Discovery of 2-((R)-4-(2-Fluoro-4-(methylsulfonyl)phenyl)-2-methylpiperazin-1-yl)-N-((1R,2s,3S,5S,7S)-5-hydroxyadamantan-2-yl)pyrimidine-4-carboxamide (SKI2852): A Highly Potent, Selective, and Orally Bioavailable Inhibitor of 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1)

Article abstract of DOI:10.1021/acs.jmedchem.6b01122

A series of picolinamide- and pyrimidine-4-carboxamide-based inhibitors of 11β-hydroxysteroid dehydrogenase type 1 was synthesized and evaluated to optimize the lead compound 9. The combination of the replacement of a pyridine ring of 9 with a pyrimidine ring and the introduction of an additional fluorine substituent at the 2-position of the phenyl ring resulted in the discovery of a potent, selective, and orally bioavailable inhibitor, 18a (SKI2852), which demonstrated no CYP and PXR liabilities, excellent PK profiles across species, and highly potent and sustainable PD activity. Repeated oral administration of 18a significantly reduced blood glucose and HbA1c levels and improved the lipid profiles in ob/ob mice. Moreover, the HbA1c-lowering effect of metformin was synergistically enhanced in combination with 18a.

Full text of DOI:10.1021/acs.jmedchem.6b01122

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Article
Discovery of 2-((R)-4-(2-Fluoro-4-(methylsulfonyl)phenyl)-2-methylpiperazin-1-
yl)-N-((1R,2s,3S,5S,7S)-5-hydroxyadamantan-2-yl)pyrimidine-4-
carboxamide (SKI2852): A Highly Potent, Selective, and Orally Bioavailable
Inhibitor of 11#-Hydroxysteroid Dehydrogenase Type 1 (11#-HSD1)
Je Ho Ryu, Jung A Lee, Shinae Kim, Young Ah Shin, Jewon Yang, Hye Young Han, Hyun Joo Son,
Yong Hyuk Kim, Joon Ho Sa, Jae-Sun Kim, Jungeun Lee, Jeeyeon Lee, and Hyeung-Geun Park
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01122 • Publication Date (Web): 31 Oct 2016
Downloaded from http://pubs.acs.org on November 2, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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However, no copyright claim is made to original U.S. Government works, or works
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Discovery of 2ꢀ((R)ꢀ4ꢀ(2ꢀFluoroꢀ4ꢀ(methylsulfonyl)phenyl)ꢀ2ꢀ
methylpiperazinꢀ1ꢀyl)ꢀNꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀ
yl)pyrimidineꢀ4ꢀcarboxamide (SKI2852): A Highly Potent, Selective, and
Orally Bioavailable Inhibitor of 11βꢀHydroxysteroid Dehydrogenase Type
1 (11βꢀHSD1)
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†
,‡
†
†,§
†
‡
†
Je Ho Ryu, Jung A Lee, Shinae Kim, Young Ah Shin, Jewon Yang, Hye Young Han,
†
†
†
†,∥
‡
Hyun Joo Son, Yong Hyuk Kim, Joon Ho Sa, JaeꢀSun Kim,
Jungeun Lee, Jeeyeon
‡
*,‡
Lee, and Hyeungꢀgeun Park
†
Life Science R&D Center, SK Chemicals, SeongnamꢀSi, BundangꢀGu, SampyeongꢀDong
686, GyeonggiꢀDo, 463ꢀ400, Korea
‡
Research Institute of Pharmaceutical Science and College of Pharmacy, Seoul National
University, Seoul, 151ꢀ742, Korea
ABSTRACT
A series of picolinamideꢀ and pyrimidineꢀ4ꢀcarboxamideꢀbased inhibitors of 11βꢀ
hydroxysteroid dehydrogenase type 1 was synthesized and evaluated to optimize the lead
compound 9. The combination of the replacement of a pyridine ring of 9 with a pyrimidine
ring and the introduction of an additional fluorine substituent at the 2ꢀposition of the phenyl
ring resulted in the discovery of a potent, selective, and orally bioavailable inhibitor, 18a
(SKI2852), which demonstrated no CYP and PXR liabilities, excellent PK profiles across
species, and highly potent and sustainable PD activity. Repeated oral administration of 18a
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significantly reduced blood glucose and HbA1c levels and improved the lipid profiles in
ob/ob mice. Moreover, the HbA1cꢀlowering effect of metformin was synergistically enhanced
in combination with 18a.
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INTRODUCTION
An estimated 285 million people worldwide suffered from diabetes in the year 2010, and this
1
number is expected to rise to 438 million by the year 2030. The majority of these cases are
defined as type 2 diabetes mellitus, which is a combination of metabolic abnormalities, such
as hyperglycemia and insulin resistance. Metabolic syndrome is a prediabetic state that
consists of central obesity, glucose intolerance, insulin resistance, dyslipidemia, hypertension,
2
and inflammatory or prothrombotic states. When left untreated, metabolic syndrome
progresses to type 2 diabetes and results in serious complications, including cardiovascular
disease, diabetic retinopathy, renal failure, nerve damage, and ischemic stroke.
Recent studies have implicated aberrant glucocorticoid signaling as a cause of the
3
development of several phenotypes that are associated with metabolic syndrome. Elevated
levels of active glucocorticoids in the liver and adipose tissue can lead to insulin resistance,
impairment of glucose uptake, enhanced hepatic gluconeogenesis, and increased lipolysis. In
this respect, an attractive therapeutic target is 11βꢀhydroxysteroid dehydrogenase type 1 (11βꢀ
4
ꢀ7
HSD1). 11βꢀHSD1 is a key enzyme that acts as an NADPHꢀdependent reductase and
converts inactive glucocorticoids (cortisone in humans and 11ꢀdehydrocorticosterone in
rodents) into the receptorꢀactive glucocorticoids (cortisol in humans and corticosterone in
rodents). This enzyme is highly expressed in metabolically active tissues, including the liver
8ꢀ11
and adipose tissue, and regulates tissueꢀspecific glucocorticoid levels.
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The connection between 11βꢀHSD1 and metabolic syndrome has been demonstrated in
several studies that use a genetic mouse model. Transgenic mice overexpressing 11βꢀHSD1 in
only the liver or adipose tissues showed features of metabolic syndrome, such as abdominal
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obesity, glucose intolerance, insulin resistance, dyslipidemia, and hypertension.
In
contrast, 11βꢀHSD1ꢀdeficient mice demonstrated a reduction in body weight, improved lipid
profiles and glucose tolerance, and increased insulin sensitivity when maintained on a highꢀfat
15,16
diet.
These findings suggest that 11βꢀHSD1 inhibition could be a promising strategy for
the treatment of metabolic syndrome as well as type 2 diabetes.
In the last decade, extensive research by the pharmaceutical industry has identified
1
7ꢀ19
various classes of 11βꢀHSD1 inhibitors,
and several compounds have reached the clinicalꢀ
2
0
development phase. Incyte took the inhibitor INCBꢀ13739 (structure not disclosed) as far as
phase 2 trials as an addꢀon therapy to metformin for patients with type 2 diabetes exhibiting
inadequate glycemic control with metformin alone. After 12 weeks of treatment, this
compound resulted in significant reductions in HbA1c (ꢀ0.6%), fasting plasma glucose (ꢀ24
2
0
mg/dl), and HOMAꢀIR (ꢀ24%) compared with placebo. Merck has progressed two
21,22
22
compounds, 1 (MKꢀ0916)
and 2 (MKꢀ0736) (Figure 1), into clinical trials. The
administration of 1 to patients with type 2 diabetes and metabolic syndrome led to modest
2
1
decreases in HbA1c (ꢀ0.3%), body weight, and blood pressure. The compound 2 modestly
improved secondary blood pressure endpoints, LDLꢀcholesterol, and body weight in
22
overweight and obese patients with hypertension. However, none of these compounds have
2
3,24
progressed beyond phase 2 trials. Other companies, for example, Pfizer (3, PFꢀ915275),
2
5
26
Amgen (4, AMGꢀ221), Astrazeneca (5, AZDꢀ4017), and Boehringer Ingelheim (6, BI
27
1
35585), have also progressed compounds into the clinic, and the structures of them are
shown in Figure 1.
