Structure-based optimization of arylamides as inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1021/jm9005302

Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties. As part of an effort to identify inhibitors

Full text of DOI:10.1021/jm9005302

5880 J. Med. Chem. 2009, 52, 5880–5895
DOI: 10.1021/jm9005302
Structure-Based Optimization of Arylamides as Inhibitors of Soluble Epoxide Hydrolase†
Anne B. Eldrup,*,‡ Fariba Soleymanzadeh,‡ Steven J. Taylor,‡ Ingo Muegge,‡ Neil A. Farrow,‡ David Joseph,§
‡
Keith McKellop, Chuk C. Man, Alison Kukulka, and Stephane De Lombaert
‡
‡
ꢀ
‡Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877, §Department of Drug Discovery
Support, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877, and Department of Analytical Sciences, Boehringer Ingelheim
Pharmaceuticals, Ridgefield, Connecticut 06877
Received April 24, 2009
Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels
of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties.
As part of an effort to identify inhibitors of sEH with high and sustained plasma exposure, we recently
performed a high throughput screen of our compound collection. The screen identified N-(3,3-diphenyl-
propyl)-nicotinamide as a potent inhibitor of sEH. Further profiling of this lead revealed short
metabolic half-lives in microsomes and rapid clearance in the rat. Consistent with these observations,
the determination of the in vitro metabolic profile of N-(3,3-diphenyl-propyl)-nicotinamide in rat liver
microsomes revealed extensive oxidative metabolism and a propensity for metabolite switching. Lead
optimization, guided by the analysis of the solid-state costructure of N-(3,3-diphenyl-propyl)-nicotin-
amide bound to human sEH, led to the identification of a class of potent and selective inhibitors. An
inhibitor from this class displayed an attractive in vitro metabolic profile and high and sustained plasma
exposure in the rat after oral administration.
Introduction
yl-ureido)dodecanoic acid (AUDA), had been extensively
utilized as tool compounds (Figure 2). Common structural
features of these inhibitors are the large hydrophobic domains
flanking their central urea pharmacophore.10-14 Reasoning
that these large hydrophobic domains may limit the “drug-
like” characteristics and pharmacokinetic properties of these
compounds, we elected to perform a high-throughput screen
of our in-house compound collection in order to identify a
structurally different starting point for an optimization pro-
cess. N-(3,3-Diphenyl-propyl)-nicotinamide (Figure 3, 1) was
identified as a potent inhibitor of sEH and selected as the
starting point for lead optimization. Optimization of 1 was
guided by the analysis of its in vitro metabolic profile and by
the close examination of its solid-state structure when bound
to human sEH. Our analysis led to the identification of a new
class of functionalized 3,3-diphenylpropylamine arylamides
that are potent inhibitiors of sEH activity in both molecular
and cell-based assays. A compound from this class, 4-cyano-
N-[3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl]-
benzamide (24), was found to display attractive stability
profiles in rat and human liver microsomes and high and
sustained plasma exposure in the rat after oral administration.
Cytochrome P450-mediated epoxidation of arachidonic acid
yields four regioisomeric epoxyeicosatrienoic acids (EETsa)
that display vasodilatory, antiinflammatory, and analgesic, as
well as migratory and proliferative properties.1-3 The intri-
guing biological properties and complex biology of EETs have
drawn attention to the role of soluble epoxide hydrolase (sEH)
as the enzyme primarily responsible for the further metabolic
conversion of these regioisomeric epoxides (Figure 1).4,5
Soluble epoxide hydrolase (sEH) promotes the hydroly-
sis of EETs to their pharmacologically less active and
more rapidly cleared dihydroxy epoxyeicosatrienoic acids
(DHETs).6 Researchers have hypothesized that the selective
inhibition of sEH will lead to an increase in circulating levels
of EETs, resulting in the potentiation of their in vivo phar-
macological properties.1,2 In accordance with this hypothesis,
several reports have been published that demonstrate efficacy
of small molecule inhibitors of sEH in cardiovascular animal
models and in models of inflammation.7-9
We recently decided to determine the potential of sEH
inhibitors as vasodilators and as cardio- or renal-protective
agents. At the onset of our studies, two potent sEH inhibitors,
1-cyclohexyl-3-dodecylurea (CDU) and 12-(3-adamantan-1-
Chemistry
†PDB files for the complexes soluble epoxide hydrolase and inhibitors
1 and 24 (PDB identifiers 3I1Y and 3I28, respectively) have been
deposited with the Protein Data Bank.
*To whom correspondence should be addressed. Phone: 203 798
5658. Fax: 203 791 6072. E-mail: anne.eldrup@boehringer-ingelheim.
com.
a Abbreviations: sEH, soluble epoxide hydrolase; EETs, epoxyeico-
satrienoic acids; DHETs, dihydroxy epoxyeicosatrienoic acids; CDU,
1-cyclohexyl-3-dodecylurea; AUDA, 12-(3-adamantan-1-yl-ureido)do-
decanoic acid; CIU, N-cyclohexyl-N0-(4-iodophenyl) urea.
The identified lead N-(3,3-diphenyl-propyl)-nicotinamide
(1) was resynthesized from nicotinic acid and (3,3-diphenyl-
propyl)-amine using hydroxybenzotriazole, 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide, and Hunig’s base in di-
methylformamide, as described in the general procedure for
amide coupling. Our initial SAR focused on structural modi-
fications to define an optimized scaffold for sEH inhibition
(Tables 1-3). Subsequent chemical functionalizations were
r
pubs.acs.org/jmc
Published on Web 09/11/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5881
Table 1. Inhibitory Potency (IC50) against sEH and Stability in Micro-
somes (t1/2) for Compounds 2-5 Compared to Compound 1a
human sEH
IC50 [nM]
rat sEH
IC50 [nM]
hLM t1/2 rLM t1/2
[min]
G
n
m
[min]
1
2
3
4
5
Ph
Ph
Ph
H
0
0
0
0
1
1
0
2
1
1
7.0
>3000
120
4.8
>3000
26
13
4
3
4
6
3
700
70
150
105
4
76
4
Ph
31.0
a Compounds were designed to examine the effect a change in the
length of the linker to right-hand side benzhydryl pharmacophore, the
importance of the benzhydryl motif, and effect of a transposition of the
left-hand side 3-pyridiyl motif.
Table 2. Inhibitory Potency (IC50) against sEH for Compounds 6 and 7
Compared to Compound 1a
A
B
human sEH IC50 [nM]
rat sEH IC50 [nM]
1
6
7
C(O)
NH
S(O)2
NH
C(O)
NH
7.0
45
4.8
18
Figure 1. Cytochrome P450-mediated epoxidation of arachidonic
acid yields four regioisomeric epoxyeicosatrienoic acids (EETs).
EETs are further metabolized by soluble epoxide hydrolase (sEH)
to yield their corresponding and generally less active vicinal dihy-
droxy epoxyeicosatrienoic acids (DHETs). Inhibition of sEH selec-
tively over the CYP 450 expoxygenases has been hypothesized to
lead to an increase in circulating EETs levels, resulting in the
potentiation of their in vivo beneficial pharmacological effects.
