Discovery of HSD-621 as a potential agent for the treatment of type 2 diabetes

Article abstract of DOI:10.1021/ml300352x

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the conversion of inactive glucocorticoid cortisone to its active form, cortisol. The glucocorticoid receptor (GR) signaling pathway has been linked to the pathophysiology of diabetes and metabolic syndrome. Herein, the structure-activity relationship of a series of piperazine sulfonamide-based 11β-HSD1 inhibitors is described. (R)-3,3,3-Trifluoro-2-(5-(((R)-4-(4- fluoro-2-(trifluoromethyl)phenyl)-2-methylpiperazin-1-yl)sulfonyl)thiophen-2-yl) -2-hydroxypropanamide 18a (HSD-621) was identified as a potent and selective 11β-HSD1 inhibitor and was ultimately selected as a clinical development candidate. HSD-621 has an attractive overall pharmaceutical profile and demonstrates good oral bioavailability in mouse, rat, and dog. When orally dosed in C57/BL6 diet-induced obesity (DIO) mice, HSD-621 was efficacious and showed a significant reduction in both fed and fasting glucose and insulin levels. Furthermore, HSD-621 was well tolerated in drug safety assessment studies.

Full text of DOI:10.1021/ml300352x

Letter
pubs.acs.org/acsmedchemlett
Discovery of HSD-621 as a Potential Agent for the Treatment of Type
2 Diabetes
Zhao-Kui Wan,* Eva Chenail, Huan-Qiu Li, Manus Ipek, Jason Xiang, Vipin Suri, Seung Hahm, Joel Bard,
Kristine Svenson, Xin Xu, Xianbin Tian, Mengmeng Wang, Xiangping Li, Christian E. Johnson,
Ariful Qadri, Darrell Panza, Mylene Perreault, Tarek S. Mansour, James F. Tobin, and Eddine Saiah
Worldwide Research and Development, Pfizer Inc., 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, United States
S
* Supporting Information
ABSTRACT: 11β-Hydroxysteroid dehydrogenase type 1 (11β-
HSD1) catalyzes the conversion of inactive glucocorticoid cortisone
to its active form, cortisol. The glucocorticoid receptor (GR)
signaling pathway has been linked to the pathophysiology of
diabetes and metabolic syndrome. Herein, the structure−activity
relationship of a series of piperazine sulfonamide-based 11β-HSD1
inhibitors is described. (R)-3,3,3-Trifluoro-2-(5-(((R)-4-(4-fluoro-
2-(trifluoromethyl)phenyl)-2-methylpiperazin-1-yl)sulfonyl)thiophen-2-yl)-2-hydroxypropanamide 18a (HSD-621) was identi-
fied as a potent and selective 11β-HSD1 inhibitor and was ultimately selected as a clinical development candidate. HSD-621 has
an attractive overall pharmaceutical profile and demonstrates good oral bioavailability in mouse, rat, and dog. When orally dosed
in C57/BL6 diet-induced obesity (DIO) mice, HSD-621 was efficacious and showed a significant reduction in both fed and
fasting glucose and insulin levels. Furthermore, HSD-621 was well tolerated in drug safety assessment studies.
KEYWORDS: 11β-hydroxysteroid dehydrogenase type I (HSD1), diet-induced obesity, type II diabetes, piperazine sufonamides
lucocorticoid hormones are key regulators of a wide range
of biological processes such as the control of immune and
11β-HSD1 activity in adipose tissue also correlates positively
with body mass index (BMI), fat percentage, and fasting
glucose and insulin levels in humans. Recent studies have
demonstrated that whole-body 11β-HSD1 activity is elevated in
obese men with type 2 diabetes, whereas liver 11β-HSD1
activity is relatively unchanged, suggesting that disease
suppression via 11β-HSD1 inhibition is likely to be more
effective in obese patients with type 2 diabetes.15
Several classes of potent and selective 11β-HSD1 inhibitors
have been reported in the literature.16−26 Some inhibitors,
including our own,16,17 have displayed in vivo efficacy in animal
models related to diabetes.27−31 Data from phase II clinical
trials for 11β-HSD1 inhibitor INCB-173932 showed improved
hepatic and peripheral insulin sensitivity and a reduction in
fasting plasma glucose and cholesterol observed after 28 days of
treatment in type II diabetic patients. Decreased triglyceride
levels and improvement in blood pressure were also reported.