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Figure 1. Chemical structures of 11βꢀHSD1 inhibitors that advanced into clinical trials
We have previously described a novel class of 11βꢀHSD1 inhibitors that contain the
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picolinamide core.
Structural modifications of the initial hit compound 7 resulted in the
discovery of the highly potent and metabolically stable compound 8. High potency was
achieved by the incorporation of a hydroxyꢀadamantyl group, and the replacement of the
piperidine ring with 1ꢀ(4ꢀcyanophenyl)piperazine led to a significant improvement in
2
8
metabolic stability. Further optimization was performed to improve both the potency and
pharmacokinetic (PK) profiles of 8. The replacement of the cyano group at the 4ꢀposition of
the phenyl ring with a methylsulfonyl group resulted in an increase in its biochemical and
cellular potencies, and modifying the piperazine ring by introducing an (R)ꢀmethyl group
substantially improved the PK properties. This lead compound 9 demonstrated excellent
activity in a mouse ex vivo pharmacodynamic (PD) model and reduced the blood glucose,
2
9
LDL cholesterol, and triglyceride levels in ob/ob mice after oral administration (Figure 2).
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Figure 2. Progress of picolinamide 11βꢀHSD1 inhibitors from hit (7) to lead (9)
However, profiling in drug metabolism studies showed that compound 9 was a moderateꢀ
toꢀstrong inhibitor of CYP3A4 and CYP2C19, exhibiting IC50 values of 0.97 ꢁM and 3.7 ꢁM,
respectively. Therefore, in this report, we attempted further structural optimization of 9 to
diminish the CYP liability while maintaining the high in vitro potency and ex vivo PD
activity. Our strategy to reduce the interaction with CYP enzymes consisted in decreasing the
lipophilicity of the molecule by introducing a more polar moiety to the leftꢀhand side or
replacing the central pyridine ring with a pyrimidine ring.
CHEMISTRY
Leftꢀhand sideꢀmodified polar picolinamide analogs 13aꢀh were synthesized via the routes
that are outlined in Scheme 1. Coupling 6ꢀbromopicolinic acid with transꢀ4ꢀaminoadamantanꢀ
1
ꢀol in the presence of HBTU yielded 6ꢀbromopicolinamide 10. A palladiumꢀcatalyzed crossꢀ
coupling reaction of 10 with (R)ꢀ4ꢀNꢀBOCꢀ2ꢀmethylpiperazine, followed by treatment with
TFA, provided intermediate 11. Carboxylic acid derivatives 12 and 13a were obtained from
the basic hydrolysis reaction of the ester intermediates, which were synthesized by the
palladiumꢀassisted coupling of 11 with methyl 4ꢀbromobenzoate or methyl 5ꢀ
3
0
bromopicolinate. Compounds 12 and 13a were then transformed into the amide derivatives
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3bꢀe via HBTU coupling with the appropriate amine. The palladiumꢀmediated reaction of 11
with 4ꢀbromobenzenesulfonamides that were protected with tertꢀbutyl groups produced the
desired Nꢀ(tertꢀbutyl) sulfonamide intermediates, which were treated with TFA to give
sulfonamide analogs 13f and 13g. The reaction of 11 with 2ꢀ(5ꢀbromopyridinꢀ2ꢀyl)propanꢀ2ꢀ
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2 3
ol in the presence of Pd (dba) and BINAP afforded derivative 13h.
Pyrimidineꢀ4ꢀcarboxamide compounds 17aꢀg and 18aꢀf were prepared as outlined in
Scheme 2. 2ꢀChloropyrimidineꢀ4ꢀcarboxylic acid was converted to the corresponding tertꢀ
3
1
butyl ester by using pꢀtoluenesulfonyl chloride and tertꢀbutanol. Subsequently, a coupling
reaction with (R)ꢀ1ꢀbenzylꢀ3ꢀmethylpiperazine yielded (R)ꢀ2ꢀ(4ꢀbenzylꢀ2ꢀmethylpiperazinꢀ1ꢀ
yl)pyrimidine 14. The TFAꢀmediated removal of the tertꢀbutyl group and the HBTUꢀassisted
coupling with transꢀ4ꢀaminoadamantanꢀ1ꢀol produced carboxamide 15, which was then
hydrogenated to give intermediate 16. The final compounds 17aꢀg and 18aꢀc were
synthesized by the palladiumꢀcatalyzed reaction of 16 with the commercially available 4ꢀ
3
2
substituted bromobenzenes.
Sulfonyl derivatives 18dꢀf, whose corresponding
bromobenzenes are not available, were prepared by the reaction of 16 with the appropriate 4ꢀ
sulfonyl fluorobenzenes at high temperatures.
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a
Scheme 1
O
O
R
HO
X
N
H
X
N
O
OH
N
O
OH
N
N
f
N
N
N
N
H
H
10
11
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3a X = CH
13b X = CH, R = H
13c R = 4-Cl R = Me
12 X = N
1
1
3d X = N, R = H
3e X = N, R = Me
d, e
O
O
S
R
N
H
O
O
OH
HN
O
N
OH
N
O
OH
Br
N
a
Br
N
b, c
N
g, h
N
N
OH
N
N
H
N
H
H
10
11
13f R = H
13g R = Me
i
HO
N
N
O
N
OH
N
N
H
13h
a
Reagents and conditions: (a) transꢀ4ꢀaminoadamantanꢀ1ꢀol, HBTU, DIPEA, ACN, 85%; (b) (R)ꢀtertꢀ
t
butyl 3ꢀmethylpiperazineꢀ1ꢀcarboxylate, Pd
2
(dba)
3
, xantphos, NaO Bu, toluene, 100 ºC, 54%; (c)
2
TFA, DCM, 92%; (d) methyl 4ꢀbromobenzoate or methyl 5ꢀbromopicolinate, Pd(OAc) , xantphos,
Cs
8%;
methylbenzenesulfonamide, Pd[P(oꢀtolyl)
DCM, 56ꢀ64%; (i) 2ꢀ(5ꢀbromopyridinꢀ2ꢀyl)propanꢀ2ꢀol, Pd
9%.
2
CO
3
, toluene, 100 ºC, 46ꢀ59%; (e) NaOH, MeOH, 74ꢀ88%; (f) RNH
(g) 4ꢀbromoꢀNꢀ(tertꢀbutyl)benzenesulfonamide or
Cl , BINAP, Cs CO
(dba)
2
, HBTU, DIPEA, ACN, 55ꢀ
7
4ꢀbromoꢀNꢀ(tertꢀbutyl)ꢀNꢀ
3
]
2
2
2
3
, toluene, 90 ºC, 38ꢀ96%; (h) TFA,
t
2
3
, BINAP, NaO Bu, toluene, 100 ºC,
2
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Scheme 2
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a
t
Reagents and conditions: (a) pꢀtoluenesulfonyl chloride, pyridine, BuOH, 62%; (b) (R)ꢀ1ꢀbenzylꢀ3ꢀ
methylpiperazine, DIPEA, ACN, 100 ºC, 94%; (c) TFA, DCM, quantitative yield; (d) transꢀ4ꢀ
aminoadamantanꢀ1ꢀol, HBTU, DIPEA, ACN, 68%; (e) H , Pd/C, MeOH, 81%; (f) bromobenzenes,
2
t
Pd (dba) , BINAP, NaO Bu, toluene, 100 ºC, 41ꢀ63% (for 17aꢀg and 18aꢀc); (g) fluorobenzenes,
2
3
K
2
CO
3
, DMF, 130 ºC, 35ꢀ69% (for 18dꢀf).
RESULTS AND DISCUSSION
The ability of all of the synthesized compounds to inhibit the 11βꢀHSD1 enzyme was
evaluated by using a cellꢀbased assay that utilized HEK293 cells stably transfected with
human 11βꢀHSD1 cDNA. The metabolic stability of the compounds was also evaluated by
determining the % remaining after a 30ꢀmin incubation with mouse liver microsomes (MLM).