>3000
>3000
a Compounds were designed to examine the effect reversing the
central amide pharmacophore or replacing it with a sulfonamide moiety.
designed to improve metabolic stability. Standard methods
for chemical synthesis and commercial starting materials were
used unless otherwise described (see below and Experimental
Section).
The effect of a change in the tether length between the
amide pharmacophore and the benzhydryl motif of N-(3,3-
diphenylpropyl)-nicotinamide 1 was investigated by the
synthesis of the 2,2-diphenylethyl and 4,4-diphenylbutyl deri-
vatives (Table 1, entries 2 and 3). 4,4-Diphenylbutylamine,
required for the synthesis of compound 3, was available
according to literature procedures.15 The importance of the
right-hand side benzhydryl motif was investigated by the
synthesis of its 3-phenylpropyl analogue in which one phenyl
is omitted (Table 1, entry 4). In addition, the effect of a
transposition of the left-hand side 3-pyridiyl pharmacophore
by insertion of a methylene spacer was investigated by the
synthesis of the 3-pyridylacetamide analogue of 1 (Table 1,
entry 5).
To define the importance of the central amide pharma-
cophore, compounds were synthesized in which the amide
configuration was either reversed (Table 2, entry 6) or
replaced by a sulfonamide (Table 2, entry 7). The prerequi-
site 4,4-diphenyl-butyric acid for the synthesis of compound 6
was prepared from 4-phenyl-butyrolactone, benzene, and
aluminum chloride according to the described literature
procedure.16
Figure 2. Recent studies of structurally simple inhibitors of soluble
epoxide hydrolase, such as 1-cyclohexyl-3-dodecylurea (CDU) and
12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) (Figure 2),
has confirmed a role for the inhibition of sEH in both cardiovas-
cular and inflammatory diseases. Common structural features of
these inhibitors are the large hydrophobic domains flanking the
central urea pharmacophore, which may limit the “drug-like”
characteristics of these compounds.
The effect of transposition or omission of the left-hand side
pyridyl heteroatom of 1 was examined by preparing its
regioisomeric 2-pyridyl and 4-pyridyl analogues (Table 3,
Figure 3. Chemical structure of the sEH inhibitor, N-(4,4-diphenyl-
propyl)-nicotinamide (1), identified through high throughput screening.

5882 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
Table 3. Inhibitory Potency (IC50) against sEH and Stability in Micro-
somes (t1/2) for Compounds 8-10 Compared to Compound 1a
Table 4. Inhibitory Potency (IC50) against sEH and Stability in Micro-
somes (t1/2) for Compounds 11-16a
human sEH
IC50 [nM]
rat sEH
IC50 [nM]
hLM t1/2 rLM t1/2
[min]
X
Y
Z
[min]
8
1
9
N
CH
CH CH
100
7.0
7.9
5.1
43
8
13
3
4
3
5
3
N
CH
N
4.8
7.6
7.2
CH CH
10 CH CH CH
5
a Compounds were designed to examine the effect of either a trans-
position or an omission of the left-hand side pyridyl heteroatom.
entries 8 and 9) and by synthesizing the corresponding
benzamide analogue in which the heteroatom is omitted
(Table 3, entry 10).
Our profiling of compounds 1-10 defined N-(3,3-diphen-
ylpropyl)-arylamide as a preferred scaffold for sEH inhibition
(Tables 1-3), and subsequent chemical functionalizations
were carried out to improve the poor in vitro metabolic
stability of this compound class (see Results and Discussion
Section). The effect of polar substituents on the left-hand side
aromatic ring system was examined through the synthesis of
the nicotinamide (11), 6-hydroxy-nicotinamide (12), 6-(2,2,
2-trifluoro-ethoxy)-nicotinamide (13), 2-(2,2,2-trifluoro-eth-
oxy)-isonicotinamide (14), 4-cyano-benzamide (15), and
4-methanesulfonyl-benzamide (16) derivatives in the context
ofa bis-(4-fluorophenyl)-modified benzhydrylright-hand side
motif (Table 4, entries 11-16). The prerequisite 3,3-bis-(4-
fluorophenyl)-propylamine for these transformations was
prepared similarly to published methods.17 Briefly, 4,40-di-
fluorobenzophenone was reacted with the anion of acetoni-
trile to give 3,3-bis-(4-fluorophenyl)-3-hydroxypropionitrile,
which upon reduction with lithium aluminum hydride and
aluminum trichloride gave the desired 3,3-bis-(4-fluoro-
phenyl)-propylamine.
On the basis of our analysis of the solid-state structure of 1
bound to human sEH, and in a further effort to improve
metabolic stability profiles for this compound class, the
synthesis of inhibitors with unsymmetrically substituted benz-
hydryl right-hand sides was undertaken (Table 5, entries
21-25). Access to the desired 3-(4-fluorophenyl)-3-(4-metha-
nesulfonyl-phenyl)-propylamine intermediate (20) proceeded
via a Pd2(dba)3 mediated Suzuki-Miyaura cross-coupling
reaction between 4-(methanesufonyl)benzeneboronic acid
and 3-chloro-3-(4-fluorophenyl)-acrylonitrile (Scheme 1, 17)
as we have previously reported.18 The resulting unsymmetri-
cally substituted acrylonitrile (18) was subjected to catalytic
hydrogenation in the presence of di-tert-butyl dicarbonate
to give the t-Boc-protected propylamino derivative (19).
Removal of the t-Boc group gave the desired propylamino
intermediate, 20, after treatment with aqueous hydrochloric
acid in methanol. The effect of this unsymmetrical functiona-
lization of the benzhydryl motif was assessed by the synthesis
of a similar set of substituted nicotinic and benzoic acids as
examined for the bis-(4-fluorophenyl) modified benzhydryl
motif (Table 5, entries 21-25).
a Compounds were designed to examine the effect of polar substitu-
ents on the left-hand side aromatic ring system in the context of a bis-
(4-fluorophenyl)-modified benzhydryl right-hand side motif.
synthesis of optically pure analogues was undertaken
(Scheme 2 and Table 5, entries S-24, R-24, S-22, and R-22).
