Synthesis and biological evaluation of picolinamides as potent inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)

Article abstract of DOI:10.1016/j.bmcl.2014.11.074

Synthesis of a series of 6-substituted picolinamide derivatives and their inhibitory activities against 11β-hydroxysteroid dehydrogenase type 1 are described. Optimization of the initial hit compound, N-cyclohexyl-6-(piperidin-1-yl)picolinamide (1) from high throughput screening of in-house library resulted in the discovery of the highly potent and metabolically stable compound 25, which was efficacious in a mouse ex vivo pharmacodynamic model and reduced the fasting blood glucose and insulin levels in a HF/STZ mouse model after oral dosing.

Full text of DOI:10.1016/j.bmcl.2014.11.074

Bioorganic & Medicinal Chemistry Letters 25 (2015) 695–700
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of picolinamides as potent
inhibitors of 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1)
Je Ho Ryu a,b, Shinae Kim a, Hye Young Han a, Hyun Joo Son a, Hyun Jung Lee a, Young Ah Shin a,
Jae-Sun Kim a, Hyeung-geun Park b,
⇑
a Life Science R&D Center, SK Chemicals, 686 Sampyeong-dong, Bundang-gu, Seongnam-si 463-400, Republic of Korea
b Research Institute of Pharmaceutical Science and College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a series of 6-substituted picolinamide derivatives and their inhibitory activities against
11b-hydroxysteroid dehydrogenase type 1 are described. Optimization of the initial hit compound,
N-cyclohexyl-6-(piperidin-1-yl)picolinamide (1) from high throughput screening of in-house library
resulted in the discovery of the highly potent and metabolically stable compound 25, which was
efficacious in a mouse ex vivo pharmacodynamic model and reduced the fasting blood glucose and
insulin levels in a HF/STZ mouse model after oral dosing.
Received 17 August 2014
Revised 25 November 2014
Accepted 27 November 2014
Available online 5 December 2014
Keywords:
11b-HSD1 inhibitor
Diabetes
Ó 2014 Elsevier Ltd. All rights reserved.
Metabolic syndrome
Picolinamide
Metabolic stability
11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is a key
enzyme that acts as an NADPH-dependent reductase and converts
inactive cortisone into active cortisol, which is an actual circulating
glucocorticoid in humans. 11b-HSD1 is mainly distributed in
specific tissues, such as liver, adipose, and brain, and regulates
tissue-specific glucocorticoid levels.1
inhibitors5 (Fig. 1), including sulfonamides (PF-915275),6 triazoles
(Merck 544),7 thiazolones (AMG-221),8 and carboxamides
(AZD-4071).9
As one of our research projects for the development of new
therapeutics for the treatment of type II diabetes, high throughput
screening of an in-house compound library utilizing the micro-
somal fractions overexpressing human 11b-HSD1 enzyme was per-
formed, and hit compound 1 was identified to be a novel inhibitor
of 11b-HSD1. Interestingly, compound 1 shared the same picolina-
mide core structure with the previously reported 11b-HSD1 inhib-
itor, BMS3010 (Fig. 2). To guide the medicinal chemistry program,
Chronic high level of glucocorticoids can result in insulin
resistance from impairment of insulin-dependent glucose uptake,
increased hepatic gluconeogenesis, and reduced insulin secretion
from the pancreas. Due to the physiological relationship between
glucocorticoids and metabolic disease risk factors, 11b-HSD1 has
been regarded to be a potential target for the treatment of meta-
bolic syndrome as well as type II diabetes.2 Several studies using
a genetic mouse model have supported this idea. For example, liver
or adipose tissue-specific overexpression of 11b-HSD1 in trans-
genic mice showed several features of metabolic syndrome, includ-
ing abdominal obesity, dyslipidemia, glucose intolerance, and
insulin resistance.3 In contrast, 11b-HSD1 knock-out mice demon-
strated a reduction in body weight, improved lipid profiles, and
increased insulin sensitivity under the conditions of a high fat
diet.4
O
S
O
N
N
N
N
NH2
N
H
NC
PF-915275
Merck 544
O
S
O
N
N
H
HO
In the last decade, extensive research in the pharmaceutical
industry has disclosed various chemical classes of 11b-HSD1
N
H
S
N
N
O
AMG-221
AZD-4071
⇑
Corresponding author. Tel.: +82 2 880 7871; fax: +82 2 872 9129.
