Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11β hydroxysteroid dehydrogenase type 1 inhibitor

Article abstract of DOI:10.1016/j.bmc.2017.04.033

A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with an IC₅₀ of 4.3?nM and in primary human adipose tissue with an IC₈₀ of 53?nM. Oral administration of 11j to cynomolgus monkey inhibited 11β HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

Full text of DOI:10.1016/j.bmc.2017.04.033

Accepted Manuscript
Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11β hydrox-
ysteroid dehydrogenase type 1 inhibitor
Linghang Zhuang, Colin M. Tice, Zhenrong Xu, Wei Zhao, Salvacion Cacatian,
Yuan-Jie Ye, Suresh B. Singh, Peter Lindblom, Brian M. McKeever, Paula M.
Krosky, Yi Zhao, Deepak Lala, Barbara A. Kruk, Shi Meng, Lamont Howard,
Judith A. Johnson, Yuri Bukhtiyarov, Reshma Panemangalore, Joan Guo, Rong
Guo, Frank Himmelsbach, Bradford Hamilton, Annette Schuler-Metz, Heike
Schauerte, Richard Gregg, Gerard M. McGeehan, Katerina Leftheris, David A.
Claremon
PII:
S0968-0896(17)30593-X
http://dx.doi.org/10.1016/j.bmc.2017.04.033
BMC 13703
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2017
24 April 2017
26 April 2017
Please cite this article as: Zhuang, L., Tice, C.M., Xu, Z., Zhao, W., Cacatian, S., Ye, Y-J., Singh, S.B., Lindblom,
P., McKeever, B.M., Krosky, P.M., Zhao, Y., Lala, D., Kruk, B.A., Meng, S., Howard, L., Johnson, J.A.,
Bukhtiyarov, Y., Panemangalore, R., Guo, J., Guo, R., Himmelsbach, F., Hamilton, B., Schuler-Metz, A., Schauerte,
H., Gregg, R., McGeehan, G.M., Leftheris, K., Claremon, D.A., Discovery of BI 135585, an in vivo efficacious
oxazinanone-based 11β hydroxysteroid dehydrogenase type 1 inhibitor, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.04.033
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Discovery of BI 135585, an in vivo efficacious
oxazinanone-based 11β hydroxysteroid
dehydrogenase type 1 inhibitor
Linghang Zhuang,*,a Colin M. Tice,a Zhenrong Xu,a Wei Zhao,a Salvacion Cacatian,a Yuan-Jie Ye,a
Suresh B. Singh,a Peter Lindblom,a Brian M. McKeever,b Paula M. Krosky,b Yi Zhao,b Deepak Lala,b
Barbara A. Kruk,b Shi Meng,b Lamont Howard,b Judith A. Johnson,b Yuri Bukhtiyarov,b Reshma
Panemangalore,b Joan Guo,b Rong Guo,b Frank Himmelsbach,c Bradford Hamilton,c Annette Schuler-
Metz,c Heike Schauerte,c Richard Gregg,b Gerard M. McGeehan,b Katerina Leftherisa and David A.
Claremona
a Medicinal chemistry, Vitae Pharmaceuticals, Inc, 502West Office Center Drive, Fort
Washington, Pennsylvania 19034, United States
b Biology, Vitae Pharmaceuticals, Inc, 502West Office Center Drive, Fort Washington,
Pennsylvania 19034, United States
c Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach, Germany
RECEIVED DATE
To whom correspondence should be address.

Phone: +1 267 454-0795. Email: linghang_zhuang@yahoo.com.
Graphical Abstract
O
O
O
N
O
N
N
F
F
F
OH
OH
O
11j
7
Human adipocyte IC50: 4.3 nM
Human adipose tissue IC80: 53 nM
Log P 2.6
Human adipocyte IC50: 2.5 nM
Human adipose tissue IC80: > 1000 nM
Log P 4.2
ABSTRACT
A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor
(11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with
an IC50 of 4.3 nM and in primary human adipose tissue with an IC80 of 53 nM. Oral
administration of 11j to cynomolgus monkey inhibited 11β HSD1 activity in adipose tissue.
Compound 11j exhibited > 1000x selectivity over other hydroxysteroid dehydrogenases, displays
desirable pharmacodynamic properties and entered human clinical trials in 2011.
KEYWORDS

