Optimization of novel di-substituted cyclohexylbenzamide derivatives as potent 11β-HSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.026

Novel 4,4-disubstituted cyclohexylbenzamide inhibitors of 11β-HSD1 were optimized to account for liabilities relating to in vitro pharmacokinetics, cytotoxicity and protein-related shifts in potency. A representative compound showing favorable in vivo pharmacokinetics was found to be an efficacious inhibitor of 11β-HSD1 in a rat pharmacodynamic model (ED₅₀ = 10 mg/kg).

Full text of DOI:10.1016/j.bmcl.2009.01.026

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1446–1450
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of novel di-substituted cyclohexylbenzamide derivatives
as potent 11b-HSD1 inhibitors q
*
Dustin L. McMinn , Yosup Rew, Athena Sudom, Seb Caille, Michael DeGraffenreid,
Xiao He, Randall Hungate, Ben Jiang, Juan Jaen , Lisa D. Julian, Jacob Kaizerman,
Perry Novak, Daqing Sun, Hua Tu, Stefania Ursu, Nigel P. C. Walker, Xuelei Yan,
Qiuping Ye, Zhulun Wang, Jay P. Powers
Amgen, Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 4,4-disubstituted cyclohexylbenzamide inhibitors of 11b-HSD1 were optimized to account for lia-
bilities relating to in vitro pharmacokinetics, cytotoxicity and protein-related shifts in potency. A repre-
sentative compound showing favorable in vivo pharmacokinetics was found to be an efficacious inhibitor
of 11b-HSD1 in a rat pharmacodynamic model (ED50 = 10 mg/kg).
Received 11 November 2008
Revised 7 January 2009
Accepted 12 January 2009
Available online 15 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
11b-HSD1
Diabetes
Metabolic syndrome
Hydroxysteroid dehydrogenase
Cardiovascular disease
Cyclohexylbenzamides
In humans, 11b-HSD1 catalyzes the reduction of the inactive
glucocorticoid cortisone to its active form, cortisol.2 Glucocorticoid
balance is checked by the hypothalamic-pituitary-adrenal (HPA)
axis3 and reversion of cortisol to cortisone by isozyme 11b-
HSD2.4 Upon binding to the glucocorticoid receptor, cortisol
positively regulates glucose production. Excessive hepatic glucose
production and the functional antagonism of glucocorticoids to-
wards insulin action contribute to the diabetic state. Therefore, aug-
mented regulation of cortisol levels via inhibition of 11b-HSD1 may
be a potential therapy for treatment of diabetes.5 Inhibitors of 11b-
HSD1 may also serve as treatment for ‘metabolic syndrome.’ Unreg-
ulated overexpession of cortisol is manifested in Cushing’s disease.
Symptoms associated with this disease are typical metabolic
syndrome indicators (visceral obesity, reduced insulin sensitivity,
dyslipidemia, and high blood pressure)6 and are reversible upon
surgical correction of glucocorticoid over-production.7
liver-specific 11b-HSD1 knock-out mice models.9 Animals in the
former experiment displayed increased levels of gluconeogenesis,
visceral obesity, and triglyceride production coupled with impaired
insulin sensitivity. In contrast, 11b-HSD1 knock-out mice showed
improved glucose tolerance and insulin sensitivity. Moreover, stud-
ies utilizing fat-specific overexpression of 11b-HSD2 in mice dem-
onstrate that such animals are resistant to diet-induced obesity.
Interestingly, studies in monozygotic twins where one of the twins
is obese suggest that relative overexpression of 11b-HSD1 in fat
tissues of the obese twin may present as an acquired defect of
endocrinology as opposed to a genetic dysfunction.10 Inhibition of
11b-HSD1 may also retard the development of atherosclerosis.11
Given their potential for treatment of metabolic syndrome, diabe-
tes, and cardiovascular disease, inhibitors of 11b-HSD1 are of active
interest to the pharmaceutical industry.12
We have previously disclosed optimization of potency and met-
abolic stability for a series of trans-4-substituted cyclohexylbenza-
mide-based 11b-HSD1 inhibitors (Table 1).13 Some members of
this series, such as compound 1, showed moderate PXR activation
in vitro.14 This liability proved manageable by increasing polarity
within the otherwise hydrophobic trans-4-substituent (com-
pounds 2, 4, and 6).13a Although 2, 4, and 6 elicited minimal PXR
activation, it was recently discovered that these compounds exhib-
ited appreciable cytotoxicity in vitro.15 In contrast, cis-isomers of
In addition to Cushing’s disease, genetic evidence for the role of
11b-HSD1 in diabetes and obesity has been illustrated in separate
experiments involving fat-specific 11b-HSD1 overexpression8 and
q
See Ref. 1.