E-mail address: hgpk@snu.ac.kr (H.-g. Park).
Figure 1. Previously reported 11b-HSD1 inhibitors.
http://dx.doi.org/10.1016/j.bmcl.2014.11.074
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

696
J. H. Ryu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 695–700
cyclohexyl moiety of 1 was located more closely to the hydropho-
O
O
bic binding pocket. The piperidine ring of 1 was positioned toward
the solvent-exposed area of the dimer interface region of the pro-
tein and interacted with hydrophobic residues (Fig. 3).
N
N
N
N
H
F
N
Despite its moderate to high inhibitory activity against 11b-
HSD1 (human 11b-HSD1, IC50 = 58 nM), compound 1 showed poor
metabolic stability in the mouse microsomal assay (% remaining at
30 min = 15%). We planned to modify cyclohexyl and piperidine
rings of 1 because these lipophilic moieties are expected to be
metabolically unstable despite their important hydrophobic
interactions with the binding site of the enzyme. In this communi-
cation, we describe our optimization efforts to improve both the
potency and metabolic stability of our hit compound 1 as an
11b-HSD1 inhibitor.
1
BMS30
Figure 2. Chemical structures of hit compound 1 and BMS30.
First of all, we planned to modify the structure of 1 in two
regions, the cyclohexyl amide position and the piperidine ring at
the 2-position of the pyridine. Additionally, the derivatization
was focused on improving not only the enzymatic inhibitory activ-
ity but also the metabolic stability by introducing polar groups,
such as a hydroxyl group or carboxylic acid group. Cyclohexyl ring
modified analogs 2–9 were prepared as outlined in Scheme 1. 1,4-
Cyclohexanedione mono-ethylene ketal was either reduced by
NaBH4 or treated with MeMgCl or TMSCF3 to yield the 4-substi-
tuted cyclohexanone ketal 26. Treatment of 26 with 1 N HCl, fol-
lowed by reductive amination with cyclopropylamine, produced
the cyclohexylamine 27, which was then coupled with 6-bromopi-
colinic acid in the presence of HBTU and separated by silica gel col-
umn chromatography to yield both cis and trans intermediates
28.11 Reaction of 28 with piperidine under microwave irradiation
afforded the corresponding amides 3–8. In a similar fashion, com-
pounds 2 and 9 were obtained simply by microwave irradiation of
piperidine and the picolinamides 29–30, which were also synthe-
sized by a HBTU-assisted coupling reaction between the appropri-
ate cycloalkylamine and 6-bromopicolinic acid.
Figure 3. Overlap of BMS30 (in brown stick) and compound 1 in the binding site of
human 11b-HSD1. The protein structure and pose of BMS30 were taken from 3CH6.
Predicted binding mode of 1 was generated by Surflex-Dock.