The prerequisite (S)- and (R)-3-(4-fluorophenyl)-3-(4-metha-
nesulfonyl-phenyl)-propylamines 34a and 34b were accessed
from 4-fluorocinnamic acid (26) by coupling to the appro-
priate chiral auxiliary, (R)-(-)-4-phenyl-2-oxazolidinone or
(S)-(þ)-4-phenyl-2-oxazolidinone (Scheme2). Couplingspro-
ceeded via theinsituformationofthe mixed anhydrides, using
pivaloylchlorideand triethylamineintetrahydrofuran, togive
the desired 3-[3-(4-fluorophenyl)-acryloyl]-(R)-4-phenyl-oxa-
zolidin-2-one (27a) and 3-[3-(4-fluorophenyl)-acryloyl]-(R)-4-
phenyl-oxazolidin-2-one (27b). Chiral functionalization to
give the (R,R)- and (S,S)-diastereoisomeric 4-methanesulfa-
nylphenyl oxazolidinone intermediates 28a and 28b pro-
ceeded from 27a and 27b via an asymmetric conjugate
addition using the in situ generated organocopper reagent
available by reaction of thioanisole magnesium bromide with
copper(I)bromide-methylsulfide complex in tetrahydro-
furan. The removal of the chiral auxiliary was performed
under standard conditions, with sodium borohydride in a
mixture of tetrahydrofuran and water, to give the optically
pure alcohols 29a and 29b. Further chemical transformation
of these alcohols to their corresponding azides 30a and 30b
was accomplished by reaction with toluenesulfonyl chloride in
the presence of triethylamine in dichloromethane, followed
by the replacement of the tosylate using sodium azide in
To fully exploit the fact that the aromatic rings of the
benzhydrylmotifexperience differentelectronicenvironments
within the protein (see Results and Discussion Section), the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5883
Table 5. Inhibitory Potency (IC50) against sEH, Cell-Based Potency
(EC50), and Stability in Microsomes (t1/2) for Compounds 21-25a
Scheme 1. Synthetic Route to 3-(4-Fluorophenyl)-3-(4-metha-
nesulfonyl-phenyl)-propyl Amine (20)a
a Reagents and conditions: (i) (4-methanesulfonyl)benzeneboronic
acid, [(t-Bu)3PH]BF3, Pd2(dba)3, and KF in tetrahydrofuran overnight
at 45°C; (ii) di-tert-butyl dicarbonate and Pd/C in methanol shaken
under hydrogen at 50 psi for 24 h at room temperature; (iii) HCl in
methanol, then p-toluenesulfonic acid 1 h at room temperature.
trienoic acid in human Hep G2 cells using an ELISA readout.
Metabolic stability was an important driver of the SAR for
this compound class, and for selected compounds, half-lives in
human and rat liver microsomes were determined as a mea-
sure of first-pass metabolism (see Experimental Section for
methods). Compound exposure in rat was assessed after oral
administration at 1, 2, 4, and 6 h post dosing (see Experi-
mental Section for methods).
a Compounds were designed to examine the effect of polar substitu-
ents on the left-hand side aromatic ring system in the context of an
unsymmetrically methanesulfonyl substituted benzhydryl right-hand
side.
In an initial set of compounds, the length of the linker
between the central amide pharmacophore and the right-hand
side benzhydryl motif was varied from ethyl through propyl to
butyl, leading to the finding that the propyl linker is optimal
for inhibitory potency (Table 1, entries 1-3). Truncation to
the ethyl-linked derivative resulted in complete loss of activity
(IC50 > 3000 nM for both human and rat sEH), whereas the
extension of the propyl linker by one methylene unit resulted
in a 17-fold decline in potency against human sEH (IC50
120 nM) and a 4-fold decline in potency against the rat sEH
isoform (IC50 26 nM). The importance of the benzhydryl
pharmacophore for potent sEH inhibition was demonstrated
by the omission of a phenyl ring from this motif to give the
3-phenyl propylamide analogue (Table 1, entry 4). This
compound displayed large declines in inhibitory potency for
bothsEHisoforms(IC50 700and70nM for the humanand rat
isoforms, respectively). The transposition of the left-hand side
pyridyl moiety by one methylene unit was examined by the
evaluation of the 3-pyridylacetamide compound, 5, which
displayed a moderate inhibitory effect on sEH activity (IC50
31 and 150 nM for the human and rat isoforms, respectively).
In another set of compounds, the amide pharmacophore of 1
was either reversed or replaced with a sulfonamide (Table 2,
entries 6 and 7). The potency of the compound in which the
amide was reversed was moderately reduced compared to 1
(IC50 45 and 18 nM, for the human and rat isoforms,
respectively). The compound in which the amide pharmaco-
phore was replaced with sulfonamide completely lost its abi-
lity to inhibit sEH activity (see below for an analysis of this
finding). Taken together, these findings (Tables 1 and 2) sug-
gest that the benzhydryl motif is an important pharmacophore
N,N0-dimethylformamide. The resulting azide intermediates
30a and 30b were treated with a mixture of aqueous hydrogen
peroxide and glacial acetic acid to give 31a and 31b, respec-
tively. Subsequent catalytic reduction in methanol gave the
methanesulfoxide-functionalized t-Boc-protected propylami-
no derivatives 32a and 32b after treatment with di-tert-butyl
dicarbonate. Oxidation of the methanesulfoxides 32a and 32b
was accomplished by the treatment with oxone in the presence
of aluminum oxide to afford the methanesulfoxide deriva-
tives 33a and 33b, which were subjected to treatment with
trifluoroaceticacidtoafford the desiredR- and S-enantiomers
of 3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-pro-
pylamine, 34a and 34b, respectively.
Results and Discussion
N-(3,3-Diphenyl-propyl)-nicotinamide (Figure 3, 1) was
identified as a potent inhibitor of sEH through high through-
put screening. Further profiling of this lead compound
revealed short half-lives in both human and rat liver micro-
somes and rapid clearance in the rat (data not shown). A lead
optimizationprocesswas undertakenwiththe goal toimprove
in vitro metabolic stability as an avenue to identify com-
pounds with high and sustained plasma exposure in the rat.
Compounds were evaluated in binding assays for their ability
to displace a rhodamine-labeled probe from either human or
rat sEH using a fluorescence polarization readout (see Experi-
mental Section for methods). Selected compounds were as-
sessed for their ability to inhibit the sEH-mediated conversion
of 14,15-epoxyeicosatienoic acid to 14,15-dihydroxyeicosa-

5884 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
Scheme 2. Synthetic Route to (S)-3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl Amine (S-20) from 4-Fluorocinnamic
Acid Using the Chiral Auxiliary (R)-(-)-4-Phenyl-2-oxazolidinonea
a (R)-3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl amine (R-20) was prepared similarly starting from the enantiomeric (S)-(þ)-4-
phenyl-2-oxazolidinone. Reagents and conditions: (i) pivaloyl chloride and triethylamine in tetrahydrofuran at 0°C; (ii) pre-formed anion of
(R)-(-)-4-phenyloxazolidinone (formed with lithium diisopropylamide (2M in tetrahydrofuran at -78°C) at -20°C to room temperature for 1 h;
(iii) CuBr-methylsulfide complex, dimethylsulfide, and thioanisole methylmagnesium bromide in tetrahydrofuran at -40°C to room temperature
overnight; (iv) sodium borohydride in water/tetrahydrofuran for 22 h at room temperature; (v) tosyl chloride and triethylamine in dichloromethane
at -10°C to room temperature overnight; (vi) sodium azide in N,N0-dimethylformamide overnight at room temperature; (vii) aq hydrogen peroxide
(30%) and glacial acetic acid at room temperature for 24 h; (viii) Pd/C in methanol, stirred under hydrogen for 4 h; (ix) Boc-anhydride, triethyl amine, 4 h
at rt; (x) Al2O3, oxone, 24 h at reflux; (xi) HCl, then p-toluenesulfonic acid.
for sEH and that both its phenyl ring systems contribute to the
molecular recognition of the enzyme. Moreover, the data
indicates that the exact position of the central amide pharma-
cophore relative to the benzhydryl motif is crucial for inhibi-
tion of sEH activity by this class of compounds, probably
reflecting a relatively tight fit of the benzhydryl moiety with
the enzyme.