* Corresponding author. Tel.: +1 650 244 2595.
E-mail address: dmcminn@amgen.com (D.L. McMinn).
Present address: ChemoCentryx, Inc., Mountain View, CA 94093, USA.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.026

D. L. McMinn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1446–1450
1447
Table 1
4-Substituted cyclohexylbenzamides 11b-HSD1 inhibitors19
CF3
HO
N
O
R
Compound
R
hHSD1 IC50 (nM)
% TO at 30 min HLM
PXR, % activ. at 2
l
M
HeLa Cytotox IC50 (lM)
1
2
3
4
5
6
7
Phenyl (trans)
3-Pyridyl (trans)
3-Pyridyl (cis)
2-Pyridyl (trans)
2-Pyridyl (cis)
4-Cyanophenyl (trans)
4-Cyanophenyl (cis)
1.3
1.7
1.6
1.6
1.6
0.76
4.9
<5
<5
94
<5
87
<5
33
31
2.2
38
11
66
11
75
>10
2.5
>10
2.3
>10
1.1
>10
these compounds (3, 5, and 7) exhibited reduced in vitro cytotox-
icity (IC50 > 10 M). The mechanism leading to cytotoxicity for
Synthesis of 4-cyclopropyl, 4-nitrile cyclohexylbenzamide 15
proceeded through monoethyleneketal protected 4-cyclopropyl,
4-cyano-cyclohexanone (30, Scheme 2). Preparation of intermedi-
ate 30 relied upon nucleophilic trapping of the 1-cyclo-
propylcyclohexyl trifluoromethanesulfonate intermediate formed
from treatment of the homoallylic alcohol 29 with triflic anhydride
in the presence of hindered base.18 Deprotection of the cyclohexa-
none was followed by reductive amination to afford the purified
stereoisomer 31. Amide coupling was performed as before to pro-
duce 15.
4-Hydroxy substituted compounds were obtained by reaction
of the appropriate carbon nucleophile with monoethylene ketal
protected cyclohexadione to provide after deprotection 4,4-disub-
stituted cyclohexanones 33 (Scheme 3). Completion of the synthe-
ses followed that of the nitriles with separation of stereoisomers
after the final amide coupling.
Geminal substituted cyclohexylbenzamide derivatives were
evaluated for inhibition of 11b-HSD1 in biochemical assays and
were assessed for relative protein-related potency shifts in cellular
assays with and without 3% human serum albumin (Table 2).19 PXR
transactivation potential, HeLa cytotoxicity, and in vitro metabolic
stability were evaluated. Regardless of cis/trans orientation, com-
pounds 8–23 showed minimal PXR activation relative to their
monosubstituted cognates. Moreover, with the exception of com-
pounds 16 and 22, HeLa IC50s for most disubstituted compounds
l
trans-substituted compounds is not fully understood. Unfortu-
nately, the improved cytotoxicity profile for 3, 5, and 7 accompa-
nied increases in the percent turnover in human liver
microsomes (HLM) and re-introduction of PXR activation. We
hypothesized that appropriately configured 4,4-disubstitution on
the cyclohexylbenzamide ring would provide metabolically stable,
non-cytotoxic 11b-HSD1 inhibitors. As cis-4-monosubstituted
cyclohexylbenzamides had thus far enabled PXR activation, we
investigated revealing the additional cis-substitution as size-mini-
mized polar functionality represented by simple nitrile or hydroxyl
substituents.
