3-Amino-N-adamantyl-3-methylbutanamide derivatives as 11β- hydroxysteroid dehydrogenase 1 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2014.01.017

Many adamantane derivatives have been demonstrated to function as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. 3-Amino-N-adamantyl-3-methylbutanamide derivatives were optimized by structure-based drug design. Compound 8j exhibited a good in vitro and ex vivo inhibitory activity against both human and mouse 11β-HSD1.

Full text of DOI:10.1016/j.bmcl.2014.01.017

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Amino-N-adamantyl-3-methylbutanamide derivatives as
11b-hydroxysteroid dehydrogenase 1 inhibitor
Younho Lee a, Yong June Shin b, Soon Kil Ahn c,
⇑
a College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 162-1 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea
b R&D Center, Ahn Gook Pharm., Gyeonggi Bio-Center, Suwon, Gyeonggi-do 443-766, Republic of Korea
c Institute for New Drug Development, Division of Life Sciences, Incheon National University, Incheon 406-772, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Many adamantane derivatives have been demonstrated to function as 11b-hydroxysteroid dehydroge-
nase type 1 (11b-HSD1) inhibitors. 3-Amino-N-adamantyl-3-methylbutanamide derivatives were opti-
mized by structure-based drug design. Compound 8j exhibited a good in vitro and ex vivo inhibitory
activity against both human and mouse 11b-HSD1.
Received 23 October 2013
Revised 2 January 2014
Accepted 6 January 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
11b-Hydroxysteroid dehydrogenase type 1
inhibitor
3-Amino-N-adamantyl-3-
methylbutanamide
Structure-based drug design
Anti-diabetes
The symptoms of Cushing’s syndrome include metabolic com-
plications such as insulin resistance, obesity, dyslipidemia, hyper-
tension, and type 2 diabetes. This disease is caused by excessive
levels of the active glucocorticoid hormone, cortisol.1 In tissues,
the cortisol level is regulated by two 11b-hydroxysteroid dehydro-
genase (11b-HSD) isozymes. The type 1 enzyme (11b-HSD1) is an
NADPH-dependent reductase that converts the inactive form glu-
cocorticoid, cortisone into the active form, cortisol in humans. This
enzyme is highly expressed in liver and adipose tissues. In contrast,
11b-HSD type 2 (11b-HSD2) performs oxidative functions and
expressed in the kidneys, colon, and sweat and salivary glands.
The excess cortisol hormone induces metabolic problem such as
increased lipid mass and insulin resistance by binding to the gluco-
corticoid receptor (GR) (Fig. 1).
Figure 1. The function of 11b-HSD and glucocorticoid.
11b-HSD1 alters its structure from a dimer to that of a tetramer
for substrate binding. The cofactor, NADPH and substrate, cortisone
are used to produce cortisol. The catalytic triad of Ser170, Tyr183,
and Lys187 catalyzes the conversion of cortisone to cortisol. Other
amino acids residues are important for the substrate change and
inhibitors binding as well.2,3 Many types of inhibitors that have
been developed in clinical phases have demonstrated improve-
ments in type 2 diabetes, obesity, and blood lipid profiles.4–9
H
N
H
N
H
N
H
N
S
Cl
S
Cl
R
O O
O O
O
O
1
hHSD1 IC50: 390nM
R = Bulky Functional Groups
⇑
Corresponding author. Tel.: +82 32 835 8248; fax: +82 32 835 0763.
E-mail address: skahn@incheon.ac.kr (S.K. Ahn).
Figure 2. Initial hit compound 1 and modification.
http://dx.doi.org/10.1016/j.bmcl.2014.01.017
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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2
Y. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
.
Table 1
(a)
(c)
HCl
H2N
(b)
(d)
H2N
OH
BocHN
OH
In vitro human and mouse 11b-HSD1 inhibitory activity of methylbutanamide
derivatives with an adamantyl group
R1
O
O
O
10
13
11
12
.
HCl
H2N
H
N
H
N
H
N
H
N
BocHN
R1
Cl
S
O
R1
R1
O
O
O
14
Compound
R1
11b-HSD1 IC50 (nM)
R2
H
N
H
N
Human
Mouse
S
R1
O
O
O
16
1
2
390
N/Da
Scheme 1. Reagents and conditions: (a) Boc2O, 0.5 N NaOH, 1,4-dioxane, rt, 24 h;
(b) BOP-Cl, TEA, DCM, rt, 12 h; (c) 4 M HCl in dioxane, ethyl acetate, rt, 2 h; (d) TEA,
DCM, benzene sulfonyl chloride derivatives, rt. R1 of 12: Cyclohexane, cycloheptae,
1-adamantane, 2-adamantane, 5-carboxylic ester 2-adamantamine or 5-hydroxy-2-
adamantamine. R2 of 16: Diverse ortho, meta, or para substituted benzenes.