11beta hydroxysteroid dehydrogenase type 1, inhibitor, oxazinanone, human adipocyte, human
adipose tissue.
Introduction
Diabetes is an abnormal state marked by inability to make sufficient insulin or inability to
appropriately respond to insulin, leading to the accumulation of glucose in blood and urine.1
When left untreated, diabetes results in serious complications including cardiovascular disease,
renal failure, and retinal damage and is often complicated by co-morbidities of obesity,
dyslipidemia, and hypertension. Current therapies include life style intervention, oral anti-
diabetes drugs, and injections of insulin or incretin mimetics, but the need for novel, safe, and
easy to administer alternatives persists due to inadequate improvement in glycemic control
and/or intolerable side effects.2 Advances have been made in recent years to understand the
fundamental biology underlying this assembly of diseases, collectively known as “metabolic
syndrome,” raising the opportunity for pharmacological intervention which directly impact their
etiology and progression.
One enzyme proposed to be critical for the development of metabolic syndrome is 11β-
hydroxysteroid dehydrogenase type 1 (11β-HSD1), a member of the short chain
dehydrogenase/reductase (SDR) superfamily,3-4 responsible for the local conversion of inactive
cortisone to the active glucocorticoid cortisol. Cortisol binds to and activates the glucocorticoid
receptor, resulting in increased expression of a wide range of genes involved in metabolism,
immune response, bone formation, memory, and reproduction. More specifically, cortisol drives
gluconeogenesis in the liver and adipogenesis in adipose tissue, which when elevated may
contribute to metabolic syndrome. Circulating levels of cortisol are tightly controlled through
multiple mechanisms; however, tissue-specific concentrations of cortisol vary from plasma
levels primarily due to the expression and activity of 11β-HSD1.5
Multiple groups have pursued inhibitors of 11β-HSD1 as potential drugs for treatment of
diabetes (Figure 1).6 Carbenoxolone (1), the hemisuccinate derivative of natural product
glycyrrhetinic acid, is a nonselective inhibitor of 11β-HSD1 and 11β-hydroxysteroid
dehydrogenase type 2 (11β-HSD2). Despite its lack of selectivity for 11-βHSD1, 1 has been
investigated in human studies where it enhanced hepatic insulin sensitivity.7-8 Recently, two

groups have disclosed the effects of 11β-HSD1 inhibitors in Phase II trials in Type 2 diabetes
patients. When added to ongoing metformin therapy, a 200 mg dose of INCB13739 (structure
not disclosed) once daily for 12 weeks reduced HbA1c by 0.6% and fasting plasma glucose by
24 mg/dL.9 A 6 mg/day dose of 2 (MK-0916) once daily for 12 weeks did not improve fasting
plasma glucose; however, modest improvements in HbA1c, body weight and blood pressure
were observed.10 These studies support the hypothesis that a selective inhibitor of 11β-HSD1
may be useful in the treatment of diabetes and other morbidities associated with metabolic
syndrome. Additional compounds have entered clinical trials.6 Phase I results have been
reported for aminothiazolone 3 (AMG-221)11 and aminopyridine sulfonamide 4 (PF-915275).12
Piperazine sulfonamide 5 (HSD-016)13-14 and carboxylic acid 6 (AZ8329) have also completed
Phase 1.15
Figure 1. Selected 11β-HSD1 inhibitors that have entered clinical trials.
Recently, we reported potent oxazinanone-based 11β-HSD1 inhibitor 7 (Figure 2).16 Compound
7 inhibited 11β-HSD1 with IC50 values of 0.8 nM in a biochemical assay and 2.0 nM in a cell-

based human adipocyte assay. It displayed > 1000x selectivity over 11β-HSD2 and two other
related hydroxysteroid dehydrogenases. Further development of 7 was abandoned due to its lack
of efficacy in an adipose tissue-specific, cynomolgus monkey PD study. Since 7 is very
hydrophobic (log P 4.2) and highly protein bounded (> 99%), we reasoned that its unfavorable
physical properties may have contributed to the loss of efficacy in the cyno PD study. We
anticipated that a compound with improved physical properties would show a better correlation
between in vitro and in vivo potency. Here we detail our efforts to reduce the hydrophobicity
and increase the water solubility of 7, leading to the discovery of a clinical candidate BI 135585
(11j).17
Ser170
Tyr183
O
O
Tyr280'
NADP
N
HO
Tyr177
F
F
Tyr284'
Met286'
F
Val180
Leu126
Ile121
Met179
Thr124
Figure 2. The structure of compound 7 and its proposed binding mode in the substrate binding
site of human 11β-HSD1 (docked in 2IRW).
Lead optimization
A structure-based approach was adopted to optimize the physical chemical properties of 7. A
number of X-ray structures of 11β-HSD1 have been published.18-19 In our work we employed
the protein portion of the publicly available, homodimeric20 structure deposited as PDB entry
2IRW.21 The modeled pose of 7 in the 2IRW protein is depicted in Figure 2. The left hand side
4-fluorophenyl ring occupies a hydrophobic pocket formed by Ile121, Thr124, Leu126, and

Val180. The side chain hydroxyl group of 7 formed a hydrogen bond with one of the phosphate
oxygens of the cofactor NADP and highlighted one region of the molecule where more polar
substituents might be tolerated. The right hand side 2,4-difluorophenyl ring fits into a
hydrophobic pocket shaped by Leu126, Val180, Met179, and Tyr177, as well as Met286’,
Tyr284’ and Tyr280’ (not shown for the clarity of picture) from the partner monomer. Since the
2,4-difluorophenyl ring is proximal to the interface of the two monomers, it seemed plausible
that this hydrophobic ring could be replaced with more polar groups. A three-pronged approach
was thus taken to reduce the hydrophobicity of 7 (Figure 3): 1) introduction of a basic nitrogen to
permit salt formation; 2) removal of the 2, 4-difluorophenyl ring to reduce the molecular weight
and number of aromatic rings, thereby increasing the ratio of polar surface area to total surface
area; 3) replacement of the 2, 4-difluorophenyl ring with aromatic heterocycles.