To better understand the molecular basis for the observed
inactivityof the sulfonamide analogue(7), this compoundwas
docked into the structure of human sEH obtained in-house
with compound 1 cocrystallized (vide supra and Figure 5a,b
for solid-state structure and Figure 4a,b for docking studies).
Compound 7 was docked into the catalytic site using the
program Glide assuming the same binding mode and exhibit-
ing the same hydrogen bonding interaction pattern with the
enzyme as observed for the benzamide 1 (Figure 4). Among a
variety of docking conformations that differed slightly in how
the sulfonamide atoms interacted with Asp 335, Tyr 383, and
Tyr 466, a high energy trans (Figure 4a) and low energy cis
(Figure 4b) sulfonamide conformation generated the highest
docking scores.19 The high energy trans sulfonamide dis-
played hydrogen bonding interaction patterns between the
sulfonamide moiety and sEH that were energetically favor-
able: one of the sulfonyl oxygen atoms engaged in hydrogen
bonds to Tyr 383 and Tyr 466. The second sulfonyl oxygen
was positioned in hydrogen bonding distance to Tyr 383, and
the sulfonamide nitrogen formed a hydrogen bond with the
Asp 335 residue. Not surprisingly, this docking pose exhibited
the highest Glide eModel score. In contrast, the energetically
favored cis sulfonamide conformation could only be assumed
by placing one of its sulfonyl oxygen atoms in close proximity
to one of the oxygen atoms of the carboxylic acid side chain
˚
moiety of Asp 335 (2.9 A), giving rise to an energetically
unfavorable contact (Figure 4b). While showing a reduced
eModel score, this binding pose exhibited the highest Glide-
score. On the basis of these docking models, we hypothesize
that the observed inactivity of 7 results from its inability to
assume a low energy conformation as well as energetically
favorable hydrogen bonding interactions with active site
residues. This hypothesis is consistent with the strong con-
formational preference foundfor aromaticcis sulfonamides in
protein-ligand complexes available in the Cambridge Crys-
tallographic Database and the Protein Data Bank.20 DFT/
B3LYP calculations with a 6-31G** basis set revealed that
there is a ∼4 kcal energy difference favoring the cis sulfon-
amide conformation over the trans conformation after opti-
mizing the geometries, holding the pyridyl/sulfonamide and
sulfur-nitrogen bonds fixed. The energy difference between

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5885
76.9% of the total metabolite area. The bis-(4-fluorophenyl)
analogue of 1 (Table 4, entry 11) was synthesized to assess the
impact of blocking the presumed metabolically vulnerable
sites with fluoro atoms. However, examination of the in vitro
metabolic profile of compound 11 revealed an apparent shift
in the major route of metabolism to hydroxylation of the
pyridine, now representing 76.1% of the total metabolite area.
The stability of 11 in liver microsomes was assessed and found
to be relatively short at 12 min in both human and rat liver
microsomes (Table 4, entry 11). Taken together, the shift to
hydroxylation of the pyridine moiety and the fact that in vitro
metabolism remained rapid for the fluorinated analogue
suggested to us a propensity for metabolite switching.
Figure 4. Putative binding modes of the inactive sulfonamide
7 (gray). (a) The high energy trans conformer displays energetically
favorable hydrogen bonding interaction patterns with one sulfonyl
oxygen engaged in hydrogen bonds (black dotted lines) to Tyr 383
and Tyr 466 and the second sulfonyl oxygen positioned in hydrogen
bonding distance to Tyr 383. The sulfonamide nitrogen forms a
hydrogen bond with Asp 335. The crystallographically obtained
binding pose of 1 has been added for comparison (cyan). (b) The low
energy cis sulfonamide conformation places one sulfonyl oxygen
atom in close proximity to one of the oxygen atoms of the carboxylic
acid side chain moiety of Asp 335, resulting in an energetically
unfavorable contact (red dotted line).
The intrinsic potency of the bis-(4-fluorophenyl) analogue
(Table 4, entry 11) was evaluated and found to be unchanged
relative to 1 (IC50s 8 and 7 nM for human sEH and rat sEH,
respectively). Hypothesizing that an increase in the overall
polarity of these molecules might decrease first-pass metabo-
lism, we decided to explore the effect of left-hand side
substitution in the context of the bis-(4-fluorophenyl) mod-
ified right-hand side. Hence, the effect of small, polar sub-
stituents such as hydroxyl, trifluoroethoxy, cyano, and
methanesulfonyl on benzhydryl moiety was examined in the
context of the 3-pyridyl, 4-pyridyl, and phenyl left-hand sides.
Potent inhibition of both human and rat sEH was observed
for these derivatives with IC50s ranging from 5 to 9 nM
(Table 4, entries 12-16). However, the examination of the
in vitro half-lives of these compounds revealed large differ-
ences in metabolic stabilities as a result of left-hand side
substitution. The inclusion of hydroxy and trifluoroethoxy
substituents resulted in significant improvements in metabolic
stabilities, most dramatically illustrated by the 6-(2,2,2-tri-
fluoro-ethoxy)-nicotinamide analogue, 13, which displayed a
half-life in excess of 300 min in both human and rat liver
microsomes. For compounds 14, 15, and 16, the improvement
in the metabolic stability in human microsomes was less
dramatic and their half-lives in rat liver microsomes remained
short at about 5 min. The identification of 13 as an in vitro
metabolically stable and potent sEH inhibitor prompted us to
assess its exposure in the rat. Following oral administration of
13 at 5 mg/kg, plasma concentrations were more sustained
over time compared to 1 but remained low, reaching only 0.3,
0.2, 0.1, and 0 μΜ at the 1, 2, 4, and 6 h time points,
respectively. Because of its poor aqueous solubility (less than
0.1 μg/mL at pH 7.4), 13 could not be evaluated for intestinal
permeability in our in-house caco-2 assay.