190
80
N/D
640
3
4
5
4
4
113
70
R2
H
N
H
N
(b)
S
(a)
NH2
O
OH
O O
O
O
OH
O
O
17
6
8
R2
OH
H
N
H
6
7
8
57
>1000
N/D
12
(c)
N
S
O
O O
O
OH
S
O
O
5
9
NH2
>1000
1
O
Scheme 2. (a) 2N-NaOH, THF/EtOH (1:1), rt, overnight; (b) aq NH4OH, HOBt, EDCI,
DCM, rt, 20 h; (c) (i) methanesulfonyl chloride, TEA, DCM, rt, 12 h; (ii) NaSCH3,
MeOH, 80 °C, 12 h; (iii) aq NaBO34H2O, AcOH, rt, 1 h. R2 of 5 and 17: Diverse ortho,
meta, or para substituted benzenes.
NH2
O
this Letter, we report the discovery of 3-amino-N-adamantyl-3-
methylbutanamide derivatives as a potent 11b-HSD1 inhibitor.
An initial hit compound 1 from an in house chemical library
was identified with a moderate potency in human liver micro-
some 11b-HSD1 (IC50: 390 nM). The activity was improved by
changing the cyclohexane functional group (Fig. 2). The inclusion
of more bulky groups such as cycloheptane and adamantane
improved the 11b-HSD1 inhibitory activity by 2- and 5-folds,
respectively (2 and 3). Next, we explored substituted adamantine
9
54
7
>1000
470
S
O
O
AMG221
—
a
N/D; not determined.
Recently, positive result from a 28-day phase IIa clinical trial
with a potent 11b-HSD1 inhibitor, INCB013739 were reported by
Incyte.10 However, many 11b-HSD1 inhibitors under development
including INCB013739 only exhibit inhibitory activity against
human 11b-HSD1 with no inhibition against the mouse enzyme.
This lack of cross-species activity causes great difficulties in pre-
clinical evaluation for the development of novel drug candidates.
An ongoing efforts to discover novel inhibitors active against
both human and mouse 11b-HSD1 involves structure based drug
design and the evaluation of their in vitro and ex vivo activity. In
derivatives. 2-Adamantane (4) exhibited
a potent 11b-HSD1
inhibitory activity in human liver microsomes. However, the com-
pound exhibited insufficient activity in mouse liver microsomes.
Therefore, efforts were focused on substitution at the 5-position
of adamantane to improve the activity in mice (Table 1). That
positional change strategy was also applied for the other
adamantane derivatives.5,8,9,11–13
Figure 3. The docking models of compound 7 and 8. (a) Compound 8 is depicted in magenta with yellow binding site residues. The dash line indicates the hydrogen bonding
interactions. (b) Compound 7 is presented in purple color. The terminal amide group exhibits steric hindrance against the A226.
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Y. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 2
We attempted to optimize the activity by attaching hydrophilic
functional groups to adamantane. Compound 5 exhibited potent
human 11b-HSD1 inhibitory activity and improved mouse activity.
In particular, the adamantane amide (8) demonstrated improved
inhibitory activity against the 11b-HSD1 of both species. However,
adamantane carboxylic acid (6) and methyl sulfone (9) decreased
the 11b-HSD1 inhibitory activity in both species.14,15
Figure 3 represents the binding model of the most active com-
pound 8. As previously mentioned, the amino acids, S170 and
Y183, are important for inhibitor binding. Two hydrogen bonding
interactions exist between the carbonyl oxygen of compound 8
and the hydroxyl groups of serine and tyrosine. There are two
van der Waals interactions. One occurs between the adamantane
group and Y183 and A226 residues. The dimethyl group and
A172 and Y177 residues also exhibit a similar interaction. In addi-
In vitro human and mouse 11b-HSD1 inhibitory activity of methylbutanamide
analogs with an adamantyl group
Compound
Structure
11b-HSD1 IC50 (nM)
Human
Mouse
H
N
H
N
4
S
4
113
Cl
O
O
O
H
N
H
N
N/Da
N/D
S
4a
4b
89
53
Cl
Cl
O O
O
O
O
H
H
N
N
S
O O
tion, phenyl group and Y-177 residue provide
p–p interaction. As
H
H
N
N
shown in our binding model, the intra-molecular hydrogen bond-
ing between the oxygen of the sulfone group and the hydrogen
of the amide group maintains the compound in V-shape conforma-
tion. The conformation fixed by hydrogen bonding helps the flexi-
ble compound bind properly to the active site pocket (Fig. 3a).16
4c
S
12
7
94
Cl
O O
AMG221
—
470
a
N/D; not determined.