We previously disclosed a series of compounds exemplified
by 1 and 2, which displayed potent and selective inhibition
against both human and rodent 11β-HSD1 in vitro. The
compounds also showed oral efficacy in the cortisone-induced
hyperinsulinemia rat model16 and in the diet-induced obesity
(DIO) mouse model17 (Figure 1). In the continuation of our
11β-HSD1 program, we are reporting on the discovery of our
clinical development candidate 18a [HSD-621, (R)-3,3,3-
G
stress responses as well as modulation of energy metabolism.
Glucocorticoids (cortisol in humans and corticosterone in mice
and rats) stimulate hepatic glucose production and suppress
insulin-mediated glucose uptake in peripheral tissues (i.e.,
adipose and muscle). 11β-Hydroxysteroid dehydrogenase type
I (11β-HSD1),1−7 a reduced β-nicotinamide adenine dinucleo-
tide phosphate (NADPH)-dependent enzyme, predominantly
acts as a reductase in vivo, converting inactive, nonreceptor
binding cortisone to active, receptor-binding cortisol in tissues
such as liver, adipose, vasculature, brain, and macrophages.8
The enzyme is a tetramer consisting of two dimers each with an
independent active site.8 It has been proposed that 11β-HSD1
reductase activity exists predominantly in metabolic tissues
because of the increased ratio of NADPH to β-nicotinamide
adenine dinucleotide phosphate (NADP) within the endoplas-
mic reticulum (ER) lumen and/or through direct interactions
with NADPH-generating hexose-6-phosphate dehydrogenase
(H6PDH), an enzyme that has been associated with cortisone
reductase deficiency in humans.9 Cortisone itself is generated
by the action of 11β-hydroxysteroid dehydrogenase type 2
(11β-HSD2) with cortisol using NADP as a cofactor. 11β-
HSD1 levels are highest in liver and adipose tissues as well as in
the central nervous system, whereas 11β-HSD2 is mainly
expressed in the kidney and colon.10
The rationale for 11β-HSD1 as a therapeutic target for the
treatment of diabetes and the metabolic syndrome is based on
data from tissue-specific overexpression in transgenic mice11,12
and mice 11β-HSD knockout experiments.13,14 In addition,
Received: October 18, 2012
Accepted: November 23, 2012
Published: November 23, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
the enzyme monomer to which the inhibitor is bound. Key
residues in this pocket include Y177 and P178.
We observed two binding modes with our 11β-HSD1
inhibitors: the “normal” binding mode and the “reversed”
binding mode, both of which have a conserved hydrogen bond
between one of the sulfonamide oxygens and the NH of A172
(Figure 2). In the “normal” binding mode, the trifluoromethyl
group on the phenyl ring (Ar2) of 1 and 2, common to all
analogues in the piperazine phenyl sulfonamide series, sits in
the lipophilic pocket A. In the “reverse” binding mode, the
inhibitors rotate 180° centered at the conserved hydrogen bond
and the phenyl ring Ar2 now occupies pocket B instead of
pocket A.
In the piperazine phenyl sulfonamide series, we had observed
that meta- versus para-substitution on the phenyl ring Ar1 of 1
had an effect on potency as well as ex vivo activity (Figure 1).16
We reasoned that a five-membered ring might offer an
intermediate positioning of substituents that the six-membered
phenyl sulfonamide scaffold could not provide. Guided by
structural information, a number of potent and selective
piperazine thiophene sulfonamides were designed and synthe-
sized (Scheme 1 and Table 1).
Figure 1. Potent and selective piperazine sulfonamides as 11β-HSD1
inhibitors.
trifluoro-2-(5-((R)-4-(4-fluoro-2-(trifluoromethyl)-phenyl)-2-
methylpiperazin-1-ylsulfonyl)thiophen-2-yl)-2-hydroxypropana-
mide]. Selected characterization and preclinical data for HSD-
621 will be disclosed.