4-Aryl/heteroaryl, 4-nitrile substituted cyclohexylbenzamides
were synthesized in straightforward fashion (Scheme 1). Using a
tandem one-pot double Michael addition-Dieckmann condensa-
tion between methyl acrylate and aryl/heteroarylacetonitriles,
16
b-keto esters 25 were efficiently obtained. Krapcho decarboxyl-
ation17 of 25 to the 4,4-disubstituted cyclohexanones 26 was fol-
lowed by reductive amination with cyclopropylamine. Separation
of stereoisomers by silica gel chromatography was performed fol-
lowing either this or the final step. Amide coupling to (S)-4-(1,1,1-
trifluoro-2-hydroxypropan-2-yl)benzoic acid13 provided com-
pounds 8–14 (Table 2).
were improved to >10 lM. Interestingly, trans-4-hydroxy/nitrile,
4-aryl/pyridylcyclohexylbenzamides exhibited compromised met-
abolic stability in rat microsomal homogenates (RLM) relative to
their direct (cis) analogues (12, 14, 17, vs 11, 13, 16, respectively).
Inhibition of 11b-HSD1 was also affected by cis/trans orientation
with cis-stereoisomers, being more potent in the biochemical as-
say. Compounds with relatively polar 4-aryl/heteroaryl groups (9,
10, 11, 18, 19) appeared to be less potent in the biochemical assay.
Methyl carbinol 20, reflective of diminished hydrophobic surface
area at the 4-position, also exhibited lower biochemical potency.
These observations indicated that favorable interactions with the
enzyme are dictated by a balance of polarity/hydrophobicity in fea-
tures pendant from the 4-position. Within the nitrile sub-series,
cyclohexylbenzamides with less polar 4-substituents showed con-
siderable potency shifts in the cellular assay (8, 13, 15). The 4-
cyclopropyl, 4-nitrile compound 15 showed a minimal protein-re-
lated shift in cellular potency and thus constituted the optimal ni-
trile containing compound. Of the more potent members of the
hydroxyl sub-series, potencies for the phenyl and methylcyclopro-
pyl analogs (16, 22) were more protein shifted [ca ꢀ 6Â] than the
smaller isopropyl (21 [ca 3Â]) and cyclopropyl (23 [ca 2Â]) com-
pounds. Although quite potent in cellular assays, 21 showed com-
CO2Me
O
HO
R
a
b
CN
c
CN
CN
24
R
R
25
26
CF3
HO
CF3
OH
HO
O
d
HN
N
CN
CN
O
R
R
27
8-14
Scheme 1. Reagents and conditions: (a) methyl acrylate, KOtBu, THF, 63–87% (b)
DMSO, NaCl, H2O, 160 °C, 71-quant% (c) cyclopropylamine, NaBH(OAc)3, DCE, AcOH,
35–98%; (d) EDC, HOAT, NaHCO3, (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-yl)ben-
zoic acid, DMF, 55 °C, 46–98% (syn/anti = 2/1–2.5/1).

1448
D. L. McMinn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1446–1450
Table 2
4,4-Disubstituted cyclohexylbenzamide 11b-HSD1 inhibitors19
CF3
HO
N
R1
O
R2
Compound
R1
R2
hHSD1
IC50 (nM)
h293 hHSD1
IC50 (nM)
h293 hHSD1 +
3% HSA IC50 (nM)
% TO at 30
min HLM
% TO at 30
min RLM
PXR,% of control
HeLa Cytotox
IC50 (lM)
at 2
l
Ma
8
9
CN
CN
CN
CN
Phenyl
1.7
43
64
17
22
3.3
11
1.5
9
150
190
34
26
5.8
4.2
14
62
82
190
41
39
48
96
36
48
>300
>300
170
97
26
27
219
< 5
6.8
<5
<5
6.9
10
13
<10
<5
15
<5
<5
<5
10
10
<5
6
<5
<5
<5
7.6
6.3
<5
12
<5
<5
<5
<5
6.1
<5
<3.4
5.9
<5
>10
>10
>10
>10
>10
>10
>10
>10
5.1
>10
>10
>10
>10
>10
2.8
4-CN-Phenyl
3-Pyridyl
2-Pyridyl
CN
3-Fluoro, 6-pyridyl
CN
Cyclopropyl
Phenyl
OH
3-Pyridyl
2-Pyridyl
CH3
Isopropyl
Methylcyclopropyl
Cyclopropyl
>300
>300
>300
>300
>300
>300
64
>300
>300
>300
>300
>300
69
<5
35
14
95
<10
55
<10
<5
>95
<5
<5
<5
69
43
7.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2-Pyridyl
CN
3-Fluoro, 6-pyridyl
CN
OH
Phenyl
OH
OH
OH
OH
OH
OH
154
121
52
>10
a
Rifampin was used as a control.