Figure 3. Selected analogs prepared as part of a three-pronged approach to improve the polarity
of 7.
Initially, we focused on the hydroxyethyl side chain because the model indicated that polar
groups might be tolerated in that region. When the sidechain hydroxyl was replaced with a
primary amine, the resulting compound 8a was 3-fold less potent in the enzyme assay and 30-
fold less potent in a cell-based adipocyte assay (Table 1). Changing the primary amine to
secondary N-methyl amine 8b further reduced potency in the enzyme assay. The tertiary N,N-
dimethylamine 8c was 3-fold less potent than the 8b. However, a cyclic tertiary amine,
morpholine 8d, was 4-fold more potent than N-methyl amine 8b and 16-fold more potent than
the N,N-dimethylamine 8c, suggesting that the basicity of the amine nitrogen in 8c may
contribute more to the loss of potency than steric effects. Although both 8d and imidazolyl
compound 8e exhibited IC50 values < 5 nM in the enzyme assay, their cellular potencies (IC50
values of 48.7 and 19.8 nM, respectively) were unacceptable. Since none of the amine-
containing compounds met our criteria for advancement, the first approach to reduce
hydrophobicity was terminated.
Table 1. SAR of side chain amines

a
a
Enzyme IC50
Adipocyte IC50
Compound
R2‘
(nM)
0.8
(nM)
2.5
OH
NH2
NHMe
NMe2
7
2.7
23.6
80
66.5
244
8a
8b
8c
8d
8e
morpholine-4-yl
1H-imidazol-1-yl
4.8
1.4
48.7
19.8
a Average of at least two replicates.
Explorations to reduce hydrophobicity and improve water solubility through reduction in
molecular weight and the number of aromatic rings started with compound 9a (Table 2), a
synthetic precursor of 7. Compound 9a was a potent inhibitor of 11β-HSD1 with IC50 values of
1.7 and 3.0 nM in the enzyme and adipocyte assays, respectively. Addition of two methyl
groups on the side chain led to a tertiary alcohol 9b, which was 2-fold more potent in the enzyme
assay and more stable in a rat liver microsome (RLM) incubation assay than the primary alcohol
9a. Compound 9b, however, inhibited recombinant CYP3A4 with an IC50 of 3.5 µM. Replacing
the primary alcohol with a carboxamide (compound 9c) resulted in a 4-fold loss of potency.
However, homologous carboxamide 9d regained potency in both the enzyme and adipocyte
assays. Compound 9d exhibited reasonable stability (t1/2 36 min) in RLM incubation and no
inhibition of CYP3A4 up to 30 µM. Nitrile (9e) and sulfonamide (9f) groups in the side chain
improved the potency. However, neither compound was sufficiently stable in RLM’s for
advancement.

The side chain amide 9d was followed up with variations on the right (R3) and left (R1) hand side
phenyl rings. When bromine on the right hand phenyl ring was replaced by fluorine, chlorine,
trifluoromethyl, or nitrile (compounds 9g, 9h, 9j and 9k) groups, reductions in potency were
observed. Replacing the bromine with methyl or methoxyl groups (compound 9i and 9l) retained
potency in the adipocyte assay, but the compounds were unstable during RLM incubation.
Consequently, difluoromethoxyl and trifluoromethoxyl groups were introduced to improve
metabolic stability. The difluoromethoxyl analog (compound 9n) showed an IC50 of 5.4 nM in
adipocyte assay, was stable in RLM incubation (t1/2 > 60 min), and only weakly inhibited
CYP3A4 (IC50 27 µM). The trifluoromethoxyl analog (compound 9m) was less potent (IC50
14.5 nM in the adipocyte assay). Removal of the fluorine from the left phenyl ring (R1) of
compound 9n led to a 2-fold more potent compound 9o in the adipocyte assay. Compound 9o
also had good RLM stability (t1/2 50.6 min) and a clean CYP3A4 profile (IC50 > 30 µM).
Difluoromethoxyl analogs with tertiary alcohol side chains (compound 9p and 9q) also were
prepared. While both compounds exhibited excellent potency and metabolic stability, they
inhibited CYP3A4 with IC50 values of 6.6 and 10 µM, respectively.
Table 2. SAR of truncated right hand side compounds
Enzmye
a
IC50
(nM)
1.7
Adipocyte
a
IC50
(nM)
3.0
RLM t1/2
(min)
CYP3A4
IC50 (µM)
Cpd
R1
F
R2
R3
Br
CH2CH2OH
25
> 30
9a

