Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas as highly potent soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.138

3,3-Disubstituted piperidine-derived trisubstituted urea entA-2b was discovered as a highly potent and selective soluble epoxide hydrolase (sEH) inhibitor. Despite the good compound oral exposure, excellent sEH inhibition in whole blood, and remarkable se

Full text of DOI:10.1016/j.bmcl.2009.07.138

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5314–5320
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas
as highly potent soluble epoxide hydrolase inhibitors
a
a
b
b
b
Hong C. Shen a, , Fa-Xiang Ding , Qiaolin Deng , Suoyu Xu , Hsuan-shen Chen , Xinchun Tong ,
Vincent Tong b, Xiaoping Zhang c, Yuli Chen c, Gaochao Zhou c, Lee-Yuh Pai d, Magdalena Alonso-Galicia d,
Bei Zhang c, Sophie Roy d, James R. Tata a, Joel P. Berger c, Steven L. Colletti a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
c Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
d Department of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
3,3-Disubstituted piperidine-derived trisubstituted urea entA-2b was discovered as a highly potent and
selective soluble epoxide hydrolase (sEH) inhibitor. Despite the good compound oral exposure, excellent
sEH inhibition in whole blood, and remarkable selectivity, compound entA-2b failed to lower blood pres-
sure acutely in spontaneously hypertensive rats (SHRs). This observation further challenges the premise
that sEH inhibition can provide a viable approach to the treatment of hypertensive patients.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 14 July 2009
Revised 28 July 2009
Accepted 29 July 2009
Available online 6 August 2009
Keywords:
Inhibitors
Ureas
sEH
Soluble epoxide hydrolase
Epoxyeicosatrienoic acids (EETs) are endogenous lipids derived
from the metabolism of arachidonic acid by cytochrome P450
epoxygenases.1 EETs exhibit important physiological effects. EETs
can dilate conduit vessels, renal afferent arterioles and coronary
resistance vessels.2 In addition, EETs increase sodium renal excre-
tion.3 Therefore, EETs have been shown to lower blood pressure
and reduce myocardial perfusion in animal models.4 Furthermore,
EETs elicit vascular protective effects by modifying vascular
smooth muscle cell migration, leukocyte adhesion, platelet aggre-
gation and thrombolysis.5 EETs are also anti-inflammatory as they
reduce cytokine-induced endothelial expression of the leukocyte
adhesion molecules, vascular adhesion molecule-1 (VCAM-1),
intercellular adhesion molecule-1 (ICAM-1), and E-selectin.6 Thus,
EETs may constitute an endogenous protective mechanism against
atherosclerosis.7 The clinical implications of short-term infusion of
EETs were demonstrated by intracoronary infusion of EETs to re-
duce infarct size in a canine ischemia-reperfusion model,8 and in-
tra-arterial infusion of EETs to reduce blood pressure.9
(DHETs) via hydrolysis of EETs, whereby the biological effects of
EETs are diminished or eliminated.10 It has been postulated that
sEH inhibition may lead to elevated levels of EETs, which could
then have a significant beneficial impact on blood pressure, inflam-
mation, and lipid and carbohydrate metabolism. In particular, the
hypothesis on the blood pressure effect of sEH inhibition was
strongly supported by the blood pressure lowering and renal pro-
tection effects of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-
dodecanoic acid (AUDA) in a salt-sensitive hypertensive animal
model.11 However, the lack of off-target activity data of AUDA does
not allow for the formal conclusion that sEH is indeed a preclinical-
ly validated hypertension target. In addition, scientists at Arete
developed an sEH inhibitor (structure unknown) that has recently
entered phase II clinical trials specifically targeted for diabetes.12
The hydrolase catalytic pocket of sEH contains two tyrosine res-
idues (Tyr381 and Tyr465) which act as hydrogen bond donors to
activate the epoxide ring opening by Asp333.13 Amide and urea
groups fit in the pocket with the carbonyl oxygen being a hydrogen
To enhance the levels of EETs thereby providing potential ther-
apeutic benefits, a straightforward approach is to inhibit soluble
epoxide hydrolase, which is also referred to as sEH or EPHX2. sEH
is an enzyme that converts EETs to dihydroxy eicosatrienoic acids
H
N
H
N
Ar
N
N
n
R
O
O
Cl
* Corresponding author. Tel.: +1 732 594 1755; fax: +1 732 594 9473.