O
O
O
O
O
O
O
O
a
b
OH
a, b
c
O
OH
OH
( )2
CN
O
O
R
c
R
O
O
28
29
30
28
32
33
d, e
CF3
HO
HN
N
CF3
HO
f
CN
CN
O
HN
N
O
15
31
d
OH
OH
R
Scheme 2. Reagents and conditions: (a) (3-benzyloxypropyl)triphenylphospho-
nium bromide, nBuLi, THF, 43%; (b) Na, EtOH, Et2O, NH3, 99%; (c) sym-collidine,
Tf2O, CH2Cl2, À78 °C, then Et2AlCN, 42%; (d) TFA, H2O, 99%; (e) cyclopropylamine,
NaBH(OAc)3, DCE, AcOH, 64% (syn/anti = 2.2/1); (f) EDC, HOAT, NaHCO3, (S)-4-
(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoic acid, DMF, 55 °C, 85%.
R
16-23
34
Scheme 3. Reagents and conditions: (a) RBr, tBuLi or RMgBr, Et2O or THF, 45–79%;
(b) 2 N HCl, THF, 71–99%; (c) cyclopropylamine, NaBH(OAc)3, DCE, AcOH, 89–98%;
(d) EDC, HOAt, (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoic acid, DMF, 15–
56% (syn/anti = 1.2/1–2.3/1).
promised in vitro rat metabolic stability. Being more metabolically
stable, compound 23 proved to be the optimal 4-hydroxyl
cyclohexylbenzamide.
[3H] cortisone. Rat 11b-HSD1 activity was then measured by detec-
tion of tritiated cortisol. A dose dependent reduction in cortisol
production was observed indicating an ED50 of ca. 10 mg/kg for
inhibition of rat 11b-HSD1 (Fig. 1). Noting the superior inhibition
A comparison of PK amongst animals with potential to serve as
pharmacodynamic models indicated superior oral exposure for hy-
droxyl substituted cyclohexylbenzamide 23 relative to nitrile 15
(Table 3). Moreover, the hydroxyl cyclohexylbenzamide was 2.4Â
less protein bound in a human protein binding assay (separation
by ultracentrifugation, fraction unbound = 0.12). Compound 23
inhibited the rat 11b-HSD1 enzyme (IC50 = 244 nM) more effec-
tively than the cynomolgus monkey enzyme (IC50 = 640 nM). It
was therefore decided to forward 23 to a pharmacodynamic model
in the rat and assess the effect of oral administration towards inhi-
bition of 11b-HSD1.20
Table 3
Pharmacokinetic data for 4,4-di-substituted cyclohexylbenzamides
Compound
Species
CL, iv (L/h/kg)
Vdss (L/kg)
AUC, po (
l
g*h/L)
% F
23
15
23
15
Rata
0.24
0.19
0.11
0.093
2.7
2.2
1.5
1.9
8900
4100
4700
1000
76
40
43
5
Ratb
Cynoa
Cynoa
Compound 23 was dosed orally in Sprague–Dawley rats at 1, 3,
10, and 30 mg/kg. Two hours post dose,21 animals were sacrificed,
epididymal fat was isolated and incubated in media containing
a
Dosed iv 0.5 mg/kg, po 2.0 mg/kg.
Dosed iv 0.7 mg/kg, po 2.0 mg/kg.
b

D. L. McMinn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1446–1450
1449
References and notes
Epididymal fat 11β-HSD1 activity
40
30
20
10
0
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Sudom, A.; Sun, D.; Tu, H.; Ursu, S.; Wang, Z.; Yan, X.; Ye, Q. Program and
Abstracts, 31st National Medicinal Chemistry Symposium, University of
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M. Endocr. Rev. 2004, 25, 831; (b) Seckl, J. R.; Walker, E. A. Trends Endocrinol.
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(mg/kg)
Figure. 1. Dose dependent inhibition of rat 11b-HSD1 and plasma exposure by oral
administration of 23.
by 23 of the human relative to the rat enzyme, the results of this
experiment indicate a reasonable potential for compounds of this
class to serve as effective orally bioavailable inhibitors of 11b-
HSD1 in humans.