F
F
F
F
F
F
F
F
F
F
F
F
F
H
F
H
CH2C(Me)2OH
CH2C(O)NH2
Br
Br
0.8
7.7
1.1
0.8
0.7
10.6
2.3
2.2
3.6
65.9
4.1
2.9
1.9
53.0
3.5
9b
9c
9d
9e
9f
9g
9h
9i
CH2CH2C(O)NH2
CH2C(Me)2CN
Br
Br
3.0
0.8
36.1
20.3
11.4
> 30
CH2CH2CH2NHSO2Me
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2CH2C(O)NH2
CH2C(Me)2OH
Br
F
Cl
Me
CF3
CN
OMe
OCF3
0.7
22.4
12.9
4.7
7.1
35.0
9j
9k
9l
9m
9n
9o
9p
9q
5.9
14.5
5.4
2.1
5.7
0.7
9.0
> 60
> 60
50.6
> 60
>60
OCHF2 2.0
OCHF2 2.0
OCHF2 1.2
OCHF2 1.3
27.0
>30
6.6
CH2C(Me)2OH
10
a Average of at least two replicates.
Based on in vitro potency, selectivity against the CYP enzymes, and stability during RLM
incubation, 9d, 9n, 9o and 9q were selected for further profiling. These compounds had good
aqueous solubility, ranging from 0.26 to 1.5 mg/mL, when tested as amorphous materials and
were stable when incubated with human liver microsomes (Table 3). When dosed to rats
intravenously, amides 9d and 9n exhibited acceptable mean residence times (MRT 1.2 and 0.84
h), albeit with high clearance rates of 49 and 67 mL/min/kg, respectively. Not surprisingly, the
des-fluoro amide 9o has a shorter mean residence time (MRT 0.55 h) and a higher clearance rate
(91 mL/min/kg) than its fluorinated counterpart 9n. The des-fluoro tertiary alcohol 9q, however,
showed a more moderate clearance rate of 23 mL/min/kg and an acceptable mean residence time
of 0.84 h. All four compounds appeared to be absorbed readily in a rat pharmacokinetic (PK)
study; bioavailability ranged from 42 to 77%. Further profiling revealed that amides 9d and 9n
inhibited human Ether-à-go-go-Related Gene (hERG) potassium ion channel with IC50 values of
6.3 and 5.3 µM, respectively, and these four compounds were not pursued further.

Table 3. Profile of selected truncated analogs 9
Rat PK
IV
MRT (mL/min
Aqueous
Solubility
(mg/mL)
HLM hERG
t1/2 IC50
(min) (µM)
Compound
IV CL
F
(%)
(h)
/kg)
49
0.30
0.46
1.5
> 90
> 90
> 90
> 90
6.3
5.3
1.2
42
77
51
57
9d
9n
9o
9q
0.84
0.55
0.84
67
91
0.26
23
Our third approach to reduce logP was to replace the distal phenyl ring in 7 with polar
heterocycles. Initial compounds were prepared with a hydroxypropyl sidechain (Table 4). All
three pyridine regioisomers (10a, 10b, and 10c) showed IC50 < 1 nM in both enzyme and cell
adipocyte assays. However, the 3- and 4- pyridinyl compounds, 10b and 10c, inhibited CYP3A4
with IC50 values of ~2 µM, while 2- pyridinyl compound 10a only marginally inhibited CYP3A4
with IC50 of 23 µM. Presumably, the more hindered environment in compound 10a prevented
the pyridine nitrogen from interacting effectively with the heme iron in CYP3A4. The same
phenomenon was observed with compound 10d, a 2,6-dimethyl analog of 10c, which did not
show any CYP3A4 inhibition up to 30 µM. Decreasing the basicity of the pyridine nitrogen was
also explored to alleviate CYP3A4 inhibition. The less basic fluoropyridine 10e (CYP3A4 IC50
10 µM) was less potent against CYP3A4 than its des-fluoro counterpart 10b (CYP3A4 IC50 2.1
µM). 2-Methoxypyridine 10f did not inhibit CYP3A4 up to 30 µM, presumably due to synergy
between electronic and steric effects. In addition, polar, but non-basic pyridinone 10g was
tolerated.
Table 4. SAR of pyridine analogs on the right hand side

Enzmye
IC50a (nM)
Adipocyte
IC50a (nM)
CYP 3A4
IC50 (µM)
Compound
R3
0.7
1.0
0.6
0.6
0.8
0.4
23 .3
2.1
10a
10b
10c
1.8
1.3
2.2
> 30
10d
0.5
2.3
0.5
4
11.0
> 30
10e
10f
1.4
5.7
10.5
10g
a Average of at least two replicates.
Although potent, these compounds generally were not stable to RLM incubation. For instance,
10d has t1/2 of 13 min in RLM. Identification of the metabolites generated during RLM

incubation implicated the propyl alcohol sidechain. To address this liability, analogs with
tertiary alcohol side chains were prepared (Table 5). Compound 11a, the tertiary alcohol
counterpart of compound 10d, exhibited IC50 values of 2.6 and 1.2 nM in enzyme and adipocyte
assays, respectively. It was stable in RLM (t1/2 > 45 min), although slightly less stable in
microsomes from cynomolgus monkeys (t1/2 38 min). The 2-methoxypyridine 11b was potent
against 11β-HSD1 (IC50 0.6 nM in enzyme assay), but inhibited CYP2C9 with an IC50 of 8 µM.
The fluoro- and cyano- pyridine analogs (compounds 11c and 11d) were potent in the adipocyte
assay and stable in liver microsome stability studies, but showed some inhibition of CYP3A4
(IC50 < 30 µM). The carboxamide 11e represented a compound which was potent, stable in rat,
monkey and human liver microsomes, and had a clean CYP profile (no inhibition up to 50 µM);
however, 11e had low bioavailability in rat (15%) and was not pursued further. Analogs with
more electron deficient heterocycles, such as pyrazine (11f), pyridazine (11g) or pyrimidine
(11h), combined potency with clean CYP profiles. While 11f suffered from a short t1/2 (16 min)
in RLM incubation, 11g and 11h showed good stability in rat, cynomolgus monkey and human
liver microsomes (t1/2 > 45 min). The pyridinones 11i and 11j and pyridazinone 11k also were
potent inhibitors of 11β-HSD1. They exhibited clean CYP profiles (IC50 > 43 µM) and were
stable in rat, cyno and human microsomes (t1/2 > 90 min, except for compound 11k in
cynomolgus monkey microsome where t1/2 > 45 min). The five member thiazole analog 11l was
less potent, with IC50 of 11.3 nM in the adipocyte assay. The bicyclic triazolopyridine 11m
inhibited CYP3A4 with an IC50 of 10 µM. Incorporation of aromatic heterocycles led to the
identification of compounds 11g-k which passed preliminary metabolic stability and drug-drug
interaction screening.
Table 5. SAR of right hand side heterocycle compounds bearing tertiary alcohol side chain