E-mail address: hong_shen@merck.com (H.C. Shen).
Figure 1. General scaffold of urea analogs.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.138

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5314–5320
5315
M)
Table 1
In vitro SAR on sEH and mEH inhibition, DHET production, CYP and ion channel inhibition
Compd
sEH IC50 (h, r)a nM
DHET, IC50 (nM)
mEH, IC50
(l
M)
CYP2C9, 2D6, 3A4 IC50
(
lM)
DLZ, IKr Nav1.5, IC50 (l
b
c
H
N
N
O
1, 5
<1
>100
—
>10, >10, <10
<10, >10, >10
>10, >10, <10
<10, >10, >10
>10, <10, <10
<10, >10, <10
<10, >10, <10
<10, ꢀ10, <10
1.7, 12, >10
19, 18, <10
15, 5, >10
0.9, 21, <10
2, 20, <10
0.7, 0.2, >1
0.9, 2, ꢀ10
0.5, 4, >10
Cl
O
1a
N
d
59, 207
550, 349
7, 6
—
1b
—
—
—
—
—
<1
>100
—
N
1c
1d
Cl
Cl
1e
<1, 3
8, 11
14, 7
1, 3
—
>100
22
CF3
1f
Cl
1g
N
47
1h
HN
7, 6
3
14
<10, ꢀ10, >10
>10, <10, <10
>10, >10, <10
<10, <10, >10
<10, ꢀ10, <10
<10, —, <10
1, 0.8, >10
2, 9, —
1i
1, 5
—
8
54
Cl
N
1j
7, 6
3
—, 5, —
1k
N
29, 26
5, 19
1, 4
13
3
>100
>100
>100
—, 13, —
—, 9, —
N
H
1l
N
N
1m
<1
10, 26, >10
1n
a
b
c
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
Studies were performed with HEK293 cells.
Human mEH was used.
d
Dashed lines indicate that compounds were not tested.
bond acceptor for Tyr381 and Tyr465, and the N–H of amide or
urea being a hydrogen bond donor to Asp333. Therefore, various
ureas and amides have been designed and prepared as selective
and orally bioavailable sEH inhibitors.14 Herein we report 3,3-
disubstituted piperidine-derived trisubstituted ureas (Fig. 1) as
highly potent and bioavailable sEH inhibitors, whose sEH and off-

5316
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5314–5320
Table 2
In vitro SAR on sEH and mEH inhibition, DHET production, CYP and ion channel inhibition
sEH IC50 (h, r)a (nM)
DHET, IC50 (nM)
mEH, IC50
(
lM)
CYP2C9, 2D6, 3A4 IC50
(
lM)
DLZ, IKr Nav1.5, IC50 (lM)
b
c
Compd
H
N
N
O
Cl
CO2H
2a
52, 398
5, 76
38
4
>100
>100
>100
>100
—
>10, >10, >10
>10, >10, >10
>10, >10, >10
>10, >10, >10
—
>30, >30, >10
>30, >30, >10
>30, >30, >10
>30, >30, >10
—
CO2H
2b
CO2H
2c
12, 176
5, 120
<1, 3
6
CO2H
<1
2d
CO2Me
d
—
2e
CN
3, 16
<1
<1
<1
<1
1.3
<1
1.4
<1
>100
>100
>100
>100
>100
>100
>100
>100
>10, >10, >10
<10, ꢀ10, >10
<10, >10, <10
>10, >10, >10
>10, <10, <10
>10, ꢀ10, <10
>10, >10, >10
ꢀ10, >10, ꢀ10
7, 10, >10
0.9, 0.07, >10
5, 6, >10
2f
OH
2g
1, 15
OH
1.3, 10
5,26
2h
CONH2
—, 7, —
2i
NMe2
12, 119
5, 59
0.1, 0.3, >10
5, 3, >10
2j
N
N
N
N
H
2k
N
N
O
N
N
H
N
H
2l
1.4, 34
5, 32
22, 27, —
O
N
H
4, 8, >10
2m
O
N
N
3, 31
—
—
—
>10, >10, <10
>10, >10, >10
1, 6, >10
2n
O
8, 100
<1
26, 29, >10
O
2o
a
b
c
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
Studies were performed with HEK293 cells.