the hit compound 1 was docked into the human 11b-HSD1-BMS30
co-crystal structure (PDB code 3CH6). The overall binding mode of
1 in the binding site of human 11b-HSD1 was similar to that of
BMS30. However, detailed analysis revealed that the carbonyl
group of 1 did not establish hydrogen bonds with the key interac-
tion residues (hydroxyl groups of Ser170 and Tyr183), whereas the
Piperidine ring-modified compounds 10–25 were synthesized
via the routes outlined in Schemes 2 and 3. Compounds 10–13 were
prepared by the reaction of picolinamide 30 with 4-substituted
HO
R
O
HO
R
d, e
a or b or c
HN
27
O
O
O
O
N
26
OH
R
OH
R
O
O
O
g
f
Br
Br
N
N
Br
N
N
OH
N
N
28
3
, R = H, trans
4, R = H, cis
h
5
6
, R = Me, trans
, R = Me, cis
7, R = CF3, trans
O
O
8
, R = CF3, cis
g
A
N
N
A
N
H
N
H
OH
29, 30
2, 9
29, 2
, A =
OH
30, 9
, A =
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, 98%, (R = H); (b) MeMgCl, THF, 59% (R = Me); (c) TMSCF3, TBAF, THF, 97% (R = CF3); (d) 1 N-HCl, THF, 62–92%; (e)
cyclopropylamine, NaBH(OAc)3, AcOH, DCM, 87–95%; (f) HBTU, DIPEA, ACN, 65–90%; (g) piperidine, ACN, 150 °C, microwave, 82–99%; (h) trans-4-aminocyclohexanol or
trans-4-aminoadamantan-1-ol, HBTU, DIPEA, ACN, 73–85%.

J. H. Ryu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 695–700
697
O
R
OH
R
OH
a or b
c
N
Bn
N
Bn
N
H
31
32
HO
R
OH
OH
O
O
d
Br
N
N
N
N
H
N
H
10 R = CF3
30
11
12
13
R = Me
R = cPr
R = Ph
e
n
X
O
OH
N
N
14
X = OH, n = 1
N
H
33 X = CO2Me, n = 1
f
f
g
g
15
16
X = CONH2, n = 1
X = CO2H, n = 1
34 X = CO2Me, n = 2
17
X = CONH2, n = 2
18 X = CO2H, n = 2
Scheme 2. Reagents and conditions: (a) TMSCF3, TBAF, THF, 89% (R = CF3); (b) RMgX, THF, 30–73% (R = Me, cPr, Ph); (c) H2, Pd/C, EtOH, 28–98%; (d) ACN, 150 °C, microwave,
21–79%; (e) piperidin-4-ylmethanol or methyl 2-(piperidin-4-yl)acetate or methyl 3-(piperidin-4-yl)propanoate, TEA, ACN, 150 °C, microwave, 69–94%; (f) formamide,
NaOMe, DMF, 100 °C, 64–79%; (g) 10% NaOH, MeOH, 62–78%.
N
O
O
OH
OH
a
Br
N
N
N
N
H
N
H
30
19
b
c, d
R
OH
X
N
O
N
O
OH
N
N
Y
N
N
N
H
X
N
H
22, X = CH, R = H
20, X = CH, Y = N
21
23
24
, X = N, R = H
, X = N, Y = CH
, X = CH, R = OH
25, X = CH, R = CN
Scheme 3. Reagents and conditions: (a) 1-benzylpiperazine, Pd2(dba)3, xantphos, NaOtBu, toluene, 100 °C, 34%; (b) 1-arylpiperazine, TEA, ACN, 150 °C, microwave, 64–84%;
(c) H2, Pd/C, MeOH, 95%; (d) bromomethyl-pyridine hydrobromide, DIPEA, DCE, 70 °C, 60–72%.
piperidines 32, which could be synthesized by the treatment of 1-
benzylpiperidin-4-one with TMSCF3 or an alkyl magnesium halide,
followed by palladium-catalyzed hydrogenolysis. Compounds 14,
33, and 34 were prepared similarly by microwave reaction of 30
with the commercially available 4-substituted piperidines. Ester
intermediates 33–34 were then simply transformed to the carbox-
ylic acid or amide derivatives 15–18 (Scheme 2). Piperazine analogs
19 and 22–25 were synthesized by either palladium-mediated ami-
nation or microwave reaction of 30 with 1-benzylpiperazine or 1-
arylpiperazines, respectively. Hydrogenolysis of 19, followed by
N-alkylation using bromomethyl-pyridines, afforded 20–21
(Scheme 3).