The solid-state structure of the co-stucture of 1 and human
sEH was determined to provide guidance for the design of
compounds with increased metabolic stability and improved
physicochemical properties while maintaining excellent bind-
ing characteristics. As shown previously, the active site in both
rat and human sEH is formed by a channel in the protein
between two large, solvent accessible pockets, with the active
site tyrosine and aspartic residues encircling the ligand.22-24
The costructure of 1 (Figure 5a) reveals the amide moiety of 1
positioned in a similar orientation to that observed previously
for urea-based inhibitors, with the carbonyl oxygen directed
toward the two tyrosines in the active site and the amide
proton directed toward the aspartic acid. The structure shows
the pyridine ring placed in the Trp 336 niche (adopting
a similar nomenclature to that of Gomez et al.24) forming a
π-stacking interaction with the tryptophan residue. The
π-stacking interaction results in the pyridine ring adopting a
similar orientation to that reported for the phenyl group in the
cis and trans sulfonamides is consistent with earlier observa-
tions.21 Allowing the pyridyl/sulfonamide to be optimized
reduces the energy difference to ∼2 kcal/mol; however, the
energetically favored orientation of the pyridyl group would
now lead to unfavorable interactions with Trp 336 (data not
shown).
The importance of the left-hand side aromatic moiety of 1
was further delineated by evaluating analogues in which the
3-pyridyl ring was replaced with 2-pyridyl, 4-pyridyl, or
phenyl (Table 3, entries 8-10). The 4-pyridyl and phenyl
derivatives 9 and 10 were both potent inhibitors of human and
rat sEH, with IC50s ranging from 5 to 8 nM. In contrast, the
2-pyridyl derivative displayed a loss in inhibitory potency
averaging 12- to 14-fold relative to 1 (IC50 100 and 43 nM for
human and rat sEH, respectively). A plausible explanation for
the reduced potency of 8 compared to 1 invokes an intramo-
lecular hydrogen bond between the amide nitrogen and the
2-pyridyl nitrogen, leading the pyridyl ring system to adopt a
coplanar conformation withthe amide, thereby positioning its
˚
2-pyridyl nitrogen in close proximity (3.3 A) to the charged
oxygen atoms of the Asp 335 side chain, resulting in a
repulsive electrostatic interaction. This rationale was sup-
ported by comparing interaction energies between 1 and 8
within human sEH using a cocrystal structure of 1 (vide supra
and Figure 5) and a model of 8 in the identical conformation
asbasisfor the calculations. Using the OPLS2005 force field, a
difference of 1.7 kcal/mol favoring 1 over 8 was calculated.
Evaluation of the in vitro metabolic stability profiles for
these first sets of compounds (Tables 1 and 3) indicated rapid
metabolism in human and rat liver microsomes (see Experi-
mental Section for method). We anticipated that the observed
rapidinvitrometabolismwould translatetorapidclearancein
vivo, hence limiting the sustained in vivo plasma exposure of
these compounds. In accordance with this hypothesis, the
plasma levels of 1 in the rat after oral administration were
quickly depleted over time (data not shown), supporting the
assumption that in vitro metabolic stability is predictive of in
vivo clearance for this compound class. Careful examination
of the in vitro metabolites generated from 1 in rat liver
microsomes confirmed the extensive hydroxylation of the
phenyl groups of the benzhydryl motif, which accounted for

5886 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
Figure 5. X-ray cocrystal structure of human sEH and 1. (a) The 2fo - fc map contoured at 0.95σ showing the orientation of 1 in the binding
site of human sEH. The catalytic residues Tyr 383, Tyr 466, and Asp 335 encircle the amide group of the ligand. Key residues involved in the
catalytic mechanism are shown, as are some of the hydrophobic residues that line the Phe 267 pocket. The distance between the ligand and the
rear of the Phe 267 pocket is indicated. (b) Surface representation illustrating how the environments of the C2 and C3 are very different. The C2
group is seen to be buried deep in the Phe 267 pocket, whereas the C3 phenyl is directed toward the solvent opening.
murine sEH N-cyclohexyl-N0-(4-iodophenyl) urea (CIU)
structure.25
is of interest to note that the C2 phenyl group does not fully
occupy this pocket, which suggests that additional potency
may be gained by more completely filling this pocket with
hydrophobic substituents. The C3 phenyl group in 1 is
directed toward the solvent opening of the Phe 267 pocket
(Figure5b). Previousinhibitor-sEHcostructureshaveshown
that this region of the protein can accommodate a variety of
substituents, including cyclohexyl and carboxylic acid groups.
In the costructure with1, the phenyl ring is positioned to allow
π-stacking interaction with His 524. This interaction results in
the ring lying close to one side of the pocket. In contrast, the
cyclohexyl and acid moieties, seen in other structures, have
tended to lie slightly more to the center of the pocket.
The trajectory of the propyl linker between the amide and
benzhydryl motif is similar to that reported for the cyclohexyl
urea compounds, with the molecule stretching from the active
site triad toward the Phe 267 pocket. At this point, compound
1 bifurcates with one phenyl group, designated here as C2,
directed toward the back of the Phe 267 pocket, and the other,
referred here as C3, pointing toward the solvent opening. The
phenyl directed to the rear of the pocket has a different
trajectory from that seen in other sEH inhibitors: the cocrystal
structure of human sEH with the CIU inhibitor does occupy
some of the same space as the phenyl group, but the 4-iodo
phenyl in that inhibitor is not directed as deeply toward the
core of the protein. In the costructure with 1, the C2 phenyl
group is surrounded by hydrophobic residues (Phe 267, Pro
268, Phe 387, Leu 397, Leu 406, Leu 417, Leu 428, Leu 463). It
The fact that the two phenyl groups in the costructure of 1
occupy very different environments within the protein, one
directed to a hydrophobic region of the protein, the other
positioned in a more polar and solvent exposed environment,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5887
Figure 6. X-ray cocrystal structure of human sEH and 24. The 2fo - fc map contoured at 0.95σ, showing that 24 binds in a similar mode to 1.
The structure reveals that the 4-fluorophenyl group is directed into the hydrophobic environment of the Phe 267 pocket, whereas the
4-methanesulphonyl substituted phenyl group is directed toward the solvent.
suggests that the introduction of asymmetry may be accom-
modated by the binding site. Asymmetric functionalization to
exploit these differences might provide a path to the improve-
ment of physicochemical properties as well as to improved
potency. In pursuit of this idea, methanesulfonyl, a polar and
nonionizable substituent, was introduced in the phenyl
4-position of the benzhydryl motif. The 4-fluoro substitution
of the phenyl was maintained to prevent oxidative metabolism
at this position. The effect of this combination of substituents
on intrinsic potency and in vitro metabolic stability was
probed by the synthesis of the racemic analogues 21-25
(Table 5) in which the left-hand side aryl pharmacophore
was either unsubstituted or substituted with hydroxyl, 2,2,
2-trifluoroethyloxy, cyano, or methanesulfonyl. The inclusion
of the methanesulfonyl substituent on the benzhydryl motif of
the nicotinamide derivative, 21, resulted in an sEH inhibitor
with largely uncompromised potency relative to 1 (IC50s 8 and
14 nM for human and rat sEH, respectively). The 6-hydro-
xynicotinamide analogue of this compound (22) displayed a
minor decrease in intrinsic potency against the human and rat
sEH isoforms (IC50s 16 and 29 nM, respectively), whereas the
2,2,2-trifluoroethoxy, cyano, and methanesulfonyl substi-
tuted analogues 23, 24, and 25 remained equipotent to 1, with
IC50s ranging from 4 to 7 nM against human sEH and from 6
to 9 nM against the rat enzyme. These results indicate that the
methanesulfonyl substitution is well accommodated by sEH.