Human mEH was used.
d
Dashed lines indicate that compounds were not tested.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5314–5320
5317
target activities, ex vivo target engagement, pharmacokinetics (PK)
and pharmacodynamics (PD) profiles are subject to detailed
discussion.
Human and rat sEH enzyme inhibition assays established the
intrinsic activity of our compounds. Potent compounds were fur-
ther evaluated in a human whole cell 14,15-DHET production assay
to assess their cellular activity. Major counterscreening targets in-
cluded microsomal epoxide hydrolase (mEH), several CYP enzymes
and selected ion channels. As mEH plays significant roles in xeno-
biotic detoxification15 and steroid metabolism,16 and certain ureas
were reported to inhibit mEH,17 mEH was selected as a major
counterscreen target. Additionally, the inhibitory properties of
compounds on CYP2C9, 2D6 and 3A4 were also measured. Of these
three CYP enzymes, CYP2C9 is of particular importance due to its
role in the production of EETs.1b Moreover, the activity of the com-
pounds against potassium (IKr),18 calcium (DLZ binding)19 and so-
dium (Nav1.5)20 channels, was also examined due to their known
blood pressure and/or cardiac effects. Importantly, to the best of
our knowledge, all previous publications on sEH inhibitors by other
research groups did not address whether they were effectors of
mEH, CYP enzymes or ion channels. It is our belief that to deter-
mine whether sEH inhibitors can provide mechanism-based blood
Figure 2. The minimized docking model of entA-2b (green) in human sEH. The
dashed lines illustrate atom pairs within hydrogen bond distance. The pictures were
prepared by PyMOL (Delano Scientific LLC, South San Francisco, CA). The model is
based on the X-ray crystallographic structure of 1ZD3 (PDB code): human soluble
epoxide hydrolase 4-(3-cyclohexyluriedo)-butyric acid complex.13
Table 3
In vitro SAR on sEH and mEH inhibition, DHET production, CYP and ion channel inhibition
sEH IC50 (h, r)a (nM)
DHET, IC50 (nM)
mEH, IC50
(
lM)
CYP2C9, 2D6, 3A4 IC50
(
lM)
DLZ, IKr Nav1.5, IC50 (lM)
b
c
Compd
H
N
N
O
Cl
CO2H
5, 69
2
>100
>100
>100
>100
>100
—
>100, >100, >100 65 for CYP2C8
>10, >10, >10
>30, >30, >10
>30, >30, >10
>30, >30, >10
>30, >30, >10
>30, >30, >10
>30, >30, >10
>30, >30, >10
>30, 21, <10
entA-2b
F3C
16, 70
12, 176
5, 120
30, 467
28, 331
19, 290
8, 413
<1
3a
d
—
>10, >10, >10
CF3
3b
CF3
<1
—
>10, >10, >10
3c
N
ꢀ10, >10, >10
3d
N
—
>10, >10, >10
3e
N
—
—
>10, >10, >10
3f
CO2H
H
N
N
<1
—
<10, >10, >10
O
F3CO
3g
a
b
c
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
Studies were performed with HEK293 cells.
Human mEH was used.
d
Dashed lines indicate that compounds were not tested.

5318
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5314–5320
pressure lowering efficacy, both high selectivity and excellent po-
tency against sEH are imperative.
As a potent and selective sEH inhibitor, racemic compound 2b
was further resolved to provide two enantiomers. The eutomer
entA-2b was 100- and 500-fold more active than the distomer
entB-2b in the sEH inhibition and DHET production assays respec-
tively. The substituent effect of the phenyl group of 2b was subse-
quently studied. Para-trifluoromethyl analog 3c was more active
against sEH than the meta- and ortho-trifluoromethyl analogs
(3a–b). Furthermore, pyridine replacement of the phenyl group
in 2b yielded compounds with weaker sEH inhibition while main-
taining excellent off-target profiles (3d–f). Biaryl compound 3g
was associated with good human but weak rat sEH activity, as well
as substantial sodium channel and CYP2C9 inhibition (Table 3).
The rat PK data of several potent analogs are summarized in
Table 4. Clearly, compound 2b was unique in its excellent bioavail-
ability, low clearance, and good oral exposure. In contrast, other
analogs of 2b (1a, 2i–k) had much higher clearance and lower oral
exposure.