All synthesized compounds were evaluated for the inhibition of
human 11b-HSD1 enzyme as well as in a cell-based assay. The bio-
chemical enzyme assay was performed using microsomal fractions
overexpressing 11b-HSD1 as the enzyme source and NADPH as
the cofactor. The cell-based assay utilized HEK293 cells stably
transfected with human 11b-HSD1 cDNA without supplementa-
tion of NADPH. 11b-HSD1 enzyme activity was determined by
measuring the conversion of cortisone to cortisol, the levels of
which were quantified by an enzyme immunoassay method.12 In
addition, the metabolic stability of the compounds was evaluated
by determining the % remaining after a 30-min incubation with
mouse liver microsomes.
For improving the metabolic stability of 1, initial optimization
was attempted by introducing a hydroxyl group at the 4-position
of the cyclohexyl ring. As shown in Table 1, the 4-hydrox-
ycyclohexyl compound 2 showed an unexpected two-fold increase
of potency in the human enzymatic assay. To increase the hydro-
phobic interaction, we then introduced a cyclopropyl group to
the amide (3 and 4) and an additional short alkyl group at the 4-
position of the cyclohexyl ring (5, 6, 7, and 8). Compared to 1
and 2, compounds 3–8 exhibited significant improvements in
human biochemical potency, indicating that lipophilicity in the

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J. H. Ryu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 695–700
Table 1
Cyclohexyl ring modification
could be acceptable in potency and might improve microsomal sta-
bility by blocking the metabolically soft position and lowering the
c LogP. In accordance with our expectations, the incorporation of a
hydroxyl or amide group (10–15, and 17) resulted in potency com-
parable to compound 9, but these compounds exhibited poor met-
abolic stability. In contrast, the introduction of a carboxylic acid
group (16 and 18) slightly increased the metabolic stability com-
pared to that of 9. However, they showed significantly lower cellu-
lar potency, suggesting that the carboxylic acid moiety is
detrimental to the cell permeability of this class of 11b-HSD1
inhibitors.
O
N
N
R
Compd
R
hHSD1
IC50 (nM)
HEK293a (%Inh.
@ 100 nM)
MLM MSTb
(%R30min
)
1
2
58
NDc
14
15
N
H
Next, we attempted to replace the piperidine ring with an N-
substituted piperazine group (Table 3). Replacing the piperidine
in compound 9 with N-benzyl-piperazine (19), N-pyridylmethyl-
piperazines (20–21), and N-aryl-piperazines (22–23) led to a slight
reduction in enzymatic potency. Moreover, compounds 19–23 did
not provide any improvement in either cellular potency or meta-
bolic stability compared to compound 9. Analogs of 22, which were
substituted with polar groups at the para-position of the phenyl
ring, were also explored (24–25). The 4-hydroxy-phenyl com-
pound 24 showed a slight increase in biochemical potency and
metabolic stability compared to 22, but showed a significant
reduction in cellular potency. In the best case, the incorporation
of a cyano group (25) improved potency in both enzymatic and
cell-based assays compared to 22. Furthermore, compound 25
showed a significant improvement in the metabolic stability com-
pared to that of 9 (88% vs 69% remaining in MLM after 30 min).
In addition to the excellent potency (human 11b-HSD1
IC50 = 2.8 nM, HEK293 IC50 = 6.7 nM) and metabolic stability, com-
pound 25 showed sufficient activity against mouse 11b-HSD1
enzyme (IC50 = 4.3 nM) and was selected for further evaluation in
a mouse ex vivo pharmacodynamic model.13 In this study, male
C57BL/6 mice were orally administered once with 50 or 100 mg/
kg of inhibitor 25. The animals were sacrificed at 2 h after admin-
istration, and liver and epididymal fat (EPF) pads were collected.
The inhibition of 11b-HSD1 activity in liver and EPF was accessed
by an ex vivo measurement of cortisone to cortisol conversion in
the tissue culture media containing cortisone using a cortisol EIA
kit. As shown in Figure 4, 11b-HSD1 enzymatic activities in liver
and EPF were substantially reduced in a dose-dependent manner.