CYPs
IC50 (µM)
uSome Stability
t1/2 (min)
Adipocyte
a
IC50
(nM)
Enzmye
IC50 a (nM)
Cpd.
R
Cyn
o
3A4 2D6 2C9 Rat
Human
2.6
1.2
> 50 > 50 21.4 > 45
38
> 45
11a
0.6
0.8
1.1
0.9
0.6
0.8
11.6 > 50
8
11b
11c
11d
24.1 > 50 20.1 > 45
> 45 > 45
> 90 > 90
N
F
12.7 > 50 22.2 > 90
1.6
1.1
> 50 > 50 > 50 > 45
> 45 > 90
11e
1.0
1.2
1.2
2.5
> 50 > 50 33
16
38
> 90
11f
> 50 > 50 > 50 > 45
> 45 > 90
> 90 > 90
11g
2.5
1.6
3.7
3.3
> 50 > 50 > 50 > 90
11h
11i
48
> 50 43
> 90
> 90 > 90

1.7
1.8
1.7
4.3
> 50 > 50 > 50 > 90
> 90 > 90
11j
11k
11l
1.0
> 50 > 50 > 50 > 90
> 45 > 90
12.3
1.4
3.8
10
> 50 > 50 > 90
> 45 > 90
11m
a Average of at least two replicates.
Compound selection
The failure of prototype compound 7 to show reasonable inhibition of 11β-HSD1 enzyme
activity in the adipose tissues of acutely dosed cynomolgus monkeys, prompted the development
of a novel assay to identify compounds more likely to succeed in vivo. To this end, an in vitro
assay was developed using primary human adipose tissue collected during surgery. Tissue
fragments were incubated in cell culture media +/- selected compounds and the conversion of
cortisone to cortisol was measured.17 Compound 7 had an IC80 > 1 µM in this assay, consistent
with minimal efficacy in the monkey pharmacodynamic (PD) study. Thus an IC80 ≤ 100 nM in
this assay was set as criteria for new compounds to advance to in vivo analysis. Other criteria
included selectivity against the hERG channel and good PK in non-human primates.

The five previously selected compounds 11g-k were filtered with these criteria. All five
compounds met the human adipose tissue criterion and two (11g and 11j) exceeded it (Table 6).
A patch-clamp assay for hERG was performed at 10 µM; 50% and 12% hERG inhibition values
were observed with compounds 11k and 11h respectively, while 11g, 11i and 11j did not
significantly inhibit hERG (< 10%). When administered intravenously to cynomolgus monkeys,
11g-j showed low clearance rates and good exposure (Table 6). Compound 11j had the best
potency in the adipose tissue assay, did not inhibit hERG, exhibited the longest MRT in
cynomolgus monkeys, and was therefore chosen for further characterization.
Table 6. Inhibition of 11β-HSD1 in human adipose tissue, hERG and cyno PK for selected
compounds
Adipose Inhibition Cyno IV
Cyno PO
MRT
tissue
a
IC80
(nM)
76
of hERG
at 10µM
(%)
Compound
dnAUC MRT
(nMh)
CL Vss
(mL/min/kg) (L/kg) (h)
F
(%)
(h)
< 10
12
< 10
< 10
50
3330
2070
1770
2500
4.8
3.5
2.7
5.5
5.2
8.1
11
1.5
1.7
1.7
2.5
5.3
4.8
4.8
8.7
70
96
39
54
11g
11h
11i
100
100
53
7.5
11j
11k
100
a Average of at least two replicates.
Further characterization
Compound 11j was further screened against three related hydroxysteroid dehydrogenases and
for other off target activities (Table 7). It exhibited > 1000x selectivity over 11β-HSD2, 3β-
hydroxysteroid dehydrogenase type 2 (3β-HSD2), 17β-hydroxysteroid dehydrogenase type 1
(17β-HSD1) and pregnane X receptor (PXR). Moreover, there were no significant findings in

the SpectrumScreen of 168 assays (Ricerca) when tested at 10 µM. Compound 11j also was
retested against the hERG channel and exhibited an IC50 > 100 µM. Regarding physical
properties, 11j had a measured logP of 2.6 and a human plasma protein binding of 91.6%,
representing a significant improvement over compound 7 (log P 4.2, protein binding > 99%).
The measured water solubility of 11j was 0.22 mg/mL, whereas the water solubility of 7 was
below the limit of detection. Compound 11j was docked in the substrate binding site of human
11β-HSD1 (2IRW, Figure 4). It adopted similar binding mode as that of compound 7, with the
polar pyridone group pointing to the dimer interface. This binding mode supported the outcome
that compound 11j maintained potency, since the interface region was expected to be more
tolerable to polar groups.
Table 7. Further characterization of 11j
>10
>10
>10
>10
>10
2.6
11β-HSD2 IC50 (µM)
3β-HSD2 IC50 (µM)
17β-HSD1 IC50 (µM)
PXR EC50 (µM)
SpectrumScreen (168 assays) IC50 (µM)
Log P
Plasma protein binding (%, human)
Hepatocyte CL (mL/min/kg, human)
hERG IC50 (µM)
91.6
0.2
> 100