Starting with rationally designed trisubstituted urea 1a as a
lead structure, the structure–activity relationship (SAR) was ex-
plored by systematically modifying the three fragments of the gen-
eral scaffold colored in purple, blue, and red respectively shown in
Figure 1.
The SAR on the left-hand moiety of the scaffold was first
examined with a 3-methyl-3-phenyl piperidine as the common
right-hand motif (Table 1). Aniline-derived ureas such as 1a in
general gave excellent sEH inhibitory activity with various para-
or meta-aniline substituents including chloro, CF3, alkyl, and phe-
nyl groups (data not shown). To minimize the potential oxidation
of the aniline moiety of the ureas, electron-rich aniline-derived
ureas were not prepared. Several bicyclic and monocyclic amino-
heterocycles, however, gave inferior sEH potency as represented
by 1b and 1c. 3-Biphenylaniline-derived urea 1d also provided
good sEH activity. Substituted benzylamine 1e or 4-aryl
benzylamine analogs 1f–i led to potent analogs, of which 1h
was the most active for both human and rat sEH enzymes. Ana-
logs bearing aryl or heteroaryl groups two or three carbon atoms
away from the urea gave a wide range of activities (1j–m). It
should be noted that with a few exceptions, this series of com-
pounds provided better activity against human sEH than rat
sEH. The major issues of these compounds (1a–m) were substan-
To assess the serum shift of the sEH inhibition, the whole cell
14,15-DHET production assay of analog entA-2b was conducted
in the presence of 10% human serum. In this study, entA-2b dem-
onstrated a 2.5-fold serum shift (IC50 = 5 nM) with respect to its
IC50 in this assay in the absence of serum (IC50 = 2 nM). Com-
pound entA-2b was further evaluated in an in vitro rat whole
blood DHET production assay in which entA-2b (1
lM) or vehicle
and substrate (14,15-EET, 10 M) were incubated for 0, 15, 30
l
tial ion channel or CYP inhibitory activity (IC50 <10
l
M). In addi-
and 60 min, and the DHET production rate was measured at each
time point (Table 5). It should be noted that the data variation
was small and reproducibility was quite good for this assay.
Throughout the 60 min period, entA-2b suppressed 14,15-DHET
production by more than 90% relative to the vehicle control, con-
sistent with the excellent sEH inhibitory activity of the compound
in whole blood.
tion to its fairly clean ion channel profile, phenyl cyclopropyl
amine-derived urea such as 1n gave remarkable inhibitory
activity against human and rat sEH enzymes, and the whole cell
DHET production. With the exception of compounds 1g–k, all
tested analogs were completely inactive against mEH (IC50
>100 lM).
Next, the R substituent of the piperidine shown in Figure 1 was
optimized (Table 2). With the ion channel issues of the compounds
in Table 1, it was envisioned that by introducing a carboxylic acid
moiety, the off-target profiles, in particular ion channel activity,
would be minimized. As expected, compounds 2a–d demonstrated
neither ion channel nor CYP inhibitory activities. The linker length
between the carboxylic acid and piperidine was crucial for the sEH
potency of compounds. For example, analogs bearing a carboxylic
acid two to four carbon atoms away from piperidine (2b, 2c and
2d) gave significantly more potent sEH inhibition than analog 2a
with one methylene linker. This observation may be explained by
invoking our model of 2b in the binding pocket of human sEH. In
particular, a potential H-bond interaction between the acid moiety
and Gln382 was identified that allowed for the presence of an acid
group based on a docking model (Fig. 2). If the carboxylic acid lin-
ker was too short, as in the case of 2a, the interaction between the
enzyme and the inhibitor would be less favorable. On the other
hand, longer linkers (2c and 2d) could adopt certain conformation
to allow the favorable interaction of the acid group with Gln382
(modeling results not shown) thereby providing better activity.
The ester, cyanide, and alcohol analogs with a two-carbon linkder
(2e–g) were even more potent against sEH than 2b presumably as
a result of H-bonds between these head groups with Gln382, but
these compounds were accompanied with ion channel activity.
