was isolated from cells using TRIzol reagent (Life Tech-
nologies) following the manufacturer’s instructions. RNA was
quantified using Synergy H1/Take 3 plate setup (BioTek). e
cDNAs were synthesized using 2 ꢁg of RNA for each sample
using high-capacity cDNA Reverse Transcription kit (Life

Journal of Chemistry
5
THR
124
ALA
NDP
ALA
302
223
226
THR
VAL
231
222
SER
125
LEU
217
ILE
VAL
227
121
O
S
NH
TYR
LEU
126
183
N
O
VAL
180
O
SER
170
TYR
ALA
172
177
LEU
LEU
171
215
GLY
216
(a)
(b)
THR
124
THR
222
ASN
123
NH2
NDP
302
VAL
N
ILE
180
S
121
LEU
TYR
126
183
N
ALA
ALA
223
226
ILE
VAL
218
227
LEU
217
(c)
(d)
Figure 1: Binding modes of native ligand and TR-04 in the X-ray crystal structure of 11ꢀ-HSD1 (PDB ID: 4K1L). (a) Superimposition of the
poses of cocrystallized native ligand (shown in yellow colored tube representation) with its binding conformation (shown in maroon colored
tube representation). (b) e interactions between inbound ligand and amino acid residues of binding pocket (4K1L). (c) e pose of TR-
04 (shown in ball-and-stick representation). (d) e interactions between TR-04 and amino acid residues of binding pocket (4K1L). Green
colored circles indicate hydrophobic amino acid residues; blue colored circles indicate polar amino acid residues.
and its docked pose along with its interactions with receptor
is shown in Figure 1. e RMSD was found to be 0.158 indi-
cating the docking protocol used in this study is appropriate
for reliable prediction of docking pose of TR-04. Its molecular
interactions with amino acid residues of the binding pocket
(PDB ID: 4K1L) were similar to those of inbound ligand.
e binding pose of TR-04 and its molecular interactions
with amino acid residues of binding pocket, facilitating its

6
Journal of Chemistry
O
Cl
N
S
N
S
NH2
NH
N
N
N
1
2
4
N
S
N
S
NH2
NH2
N
N
H3C
H
3
Figure 2: Chemical structure of tetrahydrothiazolo[5,4-c]pyridine-2-amine (TR-01) and its derivatives (TR-02–4) used in this study.
Bidirectional 11ꢀ-HSD1 activity
binding (docking score = −5.924 Kcal/mol; glide score =
−6.398 Kcal/mol), is shown in Figure 1. e –NH2 group of
TR-04 forms hydrogen bonding with THR-124 while tetrahy-
drothiazolopyridine ring occupies the hydrophobic pocket
containing ILE-121, THR-183, ALA-223, ILE-218, LEU-217,
VAL-227, ALA-226, LEU-126, and VAL-180. e docking
results demonstrated possible broad and tight interactions
between TR-04 and amino acid residues of 4K1L binding
pocket and this may result in potent activity of TR-04.
e docking studies provided insights into further modi-
fication of TR-04 to develop analogues with more potent
activity. Based on these observations, we synthesized N-
unsubstituted and N-substituted tetrahydrothiazolopyridine
derivatives TR-01–4 (Figure 2). e substitution is based on
the requirement of secondary amine (NH) group and/or
primary amine (NH2) group for preserving biological activity
as well as pharmacokinetic stability of these analogues.
e detailed synthetic routes to each compound and their
characterization are summarized in Supplementary Material,
available online at https://doi.org/10.1155/2017/3182129.
∗∗
60
50
40
30
20
10
0
∗
4
∗
8
0
8
Days of differentiation
Oxoreductase
Dehydrogenase
0
4
Figure 3: Bidirectional oxoreductase (conversion of cortisone
to active cortisol, black bars) and dehydrogenase (conversion of
cortisol to inactive cortisone, open bars) activity of the 11ꢀ-HSD1
enzyme in 3T3-L1 adipocytes. Cells were grown to confluence and
differentiated into adipocytes by day 8 of the treatment. Cells
were incubated in DMEM containing cortisone or cortisol (1 ꢁM)
for 20 min and the respective metabolites/conversion ratios were
3.2. 11ꢀ-HSD1 Activity in 3T3-L1 Adipocytes. Undifferenti-
ated confluent 3T3-L1 cells showed a small amount of the
11ꢀ-HSD1 oxoreductase activity (8% of cortisone to corti-
sol conversion in 2 h), while the 11ꢀ-HSD1 dehydrogenase
activity (i.e., cortisol to cortisone conversion) was negligible
in preadipocytes. As adipocyte differentiation progressed
from day 0 to day 4, we observed a steady increase in
11ꢀ-HSD1 activity. Near-complete adipogenic differentiation
(day 8) was associated with a significant increase in the
oxoreductase activity with up to 48% cortisone to cortisol
conversion afer mature adipocytes were incubated with
1 ꢁM of steroid for 2 h (Figure 3, black bars). e 11ꢀ-HSD1
dehydrogenase activity remained relatively low throughout
measured by LC-MS. Data are the mean SEM (ꢉ = 3). ∗ < 0.05
ꢈ
and ∗∗ < 0.01 versus vehicle by Dunnett’s test subsequent to one-
ꢈ
way ANOVA.