Figure 4. The docked binding pose of compound 11j in the substrate binding site of human 11β-
HSD1 (docked in 2IRW).
Cynomolgus monkey PD study
As described elsewhere, compound 11j was further evaluated in a cynomolgus monkey PD
model measuring conversion of cortisone to cortisol, ex vivo.17 Briefly, treatment with 11j at
doses of 1 mg/kg and 3 mg/kg po, inhibited 67% and 90% of enzyme activity respectively in
perirenal adipose tissue. In abdominal subcutaneous adipose tissue, 67% and 95% reductions of
cortisol were observed, relative to vehicle treated animals. Thus, 11j is effective in lowering
11β-HSD1 activity in adipose tissues in non-human primates when dosed orally. After
successfully completing safety assessment, 11j entered human clinical trials in 2011.
Chemistry
Compounds of general structure 8 (Table 1) were prepared as shown in Scheme 1. The primary
hydroxyl group in compound 7 was mesylated, displaced with sodium azide and reduced with
triphenylphosphine to give primary amine 8a. Boc protection of 8a, followed by methylation of
the resultant carbamate, and deprotection, provided methylamine 8b. Heating 8a with
formaldehyde and formic acid gave dimethylamine 8c. The mesylate of compound 7 could also
be directly displaced with morpholine or imidazole to give compound 8d and 8e.

Scheme 1. Synthesis of side chain amine 8.
a
Reagents and conditions. (i) MsCl, Et3N, CH2Cl2, 0 °C to rt; (ii) NaN3, DMF, 70 °C; (iii) PPh3, 20:1
THF/H2O, rt; b For 8b (i) Boc2O, Et3N, CH2Cl2, 0 °C to rt; (ii) NaH, MeI, THF, 0 °C to rt; (iii) 20% TFA/
CH2Cl2, 0 °C; For 8c HCHO, HCO2H, 90 °C; c (i) MsCl, Et3N, CH2Cl2, 0 °C to rt; (ii) For 8d
morpholine, Et3N, CH2Cl2, 0 °C to rt; For 8e: imidazole, K2CO3, MeCN, reflux.
Compound 9a (Table 2) was prepared as described previously.16 Preparations of other
compounds of general structure of 9 are depicted in Scheme 2. Compounds 9b, 9p and 9q were
prepared from the respective allyl oxazinanones 12b, 12p and 12q. 16 The allyl oxazinanones
were subjected to Wacker oxidation to give inseparable mixtures of methyl ketone 13 and
aldehydes. The mixtures were then treated with sodium chlorite which oxidized the aldehydes to
carboxylic acids which were soluble in the aqueous layer, allowing separation from 13. Methyl
Grignard addition to ketone 13 provided tertiary alcohols 9b, 9p and 9q. Compound 9c was
prepared from 12b via a sequence of dihydroxylation, oxidative cleavage of the resultant diol to
an acid and amide coupling. Hydroboration of 12b provided alcohol 15b, which was oxidized to
the carboxylic acid and coupled with ammonia to give carboxamide 9d. Compounds 9g-o were
prepared using the same reaction sequence used for 9d. Cobalt catalyzed hydrocyanation of

olefin 12b provided nitrile 16, which upon methylation gave gem-dimethylnitrile 9e. Compound
9f was obtained from alcohol 15b via mesylation then methanesulfonamide displacement.
Compounds with general structure of 10 (Table 4) were prepared according to Scheme 3.
Hydroboration of olefin 12a provided propylalcohol 15a, which was converted to boronic ester
18. Suzuki coupling of bromide 15a with various heteroaryl boronic acids or 18 with various
heterocyclic halides gave compounds 10a-g. Similarly, compounds 11a-m (Table 5) were
prepared by Suzuki couplings with bromide 9r or boronic ester 19 (Scheme 5). Following the
selection of 11j as a clinical candidate an enantioselective synthesis was developed.22

Scheme 2. Synthesis of truncated right hand side compounds 9.

a
Reagents and conditions. (i) PdCl2, CuCl, O2, DMF, H2O, rt; (ii) NaClO2, NaH2PO4, 2-methyl-2-
butene, t-BuOH, H2O; b MeMgBr, THF, -78 °C; c OsO4, NMO, t-BuOH, CH2Cl2, rt; d (i) RuCl3, Oxone,
e
NaHCO3, H2O/CH3CN/EtOAc, rt; (ii) HATU, NH3, DIPEA, DMF, rt; (i) BH3THF, THF, 0 °C; (ii)
NaOH, H2O2, rt; ; f CrO3, H2SO4, H2O/acetone, 0 °C; g [N,N’-(1,1,2,2-tetramethylethylene)bis(3,5-di-
i
tert-butylsalicylideneiminato)]cobalt(II), TsCN, PhSiH3, EtOH, rt; h LiHMDS, MeI, THF, -78 °C;
j
MsCl, Et3N, CH2Cl2, 0 °C to rt; MeSO2NH2, K2CO3, CH3CN, rt.
O
O
O
O
N
O
N
O
N
b
a
O
Br
Br
B
O
OH
OH
18
15a
12a
c
c
N
=
N
N
N
O
N
N
O
10a
10b
10c
10d
F
N
N
O
N
N
OMe
OH
10
10e
10f
10g
Scheme 3. Synthesis of right hand side heterocycle compounds 10.
a
Reagents and conditions. (i) BH3.THF, THF, 0 °C; (ii) NaOH, H2O2, rt; b PdCl2, pinacolborane,
KOAc, DMSO, 90 °C; c Pd(PPh3)2Cl2, ArBr or ArB(OH)2, Cs2CO3, 1,4-dioxane, 120 °C.