As might be anticipated, the in vitro metabolism of the
nicotinamide derivative, 21, which lacks a left-hand side
substituent, remained rapid in both human and rat liver
microsomes, with half-lives of 6 and 12 min, respectively. In
contrast, the in vitro half-lives for the analogues with polar
left-hand side substituents (Table 5, 22-25) ranged from a
minimum value of 150 min to greater than 300 min, demon-
strating that highly in vitro metabolically stable inhibitors can
be achieved with this scaffold. The plasma levels of the sEH
inhibitor 24 were determined in the rat after oral administra-
tion at 5 mg/kg (see Experimental Section for methods).
Compound plasma exposure was 3.8, 3.5, 3.6, and 2.9 μM
at the 1, 2, 4, and 6 h time-points, respectively, identifying this
compound as a potent, in vitro metabolically stable sEH
inhibitor, with sustained exposure in the rat.
To confirm that the unsymmetrically substituted analogue
24 bound in the expected orientation, a cocrystal structure
with human sEH was obtained (Figure 6). The structure
obtained confirmed the anticipated binding mode with very
little difference in the position of the central amide core of the
molecule in relation to the protein compared to the costruc-
ture with 1. In the Trp 336 niche, the phenyl group of the
4-cyano-phenyl lies in a similar orientation to the 3-pyridyl of
1. The cyano group is well accommodated within the large
solvent-filled cavity of the Trp 336 niche, with Met 339
moving to accommodate the substituent. As expected, the
4-fluorophenyl group occupies the same space as the C2
phenyl in the costructure with 1, the phenyl group lying in a
very similar plane. It was noted earlier in the context of the
costructure of 1 that the C2 phenyl group did not fully occupy
the deep hydrophobic pocket. As anticipated, the 4-fluoro-
phenyl group of 24 is accommodated by the pocket. The
4-methanesulfonyl substituted phenyl group, which was de-
signed to capitalize on the more hydrophilic nature of the C3
pocket, lies in a similar orientation to the C3 group of 1, with
the methanesulfonyl substituent in close proximity to several
polar groups including the side chains of Ser 425 and Asp 496
and the backbone carbonyl of Ser 412.
Our evaluation of the unsymmetrically substituted inhibi-
tors 21-25 (Table 5) indicate that the polar methanesulfonyl
substituent is well accommodated by sEH and that the
observed improvements in microsomal stabilities lead to
decreased in vivo clearance. To better understand the impor-
tance of asymmetry for the molecular recognition of sEH, a
small set of enantiopure analogues was synthesized (Table 6,
entries S-24, R-24, S22, and R-22). The corresponding en-
antiomers of 24 displayed a 2- to 4-fold difference in potency
in favor of the S-enantiomer S-24 (IC50 8 and 6 nM, for
human and rat isoforms, respectively) over the R-enantiomer
R-24 (IC50 14 and 24 nM, for human and rat isoforms,
respectively). These results are in agreement with our analy-
sis of the solid-state structure, which predicts that the

5888 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
Table 6. Inhibitory Potency (IC50) against sEH and Stability in Micro-
somes (t1/2) for Compounds S-24, R-24, S-22, and R-22a
potent sEH inhibitor with attractive in vitro stability profiles
in rat and human liver microsomes and good oral exposure in
the rat. The structure-activity relationship of the lead, 1,
reveals a relatively strict requirement to the distance between
the central amide and benzhydryl motif, emphasizing the
importance of the amide as a recognition element for sEH
and suggesting a tight fit between the benzhydryl pharmaco-
phore and the enzyme. In contrast, the structure-activity
relationship reveals no particular structural requirements to
the left-hand side aryl pharmacophore. Although not pre-
ferred, the transpositionofthispharmacophore is allowed and
substitution is well tolerated, as is the replacement of the 3-
pyridyl pharmacophore with other (hetero)aryl groups. The
above observations are corroborated by the costructure of 1
with sEH, in which the C3 phenyl appears to participate in a
π-stacking interaction with His 524, and the C2 phenyl is in
relatively close contact with several hydrophobic residues.
The importance of the interaction between the central amide
pharmacophore and the Tyr 466, Tyr 383, and Asp 335
residues can not be overemphasized and is illustrated by the
diminished activity of the compound in which the amide
configuration is reversed and the complete abrogation of
activity for the sulfonamide derivative.
Optimization of the in vitro and in vivo metabolic para-
meters was accomplished using an integrated approach of
metabolite identification and structure-based drug design
aimed at preserving the inherently high inhibitory activity of
this lead class. The identification of metabolically vulnerable
sites and subsequent substitution with fluoro atoms led to the
identification of an in vitro metabolically stable inhibitor, 13.
However, good in vitro metabolic stability for this compound
is likely attained at the expense of suitable physicochemical
properties and efficient in vivo absorption. Guided by the
analysis of the costructure of 1 with human sEH, one fluorine
atom was substituted for an additional polar substituent on
the right-hand side benzhydryl motif, leading to the identi-
fication of a class of potent and in vitro metabolically
stable sEH inhibitors. Compound 24 from this class was
profiled extensively (Scheme 3) and was found to have a low
potential for drug-drug interactions, good selectivity against
EETs-generating CYP epoxygenases such as CYP2J2,
CYP2C8, and CYP2C9, excellent in vitro microsomal stabi-
lity, and high permeability in caco-2 cells (data not shown).
The oral administration of 24 in the rat led to sustained
compound exposure in plasma in the 3-4 μM range. Con-
sidering its overall profile, 24 is an appropriate candidate for
in vivo pharmacological profiling.
a Enantiopure compounds were synthesized to examine the impor-
tance of the asymmetry for the inhibition of sEH.
S-enantiomer will position the polar methanesulfonyl subtitu-
ent in the C3 pocket, leaving the 4-fluorophenyl group to
occupy the relatively hydrophobic C2 pocket. The evaluation
of the enantiomers of the intrinsically less potent 22, S-22, and
R-22 confirmed the preference for placement of the hydrophilic
methanesulfonyl substituent in the C3 binding pocket, in this
case suggesting a greater, 4- to 12-fold, difference between the
intrinsic potency of the S-enantiomer S-22 (IC50s 6 and 7 nM,
for human and rat sEH) over the R-enantiomer R-22 (IC50s 23
and 87 nM, for human and rat sEH). Because of a limitation
imposed by the affinity of the probe, the binding assay used in
our studies can not provide an accurate ranking of compounds
with IC50s less than approximately 5 nM. To get a more
accurate measure of the relative potencies of the advanced
compounds 21-25 (Tables 5 and 6), their profiling was ex-
tended to include a cell-based assay in Hep G2 cells. Evaluation
of the unsymmetrically substituted sEH inhibitors (Table 5,
21-25) in the cell-based assay confirmed the ranking observed
in the fluorescence polarization assay (IC50s 4, 6, 7 nM,
respectively) and revealed that compounds 23, 24, and 25 are
in fact of equal potency (EC50s 0.3, 0.3, and 0.4 nM, re-
spectively). In contrast, the evaluation of the enantiopure
derivatives (Table 6, S-24, R-24, S-22, and R-22), revealed
larger differences in the potency of the S- and R-enantiomers
than estimated from the profiling in the fluorescence polariza-
tion assay. In fact, the data acquired in the cell-based assay
suggests 14-20 fold differences in potency in favor of the
S-enantiomers relative to the R-enantiomers with an EC50 of
0.14 nM for the S-enantiomer of 24 (S-24).