3.3. Tetrahydrothiazolopyridine Derivatives Inhibit 11ꢀ-HSD1.
11ꢀ-HSD1 oxoreductase assays in mature adipocyte cells
supplemented with 1 ꢁM cortisone for 2 h showed significant
reduction of the oxoreductase activity following incubation
the differentiation process and reached maximum 6% cortisol
with 0.1–100 ꢁM of TR-01–4. Parent pharmacophore TR-01
to cortisone oxidation on day 8 following the induction of
showed moderate efficacy at inhibiting 11ꢀ-HSD1 oxoreduc-
adipogenesis (Figure 3, open bars). As 11ꢀ-HSD1 dehydro-
tase in a dose-dependent manner (Figure 4(a)). Addition of
genase activity was minimal in these cells, the next set of
experiments focused on 11ꢀ-HSD1 oxoreductase activity as
the main measure of thiazolopyridine-induced inhibition of
11ꢀ-HSD1.
benzyl group at 5th position and 4-chloro-ꢊ-cinnamoyl moi-
ety at 2nd position in TR-02 increased 11ꢀ-HSD1 potency as
compared to the original molecule TR-01. Unsubstituted TR-
03, on the other hand, exhibited decreased inhibitory activity

Journal of Chemistry
7
100
120
100
80
60
40
20
0
75
50
25
0
∗
−7.0
−6.5
−6.0
−5.5
−5.0
−4.5
−4.0
0.0
0.1
1.0
10.0
100.0
Log (M)
Tr-04, ꢁM
TR-01
TR-02
TR-03
TR-04
(a)
(b)
Figure 4: Tetrahydrothiazolopyridine derivatives TR-01–4 inhibited 11ꢀ-HSD1 oxoreductase activity in the mature 3T3-L1 adipocytes. Cells
were treated for 2 h with 0–100 ꢁM of the test compounds and incubated in DMEM containing cortisone (1 ꢁM) for 20 min. e resulting
∗
ꢈ
metabolite/conversion ratios were measured by LC-MS. Data are the mean SEM (ꢉ = 3).
< 0.05 versus vehicle by Dunnett’s test
subsequent to one-way ANOVA.
for 11ꢀ-HSD1. In order to understand the disparity between
TR-02 and TR-03, a methyl analogue TR-04 was synthesized.
Treatment with TR-04 resulted in a total inhibition of 11ꢀ-
HSD1 oxoreductase activity when tested at the concentrations
adipocytes (Figure 5(d) and 5(e)). Size and degree of lipid
droplets accumulation in the presence of the different con-
centrations of TR-04 (0–10 ꢁM) indicated dose-dependent
suppression of adipogenesis in response to treatment with
thiazolopyridine derivatives (Figure 5(f)).
of >10 ꢁM with a corresponding IC value of 0.095 ꢁM. No
50
cytotoxicity was observed in MTT assay at concentrations
lower than 10 ꢁM for TR-04 (Figure 4(b)).
3.5. Gene Expression of Adipogenic and Insulin Sensitivity
Markers. 3T3-L1 cells differentiated for 8 days in the presence
of cortisone showed increased expression of markers of lipid
metabolism (FAS, HSL, and LPL), insulin signaling (GLUT4),
adipocyte function (Lep), and differentiation (PPAR-ꢂ, aP2);
however, the levels of adiponectin (AdiQ) cytokine that
regulates energy metabolism and insulin sensitivity were
drastically reduced. Coincubation of 3T3-L1 cells with 100 nM
TR-04 normalized mRNA levels of biomarkers of lipid
metabolism and adiponectin (1.6-fold versus 0.2-fold in
cortisone-treated cells, ꢈ < 0.01) but had little effect on
expression levels of insulin-inducible GLUT4 transporter
(Figure 6). Reduced mRNA levels of HSL and LPL were
also observed. is is in agreement with previous studies
that showed that HSL knockout mice had lower weight
gains and adiposity, likely due to compensatory decrease in
the reesterification of fatty acids that results in decreased
resynthesis of TAG and increased liberation of fatty acids
to the vasculature [27]. LPL expression followed the same
pattern, although its connection to adipogenesis was not clear
and warrants further investigation.