Scheme 4. Synthesis of right hand side heterocycle compounds 11.
a
Reagents and conditions. Reagents and conditions. (i) PdCl2, CuCl, O2, DMF, H2O, rt; (ii) NaClO2,
c
NaH2PO4, 2-methyl-2-butene, t-BuOH, H2O; b MeMgBr, THF, -78 °C; PdCl2, pinacolborane, KOAc,
DMSO, 90 °C;d Pd(PPh3)2Cl2, ArX or ArB(OH)2, Cs2CO3, 1,4-dioxane, 120 °C.
Conclusion
We have described our efforts to improve the efficacy of compound 7 through modulation of its
physical properties. To this end, we have successfully introduced polar groups into 7 and
identified 11j which has lower hydrophobicity (log P 2.6) and protein binding (91.6%).
Compound 11j is a potent inhibitor of 11β-HSD1 in human adipocytes with > 1000x selectivity
over three other hydroxysteroid dehydrogenases, including 11β-HSD2. More importantly, 11j

inhibits the conversion of cortisone to cortisol in primary human adipose tissue with IC80 values
of 53 nM, compared to IC80 > 1 µM for 7. Oral administration of 11j to cynomolgus monkey
inhibited 11β-HSD1 activity in adipose tissues. Compound 11j displays desirable
pharmacodynamic properties, meets safety assessment criteria, and entered human clinical trials
in 2011.
Supporting Information Available
Synthetic procedures as well as NMR and LC-MS data on new compounds; assay protocols.
1.
http://www.who.int/diabetes/global-report/en/ (accessed 02/15/2017).
2. Mohler, M. L.; He, Y.; Wu, Z.; Hwang, D. J.; Miller, D. D., Recent and emerging anti-
diabetes targets. Med. Res. Rev. 2009, 29, 125-195.
3. Tanaka, N.; Nonaka, T.; Nakamura, K. T.; Hara, A., SDR: structure, mechanism of
action, and substrate recognition. Curr. Org. Chem. 2001, 5, 89-111.
4. Bray, J. E.; Marsden, B. D.; Oppermann, U., The human short-chain
World Health Organization Global report on diabetes.
dehydrogenase/reductase (SDR) superfamily: A bioinformatics summary. Chem.-Biol. Interact.
2009, 178, 99-109.
5.
dehydrogenase type 1 and metabolic disease. Ann. N. Y. Acad. Sci. 2006, 1083, 165-184.
6. Scott, J. S.; Goldberg, F. W.; Turnbull, A. V., Medicinal Chemistry of Inhibitors of 11β-
Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1). J. Med. Chem. 2014, 57, 4466-4486.
7. Walker, B. R.; Connacher, A. A.; Lindsay, R. M.; Webb, D. J.; Edwards, C. R. W.,
Walker, B. R.; Andrew, R., Tissue production of cortisol by 11beta-hydroxysteroid
Carbenoxolone increases hepatic insulin sensitivity in man: a novel role for 11-oxosteroid
reductase in enhancing glucocorticoid receptor activation. J. Clin. Endocrinol. Metab. 1995, 80,
3155-9.
8. Taylor, A.; Irwin, N.; McKillop, A. M.; Flatt, P. R.; Gault, V. A., Sub-chronic
administration of the 11beta -HSD1 inhibitor, carbenoxolone, improves glucose tolerance and
insulin sensitivity in mice with diet-induced obesity. Biol. Chem. 2008, 389, 441-445.
9.
Rosenstock, J.; Banarer, S.; Fonseca, V. A.; Inzucchi, S. E.; Sun, W.; Yao, W.; Hollis,
G.; Flores, R.; Levy, R.; Williams, W. V.; Seckl, J. R.; Huber, R., The 11-beta-hydroxysteroid
dehydrogenase type 1 inhibitor INCB13739 improves hyperglycemia in patients with type 2
diabetes inadequately controlled by metformin monotherapy. Diabetes Care 2010, 33, 1516-
1522.