Relative to the vehicle controls, 11b-HSD1 activity in animals trea-
ted with 100 mg/kg of 25 was reduced by 65% and 80% in liver and
EPF, respectively, demonstrating that the compound was effi-
ciently absorbed and distributed to the target tissues.
OH
OH
27
21
22
23
N
H
3
4
17
70
N
N
OH
1.8
6.4
26
27
Me
OH
5
6
7
22
39
26
N
N
N
N
OH
Me
4.6
3.7
39
17
CF3
OH
OH
CF3
8
9
13
19
99
9
OH
1.7
69
N
H
On the basis of the favorable ex vivo inhibition, compound 25
was examined for in vivo efficacy using the HF/STZ mouse model.
The HF/STZ model manifests hyperglycemia, glucose intolerance,
insulin resistance, and a loss of insulin-secreting b cells, all of
which are characteristics of type 2 diabetes in human patients.14
As illustrated in Figure 5A, after 10 days of treatment with
50 mg/kg of 25 twice daily by oral administration, fasting glucose
levels in HF/STZ mice were reduced by 46% compared to those in
the vehicle control. Moreover, plasma insulin levels were also sig-
nificantly reduced in 25-treated mice (Fig. 5B). Effects on food
intake and body weight were not observed, and the mice appeared
healthy with no visible abnormalities. These results revealed that
the 11b-HSD1 inhibitor 25 could improve both blood glucose con-
trol and insulin sensitivity, showing similar efficacy as rosiglitaz-
one (RG) in HF/STZ mice.
All assay data are reported as the average of at least two determinations.
a
HEK293 cells stably transfected with human 11b-HSD1 cDNA.
Metabolic stability test using mouse liver microsomes.
Not determined.
b
c
right-hand side of the compounds was beneficial to 11b-HSD1
inhibition. However, the metabolic stability of 2–8 was not notably
improved (<40% remaining in MLM after 30 min), and the cellular
potency was also low (<40% 11b-HSD1 inhibition at 100 nM),
except for 4. Interestingly, the incorporation of a hydroxy-adaman-
tyl ring (9) led to as much as a 16-fold increase in enzymatic
potency compared to 2. Moreover, compound 9 showed improved
metabolic stability despite its higher c LogP value than that of 2
(2.42 vs 1.79, respectively) and was highly potent in the cell-based
assay.
We then turned our attention to modification of the left-hand
side by substitution on the piperidine ring (Table 2). Based on
the high potency and acceptable metabolic stability of 9, the
hydroxy-adamantyl group was maintained in subsequent deriva-
tives. Because the piperidine ring of 1 was located in the solvent
accessible area in our docking study, we expected that the intro-
duction of polar groups at the 4-position of the piperidine ring
In conclusion, we have identified a series of 6-substituted pico-
linamide derivatives as novel and potent inhibitors of 11b-HSD1.
Based on the docking results of our initial hit compound 1, SAR
studies were performed and resulted in a significant improvement
of both potency and metabolic stability. High potency toward
human 11b-HSD1 was achieved by the incorporation of
a
hydroxy-adamantyl group. In addition, the replacement of piperi-

J. H. Ryu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 695–700
699
Table 2
Piperidine ring substitution with polar functional groups
R1
R2
O
OH
N
N
N
H
Compd
R1
R2
hHSD1 IC50 (nM)
HEK293a (%Inh. @ 100 nM)
MLM MSTb (%R30min
)
9
10
11
12
13
14
15
16
17
18
H
OH
OH
OH
H
1.7
2.7
3.9
3.2
3.2
1.9
6.6
18
99
97
90
98
96
97
28
12
83
25
69
8
46
3
CF3
Me
cPr
Ph
H
H
H
H
H
OH
0
CH2OH
CH2CONH2
CH2CO2H
CH2CH2CONH2
CH2CH2CO2H
29
47
71
63
82
4.1
8.7
All assay data are reported as the average of at least two determinations.
a
HEK293 cells stably transfected with human 11b-HSD1 cDNA.