Among four amides (2i, 2m–o), 2o was the least active for sEH,
but was also the cleanest for CYP and ion channels. Interestingly,
the tetrazole replacement of carboxylic acid led to more ion chan-
nel activity (2k). In contrast, a tetrazole amide (2l) was an effective
replacement for the acid providing sEH inhibition and an off-target
profile similar to 2b. Moderately active against sEH, amine analog
(2j) was a potent inhibitor of calcium and potassium channels. Cor-
relating well with their enzyme inhibitory activity, low to subn-
anomolar inhibition of whole cell DHET production was observed
for analogs 2d–o.
To ensure its selectivity for sEH, compound entA-2b was tested
against 167 other biological targets. Remarkably, the compound
showed minimal activity in all cases (IC50 >10 lM).
With extraordinarily selective sEH inhibition, excellent DHET
production inhibition in whole blood, and a good PK profile,
entA-2b was evaluated for its blood pressure effect in the sponta-
neously hypertensive rat (SHR) model (50 mpk, po), in which sys-
tolic and diastolic blood pressure, heart rate, and pulse pressure
were measured via a telemetry method over 8 h post dose. No
blood pressure effects were observed in this study for entA-2b
(Fig. 3). In addition, several other compounds with significantly
Table 4
Rat PKa
Compds
F
(%)
Cl (mL/min/
kg)
Vdss (L/
kg)
T1/2
(h)
AUCNpo
mg)
(lM h kg/
1a
2b
2i
2j
2k
13
84
13
52
43
4
116
59
12
3.9
3.2
4.4
0.66
0.39
0.15
10
0.05
0.37
ꢀ0
0.8
0.88
1.4
1.4
0.17 124
a
Formulations: 1 mg/mL ethanol:PEG:water (20:40:40). IV dose: 1 mg/kg (n = 2).
PO dose: 2 mg/kg (n = 3). Blood concentration was determined by LC/MS/MS fol-
lowing protein precipitation with acetonitrile.
Table 5
Inhibition of in vitro 14,15-DHET production in whole rat blood by entA-2ba
Incubation time (min)
0
15
30
60
DHET production rate (% of control)
Deviation (%)
6.3
0.1
3.4
0.4
5.3
0.0
8.1
0.8
a
14,15-DHET production was measured at the indicated time point post dosing.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5314–5320
5319
procedure.23 The subsequent urea formation, chiral separation of
two enantiomers, and hydrolysis sequence provided entA-2b.24
In conclusion, we have identified entA-2b, a trisubstituted urea
containing a 3,3-disubstituted piperidine motif, as a potent, selec-
tive sEH inhibitor with good target engagement and PK. However,
this compound failed to demonstrate any significant blood pres-
sure lowering activity in the SHR model. This is the third distinct
sEH inhibitor we have reported on thus far that failed to exhibit
an antihypertensive effect.21 This most recent observation rein-
forces our conclusion that sEH may not be a validated antihyper-
tensive target in the SHRs, a bona fide predictive model of the
human activity of numerous anti-hypertensive therapeutics. De-
spite its lack of blood pressure lowering activity in SHRs, entA-2b
may still find utility in other indications.
Panel A
20
10
Vehicle (Im:Tw)
entA-2b 50 mpk
0
-10
-20
-30
0
1
2
3
4
5
6
7
8
Time (h)
Panel B
PO dose
Supplementary data
20
10
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.138.
0
References and notes
-10
-20
-30
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Vehicle (Im:Tw)
entA-2b 50 mpk
0
1
PO dose
systolic and panel
2
3
4
5
6
7
8
Time (h)
Figure 3. Panel
A
B
diastolic blood pressure change from
baseline (mmHg) of entA-2b (50 mpk, po) in SHRs over 8 h (pink curve: entA-2b;
green curve: vehicle (Imwitor/Tween)).
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different structures, good ex vivo target engagement and clean off-
target profiles also failed to lower the blood pressure in SHR.21
These results contradict with a previous report in which an sEH
inhibitor acutely lowered blood pressure in SHRs.22 It is conceiv-
able that potential off-target activities of the previously communi-
cated compound might have contributed to the observed anti-
hypertensive effect.
12. http://www.aretetherapeutics.com.
13. Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D. W. Protein
Science 2006, 15, 58.
The synthesis of the trisubstituted urea entA-2b is shown in
Scheme 1. Intermediate 7 was prepared following a literature
14. (a) Delombaert, S.; Eldrup, A. B.; Kowalski, J. A.; Mugge, I. A.; Soleymanzadeh,
F.; Swinamer, A. D.; Taylor, S. J. WO2007106705 A1; (b) Ingraham, R. H.