10.
Feig, P. U.; Shah, S.; Hermanowski-Vosatka, A.; Plotkin, D.; Springer, M. S.; Donahue,
S.; Thach, C.; Klein, E. J.; Lai, E.; Kaufman, K. D., Effects of an 11β-hydroxysteroid
dehydrogenase type 1 inhibitor, MK-0916, in patients with type 2 diabetes mellitus and
metabolic syndrome. Diabetes, Obes. Metab. 2011, 13, 498-504.
11.
Gibbs, J. P.; Emery, M. G.; McCaffery, I.; Smith, B.; Gibbs, M. A.; Akrami, A.; Rossi, J.;
Paweletz, K.; Gastonguay, M. B.; Bautista, E.; Wang, M.; Perfetti, R.; Daniels, O., Population
pharmacokinetic/pharmacodynamic model of subcutaneous adipose 11.beta -hydroxysteroid
dehydrogenase type 1 (11.beta -HSD1) activity after oral administration of AMG 221, a selective
11.beta -HSD1 inhibitor. Journal of Clinical Pharmacology 2011, 51, 830-841.
12.
Courtney, R.; Stewart, P. M.; Toh, M.; Ndongo, M.-N.; Calle, R. A.; Hirshberg, B.,
Modulation of 11beta -Hydroxysteroid dehydrogenase (11beta HSD) activity biomarkers and
pharmacokinetics of PF-00915275, a selective 11beta HSD1 inhibitor. J. Clin. Endocrinol.
Metab. 2008, 93, 550-556.
13. Wan, Z.-K.; Chenail, E.; Li, H.-Q.; Kendall, C.; Wang, Y.; Gingras, S.; Xiang, J.;
Massefski, W. W.; Mansour, T. S.; Saiah, E., Synthesis of potent and orally efficacious 11.beta -
hydroxysteroid dehydrogenase type 1 inhibitor HSD-016. J. Org. Chem. 2011, 76, 7048-7055.
14. Wan, Z. K. Development of HSD-016 and HSD-621 as potential agents for the treatment
of type 2 diabetes, 4th World Congress on Diabetes & Metabolism, Chicago, IL, August 14-16;
2013.
15.
Wang, Y.; Zhang, Y.; Wang, X.; Liu, Y.; Xia, M., Cyanidin-3-O-β-glucoside induces
oxysterol efflux from endothelial cells: Role of liver X receptor alpha. Atherosclerosis
(Amsterdam, Neth.), Ahead of Print.
16.
Xu, Z.; Tice, C. M.; Zhao, W.; Cacatian, S.; Ye, Y.-J.; Singh, S. B.; Lindblom, P.;
McKeever, B. M.; Krosky, P. M.; Kruk, B. A.; Berbaum, J.; Harrison, R. K.; Johnson, J. A.;
Bukhtiyarov, Y.; Panemangalore, R.; Scott, B. B.; Zhao, Y.; Bruno, J. G.; Togias, J.; Guo, J.;
Guo, R.; Carroll, P. J.; McGeehan, G. M.; Zhuang, L.; He, W.; Claremon, D. A., Structure-Based
Design and Synthesis of 1,3-Oxazinan-2-one Inhibitors of 11.beta -Hydroxysteroid
Dehydrogenase Type 1. J. Med. Chem. 2011, 54, 6050-6062.
17.
Hamilton, B. S.; Himmelsbach, F.; Nar, H.; Schuler-Metz, A.; Krosky, P.; Guo, J.; Guo,
R.; Meng, S.; Zhao, Y.; Lala, D. S.; Zhuang, L.; Claremon, D. A.; McGeehan, G. M.,
Pharmacological characterization of the selective 11β-hydroxysteroid dehydrogenase 1 inhibitor,
BI 135585, a clinical candidate for the treatment of type 2 diabetes. European Journal of
Pharmacology 2015, 746, 50-55.
18.
Pharmaceutical Biotechnology 2010, 11, 779-791.
19. Thomas, M. P.; Potter, B. V. L., Crystal structures of 11.beta -hydroxysteroid
Singh, S. B.; Tice, C. M., Structure based design of 11beta -HSD1 inhibitors. Current
dehydrogenase type 1 and their use in drug discovery. Future Medicinal Chemistry 2011, 3, 367-
390.
20. Maser, E.; Voelker, B.; Friebertshaeuser, J., 11beta -Hydroxysteroid Dehydrogenase
Type 1 from Human Liver: Dimerization and Enzyme Cooperativity Support Its Postulated Role
as Glucocorticoid Reductase. Biochemistry 2002, 41, 2459-2465.
Patel, J. R.; Shuai, Q.; Dinges, J.; Winn, M.; Pliushchev, M.; Fung, S.; Monzon, K.;
21.
Chiou, W.; Wang, J.; Pan, L.; Wagaw, S.; Engstrom, K.; Kerdesky, F. A.; Longenecker, K.;
Judge, R.; Qin, W.; Imade, H. M.; Stolarik, D.; Beno, D. W. A.; Brune, M.; Chovan, L. E.;
Sham, H. L.; Jacobson, P.; Link, J. T., Discovery of adamantane ethers as inhibitors of 11beta-
HSD-1: Synthesis and biological evaluation. Bioorg. Med. Chem. Lett. 2007, 17, 750-755.

22.
Zhang, Y.; Wu, J.-P.; Li, G.; Fandrick, K. R.; Gao, J.; Tan, Z.; Johnson, J.; Li, W.;
Sanyal, S.; Wang, J.; Sun, X.; Lorenz, J. C.; Rodriguez, S.; Reeves, J. T.; Grinberg, N.; Lee, H.;
Yee, N.; Lu, B. Z.; Senanayake, C. H., An Enantioselective Synthesis of an 11-β-HSD-1
Inhibitor via an Asymmetric Methallylation Catalyzed by (S)-3,3′-F2-BINOL. The Journal of
Organic Chemistry 2016, 81, 2665-2669.