Potent compounds (IC50 values below 10 nM in a cellꢀbased assay) with an acceptable
metabolic stability (>50% remaining in MLM after 30 min) were selected for a mouse ex vivo
3
3
PD assay. In this study, male C57BL/6 mice were orally administered 10 mg/kg of
compound, and the inhibition of 11βꢀHSD1 activity in liver and adipose tissues was assessed
by using the liver and epididymal fat (EPF) pads that were collected at 2 h after dosing. In
both the cellꢀbased assay and ex vivo PD assay, the 11βꢀHSD1 enzyme activity was
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determined by measuring the conversion of cortisone to cortisol, the levels of which were
quantified by an enzymeꢀlinked immunosorbent assay (ELISA) method.
First, the introduction of a polar functional group, such as a carboxylic acid, amide, and
sulfonamide, at the 4ꢀposition of the phenyl ring was attempted in order to reduce the CYP
liability of compound 9 (Table 1). Because the leftꢀhand side of this series of 11βꢀHSD1
10
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inhibitors was positioned to face the solventꢀexposed area in our previous docking study, we
expected that the introduction of a hydrophilic group on the phenyl ring would retain potency.
As expected, the incorporation of an amide (13b and 13c) or sulfonamide group (13f and 13g)
resulted in a cellular potency that was comparable to compound 9 (IC50 values below 10 nM).
However, these compounds were metabolically less stable than 9 and exhibited poor
pharmacodynamic activity in the mouse ex vivo PD assay. Interestingly, analog 13a
demonstrated a significantly reduced cellular potency, but we found that the biochemical
potency of 13a (hHSD1 IC50 = 3.2 nM) was similar to that of 9 (hHSD1 IC50 = 2.2 nM) in the
human 11βꢀHSD1 enzyme assay. These results suggest that the carboxylic acid moiety is
detrimental to the cell permeability of this class of 11βꢀHSD1 inhibitors.
We then planned a further modification of the leftꢀhand side, replacing the phenyl ring
with a pyridinꢀ3ꢀyl ring, and prepared a small set of derivatives. As shown in Table 1, the
picolinamide analogs 13d and 13e were 2ꢀ3 times more potent in the cellꢀbased assay than the
corresponding benzamide compounds 13b and 13c, respectively. The cellular potency of the
2ꢀ(pyridinꢀ2ꢀyl)propanꢀ2ꢀolꢀcontaining compound 13h was also similar to those of 13d, 13e,
and 9. Except for compound 13d, the metabolic stability of which was very poor, the
derivatives 13e and 13h were evaluated in the ex vivo PD assay and substantially reduced the
11βꢀHSD1 enzymatic activities, showing an efficacy that was similar to that of 9 (>50% and
>
80% inhibition at 10 mg/kg in the liver and EPF, respectively), 2 h after oral administration.
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
However, the CYP issue was not clearly solved with these compounds. A CYP inhibition
assay using human liver microsomes showed that analog 13e was a much weaker CYP3A4
inhibitor than 9 but was a potent inhibitor of CYP2C19 metabolic activity (CYP2C19 IC50
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.7 ꢁM). On the other hand, compound 13h was still a moderate CYP3A4 inhibitor with an
IC50 value of 5.4 ꢁM, although it did not inhibit CYP2C19 (Table 3).
a
Table 1. In vitro and ex vivo data for picolinamide derivatives
R
X
N
O
OH
N
N
N
H
d
Mouse PD
%inh. at 2 h)
b
c
HEK293 IC50 MLM MST
(
Compd
R
X
(
nM)
(%R30min)
Liver
70
EPF
86
9
SO
2
Me
CH
CH
CH
CH
N
3.1 ± 0.6
97 ± 2
1
3a
CO
CONH
CONHMe
CONH
CONHMe
2
H
97 ± 23
71 ± 11
57 ± 5
61 ± 6
23 ± 13
66 ± 5
65 ± 7
83 ± 4
76 ± 6
13
b
c
d
e
f
2
8.2 ± 1.8
9.2 ± 1.4
3.7 ± 0.7
2.8 ± 0.8
9.8 ± 2.7
4.3 ± 1.1
3.5 ± 0.9
0
0
26
34
1
1
3
3
2
13
N
52
0
85
11
33
87
1
1
1
3
3
3
SO
2
NH
NHMe
OH
2
CH
CH
N
g
h
SO
2
12
63
C(CH
3
)
2
a
Cellular potency and metabolic stability data are reported as an average of at least three replicates ±
b
standard error of the mean (SEM). Cellular assay utilizing HEK293 cells stably transfected with
c
d
human 11βꢀHSD1 cDNA. Metabolic stability test using mouse liver microsomes. Dosed po at 10
mg/kg; 0.5% methylcellulose and 1% Tween80 was used as vehicle.
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9
We next attempted to replace the central pyridine ring of 9 with a pyrimidine ring (Table
). The hydroxyꢀadamantyl group and (R)ꢀ2ꢀmethylꢀ4ꢀphenylpiperazinꢀ1ꢀyl moiety were
2
10
11
12
13
14
15
16
17
18
19
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maintained in the novel pyrimidineꢀ4ꢀcarboxamide derivatives, and diverse functional groups
were introduced at the 4ꢀposition of the phenyl ring (17aꢀf). Similar to the results of our
29
previous SAR study on picolinamide analogs, the compounds that were substituted with
hydrophilic groups (17dꢀf) were approximately 2 times more potent in the cellular assay than
the derivatives with hydrophobic substituents (17aꢀc). Among them, compound 17e showed
the highest cellular potency, good metabolic stability, and acceptable activity in the ex vivo
PD assay (48% and 81% inhibition at 10 mg/kg in the liver and EPF, respectively). Moreover,
17e did not inhibit 5 major human CYP isozymes, suggesting that the pyridine ring was the
main contributor to the CYP interaction in compound 9 (Table 3).
In the human 11βꢀHSD1 enzyme assay, the pyrimidineꢀ4ꢀcarboxamide 17e exhibited
comparable potency (hHSD1 IC50 = 2.7 nM) to the corresponding picolinamide inhibitor 9.
However, the cellular potency and oral bioavailability of 17e (HEK293 IC50 = 8.5 nM and F =
25% in mouse) were inferior to those of compound 9 (HEK293 IC50 = 3.1 nM and F = 41% in
mouse). We hypothesized that the much lower cLogP value of 17e (cLogP = 1.73) relative to
that of 9 (cLogP = 2.33) caused a reduction in cell permeability and oral bioavailability,
resulting in lower PD activity compared to 9. Increasing the lipophilicity of derivative 17f by
incorporating an additional fluorine substituent on the phenyl ring (17g) improved both the
potency in the cellꢀbased assay and ex vivo PD activity. Encouraged by this result, we sought
to introduce a small lipophilic group, such as a halogen (18a and 18b) or methyl group (18c),
at the 2ꢀposition of the phenyl ring of compound 17e. To further increase the lipophilicity of
17e, difluorinated derivatives (18d and 18e) and an ethylsulfonyl analog with a fluorine group
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
18f) were also explored. As shown in Table 2, these compounds, except for 18e, were more
potent than 17e in the cellular assay and demonstrated improved PD activities, especially in
the liver. In the best case, the incorporation of a single fluorine atom (18a) led to an
approximately 2ꢀfold increase in cellular potency (IC50 = 4.4 nM) and an improvement in the
metabolic stability (98% remaining in MLM after 30 min) and PD activity (86% and 88%
inhibition at 10 mg/kg in the liver and EPF, respectively) in comparison with those of
compound 17e. The PD activity of 18a was even superior to the picolinamide lead compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
, despite having a lower cLogP value (cLogP = 1.98) than that of 9. Moreover, the
pyrimidineꢀ4ꢀcarboxamide compound 18a neither inhibited 5 major CYP isozymes nor
activated PXR, a nuclear receptor that upregulates genes involved in drug metabolism (e.g.,
34
CYP3A4), suggesting that 18a has a low potential for CYPꢀmediated drugꢀdrug interactions
(Table 3).