Table 3
In vitro human and mouse 11b-HSD1 inhibitory activity of sulfonyl methylbutanamide derivatives with adamantyl groups
H
N
H
N
1
R
S
NH
2
O
O
O
O
Compound
R1
11b-HSD1 IC50 (nM)
Mouse
Metabolic Stability (Remain% @ 30 min)
Human
1
8
12
10
77
91
99
40
Cl
Cl
8a
1
1
F
8b
8c
9
76
96
5
Cl
F
Cl
1
1
7
22
25
F
8d
8e
9
91
8
F
F
F
F
99
100
14
F
8f
2
2
1
16
196
28
86
96
94
24
31
14
F
F
8g
8h
F
F
F
F
(continued on next page)
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Y. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3 (continued)
Compound
R1
11b-HSD1 IC50 (nM)
Metabolic Stability (Remain% @ 30 min)
Human
Mouse
8i
1
46
99
93
97
57
99
13
Cl
8j
2
2
21
28
F
8k
F
F
8l
1
7
24
94
—
26
—
AMG221
—
470
Table 4
Selectivity and toxicity test results of 8j including the mouse ex vivo results
11b-HSD2 (
lM)
MTT (293) (
lM)
CYP450a
>10
(l
M)
hERG (
>100
lM)
Ex vivo
Liver (%)
68
Fat (%)
11
8j
>10
>50
a
CYP450 1A2, 2D6, 2C9, 2C19, and 3A4 were tested.
The 5-amide-2-adamantamine has two diastereomers, the Z
in in vitro test. The ex vivo effect on mouse tissue was approxi-
mately 68% inhibition in mouse liver tissue at a single gavage dose
of 45 mg/kg, while weak in fat tissue (Table 4).21
In conclusion, the 3-amino-N-adamantyl-3-methylbutanamide
derivatives exhibit good inhibitory effects against both human
and mouse 11b-HSD1 and no toxic effects. Preclinical development
is underway for a number of the derivatives.
and E forms (7 and 8). The E form is more active than the Z form.
Whereas the E form occupies the 11b-HSD1 pocket properly, the
5-substituted amide group of the Z form exhibits a steric hindrance
against the A226 residue of the binding site (Fig. 3b).
Compound 1 and its derivatives were synthesized as shown in
Scheme 1.17 A Boc-protected amine (11) obtained from commer-
cially available 3-amino-3-methylbutanoic acid (10) was coupled
with diverse amines such as cyclic amine, adamantamine, 5-hydro-
xy-2-adamantamine or 5-carboxylic ester 2-adamantamine. The
compound 4 and derivatives (4a–c) were made by coupling with
substituted benzene sulfonylchloride after Boc-deprotection
(Scheme 1). Compound 8 and 9 were synthesized from the
5-carboxylic ester 2-adamantamine derivative (17) or 5-hydroxy-
2-adamantamine derivative (5), respectively (Scheme 2).18,19
Next we evaluated the effect of the dimethyl group of com-
pound 4 on compound’s inhibitory effects against 11b-HSD1. Com-
pounds that had the (S) and (R) form of the methyl group (4a and
4b) demonstrated approximately 10–20 folds decreased activities.
However, a compound containing a cyclopropyl group 4c exhibited
only slightly reduced effects in human microsomes and similar
activity in those of mice. From these results, we can see fully occu-
pied groups within the hydrophobic pocket (around A172, Y177)
are preferred for the inhibitor binding. The most active compound
4 in humans, was used for further optimization (Table 2).
Finally we sought to improve the 11b-HSD1 inhibitory activity
by adopting several substituted benzene derivatives (8a–8l). All
analogs demonstrated one digit nano-molar IC50 values against
human 11b-HSD1. However, the mouse 11b-HSD1 inhibitory activ-
ities did not correlate with the compound structure. We doubted
the lack of correlation and reached a tentative conclusion. Our
assay was conducted using liver microsomes as a source of
11b-HSD1. However, liver microsomes contain many other
enzymes such as CYP450 responsible for the degradation of xeno-
biotics, which affect the stability of compounds and the inhibitory
activity of tested compounds (Table 3).20
Acknowledgments
This work was supported by the Incheon National University
Research Grant in 2013.