Experimental Section
General Procedures. 1H NMR spectra were recorded on a
Bruker Avance (400 MHz) spectrometer. Chemical shifts are
reported in ppm from tetramethylsilane with the solvent reso-
nance as the internal standard (CDCl3: 7.26 ppm). Data are
reported as follows: chemical shifts, multiplicity (s=singlet, d=
doublet, t = triplet, q = quartet, br = broad, m = multiplet),
coupling constants (Hz), and integration. Liquid chromatogra-
phy was performed using prepacked silica gel columns (Analogix
Flash chromatograph RS-12 cartridges) of the indicated solvent
system. Separation was achieved with a CombiFlash SQ16X
chromatographer by Isco. All compounds described were of
>95% purity. Purity was confirmed by analytical LC/MS re-
corded on a system consisting of a Waters ZQ mass spectrometer,
an Agilent 1100 DAD, Sedex 85 ELS detector, and an Agilent
1100 HPLC system, equipped with a Zorbax SB-C18 rapid
Conclusions
In conclusion, 4-cyano-N-[3-(4-fluorophenyl)-3-(4-metha-
nesulfonyl-phenyl)-propyl]-benzamide, 24, was identified as a

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5889
Scheme 3. SAR Advancement, Illustrated by the Profiles of Compounds 1, 13, and 24a
a The original screening hit 1 displayed high potency against both human and rat sEH and acceptable selectivity towards EETs forming CYPs but
lacked in vitro metabolic stability. SAR to define an optimal scaffold for sEH inhibition, paired with substitution of the right-hand side benzhydryl motif
with fluoro atoms, led to the identification of an in vitro metabolically stable inhibitor, 13. However, good in vitro metabolic stability for this compound
was attained at the expense of efficient in vivo absorption. Guided by the analysis of the co-structure of 1 with human sEH, one fluorine atom was
substituted for an additional polar substituent, leading to the identification of compound 24. This compound was found to have a low potential for
drug-drug interactions, good selectivity against EETs-generating CYP epoxygenases such as CYP2J2, CYP2C8, and CYP2C9, excellent in vitro
microsomal stability, and high and sustained compound exposure in the rat.
resolution cartridge (4.6 mm ꢀ 30 mm, 3.5 um). Elution started
with 95% water (0.1% formic acid)/5% acetonitrile (0.1% formic
acid) and ended with 95% acetonitrile (0.1% formic acid)/5%
water (0.1% formic acid) and used a linear gradient at a flow rate
of 2.5 mL/min. Final compounds were analyzed by high resolu-
tion mass spectrometry acquired on an Agilent LC/MSD TOF
mass spectrometer equipped with an Agilent 1100 binary pump,
WPALS auto sampler, Colcom oven, and DAD detector. Direct
loop injection was performed using a 5 μL injection volume at a
flow rate of 0.8 mL/min. Samples were dissolved in mobile phase,
which consisted of 70/30 (A:B) (isocratic) with A 0.05% formic
acid water (v/v) and B 0.05% formic acid in methanol (v/v).
Detection was performed using positive ion ESI with a routine
resolution of 6500 at m/z 322. Positive ion electrospray mass
spectra of all final compounds were consistent with the proposed
structures and molecular formula.
The following compounds were synthesized according to avail-
able literature procedures: 4,4-diphenyl-butylamine (Caldirola,
1992), 3,3-bis-(4-fluorophenyl)-propylamine (Buschauer, 1992),
4,4-diphenylbutyriuc acid (Miyano, 1990). The following chemi-
cals were used as received: Hydroxybenzotriazole (Aldrich 36,244-
1), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimde hydrochlor-
ide (Aldrich 16,146-2), diisopropylethylamine (Aldrich 38,764-9),
nicotinic acid (Aldrich N785-0), 3,3-diphenyl-propylamine
(Aldrich 13,629-8), 2,2-diphenyl-ethylamine (Aldrich D,20,670-
9), phenethylamine (Aldrich 40,726-7), pyridine-2-carboxylic acid
(picolinic acid) (Aldrich P4,280-0), 3-aminopyridine (Aldrich
A-7,820-9), isonicotinic acid (Aldrich I-1,750-8), benzensulfonyl
chloride (Aldrich 10,813-8), isonicotinic acid (Aldrich I-1,750-8),
2-(pyrdin-3-yl)-acetic acid hydrochloride (Aldrich P6,580-0),
6-hydroxynicotinic acid (Aldrich 12,875-9), 6-(2,2,2-tri-
fluoro-ethoxy)-isonicotinic acid (Ryan Scientific RF 04860),
2-(2,2,2-trifluoro-ethoxy)-isonicotinic acid (Oakwood 017206),
4-cyanobenzoic acid (Aldrich C-8,980-3), 4-(methanesulfonyl)-
benzoic acid (Aldrich 13,641-7), 4-fluorocinnamic acid (Aldrich
22,272-0), (R)-4-phenyl-2-oxazolidinone (Aldrich 40,245-1), (S)-
4-phenyl-2-oxazolidinone (Aldrich 37,669-8), lithium diisopropyl
amide (2 M in heptane/tetrahydrofuran/ethylbenzene) (Aldrich
36,179-8), copper(I)bromide-methylsulfide complex (Aldrich
23,050-2), methylsulfide (Aldrich 47,157-7), thioanisole magne-
sium bromide (0.5 M in tetrahydrofuran) (Aldrich 56,176-2).
General Procedure for Amide Bond Formation. To a solution
of the carboxylic acid (1 equiv) in N,N0-dimethylformamide was
added the amine (1.0 equiv), followed by the addition of 1-
hydroxybenzotriazole (2.0 equiv), 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimde hydrochloride (2.0 equiv), and diisopropy-
lethylamine (3.0 equiv). The reaction was stirred overnight. The
mixture was diluted with water and extracted twice with dichloro-
methane. The organic phase was evaporated in vacuo, and
the crude product was purified by silica gel chromatography
(0-5% methanol in dichloromethane) to provide desired
product.