On the basis of the profile of our earlier compounds,16,17 we
imposed additional requirements when selecting compounds to
move forward in our screening cascade. Specifically, we were
interested in compounds with the following profile: (i) potent
cellular activity, (ii) good microsomal stability, and (iii) potent
adipose ex vivo activity. Among these considerations, adipose
ex vivo activity became the most significant driver in terms of
compound selection for in vivo efficacy studies. This stemmed
from the earlier observation of poor in vivo results from
compounds possessing high liver ex vivo activity alone.17 This
finding is in line with the report that liver-specific over-
expression of 11β-HSD1 in transgenic mice causes insulin
resistance without obesity,33 possibly indicating the importance
of targeting 11β-HSD1 outside of the liver, particularly in the
adipose.15
a
Scheme 1
Our earlier work taught us that the active site of 11β-HSD1
provides opportunities for both electrostatic and van der Waals
interactions (e.g., 116 and 217). Among these interactions was a
hydrogen bond between one of the sulfonamide oxygens with
the backbone nitrogen of A172 (Figure 2). This was conserved
a
Reagents and conditions: (a) n-BuLi, CF3(CO)Me, THF (chiral
separation for 9 to give 9a and 9b). (b) Lithium diisopropylamide
(LDA), CF3(CO)Me. (c) KOH, t-BuOH, then chiral separation to
give 10a and 10b. Note: 3, 4, and 6 were synthesized analogous to 5.17
We began by replacing the left-hand side phenyl group Ar1 in
compounds 1 and 2 with five-membered rings as exemplified by
imidazole and triazole analogues 7 and 8 (Table 1 and Scheme
1). These compounds bear a lipophilic trifluoromethyl propan-
2-ol moiety, an optimally refined group in our first clinical
compound HSD-016.34 Both compounds were easily synthe-
sized by halogen/lithium exchange at the C2 positions followed
by quenching by trifluoroacetone. Encouragingly, both
compounds were found to be active against both human and
mouse 11β-HSD1 enzymes with moderate fat ex vivo activity of
33 and 38%, respectively, 5 h after oral dosing at 5 mg/kg.
Continuing efforts led to the discovery of the thiophene
analogue 9 (Table 1). In our cell-based assays, 9 was very
potent against human and mouse 11β-HSD1 enzymes (15 and
10 nM, respectively) and displayed reasonable stability against
both human liver microsomes (HLM, >30 min) and mouse
liver microsomes (MLM, 17 min). More encouragingly, a 44%
inhibition of 11β-HSD1 in epididymal fat (epi fat) was
observed in the ex vivo assay. Interestingly, single diasteromers
9a and 9b had distinct biological profiles, in which the S-isomer
9a demonstrated much higher inhibition than the R-isomer 9b
(80 vs 5%, Table 1).
Figure 2. “Normal” and “reverse” binding modes of piperazine
sulfonamides as 11β-HSD1 inhibitors in human 11β-HSD1 enzyme.
among all compounds in our series. The overall binding site can
be described as two pockets, one on either side of this
conserved hydrogen bond. Pocket A contains the active site of
the enzyme as well as NADP. It is a lipophilic region formed by
the side chains of residues I121, T124, L126, V180, and Y183.
Some inhibitors were shown to reach outside the hydrophobic
pocket and interact with the cofactor NADP. The second side
of the binding pocket (pocket B) holds the remainder of the
steroid substrate and is formed, in part, by the dimer partner of
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(Scheme 2 and Table 2). This substitution was predicted by
molecular modeling to yield compounds with hydrogen bond
Table 1. Inhibitory Activity, Microsomal Stability, and
Epididymal Fat ex Vivo Inhibition of Selected Compounds
a
Scheme 2
a
Reagents and conditions: (a) n-BuLi, CF3(CO)CO2Me. (b) NH3,
MeOH.
Table 2. Inhibitory Activity, Microsomal Stability, and
Epididymal Fat ex Vivo Inhibition of Compounds Targeting
the “Reverse” Binding Mode
a
The assay was performed in duplicate; the compounds were tested in
11-point 2-fold dilutions, and the standard error of IC50 was <10%; the
b
lower threshold of this assay was 10 nM. 10% (v/v) human serum
c
was added to the cell culture media. po dosed at 10 mg/kg; results
d
obtained 5 h after dosing. Not significant.
It is known that engaging the cofactor NADP is capable of
modulating the binding efficiency.35 Molecular modeling
suggested that a favorable hydrogen bond could exist between
a carboxamide group off the phenyl ring (Ar2) and the
phosphate backbone of the NADP in the “normal” bind mode,
and this was borne out subsequently in crystal structures of
related compounds (data not shown). Thus, the fluoro group at
the phenyl C4 position in compound 9 was replaced by a
carboxamide group to give 11a and 11b after chiral separation
(Scheme 1 and Table 1). While no measurable improvement in
cell-based potency was observed due to the 10 nM assay
threshold, potent fat ex vivo inhibition was observed for both
compounds 11a and 11b (87 and 86%, respectively).
Unfortunately, compounds 11a and 11b have relatively short
half-lives in both human and mouse microsomes (17 min/17
and 10 min/17 min, respectively). Additionally, both
compounds displayed inhibition against the cytochrome P450
isozyme 2C9 with IC50 values in the 100 nM range. In
comparison, compounds 9 and 9a were not substrates of P450
enzymes.