WO2007106706 A1; (c) Hammock, B. D.; Jones, P. D.; Morisseau, C.; Huang, H.;
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H
N
O
c
a
CN
b
CN
CO2Me
CO2Me
CO2Me
O
5
6
4
H
N
O
N
N
d
e
N
Cl
N
H
Cl
*
H
CO2Me
CO2Me
2e
CO2Me
7
8
O
N
f
N
Cl
*
H
CO2H
entA-2b
Scheme 1. Reagents and conditions: (a) Methyl acrylate, triton B, t-butanol, reflux,
4 h; (b) H2 (50 psi), PtO2, HOAc, rt, 5 h; (c) BH3 dimethyl sulfide complex, THF, rt,
3 h, 48% over 3 steps; (d) parachlorophenyl isocyanate, DCM, rt, 14 h, rt, 72%; (e)
SFC, 40%; (f) LiOH (1 N), THF/MeOH/water (3:1:1), rt, 2 h, 95%.

5320
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5314–5320
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24. The procedures of the preparation of entA-2b: to the boiling solution 4 (12 g,
102 mmol) and methyl acrylate (29 mL) in tert-butanol (30 mL) was added a
solution of triton (40% in methanol, 9.5 mL) in tert-butanol (14 mL) through
condenser dropwise. The solution was heated at reflux for 4 h. After distillation
of solvents, the residue was dissolved in CHCl3 (200 mL), washed with 1 N HCl
(100 mL), water (200 mL), dried over Na2SO4 to give 30.6 g of 5 as a crude oil.
The solution of 5 (4.7 g, 16.2 mmol) and PtO2 (0.61 g) in acetic acid (50 mL) was
subjected to hydrogenation at 50 psi for 5 h. After filtration, the filtrate was
concentrated and the residue was dissolve in EtOAc (200 mL), washed with
satd NaHCO3 (200 mL), dried over Na2SO4 to give 4.5 g of 6 as an oil. To the
solution of 6 (4.5 g, 17.2 mmol) in THF (100 mL) was added BH3 (20 mL,
40 mmol, 2 M in THF) at 0 °C, The resulting solution was stirred at rt for 3 h
before quenched with 3 N HCl (30 mL) at 0 °C. After removing the solvent, the
residue was partitioned between water (200 mL) and EtOAc (200 mL), the
aqueous phase was then neutralized by 1 N NaOH, extracted with 30%
isopropanol/chloroform (2 Â 100 mL). The combined organic phase was dried
over Na2SO4, and concentrated to give 1.89 g of 7 as a crude oil with an overall
yield of 48% over three steps. To the solution of 7 (1.57 g, 6.3 mmol) in DCM
(100 mL) was added 4-chlorophenyl isocyanide (1.024 g, 6.7 mmol) and the
resulting solution was stirred at rt for 16 h. After concentration, the residue
was purified on silica gel using 25% EtOAc/hexane to get 1.83 g of 2e as a
colorless oil (72% yield). The racemic 2e (4.2 g, 10.9 mmol) was subjected to
chiral separation (SFC) to give a single enantiomer 8 (1.7 g, >99% ee, 40% yield).
To the solution of 8 (1.7 g, 4.4 mmol) in THF/MeOH/water (3:1:1, 350 mL) was
added LiOH (1 N, 50 mL) dropwise and the resulting solution was stirred for
1 h. After removing the volatile solvents, the aqueous phase was acidified to pH
4 using 1 N HCl, and then extracted with EtOAc (2 Â 200 mL), the combined
EtOAc phase was washed with brine (2 Â 200 mL), dried over Na2SO4, and
concentrated to give entA-2b (1.56 g, 95% yield) as a colorless oil. 1H NMR
(acetone-d6, 500 MHz): d 8.11 (1H, s), 7.53 (2H, d), 7.46 (2H, d), 7.35 (2H, t),
7.23 (3H, m), 4.01 (1H, d), 3.64 (1H, d), 3.56 (2H, m), 2.16–1.87 (6H, m), 1.71
(1H, m), 1.52 (1H, m); LC–MS m/z: 387 (M++1).