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compound did not inhibit 11b-HSD type 2 and had no toxic effects
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14. The in vitro activity of 11b-HSD1 derived from microsomal fractions was
measured using the HTRF assay (Cisbio, Marcoule, France) according to the
manufacturer’s instructions. Briefly, different concentrations of compounds
were added to 96-well plates, followed by the addition of TE buffer (20 mM
Tris buffer and 5 mM ethylenediaminetetraacetic acid, pH 6.0) containing
adamantane amide inhibitor in the X-ray crystal complex. The initial
complex was optimized with 1000 steps of steepest decent and 3000 steps
of conjugate gradient while holding the 11b-HSD1 heavy atoms restrained to
their initial positions by means of
a harmonic force constant of
1 kcal molÀ1 ÅÀ2 using CHARMm in Accelrys Discovery Studio 2.5.
17. Yu, J.; Liu, H.; Xia, G.; Liu, L.; Xu, Z.; Chen, Q.; Ma, C.; Sun, X.; Xu, J.; Li, H.; Li, P.;
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Longenecker, K.; Judge, R.; Qin, W.; Imade, H. M.; Stolarik, D.; Beno, D. W. A.;
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20. All novel synthetic compounds gave satisfactory analytical and spectral data.
Selected data for 8j: 1H NMR (400 MHz, CDCl3) d 7.88–7.92 (m, 1H), 7.54–7.60
(m, 1H), 7.23–7.29 (m, 1H), 7.18–7.22 (m, 1H), 6.36 (d, J = 8.0 Hz, 1H), 5.82 (s,
1H), 5.60 (br s, 1H), 5.30 (br s, 1H), 4.08 (t, J = 3.8 Hz, 1H), 2.49 (s, 2H), 2.11 (s,
2H), 1.96–2.05 (m, 5H), 1.87–1.91 (m, 4H), 1.63 (d, J = 13.2 Hz, 2H), 1.25 (s, 6H);
13C NMR (100 MHz, DMSO-d6) d 179.2, 170.1, 159.5, 157.8, 135.5, 131.6, 129.6,
125.3, 117.6, 55.8, 55.4, 52.9, 47.8, 39.2, 39.0, 31.7, 30.5, 27.4, 27.2; Anal. Calcd
for C22H30FN3O4S: C, 58.52; H, 6.70; N, 9.31. Found: C, 58.63; H, 6.72, N, 9.28.
21. For ex vivo 11b-HSD1 activity analysis, male C57BL/6J mice were randomly
assigned to 2 groups based on body weight. Compound 8j was suspended in
0.5% carboxymethylcellulose (CMC) and given as a single gavage dose of
45 mg kgÀ1; control animals were given vehicle (0.5% CMC) only. The animals
were sacrificed 2 h after dosing, and the liver and epididymal fat tissues were
isolated immediately, frozen in liquid nitrogen, and stored at À80 °C. At the
time of the assay, frozen tissues were partially thawed and dissected into 30–
40 mg samples and placed directly into 24-well plates containing pre-warmed
200
were initiated by the addition of human (20
(320
l
M NADPH and 160 nM cortisone (Sigma, St. Louis, MO). The reactions
l
g), mouse (20 g), or monkey
l
l
g) microsomal fractions, and were allowed to incubate for 2 h at 37 °C.
Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to
each well and incubated for an additional 2 h at room temperature. The cortisol
concentration was calculated using a calibration curve; data were obtained
from at least 2 determinations. The IC50 values were calculated from dose
response curves using GraphPad Prism software.
To measure 11b-HSD2 activity, human kidney microsomes were incubated in
96-well plates in the presence of cortisol (Sigma, St. Louis, MO) with or without
compounds. Enzyme activity was determined by measuring the amount of
cortisone product using LC–MS.
A luminescent CYP inhibition assay was performed using P450-Glo assay
system according to the manufacturer’s instructions (Promega, Madison, WI,
USA).
Human and mouse liver microsome stabilities were determined as follows. The
compounds were incubated with liver microsomes. At 30 min time points,
aliquots were quenched and analyzed by LC–MS.
15. All data are presented as mean SEM. Differences between groups were
determined by one-way ANOVA followed by Duncan’s multiple comparison
test. Data were considered to be statistically significant at p < 0.05.
16. The binding models of the 11b-HSD1/compound 7 and 8 complexes were
described in Figure 3. In docking studies, the structure of 11b-HSD1 was taken
from PDB 2ILT (http://www.rcsb.org), in which the structure was solved in a
complex bound to adamantane sulfone inhibitor. Based on the adamantane
amide coordinates, the compound structure was superimposed to an
DMEM supplemented with 100 lM NADPH and 1 lM cortisone. After 3-h
incubation at 37 °C, the cortisol concentration in the media was measured
using the HTRF cortisol assay; inhibition of 11b-HSD1 activity in tissues was
determined relative to that in vehicle-treated mice.
Please cite this article in press as: Lee, Y.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.01.017