A 2.65 Å resolution X-ray co-crystal structure of 23 bound to
human 11b-HSD1 containing NADP was acquired (Fig. 2).22 Similar
to previously obtained structures from this13 and closely related
compound classes,12c 23 binds in a V-shaped conformation in the
active site of the enzyme (Fig. 2a). A H-bond from the carbonyl
of the amide to Y183 disrupts the catalytic triad (Fig. 2b). Also, a
potential VDW contact exists between the aminocyclopropyl group
and Y177 (Fig. 2d). The cyclohexanol hydroxyl group makes a
hydrogen bond to Y280 (Fig. 2c). The trans-isomer of 23 would
likely fail to achieve this interaction with Y280 post-desolvation.
Not surprisingly, the trans-isomer displays a compromised inhibi-
tion of the enzyme (hHSD1 IC50 = 90 nM).
In conclusion, 4,4-disubstituted cyclohexylbenzamide 11b-
HSD1 inhibitors were optimized to eliminate in vitro cytotoxicity
and PXR transactivation potential. Replacement of 4-aryl/hetero-
aryl features with a cyclopropyl group served to minimize pro-
tein-related shifts in potency resulting in an orally bioavailable
compound capable of inhibiting 11b-HSD1 activity in animal tis-
sues. These early developments suggest that additional effort with-
in this sub-series is warranted. Efforts within this chemical
archetype are ongoing.
13. (a) Julian, L.D.; Bostick, T.; Caille, S.; Chan, H.; DeGraffenreid, M.; He, X.;
Hungate, R.W.; Jaen, J. C.; Jiang, B.; Kaizerman, J.; Liu, J.; McMinn, D.; Powers,
J.P.; Rew, Y.; Sudom, A.; Sun, D.; Tu, H.; Ursu, S.; Wang, Z.; Yan, X.; Ye, Q. Poster
presented at 234th ACS National Meeting, Boston, MA, 2007.; (b) Julian, L. D.;
Wang, Z.; Bostick, T.; Caille, S.; Choi, R.; DeGraffenreid, M.; Di, Y.; He, X.;
Hungate, R. W.; Jaen, J. C.; Liu, J.; McMinn, D.; Monshouwer, M.; Rew, Y.;
Sudom, A.; Sun, D.; Tu, H.; Ursu, S.; Walker, N.; Yan, X.; Ye, Q.; Powers, J. P. J.
Med. Chem. 2008, 51, 3953.
14. PXR activation relative to percent of control (rifampin) was determined using
HepG2 cells transfected with a luciferase reporter construct driven by human
PXR cDNA with luciferase activity determined by chemiluminescence.
15. IC50 for inhibition of HeLa cell growth at 72 h, Alamar Blue fluorescence based
detection of cell viability.
16. DeGraffenreid, M. R.; Bennett, S.; Caille, S.; Gonzalez- Lopez de Turiso, F.;
Hungate, R. W.; Julian, L. D.; Kaizerman, J. A.; McMinn, D. L.; Rew, Y.; Sun, D.;
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19. All compounds were characterized by NMR and LCMS and found to be >95%
purity. As described in reference 12c, 11b-HSD1 enzyme activity was
determined by measuring the reduction of [3H]-cortisone to [3H]-cortisol.
Figure. 2. Co-crystal structure of compound 23 in human 11b-HSD1. Compound
carbons are color coded purple, NADP carbons are grey. Hydrogen bonds are shown
as a dashed magneta line.

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[3H]-cortisol was captured by an anticortisol monoclonal antibody conjugated
to scintillation proximity assay (SPA) beads and quantified by scintillation
inhibition were calculated with a dose response curve fitting algorithm with at
least duplicate sets of samples.
detection. Biochemical enzyme assays were performed with baculovirus-
produced recombinant full-length human, rat, or cyno11b-HSD1 as the enzyme
source and NADPH as cofactor. Cell-based enzyme assays employed HEK293
cells stably expressing recombinant human full-length 11b-HSD1 as the
enzyme source without supplementation of NADPH. IC50 values for enzyme
20. Details for an analogous experiment carried out in cynomolgus monkeys is
described in Ref. 13.
21. Time point chosen to coincide with Tmax (ca. 2 h) of the compound upon oral
administration.
22. PDB database deposition code 3FCO.