Although compounds 11a and 11b failed to meet our criteria
for further advancement, they prompted us to further explore
alternative electrostatic interactions with NADP through the
“reverse” binding mode. In addition to maintaining the key
hydrogen bond at the center of the molecule, we incorporated
functionalities capable of forming hydrogen bond interactions
with NADP with a donor group on the aryl sulfonamide part of
the molecule while keeping constant the 2-trifluoromethyl-4-
fluorophenyl group to maintain the lipophilic interactions with
pocket B (Figure 2).
a
The assay was performed in duplicate; the compounds were tested in
11-point 2-fold dilutions, and the standard error of IC50 was <10%; the
lower threshold of this assay was 10 nM. 10% (v/v) human serum
was added to the cell culture media. po at 10 mg/kg otherwise noted,
results obtained 5 h after dosing.
b
c
interactions between the carboxamide moiety and the cofactor
NADP in the “reverse” binding mode. Increased microsomal
stability was also predicted due to the overall increase in the
polarity of the compounds. In addition, we also noticed that the
dehydration of tertiary alcohol in 9a occurs under acid
conditions. Removal of the α-protons with a C(O)NH2
group was anticipated to mitigate the problem.
Treatment of 12 and 13 with n-BuLi followed by the
addition of methyl 4,4,4-trifluoro-2,3-dioxobutanoate gave the
desired ester intermediate, which was then amidated with
concentrated NH3 to give compounds 15a,b and 16a,b,
respectively (Scheme 2). The difference in ex vivo efficacy for
these two substitution patterns was found to be consistent with
our previous observations, the meta-substitution being
favored16 as shown in compounds 16a and 16b (Table 2).
We began this effort by replacing the methyl group of the
trifluoromethylpropan-2-ol moiety in 9 with a carboxamide
group in the phenyl sulfonamide moiety, leading to the para-
and meta-substituted phenyl analogues 15a,b and 16a,b
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The R-isomer 16a showed more robust ex vivo inhibition (82
vs 38%) as well as increased human and mouse microsomal
stability (18 and 30 min vs 2 and 2 min, respectively).
hydrogen bond with the phosphate oxygen (N−O: 3.3 Å). In
this “reversed” binding mode, the lipophilic binding pocket A is
satisfied by the trifluoromethyl group (CF3) at the C5 position
of the thiophene ring. 2-Trifluoromethyl-4-fluorophenyl group
now interacts with Y177 (edge-to-face π-stacking) and P178 as
well as the C-terminal helix of the dimer partner chain.
A number of analogues bearing amide isosteres were
designed and synthesized as exemplified in compound 19.
The cell-based activity and mouse epididymal fat ex vivo activity
were comparable to those of HSD-621 (Table 2). Unfortu-
nately, this compound had poor rat liver microsomal stability.
As shown in Tables 1 and 2, compounds 9a, 16a, and HSD-
621 have good cell-based activity, microsomal stability, and
epididymal fat ex vivo potency. Selectivity over 11β-HSD2 was
also found to be excellent with IC50 values greater than 100 μM
for all three compounds. These compounds were advanced into
pharmacokinetic (PK) studies as summarized in Table 3.
In a side-by-side comparison in the male C57B6 mouse PK
studies, compound HSD-621 demonstrated a more balanced
overall PK profile than compounds 9a and 16a. This compound
has a low clearance and good in vivo half-life and oral
bioavailability (Table 3, 67%). Therefore, HSD-621 was taken
forward into further PK studies in male Sprague−Dawley (SD)
rats and male beagle dogsthe species used for safety
evaluations. As illustrated in Table 3, clearance of HSD-621
was low as compared to the hepatic blood flow rate in these
animal species, resulting in a long t1/2. The oral bioavailability
was greater than 50% in both species tested (Table 3, 61 and
54%, respectively).
The preference for the meta-substitution prompted us to
revisit the five-membered ring heterocycles as exemplified in
the thiazole and thiophene analogues in Table 2. Thiazole
analogue 17a showed 83% ex vivo inhibition along with
moderate microsomal stability against human and mouse liver
microsomes (Table 2, 21 and 18 min, respectively). Thiophene
analogues 18a (HSD-621)36 and 18b also displayed robust ex
vivo inhibition in the adipose. As observed in the previous
examples, the R-isomer (HSD-621) displayed a more desirable
overall profile when compared to the S-isomer 18b. HSD-621
demonstrated more potent inhibition against human 11β-
HSD1 (10 vs 145 nM), longer half-life when incubated with
human microsomes (>30 vs 18 min), and more robust
inhibition in the mouse epi fat ex vivo assays when orally
dosed at 10 mg/kg (75 vs 50%).