3.4. Adipogenesis in 3T3-L1 Cells Incubated with Tetrahy-
drothiazolopyridine Derivatives. 3T3-L1 cells differentiated
for 8 days with 1 ꢁM cortisone (instead of 0.25 ꢁM dex-
amethasone used in the standard adipogenesis protocols)
showed increased 11ꢀ-HSD1 reductase activity of 22.4
9.4% cortisone to cortisol conversion, a 2-fold decrease over
the standard differentiation protocol (Figure 5(a)). Similarly,
cortisone-induced adipocytes expressed higher levels of 11ꢀ-
HSD1 mRNA (11.1-fold versus control, ꢈ < 0.01) that also
did not reach 11ꢀ-HSD1 mRNA levels observed afer dexam-
ethasone-stimulated differentiation (20.2-fold versus control,
ꢈ < 0.001, Figure 5(b)). e change in adipogenesis and lipid
accumulation was quantified through staining the cells with
Oil red O afer 8 days of differentiation. A marked increase
in the number of red-stained cells was observed in samples
differentiated with dexamethasone (39.0-fold versus control,
ꢈ < 0.001) or cortisone (13.3-fold versus control, ꢈ < 0.01)
(Figure 5(c)).
Coincubation with 0.1–10 ꢁM compounds TR-01–4 abol-
ished these effects to a various extent. 3T3-L1 adipocytes
differentiated with cortisone and TR-04 at doses as low as
4. Discussion
100 nM had significantly lower 11ꢀ-HSD1 mRNA levels (0.6-
fold versus cortisone alone, ꢈ < 0.05, Figure 5(d)) and lipid
content (54.8% of cortisone alone, ꢈ < 0.05, Figure 5(e)). At
highest concentration tested, compound 4 nearly abolished
11ꢀ-HSD1 oxoreductase activity (Figure 5(d)) and suppressed
adipogenesis and lipid accumulation in cortisone-stimulated
e present study demonstrated a potent inhibition of the
11ꢀ-HSD1 oxoreductase activity (conversion of inactive corti-
sone to active cortisol) by tetrahydrothiazolopyridine deriva-
tives in the 3T3-L1 adipocyte cell culture. 11ꢀ-HSD1 acts as
a tissue-specific regulator of cortisol exposure in obesity and

8
Journal of Chemistry
Oil red O stain
11ꢀ-HSD1 mRNA
11ꢀ-HSD1 oxoreductase
∗∗∗
55
50
45
40
35
30
25
20
15
10
5
25
20
15
10
5
45
40
35
30
25
20
15
10
5
∗∗∗
∗∗∗
∗∗∗
∗∗
∗∗
0
0
0
C
Dex
Treatments
(a)
Csn
C
Dex
Treatments
(b)
Csn
C
Dex
Treatments
Csn
(c)
Oil red O stain
11ꢀ-HSD1 mRNA
140
120
100
80
1.4
1.2
1.0
0.8
0.6
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗∗
∗∗
60
∗∗
∗∗
40
0.4
0.2
0.0
∗∗
∗∗
20
0
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10
Concentration (ꢁM)
Concentration (ꢁM)
TR-03
TR-04
TR-03
TR-04
TR-01
TR-02
TR-01
TR-02
(d)
(e)
Control
TR-04 (0.1ꢁM)
TR-04 (1ꢁM)
TR-04 (10 ꢁM)
(f)
Figure 5: Tetrahydrothiazolopyridine derivatives TR-01–4 reduced adipogenesis in the 3T3-L1 adipocytes in a dose-dependent manner.
Adipocyte differentiation in the presence of 1 ꢁM cortisone (Csn) was half less efficient than standard 0.25 ꢁM dexamethasone (Dex) induction
as evident from induction of the (a) 11ꢀ-HSD1 activity, (b) 11ꢀ-HSD1 expression, or (c) lipid accumulation. Dosing of cortisone-treated
adipocytes with 0–10 ꢁM of TR-01–4 further decreased (d) lipid accumulation as quantified with Oil red O staining and (e) 11ꢀ-HSD1 mRNA
levels as measured by qPCR. (f) Oil red O staining of adipocytes differentiated with different concentrations of TR-04 for 8 days. Data are the
∗∗
∗∗∗
mean SEM (ꢉ = 3), ∗ < 0.05,
< 0.01, and
< 0.001 versus vehicle control by Dunnett’s test subsequent to one-way ANOVA.