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9
a
Table 2. In vitro and ex vivo data for pyrimidineꢀ4ꢀcarboxamide derivatives
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
d
Mouse PD
%inh. at 2 h)
b
c
HEK293 IC50
MLM MST
(%R30min)
(
Compd
R
(
nM)
Liver
EPF
17a
4ꢀF
22 ± 3
82 ± 5
78 ± 9
90 ± 5
86 ± 7
92 ± 3
99 ± 2
98 ± 3
98 ± 2
96 ± 4
90 ± 2
92 ± 5
95 ± 3
91 ± 6
1
1
7b
7c
4ꢀCl
4ꢀCF
21 ± 4
3
18 ± 2
17d
4ꢀCN
4ꢀSO
4ꢀC(CH
2ꢀF, 4ꢀC(CH
2ꢀF, 4ꢀSO Me
2ꢀCl, 4ꢀSO Me
2ꢀMe, 4ꢀSO Me
9.7 ± 1.3
8.5 ± 0.6
9.2 ± 0.8
8.8 ± 0.7
4.4 ± 0.5
7.6 ± 0.9
8.1 ± 1.1
7.5 ± 1.5
12 ± 3
6
12
81
46
74
88
82
77
85
1
1
7e
7f
2
Me
48
32
43
86
77
64
80
3 2
) OH
17g
3 2
) OH
1
1
8a
8b
2
2
18c
2
1
1
8d
8e
2,5ꢀF, 4ꢀSO
2
Me
Me
2,6ꢀF, 4ꢀSO
2
18f
2ꢀF, 4ꢀSO
2
Et
4.8 ± 0.6
79
82
a
Cellular potency and metabolic stability data are reported as an average of at least three replicates ±
b
standard error of the mean (SEM). Cellular assay utilizing HEK293 cells stably transfected with
c
d
human 11βꢀHSD1 cDNA. Metabolic stability test using mouse liver microsomes. Dosed po at 10
mg/kg; 0.5% methylcellulose and 1% Tween80 was used as vehicle.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. CYP inhibition and PXR activation data for selected compounds
a
b
CYP IC50 (ꢁM)
PXR
Compd
(% activation
at 10 ꢁM)
1
A2
2C9
>10
>10
>10
>10
>10
2C19
3.7
2D6
>10
>10
>10
>10
>10
3A4
0.97
>10
5.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
1
1
1
1
>10
>10
>10
>10
>10
32.7
3e
3h
7e
8a
0.7
>10
>10
>10
>10
>10
0.0
a
b
Assays were performed in pooled human liver microsomes. Data are expressed as a percent of
control where 100% of control is equivalent to the activity achieved with 10 ꢁM rifampicin.
To examine the binding mode of 18a in the active site of 11βꢀHSD1, we carried out a
docking study using the flexible molecular docking program SurflexꢀDock. As shown in
Figure 3, compound 18a docked into the active site cavity of 11βꢀHSD1 well with a Vꢀshape
binding mode. The amide carbonyl group of 18a established a central hydrogen bond
interaction with the hydroxyl side chain of Ser170, one of the key residues (Ser170, Tyr183,
3
5,36
and Lys 187) that define the catalytic triad for 11βꢀHSD1 activity.
11βꢀHSD1 has large
hydrophobic pockets (pocket I and II) on both sides of these key hydrogenꢀbonding residues.
The adamantyl ring of 18a occupied hydrophobic pocket I, and the hydroxyl group attached to
the adamantyl ring formed a hydrogen bond with the carbonyl oxygen of the backbone of
Thr124. The phenylꢀpiperazine moiety of 18a formed hydrophobic interactions with pocket II
consisting of Leu126, Tyr177, Val227, and Val231. As expected, the methylsulfonyl group
was positioned toward the solventꢀexposed area of the dimer interface region of the protein.
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41
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43
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49
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53
54
55
56
57
58
59
60
Figure 3. Docking pose of compound 18a in the active site of human 11βꢀHSD1. The key hydrogen
bonding interactions of 18a to the enzyme are shown with the green dashed lines. Purple and pink
dashed lines indicate πꢀσ and alkyl hydrophobic interactions, respectively. The protein structure was
taken from 2ILT. The predicted binding mode of 18a was generated by SurflexꢀDock.
Based on the results of the SAR study, compound 18a was selected for further biological
evaluations, which included human and mouse 11βꢀHSD1 enzyme assays, in vitro selectivity
assays, in vivo PK studies, and a doseꢀdependent PD study. The biochemical enzyme assays
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9
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were performed using microsomal fractions that overexpressed 11βꢀHSDs as the enzyme
sources, and 18a showed inhibition at low nanomolar concentrations for human (hHSD1) and
mouse 11βꢀHSD1 (mHSD1) enzyme activities (hHSD1 IC50 = 2.9 nM, mHSD1 IC50 = 1.6
nM) in accordance with its nanomolar cellular potency. On the other hand, 18a demonstrated
only negligible inhibitory activity for human 11βꢀHSD2 (hHSD2), with 6% and 17%
inhibition at 1 and 10 ꢁM concentrations, respectively (hHSD2 IC50 > 10 ꢁM). These results
indicate an over 3000ꢀfold selectivity for 11βꢀHSD1 over 11βꢀHSD2, where inhibition of the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
latter can result in undesirable sodium retention, hypokalemia, and hypertension. Compound
8a showed weak activity against the glucocorticoid and mineralocorticoid receptors in both
1
agonist and antagonist formats (EC50 values above 5 ꢁM). A good selectivity profile of 18a
was also observed versus a panel of 82 enzymes related to adverse reactions (MDS Pharma
Services), with only three weak hits (UGT1A1 IC50 = 5.6 µM, PDE3 IC50 = 5.4 µM, PDE4
IC = 5.2 µM).
5
0
The pharmacokinetic profile of compound 18a was examined in other species (mouse,
rat, and dog), and the results are summarized in Table 4. As expected from its high activity in
the ex vivo mouse PD assay, the PK study of 18a in mice showed an oral bioavailability of
96% with a high systemic exposure of 11.26 ꢁg × h/mL after oral administration of a 5 mg/kg
dosage. Moreover, across all three species, 18a exhibited consistently good PK properties
characterized by moderate clearances, high exposures, and excellent oral bioavailabilities (F >
60%). To determine if compound 18a can reach the tissues where 11βꢀHSD1 is highly
expressed, we performed a tissue distribution study with 18a in mice. C57BL/6 mice received
a single oral dose of 18a (10 mg/kg) and were sacrificed 2 h after dosing. To measure the
compound exposure levels, brain, liver, adipose, and plasma samples were collected from the
animals. Consistent with the observed ex vivo PD effect, 18a was well distributed in the liver
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and adipose, key target tissues for the treatment of metabolic diseases (Table 5). In contrast,
the concentration of 18a in the brain was relatively low, although the extent of free brain
penetration cannot be assessed due to the lack of data concerning the free brainꢀtoꢀfree plasma
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
8
concentration ratios (Kp,uu). The total plasma compound concentration was over 10 ꢁM, and
we have previously determined that 99% of 18a is protein bound in both human and mouse
plasma. Hence, the free compound level in plasma was approximately 100 nM, which is >50ꢀ
fold higher than the IC50 value of 18a against mouse 11βꢀHSD1. This is also consistent with
the >80% enzyme inhibition at 10 mg/kg in the target tissues.
Table 4. In vivo PK data for compound 18a
a
b
IV
PO
Species
CL
V
ss
t
1/2
AUC
C
max
t
max
AUC
F (%)
(
L/kg/h) (L/kg)
(h) (ꢁg×h/mL)
(ꢁg/mL)
(h)
1.0
1.3
2.1
(ꢁg×h/mL)
c
Mouse
0.42
0.93
0.36
1.1
2.1
2.4
1.7
2.35
1.12
1.47
2.21
11.26
96
60
98
c
Rat
1.8
4.7
1.02
3.39
d
Dog
1.12
11.52
a
b
1
0% hydroxylpropylꢀβꢀcyclodextrin was used as vehicle. 0.5% methylcellulose and 1% Tween80
c
d
was used as vehicle. Dosed iv at 1 mg/kg, po at 5 mg/kg. Dosed iv at 0.5 mg/kg, po at 4 mg/kg.
a
Table 5. Tissue distribution of compound 18a in mice
Tissue
Brain
Liver
Adipose
Plasma
Concentration (nmol/g)
Tissue/Plasma ratio
0.41
0.04
31.0
2.94
6.64
0.62
10.6
1
a
Animals were dosed po with 10 mg/kg dissolved in 0.5% methylcellulose and 1% Tween80. Tissues
were collected upon sacrifice at 2 h postꢀdose.