The crystal structure of HSD-621 presents a “reversed”
binding mode as predicted with the carboxamide on the
thiophene side of the molecule directly interacting with the
NADP through a hydrogen bond with the phosphate while
maintaining a hydrogen bond with backbone A217 (O−N: 3.3
Å). The amide carbonyl oxygen of HSD-621 interacts with
Y183 OH (Figure 3; O−O: 2.8 Å), and the amide NH forms a
Because of its desirable overall pharmaceutical profile, HSD-
621 was selected for further safety screens. In addition to its
selectivity over 11β-HSD2, excellent selectivity against
peroxisome proliferator-activated receptors (PPAR) α, β, and
γ was also observed (>100 μM). In the in vitro metabolism
studies, there were no major metabolites detected, and half-lives
were >30 min in rat, mouse, and human liver microsomes. No
̀
the human ether-a-go-go-related gene (hERG) ion channel
activity was observed at 33 μM. HSD-621 was also clean in
genotoxicity assay (AMES test) and clean in the nova screen
against 134 targets at 5 μM. In 14 day safety assessment and
metabolism studies, HSD-621 was well tolerated in rats dosed
at 200, 600, and 2000 mg/kg and dogs dosed at 100, 300, and
1000 mg/kg.
To evaluate the in vivo efficacy of HSD-621, we administered
this compound to male C57B6 mice (5.5−7 months old and on
a 35.5% fat diet for 4 months) mixed in food at 0.3 mg drug/g
food. This dose provides an AUC equivalent to that of an oral
dosing at 2 mg/kg. HSD-621 treatment of C57B6 DIO mice
Figure 3. Crystal structure of HSD-621 bound to human 11β-HSD1.
The inhibitor is shown with yellow carbons, and the NADP cofactor
and residues containing atoms within 4 Å of the inhibitor are shown in
green. The figure was generated with Pymol.
Table 3. Selected PK Profile of Compounds 9a, 16a, and HSD-621
a
a
iv
po
compd
9a
species
Cl (mL/min/kg) Vss (L/kg) AUC (h ng/mL) t1/2 (h) Cmax (ng/mL) tmax (h) AUC (h ng/mL) t1/2 (h) F (%)
b
mouse
mouse
mouse
9.0
5.4
9.0
8.0
1.0
5.8
1.8
4.2
3.8
1.8
3588
6131
9.5
4.2
449
1554
987
0.5
2.0
2.0
1.8
2.8
3403
14005
11700
6542
6.0
2.8
19
46
67
61
54
b
b
16a
HSD-621
HSD-621
HSD-621
3600
6.1
4.8
c
rat
4310
8.6
656
3.3
d
dog
32358
20.3
1444
43488
17.2
a
b
See the Experimental Procedures for dosing and analytical protocols. C57B6 mice: iv dosed at 2 mg/kg in 50% DMSO/50% PEG200 and po
c
dosed at 10 mg/kg in 2% Tween/0.5% methyl cellulose in water. SD rat: iv dosed at 2 mg/kg in 50% DMSO/50% PEG200 and po dosed at 10 mg/
kg in 2% Tween/0.5% methyl cellulose in water. Male dog: iv dosed at 2 mg/kg in 50% DMSO/50% PEG200 and po dosed at 5 mg/kg in 2%
d
Tween/0.5% methyl cellulose in water.
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observed during the course or at the end of the study. There
was no body weight loss, no reduction of food intake, and no
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ASSOCIATED CONTENT
* Supporting Information
Biological assays and experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-665-5635. E-mail: Zhao-Kui.Wan@pfizer.com.
■
Notes
The authors declare no competing financial interest.
(16) Selected recent reports on 11β-HSD1 inhibitors: Xiang, J.; Wan,
Z.-K.; Li, H.-Q.; Ipek, M.; Binnun, E.; Nunez, J.; Chen, L.; McKew, J.
C.; Mansour, T. S.; Xu, X.; Suri, V.; Tam, M.; Xing, Y.; Li, X.; Hahm,
S.; Tobin, J.; Saiah, E. Piperazine sulfonamides as potent, selective, and
orally available 11β-hydroxysteroid dehydrogenase type 1 inhibitors
with efficacy in the rat cortisone-induced hyperinsulinemia model. J.
Med. Chem. 2008, 51, 4068−4071.
ACKNOWLEDGMENTS
■
We thank Nelson Huang, Ning Pan, Peter Tate, Walter
Massefski, and Li Di for coordinating and obtaining analytical
data. We also thank Dr. Katherine Lee for proofreading this
manuscript.
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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.;
Saiah, E. Efficacious 11β-hydroxysteroid dehydrogenase type 1
inhibitors in the diet-induced obesity mouse model. J. Med. Chem.
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type 1 inhibitor for clinical study: Identification of (S)-2-((1S,2S,4R)-
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