ꢈ
ꢈ
ꢈ

Journal of Chemistry
9
TR-04 (100 nM)
adipogenesis across all adipose tissue depots, or this effect
was limited to the cutaneous lineages similar to the 3T3-
L1 adipocytes used in this study. Further preclinical studies
are needed to explore this regulation and its application to
metabolic phenotypes. However, pharmacological activity of
TR-04 in vitro compared favorably to salicylates that reduced
11ꢀ-HSD1 mRNA levels in fully differentiated human SGBS
adipocytes in the dose range of 10–100 ꢁM [17].
8
7
6
5
4
3
We also demonstrated that reduced adipogenesis data
was consistent with changes in expression of key adipogenic
genes associated with lipid metabolism and insulin resistance.
Simultaneous increase in adiponectin and decrease in leptin
expression suggested that treatment with TR-02 and TR-04
might be effective at improving regulation of insulin resis-
tance and energy homeostasis in obese subjects [23]. At the
same time, the observed reduction in the levels of FAS, HSL,
and LPL in tetrahydrothiazolopyridine-treated adipocytes
indicated reduced deposition of fatty acids in adipose cells.
is finding was in a total agreement with Oil red O staining
that quantifies the presence of fat or lipids in adipocytes.
∗∗
∗∗
2
1
0
∗
∗
∗
∗∗
Treatments
AdiQ
HSL
LPL
PPARg
aP2
Lept
Glut4
FAS
Figure 6: Effect of tetrahydrothiazolopyridine derivative TR-04
on the expression levels of adipocyte genes associated with lipid
metabolism and insulin resistance. Adipocytes were differentiated in
the presence of 1 ꢁM cortisone and 100 nM of TR-04 and extracted
with TRIzol for mRNA measurements by qPCR. Data are the mean
5. Conclusions
Taken together, our findings suggested that tetrahydrothia-
zolopyridine derivatives TR-02 and TR-04 are the strong 11ꢀ-
HSD1 inhibitors and attractive preclinical candidates for the
treatment of obesity and type 2 diabetes mellitus.
SEM (ꢉ = 3), normalized with respect to the expression of the
∗∗
ꢀ-actin of undifferentiated control cells. ∗ < 0.05 and
< 0.01
ꢈ
ꢈ
versus vehicle control by two-tailed Student’s ꢇ-test.
Conflicts of Interest
e authors declare that there are no conflicts of interest
regarding the publication of this paper.
type 2 diabetes and presents an attractive therapeutic strategy
to alleviate multiple complications in patients with metabolic
syndrome and excess weight. As expected, undifferentiated
3T3-L1 preadipocytes showed low levels of both 11ꢀ-HSD1
oxoreductase and dehydrogenase activity due to bidirectional
nature of the 11ꢀ-HSD1 enzyme. 11ꢀ-HSD1 is a lower-affinity
NADP(H)-dependent enzyme that acts as oxoreductase that
regenerates active glucocorticoids from their inactive keto-
glucocorticoid precursors in key metabolic tissues [7]. e
opposing dehydrogenase activity of 11ꢀ-HSD1 (conversion of
cortisol to cortisone) becomes evident only in cell culture
and tissue homogenates or upon purification due to decrease
of ER luminal cofactor NADP(H) generated by hexose-
6-phosphate dehydrogenase [8]. Mature 3T3-L1 adipocytes
exhibited an expected switch predominantly to 11ꢀ-HSD1
oxoreductase activity [28] by day 8 of differentiation.
Acknowledgments
is work was supported in part by Exploratory Research
Grant Scheme, Ministry of Education, Malaysia, Grant
ERGS/1/2013/SKK02/IMU/03/1; International Medical Uni-
versity Seed Grant IMU 263/2012; NCSU Office of Inter-
national Affairs Grant 2013-1705; and the Cell Culture and
Phenotyping Core of the Plants for Human Health Institute,
North Carolina State University. e authors would like to
thank Mr. Selvam Paramasivan (India) for helping with NMR
and mass spectral data.
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Volume 2014
Volume 2014
Volume 2014
Volume 2014
Volume 2014