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9
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To evaluate the detailed pharmacodynamic profile of compound 18a, C57BL/6 mice were
orally administrated 3, 10, or 30 mg/kg of 18a, once, and sacrificed at 2 or 6 h postꢀdose for
an ex vivo analysis. As shown in Table 6, 18a significantly reduced the 11βꢀHSD1 enzymatic
activities in both the liver and EPF after 2 and 6 h in a doseꢀdependent manner. The activity of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11βꢀHSD1 was reduced by over 90% in animals that were treated with 30 mg/kg of 18a and
was inhibited in both tissues, even at 3 mg/kg (74% and 86% inhibition in the liver and EPF,
respectively) after 2 h. Moreover, these PD activities were maintained for up to 6 h after
administration. The prolonged PD activity of 18a was beyond that expected based on its
pharmacokinetic profile (t1/2 value of 1.7 h in mouse), and these results suggest that 18a is a
potent 11βꢀHSD1 inhibitor with a long duration of action in target tissues that is suitable for
onceꢀdaily dosing.
Table 6. Ex vivo mouse PD data for compound 18a
a
Ex vivo mouse PD (%inh.)
Dose
Liver
EPF
2
h
6 h
92
87
71
2 h
92
88
86
6 h
93
93
84
3
0 mg/kg
0 mg/kg
91
86
74
1
3
mg/kg
a
Dosed po at indicated dose; 0.5% methylcellulose and 1% Tween80 was used as vehicle.
In the safety pharmacology studies of 18a, no abnormal changes were observed in the
cardiovascular and respiratory systems of telemetered dogs that were given oral doses of up to
400 mg/kg, or in the central nervous system of mice that were given oral doses of up to 1000
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mg/kg. The wholeꢀcell patchꢀclamp technique was used to investigate the effects of 18a on
hERG channels stably expressed in CHO cells, and the IC50 value was estimated to be 8.0
µM. Considering that the human and mice plasma protein binding levels of 18a are both 99%,
the expected unbound Cmax of 18a at the effective dose is >30ꢀfold lower than 8.0 µM,
indicating a low risk of cardiac toxicity. The genotoxic potential of 18a was found to be
negative in a reverse mutation assay, a chromosome aberration assay, and an in vivo mouse
bone marrow erythrocyte micronucleus test. In a 4ꢀweek repeated oralꢀdose toxicity study in
mice, 18a was administered once daily at doses of 40, 100, 250, and 625 mg/kg, and the
NOAEL was 625 mg/kg in both males and females. In a 4ꢀweek repeated oralꢀdose toxicity
study in dogs, 18a was administered once daily at doses of 25, 100, and 400 mg/kg, and the
NOAEL was 400 mg/kg in both sexes. Toxicokinetic studies showed that systemic exposures
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(AUC and Cmax) of 18a were increased in doseꢀdependent manners in both mice and dogs,
although they were not dose proportional (Supporting Information).
On the basis of its favorable in vitro, pharmacokinetic, pharmacodynamic, and
39
toxicologic profiles, compound 18a (SKI2852) was selected as our final candidate. To
examine the therapeutic efficacy of 18a for type 2 diabetes and metabolic syndrome, we
3
9
conducted various in vivo disease model studies, and the results were previously reported.
Repeated oral administrations of 18a significantly lowered bodyꢀweight gains in ob/ob mice
y
and partially improved lipid profiles in DIO, ob/ob, and KKꢀA mice models. It also
efficiently reduced postprandial glucose and/or blood HbA1c levels in these mice and
suppressed hepatic mRNA levels of gluconeogenic enzymes in DIO mice. Moreover, 18a
clearly enhanced hepatic and wholeꢀbody insulin sensitivities in a hyperinsulinemicꢀ
3
9
euglycemic clamp experiment in DIO mice.
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6
Metformin is a firstꢀline oral antiꢀdiabetic agent with a documented glucoseꢀlowering
40
effect in subjects with type 2 diabetes, and it is commonly prescribed in combination with
4
1
other antiꢀdiabetic drugs, such as sulfonylureas, thiazolidinediones, and DPPꢀ4 inhibitors.
0
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9
0
To evaluate the effects of the potent 11βꢀHSD1 inhibitor 18a, alone or combined with
metformin, on glycemic control and lipid profiles, we performed an additional
pharmacological study using the ob/ob mice model. As illustrated in Figure 4, after 25 days of
onceꢀdaily oral administration of the compounds, a significant effect of 18a and metformin on
glycemic control was observed; the glycosylated hemoglobin (HbA1c) level was decreased by
0
.70%, 1.20%, and 1.67% (vehicle corrected) in the metformin (300 mg/kg), 18a (20 mg/kg),
and combined 18a plus metformin treatment groups, respectively. Compound 18a alone or in
combination with metformin significantly reduced the nonꢀfasting blood glucose levels by
2
6% and 33%, respectively, compared with the vehicle control group, whereas metformin
alone did not change the level. Although there were no statistically significant difference
between the 18a and combined groups, the effect of metformin on glycemic control was
significantly enhanced in combination with compound 18a. The LDL and total cholesterol
levels were also significantly decreased in all treatment groups and were much lower in mice
that were treated with compound 18a or 18a plus metformin than in the metformin
monotherapy group (Table 7). These results suggested that the coadministration of 18a and
metformin could have an additive or synergistic effect on metabolic disorders in animal
models of type 2 diabetes, which could further expand the concept of combination therapy
with an 11βꢀHSD1 inhibitor and metformin in clinical practice.
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Figure 4. The effect of metformin, compound 18a, and the combination thereof on glucose levels in
ob/ob mice. Compounds were orally administrated to ob/ob mice, at the indicated dose (mg/kg), once
daily for 25 days. Sterile water containing 0.5% methylcellulose and 1% Tween80 was used as
vehicle. Data are expressed as the means ± SE of n = 10. Statistical analysis was performed by oneꢀ
way ANOVA followed by the Tukey test. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle, ## P <
0.01, ### P < 0.001 vs. metformin.
Table 7. The effect of metformin, compound 18a, and the combination thereof on lipid profiles in
a
ob/ob mice
LDL cholesterol
mg/dl)
Total cholesterol
(mg/dl)
Triglyceride
(mg/dl)
Group
(
Vehicle
30.3 ± 0.8
286.6 ± 6.3
90.9 ± 5.5
99.9 ± 6.3
83.4 ± 6.5
84.1 ± 5.7
Metformin
23.9 ± 0.9***
17.1 ± 1.2***
17.2 ± 0.7***
252.6 ± 7.4**
230.6 ± 8.3***
235.0 ± 5.6***
Compd 18a
Metformin + Compd 18a
a
Data are expressed as the means ± SE of n = 10. Statistical analysis was performed by oneꢀway
ANOVA followed by the Tukey test. ** P < 0.01, *** P < 0.001 vs. vehicle.
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CONCLUSION
In order to diminish the CYP liability, further structural modifications of the lead compound 9
were performed and resulted in the discovery of a series of pyrimidineꢀ4ꢀcarboxamide
derivatives as novel 11βꢀHSD1 inhibitors. The replacement of the pyridine ring of 9 with a
pyrimidine ring led to a slight decrease in the cellular potency and PD activity, which were,
however, substantially improved by introducing an additional fluorine substituent at the 2ꢀ
position of the phenyl ring. This compound, 2ꢀ((R)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀ(methylsulfonyl)phenyl)ꢀ2ꢀ
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9
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9
0
methylpiperazinꢀ1ꢀyl)ꢀNꢀ((E)ꢀ5ꢀhydroxyadamantanꢀ2ꢀyl)pyrimidineꢀ4ꢀcarboxamide
(18a),
was identified as a potent and selective inhibitor of 11βꢀHSD1 with no CYP and PXR issues.
The compound 18a also exhibited excellent PK profiles across species and a doseꢀdependent,
highly potent, and sustainable PD activity that was superior to that of 9. Finally, 18a
significantly reduced the blood glucose and HbA1c levels and improved the lipid profiles in
ob/ob mice after oral administration. Moreover, the HbA1cꢀlowering effect of metformin was
synergistically enhanced when combined with 18a in these mice. Compound 18a was selected
as the final candidate for INDꢀenabling studies. Further details of these investigations will be
reported in due course.