Metabolic stability test using mouse liver microsomes.
b
Table 3
Piperidine replacements with N-substituted piperazine rings
R
N
O
OH
N
N
N
H
Compd
R
hHSD1 IC50 HEK293a (%Inh. @
MLM MSTb
(nM)
100 nM)
(%R30min
)
19
20
6.8
91
21
9.5
7.2
87
89
39
60
N
21
22
23
Figure 4. Ex vivo data for compound 25. Acute effects of 25 were assessed by
ex vivo cortisone to cortisol turnover in liver and epididymal fat (EPF). The
compound was orally administered at 50 mg/kg and 100 mg/kg to lean C57BL/6
mice (vehicle = 0.5% MC/1% Tween 80 in water). Animals were sacrificed at 2 h post
dose and the tissues were removed to measure 11b-HSD1 activity. Values are
expressed as the mean SEM of n = 4. Statistical analysis was performed by one-
way ANOVA, followed by Tukey. ⁄⁄p <0.01, ⁄⁄⁄p <0.001 versus vehicle.
N
N
3.8
3.7
97
96
44
45
HO
24
25
3.1
2.8
7
74
88
NC
99
All assay data are reported as the average of at least two determinations.
a
HEK293 cells stably transfected with human 11b-HSD1 cDNA.
Metabolic stability test using mouse liver microsomes.
b
dine ring with 1-(4-substituted phenyl)piperazine led to a substan-
tial improvement in metabolic stability. The selected compound 25
also possessed desirable potency in the mouse 11b-HSD1 enzyme
assay and demonstrated oral efficacy in lowering liver and adipose
11b-HSD1 activities in a mouse ex vivo model. Finally, 25 exhibited
good in vivo efficacy in HF/STZ mice and reduced fasting blood glu-
cose and insulin levels after oral dosing. 25 was identified to be
advanced lead, and further optimization of the N-phenylpiperazine
moiety has been performed. These results will be reported in sub-
sequent publications.
Figure 5. Effect of compound 25 on fasting blood glucose and insulin levels in HF/
STZ mice. 5 mg/kg of rosiglitazone (RG) and 50 mg/kg of 25 were orally adminis-
tered to HF/STZ mice twice daily for 10 days. On the last day, blood glucose and
insulin levels were measured after over-night fasting. Values are expressed as the
mean SEM of n = 5. Statistical analysis was performed by one-way ANOVA,
followed by Tukey. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 versus vehicle.

700
J. H. Ryu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 695–700
Stewart, P. M.; Toh, M.; Ndongo, M.; Calle, R. A.; Hirshberg, B. J. Clin. Endocrinol.
Acknowledgment
Metab. 2008, 93, 550; (c) Siu, M.; Johnson, T. O.; Wang, Y.; Nair, S. K.; Taylor, W.
D.; Cripps, S. J.; Matthews, J. J.; Edwards, M. P.; Pauly, T. A.; Ermolieff, J.; Castro,
A.; Hosea, N. A.; LaPaglia, A.; Fanjul, A. N.; Vogel, J. E. Bioorg. Med. Chem. Lett.
2009, 19, 3493.
We thank Dr. Gildon Choi for generating and providing HEK293
cells stably transfected with 11b-HSD1 cDNAs.
7. Olson, S.; Aster, S. D.; Brown, K.; Carbin, L.; Graham, D. W.; Hermanowski-
Vosatka, A.; LeGrand, C. B.; Mundt, S. S.; Robbins, M. A.; Schaeffer, J. M.;
Slossberg, L. H.; Szymonifka, M. J.; Thieringer, R.; Wright, S. D.; Balkovec, J. M.
Bioorg. Med. Chem. Lett. 2005, 15, 4359.
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12. The biochemical enzyme assay was performed using microsomal fractions
from HEK293 cell lysates overexpressing 11b-HSD1. 0.1 ng/ml cell lysates were
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a
commercially available cortisol enzyme-linked
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