EXPERIMENTAL SECTION
Chemistry. General methods. All commercial solvents and chemicals were of reagent
grade and were used without further purification. All reactions were monitored for completion
by TLC using Merck precoated TLC plates (Kieselgel 60 F254, 0.2 mm). Flash column
chromatography was carried out using Merck silica gel (Kieselgel 60, 230ꢀ400 mesh) or
1
prepacked silica cartridges from Redisep with an Isco Companion system. H NMR spectra
were recorded on a Varian UNITY 300 (300 MHz), a Bruker AMX 500 (500 MHz), JEOL
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9
JNMꢀECA 600 (600 MHz), or Bruker AMX 800 (800 MHz) instruments and were
determined in CDCl
3 6
or DMSOꢀd . Chemical shifts are reported in parts per million (ppm)
relative to tetramethylsilane (0.00 ppm) or solvent peaks as the internal reference, and
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1
3
coupling constant (J) values are reported in hertz (Hz). C NMR spectra were recorded on a
Bruker AMX 500 (125 MHz), JEOL JNMꢀECA 600 (150 MHz), or Bruker AMX 800 (200
13
MHz) instrument. All C NMR spectra were determined in DMSOꢀd
6
and assigned in ppm
relative to the central DMSOꢀd
6
peak (39.50 ppm). Highꢀresolution mass spectra (HRMS)
were measured on a JEOL JMS 700 or JEOL JMS 600ꢀW spectrometer. Melting points were
measured on a Büchi Bꢀ540 melting point apparatus and were not corrected. The purity of all
final compounds was determined to be >95% by HPLC (Waters ACQUITY UPLC® BEH
C18 column, 2.1 x 100 mm, 1.7 ꢁm) with gradient of 30ꢀ80% acetonitrile in 0.01 M disodium
hydrogen orthophosphate buffer adjusted to pH 7.6 with phosphoric acid.
6
ꢀBromoꢀNꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀyl)picolinamide (10). To a solution
of 6ꢀbromopicolinic acid (17.5 g, 86.6 mmol) and transꢀ4ꢀaminoadamantanꢀ1ꢀol (17.4 g, 104
mmol) in acetonitrile (500 mL) were added N,Nꢀdiisopropylethylamine (18.1 mL, 104 mmol)
and HBTU (39.4 g, 104 mmol). The reaction mixture was stirred at ambient temperature for
1
5 h and then concentrated under reduced pressure. The mixture was diluted with EtOAc (600
mL), washed with saturated aqueous NH Cl (200 mL) and brine (200 mL), dried over
Na SO , filtered, and evaporated to dryness under reduced pressure. The residue was purified
4
2
4
by flash column chromatography (SiO
2
, 70% EtOAc in hexanes) to afford 10 (26.1 g, 85%) as
) δ 8.16 (dd, J = 0.9, 7.5 Hz, 1H), 8.12 (brs, 1H),
1
a white solid. H NMR (300 MHz, CDCl
3
7.72 (dd, J = 7.5, 7.8 Hz, 1H), 7.62 (dd, J = 0.9, 7.8 Hz, 1H), 4.22ꢀ4.16 (m, 1H), 2.25 (brs,
3
H), 1.99ꢀ1.88 (m, 3H), 1.86ꢀ1.78 (m, 5H), 1.61ꢀ1.53 (m, 2H), 1.43 (s, 1H). MS (ESI) m/z:
+
3
51 [M+H] .
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Nꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)ꢀ6ꢀ((R)ꢀ2ꢀmethylpiperazinꢀ1ꢀ
yl)picolinamide (11). A mixture of 10 (1.0 g, 2.85 mmol), (R)ꢀtertꢀbutyl 3ꢀmethylpiperazineꢀ
1
ꢀcarboxylate (855 mg, 4.27 mmol), Pd (dba) (52 mg, 0.057 mmol), xantphos (99 mg, 0.171
2 3
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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4
5
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8
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2
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2
3
4
5
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8
9
0
mmol), sodium tertꢀbutoxide (410 mg, 4.27 mmol), and toluene (20 mL) was stirred at 100 ºC
for 3 h under a nitrogen atmosphere. The reaction mixture was cooled to room temperature,
and saturated aqueous NH
), and the combined organic layer was dried over Na
under reduced pressure. The residue was purified by flash column chromatography (SiO
4
Cl was added. The mixture was extracted with CH
2 2
Cl (40 mL x
2
2
SO , filtered, and evaporated to dryness
4
2
,
9
0% EtOAc in hexanes) to afford (R)ꢀtertꢀbutyl 4ꢀ(6ꢀ(((1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ
2
ꢀyl)carbamoyl)pyridinꢀ2ꢀyl)ꢀ3ꢀmethylpiperazineꢀ1ꢀcarboxylate (720 mg, 54%) as a pale
+
yellow solid. MS (ESI) m/z: 471 [M+H] .
To
yl)carbamoyl)pyridinꢀ2ꢀyl)ꢀ3ꢀmethylpiperazineꢀ1ꢀcarboxylate (715 mg, 1.52 mmol) in CH
10 mL) was added trifluoroacetic acid (10 mL). After being stirred at ambient temperature
for 3 h, the reaction mixture was poured into water (30 mL) and extracted with CH Cl (40
a
solution of (R)ꢀtertꢀbutyl 4ꢀ(6ꢀ(((1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀ
2
Cl
2
(
2
2
mL x 2). The organic layers were discarded, and the aqueous layer was basified with 5 N
aqueous NaOH and extracted with 5% MeOH in CH Cl (40 mL x 3). The combined organic
, filtered, and concentrated in vacuo to afford 11 (516 mg, 92%)
) δ 8.35 (d, J = 9.0 Hz, 1H), 7.62 (dd, J = 7.2, 8.7
2
2
2 4
layer was dried over Na SO
1
as a white solid. H NMR (300 MHz, CDCl
3
Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 6.77 (d, J = 8.7 Hz, 1H), 4.44ꢀ4.37 (m, 1H), 4.22ꢀ4.15 (m,
H), 3.95ꢀ3.88 (m, 1H), 3.19ꢀ3.08 (m, 3H), 3.00ꢀ2.89 (m, 2H), 2.23 (brs, 3H), 1.99ꢀ1.92 (m,
1
2H), 1.85ꢀ1.77 (m, 6H), 1.62ꢀ1.52 (m, 2H), 1.26 (d, J = 6.6 Hz, 3H). MS (ESI) m/z: 371
+
[
M+H] .
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5ꢀ((R)ꢀ4ꢀ(6ꢀ(((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)carbamoyl)pyridinꢀ2ꢀyl)ꢀ3ꢀ
methylpiperazinꢀ1ꢀyl)picolinic acid (12). A mixture of 11 (190 mg, 0.513 mmol), methyl 5ꢀ
bromopicolinate (133 mg, 0.615 mmol), Pd(OAc) (7 mg, 0.0308 mmol), xantphos (18 mg,
2
10
11
12
13
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0.0308 mmol), cesium carbonate (251 mg, 0.769 mmol), and toluene (8 mL) was stirred at
1
00 ºC for 15 h under a nitrogen atmosphere. The reaction mixture was cooled to room
temperature, filtered, and concentrated under reduced pressure. The residue was purified by
flash column chromatography (SiO , 0ꢀ3% MeOH in CH Cl , gradient elution) to afford
2
2
2
methyl 5ꢀ((R)ꢀ4ꢀ(6ꢀ(((1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀyl)carbamoyl)pyridinꢀ2ꢀyl)ꢀ3ꢀ
+
methylpiperazinꢀ1ꢀyl)picolinate (119 mg, 46%) as a yellow solid. MS (ESI) m/z: 506 [M+H] .
To
a
solution
of
5ꢀ((R)ꢀ4ꢀ(6ꢀ(((1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀ
yl)carbamoyl)pyridinꢀ2ꢀyl)ꢀ3ꢀmethylpiperazinꢀ1ꢀyl)picolinate (100 mg, 0.198 mmol) in
MeOH (2 mL) was added 2 N aqueous NaOH (0.5 mL). After being stirred at ambient
temperature for 20 h, the pH of the reaction mixture was adjusted to 6ꢀ7 with 1 N aqueous
2 2
HCl. The mixture was extracted with CH Cl (10 mL x 4), and the combined organic layer
was dried over MgSO , filtered, and evaporated to dryness under reduced pressure. The
4
residue was purified by flash column chromatography (SiO , 5ꢀ15% MeOH in CH Cl ,
2
2
2
1
gradient elution) to afford 12 (86 mg, 88%) as a pale yellow solid. H NMR (300 MHz,
DMSOꢀd ) δ 8.39 (d, J = 3.0 Hz, 1H), 8.29 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.74
dd, J = 7.2, 8.7 Hz, 1H), 7.38 (dd, J = 3.0, 9.0 Hz, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.08 (d, J =
6
(
8.7 Hz, 1H), 4.62ꢀ4.52 (m, 1H), 4.50 (s, 1H), 4.12ꢀ4.03 (m, 1H), 4.02ꢀ3.90 (m, 3H), 3.49ꢀ3.16
(m, 3H), 2.06 (brs, 3H), 1.79ꢀ1.64 (m, 8H), 1.54ꢀ1.45 (m, 2H), 1.21 (d, J = 6.3 Hz, 3H). MS
+
(ESI) m/z: 492 [M+H] .
4ꢀ((R)ꢀ4ꢀ(6ꢀ(((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)carbamoyl)pyridinꢀ2ꢀyl)ꢀ3ꢀ
methylpiperazinꢀ1ꢀyl)benzoic acid (13a). Compound 13a was prepared according to the
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6
procedure of 12 from intermediate 11 and methyl 4ꢀbromobenzoate in 44% yield (two steps).
1
Mp: 278 °C. H NMR (500 MHz, DMSOꢀd
6
) δ 8.31 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 8.8 Hz,
2H), 7.72 (dd, J = 7.2, 8.7 Hz, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.06 (d, J = 8.7 Hz, 1H), 6.98 (d,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 8.8 Hz, 2H), 4.60ꢀ4.52 (m, 1H), 4.50 (brs, 1H), 4.10ꢀ4.03 (m, 1H), 3.98ꢀ3.94 (m, 1H),
3
8
1
.91ꢀ3.82 (m, 2H), 3.45ꢀ3.29 (m, 2H), 3.18ꢀ3.09 (m, 1H), 2.11ꢀ2.03 (m, 3H), 1.80ꢀ1.64 (m,
13
H), 1.54ꢀ1.45 (m, 2H), 1.20 (d, J = 6.5 Hz, 3H). C NMR (125 MHz, DMSOꢀd
6
) δ 167.29,
62.94, 156.54, 153.66, 147.61, 138.96, 130.92, 118.90, 112.64, 110.24, 110.16, 65.36,
51.66, 50.45, 48.10, 46.18, 45.19, 44.26, 33.49, 30.50, 29.08, 14.61. MS (FAB) m/z: 491
+
[
M+H] . HRMS (FAB) m/z: calcd for C H N O 491.2658, found 491.2654.
2
8
35
4
4
6ꢀ((R)ꢀ4ꢀ(4ꢀCarbamoylphenyl)ꢀ2ꢀmethylpiperazinꢀ1ꢀyl)ꢀNꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀ
hydroxyadamantanꢀ2ꢀyl)picolinamide (13b). To a solution of 13a (60 mg, 0.122 mmol) in
acetonitrile (2 mL) were added ammonia (0.5 M solution in dioxane, 0.49 mL, 0.244 mmol),
N,Nꢀdiisopropylethylamine (0.043 mL, 0.244 mmol), and HBTU (56 mg, 0.146 mmol). The
reaction mixture was stirred at ambient temperature for 3 h, and saturated aqueous NH
4
Cl was
added. The mixture was extracted with 10% MeOH in CH Cl (15 mL x 2), and the combined
2
2
2 4
organic layer was dried over Na SO , filtered, and evaporated to dryness under reduced
2
pressure. The residue was purified by flash column chromatography (SiO , 5% MeOH in
1
CH
2
Cl
2
) to afford 13b (47 mg, 78%) as a pale yellow solid. Mp: 261 °C. H NMR (500 MHz,
DMSOꢀd
6
) δ 8.31 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.73 (dd, J = 7.2, 8.6 Hz, 1H),
.71 (brs, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 7.00 (brs, 1H), 6.97 (d, J =
.8 Hz, 2H), 4.61ꢀ4.54 (m, 1H), 4.50 (s, 1H), 4.12ꢀ4.05 (m, 1H), 3.98ꢀ3.93 (m, 1H), 3.90ꢀ3.79
7
8
(m, 2H), 3.42ꢀ3.33 (m, 1H), 3.25ꢀ3.20 (m, 1H), 3.07ꢀ3.00 (m, 1H), 2.12ꢀ2.03 (m, 3H), 1.79ꢀ
13
1
.64 (m, 8H), 1.54ꢀ1.46 (m, 2H), 1.21 (d, J = 6.5 Hz, 3H). C NMR (125 MHz, DMSOꢀd
6
) δ
167.63, 162.94, 156.61, 152.79, 147.60, 138.96, 128.90, 123.08, 112.99, 110.33, 110.17,
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9
65.35, 51.65, 51.14, 47.92, 46.60, 45.19, 44.26, 33.48, 30.50, 29.07, 14.30. MS (FAB) m/z:
+
4
90 [M+H] . HRMS (FAB) m/z: calcd for C28
H
36
N
5
O
3
490.2818, found 490.2820.
Nꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)ꢀ6ꢀ((R)ꢀ2ꢀmethylꢀ4ꢀ(4ꢀ
(methylcarbamoyl)phenyl)piperazinꢀ1ꢀyl)picolinamide (13c). Compound 13c was prepared
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
according to the procedure of 13b from benzoic acid 13a and CH
3
NH
2
(2.0 M solution in
1
THF) in 70% yield. Mp: 227 °C. H NMR (800 MHz, DMSOꢀd
6
) δ 8.31 (d, J = 8.3 Hz, 1H),
8
.14 (q, J = 4.5 Hz, 1H), 7.74ꢀ7.72 (m, 3H), 7.29 (d, J = 7.2 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H),
6.98 (d, J = 8.9 Hz, 2H), 4.60ꢀ4.55 (m, 1H), 4.50 (s, 1H), 4.11ꢀ4.04 (m, 1H), 3.98ꢀ3.94 (m,
H), 3.88ꢀ3.84 (m, 1H), 3.83ꢀ3.79 (m, 1H), 3.39ꢀ3.35 (m, 1H), 3.23ꢀ3.20 (m, 1H), 3.05ꢀ3.00
m, 1H), 2.75 (d, J = 4.5 Hz, 3H), 2.10 (brs, 1H), 2.06 (brs, 2H), 1.78ꢀ1.71 (m, 4H), 1.69ꢀ1.65
1
(
1
3
(m, 4H), 1.53ꢀ1.47 (m, 2H), 1.21 (d, J = 6.6 Hz, 3H). C NMR (200 MHz, DMSOꢀd
6
) δ
1
6
66.28, 162.94, 156.61, 152.66, 147.60, 138.97, 128.33, 123.43, 113.16, 110.36, 110.18,
5.36, 51.65, 51.22, 47.87, 46.65, 45.19, 44.26, 33.48, 30.50, 29.07, 26.09, 14.25. MS (FAB)
+
m/z: 504 [M+H] . HRMS (FAB) m/z: calcd for C29
H
38
N
5
O
3
504.2975, found 504.2979.
6ꢀ((R)ꢀ4ꢀ(6ꢀCarbamoylpyridinꢀ3ꢀyl)ꢀ2ꢀmethylpiperazinꢀ1ꢀyl)ꢀNꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀ
hydroxyadamantanꢀ2ꢀyl)picolinamide (13d). Compound 13d was prepared according to the
procedure of 13b from picolinic acid 12 and ammonia (0.5 M solution in dioxane) in 78%
1
yield. Mp: 237 °C. H NMR (500 MHz, DMSOꢀd
6
) δ 8.32ꢀ8.27 (m, 2H), 7.86 (d, J = 8.8 Hz,
1
1
4
H), 7.77 (brs, 1H), 7.73 (dd, J = 7.1, 8.7 Hz, 1H), 7.43 (dd, J = 2.7, 8.8 Hz, 1H), 7.31 (brs,
H), 7.30 (d, J = 7.1 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H), 4.61ꢀ4.55 (m, 1H), 4.50 (s, 1H), 4.12ꢀ
.06 (m, 1H), 3.98ꢀ3.87 (m, 3H), 3.46ꢀ3.39 (m, 1H), 3.36ꢀ3.31 (m, 1H), 3.18ꢀ3.11 (m, 1H),
1
3
2.12ꢀ2.03 (m, 3H), 1.79ꢀ1.64 (m, 8H), 1.54ꢀ1.46 (m, 2H), 1.21 (d, J = 6.5 Hz, 3H). C NMR
125 MHz, DMSOꢀd ) δ 166.27, 162.94, 156.55, 148.09, 147.62, 139.33, 138.97, 134.42,
22.47, 120.07, 110.33, 110.26, 65.35, 51.68, 50.32, 47.90, 46.06, 45.18, 44.25, 33.46, 30.50,
(
6
1
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Journal of Medicinal Chemistry
Page 28 of 51
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6
+
29.07, 14.40. MS (FAB) m/z: 491 [M+H] . HRMS (FAB) m/z: calcd for C27
H
35
N
6
O
3
491.2771, found 491.2765.
Nꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)ꢀ6ꢀ((R)ꢀ2ꢀmethylꢀ4ꢀ(6ꢀ
0
1
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5
6
7
8
9
0
1
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4
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9
0
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0
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7
8
9
0
(methylcarbamoyl)pyridinꢀ3ꢀyl)piperazinꢀ1ꢀyl)picolinamide (13e). Compound 13e was
3 2
prepared according to the procedure of 13b from picolinic acid 12 and CH NH (2.0 M
1
solution in THF) in 55% yield. Mp: 163 °C. H NMR (500 MHz, DMSOꢀd
6
) δ 8.39 (d, J = 4.8
Hz, 1H), 8.32ꢀ8.27 (m, 2H), 7.85 (d, J = 8.8 Hz, 1H), 7.73 (dd, J = 7.2, 8.7 Hz, 1H), 7.43 (dd,
J = 2.7, 8.8 Hz, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H), 4.62ꢀ4.55 (m, 1H),
4
.50 (s, 1H), 4.12ꢀ4.04 (m, 1H), 3.98ꢀ3.86 (m, 3H), 3.46ꢀ3.38 (m, 1H), 3.35ꢀ3.29 (m, 1H),
3
.17ꢀ3.09 (m, 1H), 2.79 (d, J = 4.8 Hz, 3H), 2.11ꢀ2.03 (m, 3H), 1.79ꢀ1.63 (m, 8H), 1.54ꢀ1.45
1
3
(m, 2H), 1.21 (d, J = 6.5 Hz, 3H). C NMR (125 MHz, DMSOꢀd
6
) δ 164.62, 162.95, 156.56,
1
4
48.07, 147.62, 139.40, 138.97, 134.46, 122.22, 120.23, 110.34, 110.28, 65.36, 51.69, 50.40,
+
7.86, 46.13, 45.18, 44.25, 33.47, 30.50, 29.07, 25.80, 14.38. MS (FAB) m/z: 505 [M+H] .
505.2927, found 505.2937.
37 6 3
HRMS (FAB) m/z: calcd for C28H N O
Nꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)ꢀ6ꢀ((R)ꢀ2ꢀmethylꢀ4ꢀ(4ꢀ
sulfamoylphenyl)piperazinꢀ1ꢀyl)picolinamide (13f). A mixture of 11 (30 mg, 0.081 mmol), 4ꢀ
3 2 2
bromoꢀNꢀ(tertꢀbutyl)benzenesulfonamide (28 mg, 0.097 mmol), Pd[P(oꢀtolyl) ] Cl (1 mg,
0.00081 mmol), BINAP (3 mg, 0.0049 mmol), cesium carbonate (26 mg, 0.081 mmol), and
toluene (5 mL) was stirred at 90 ºC for 15 h under a nitrogen atmosphere. The reaction
mixture was cooled to room temperature, filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (SiO , 5% MeOH in CH Cl ) to
2
2
2
afford
6ꢀ((R)ꢀ4ꢀ(4ꢀ(Nꢀ(tertꢀbutyl)sulfamoyl)phenyl)ꢀ2ꢀmethylpiperazinꢀ1ꢀyl)ꢀNꢀ
(
(1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀyl)picolinamide (45 mg, 96%) as a pale yellow
+
solid. MS (ESI) m/z: 582 [M+H] .
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Journal of Medicinal Chemistry
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To a solution of 6ꢀ((R)ꢀ4ꢀ(4ꢀ(Nꢀ(tertꢀbutyl)sulfamoyl)phenyl)ꢀ2ꢀmethylpiperazinꢀ1ꢀyl)ꢀNꢀ
(
2 2
(1R,2s,3S,5S,7S)ꢀ5ꢀhydroxyadamantanꢀ2ꢀyl)picolinamide (40 mg, 0.069 mmol) in CH Cl (2
mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred at ambient
temperature for 15 h and then concentrated under reduced pressure. The mixture was diluted
10
11
12
13
14
15
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with water (5 mL) and extracted with CH
the aqueous layer was basified with saturated aqueous NaHCO
MeOH in CH Cl (10 mL x 2). The combined organic layer was dried over Na
2
Cl
2
(5 mL). The organic layer was discarded, and
and extracted with 10%
SO , filtered,
3
2
2
2
4
and evaporated to dryness under reduced pressure. The residue was purified by flash column
chromatography (SiO , 5% MeOH in CH Cl ) to afford 13f (23 mg, 64%) as a pale yellow
2
2
2
1
solid. Mp: 243 °C. H NMR (500 MHz, DMSOꢀd ) δ 8.31 (d, J = 8.3 Hz, 1H), 7.73 (dd, J =
6
7.2, 8.4 Hz, 1H), 7.64 (d, J = 8.9 Hz, 2H), 7.30 (d, J = 7.2 Hz, 1H), 7.08ꢀ7.03 (m, 5H), 4.60ꢀ
4
3
1
.53 (m, 1H), 4.50 (s, 1H), 4.11ꢀ4.04 (m, 1H), 3.98ꢀ3.94 (m, 1H), 3.91ꢀ3.82 (m, 2H), 3.44ꢀ
.37 (m, 1H), 3.33ꢀ3.27 (m, 1H), 3.14ꢀ3.06 (m, 1H), 2.12ꢀ2.03 (m, 3H), 1.79ꢀ1.64 (m, 8H),
1
3
6
.54ꢀ1.46 (m, 2H), 1.20 (d, J = 6.5 Hz, 3H). C NMR (125 MHz, DMSOꢀd ) δ 162.94,
156.55, 152.73, 147.60, 138.97, 132.17, 127.17, 112.93, 110.29, 110.19, 65.36, 51.66, 50.73,
+
48.02, 46.40, 45.18, 44.26, 33.48, 30.50, 29.07, 14.48. MS (FAB) m/z: 526 [M+H] . HRMS
(FAB) m/z: calcd for C27
36 5 4
H N O S 526.2488, found 526.2487.
Nꢀ((1R,2s,3S,5S,7S)ꢀ5ꢀHydroxyadamantanꢀ2ꢀyl)ꢀ6ꢀ((R)ꢀ2ꢀmethylꢀ4ꢀ(4ꢀ(Nꢀ
methylsulfamoyl)phenyl)piperazinꢀ1ꢀyl)picolinamide (13g). Compound 13g was prepared
according to the procedure of 13f from intermediate 11 and 4ꢀbromoꢀNꢀ(tertꢀbutyl)ꢀNꢀ
1
methylbenzenesulfonamide in 21% yield (two steps). Mp: 264 °C. H NMR (800 MHz,
DMSOꢀd
6
) δ 8.31 (d, J = 8.3 Hz, 1H), 7.73 (dd, J = 7.2, 8.5 Hz, 1H), 7.58 (d, J = 9.0 Hz, 2H),
7
1
.30 (d, J = 7.2 Hz, 1H), 7.10ꢀ7.07 (m, 4H), 4.59ꢀ4.55 (m, 1H), 4.50 (s, 1H), 4.09ꢀ4.05 (m,
H), 3.98ꢀ3.94 (m, 1H), 3.91ꢀ3.83 (m, 2H), 3.45ꢀ3.40 (m, 1H), 3.36ꢀ3.31 (m, 1H), 3.16ꢀ3.12
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