N-(3,3-Diphenyl-propyl)-nicotinamide (1). This compound
was synthesized on 0.47 mmol scale from nicotinic acid and
3,3-diphenyl-propylamine according to the general procedure
for amide coupling to give the desired compound (110 mg,
73.3%). 1H NMR δ: 8.75 (m, 1H), 8.71 (m, 1H) 8.06-8.01 (m,
1H), 7.40 (m, 1H), 7.29-7.35 (m, 8 H), 7.27-7.19 (m, 2H), 6.22
(brs, 1H), 4.06 (t, J=7.7 Hz 1H) 3.55 (m, 2H), 2.47 (q, J=7.1 Hz,
2H). HRMS: calcd for C21H20N2O þ Hþ 317.1654; found
317.1663.
N-(2,2-Diphenyl-ethyl)-nicotinamide (2). This compound was
synthesized on 1.63 mmol scale from nicotinic acid and 2,2-
diphenyl-ethylamine according to the general procedure for
amide coupling to give the desired compound (333 mg,
67.8%). 1H NMR δ: 8.80 (m, 1H), 8.69 (m, 1H), 8.05 (m, 1H),
7.30-7.39 (m, 11 H), 6.25 (brs, 1H), 4.37 (t, J=8.07 Hz, 1H),
4.15 (q, J=8.10 Hz, 2H). HRMS: calcd for C20H18N2O þ Hþ
303.1497; found 303.1499.
N-(4,4-Diphenyl-butyl)-nicotinamide (3). This compound was
synthesized on 1.40 mmol scale from nicotinic acid and 4,4-
diphenyl-butylamine according to the general procedure for
amide coupling to give the desired compound (314 mg,
68.0%). 1H NMR δ: 8.90 (m, 1H), 8.61 (m, 1H), 8.05 (m, 1H),
7.32 (m, 1H), 7.12-7.29 (m, 10H), 6.28 (brs, 1H), 3.86 (t, J=7.8
Hz, 1H), 3.41 (q, J=7.2 Hz, 2H), 2.06 (m, 2H), 1.54 (m, 2H).
HRMS: calcd for C22H22N2 O þ Hþ 331.1810; found 331.1802.
N-(3-Phenyl-propyl)-nicotinamide (4). This compound was
synthesized on 1.63 mmol scale from nicotinic acid and phe-
nethylamine according to the general procedure for amide
1
coupling to give the desired compound (257 mg, 65.8%). H
NMR δ: 8.88 (m, 1H), 8.72 (m, 1H), 8.06 (m, 1H), 7.30-7.39 (m,
6 H), 6.25 (brs, 1H), 3.52 (q, J=6.89 Hz, 2H), 2.77 (t, J=7.42 Hz,
2H), 2.02 (m, 2H). HRMS: calcd for C15H16N2 O þ Hþ
241.1341; found 241.1339.

5890 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
N-(3,3-Diphenyl-propyl)-2-pyridine-3-ylacetamide (5). This
compound was synthesized on 1.15 mmol scale from
2-(pyridin-3-yl)-acetic acid and 3,3-diphenyl-propylamine ac-
cording to the general procedure for amide coupling to give the
desired compound (309 mg, 81.2%). 1H NMR δ: 8.55 (m, 2H),
7.71 (m, 1H), 7.29 (m, 6H), 7.20 (m, 5H), 5.25 (brs, 1H), 3.91 (t,
J=7.83 Hz, 1H), 3.40 (s, 2H), 3.24 (q, J=6.36 Hz, 2H), 2.27 (m,
2H). HRMS: calcd for C22H22N2O þ Hþ 331.1810; found
331.1809.
N-[4,4-Bis-(4-fluorophenyl)-propyl]-6-hydroxy-nicotinamide (12).
This compound was synthesized on 2.02 mmol scale from
6-hydroxynicotinic acid and 3,3-bis-(4-fluorophenyl)-propyla-
mine17 according to the general procedure for amide coupling to
give the desired product (480 mg, 64.4%). 1H NMR δ: 8.17 (s, 1H),
7.80 (d, J=8.8 Hz, 1H), 7.08 (q, J=5.44 and 3.27 Hz, 4H), 6.96 (brs,
1H), 6.87 (t, J=9.12 Hz, 4H), 6.55 (d, J=9.12 Hz, 1H), 3.88 (t,
J=8.18 Hz, 1H), 3.31-3.22 (m, 2H), 2.23 (q, J=6.95 and 7.77 Hz,
2H). HRMS: calcd for C21H18F2N2O2 þ Hþ 369.1415; found
369.1396.
4,4-Diphenyl-N-(pyridin-3-yl)-butyramide (6). To 4,4-diphe-
nylbutyric acid16 (243 mg, 1.00 mmol) in dichloromethane
(2 mL) was added oxalyl chloride (0.18 mL) and one drop of
N,N0-dimethylformamide. The mixture was stirred at room
temperature for 1 h, evaporated in vacuo, and redissolved in
dichloromethane (2.50 mL). To this solution was added
3-aminopyridine (94 mg, 1.06 mmol) and triethylamine (0.25
mL, 1.80 mmol). The mixture was stirred at room temperature
for 16 h, evaporated in vacuo, and purified on silica using
dichlormethane/methanol (96:4) as the eluent. Fractions con-
taining the product were pooled and evaporated in vacuo to give
the desired product (186 mg, 55.3%). 1H NMR δ: 8.53 (m, 1H),
8.27 (m, 2H), 7.44 (brs, 1H), 7.22-7.31 (m, 9H), 7.21 (m, 2H),
4.01 (t, J=7.94 Hz, 1H), 2.53 (m, 2H), 2.39 (t, J=7.69 Hz, 2H).
HRMS: calcd for C21H20N2O þ Hþ 317.1654; found 317.1632.
Pyridine-3-sulfonic Acid 3,3-(Diphenylpropyl)-amide (7). To a
solution of benzenesulfonyl chloride (213 mg, 1.00 mmol) in
dichloromethane (5 mL) was added 3,3-(diphenylpropyl)-amine
(211 mg, 1.00 mmol) followed by triethylamine (0.28 mL, 2.00
mmol). The heterogeneous mixture was stirred at room tem-
perature overnight, evaporated in vacuo, and purified on silica
using silica using dichlormethane/methanol (95:5) as the eluent.
Fractions containing the product were pooled and evaporated in
vacuo to give the desired product (167 mg, 47.9%). 1H NMR δ:
8.84 (m, 1H), 8.62 (m, 1H), 7.87 (m, 1H), 7.25 (m, 1H) 7.16-6.94
(m, 10H), 4.29 (t, 1H), 3.77 (t, 1H), 2.82 (q, 2H), 2.10 (q, 2H).
HRMS: calcd for C20H20N2O2S þ Hþ 353.1324; found
353.1318.
N-[3,3-Bis-(4-fluorophenyl)-propyl]-6-(2,2,2-trifluoro-ethoxy)-
nicotinamide (13). This compound was synthesized on 0.20
mmol scale from 6-(2,2,2-trifluoro-ethoxy)-nicotinic acid and
3,3-bis-(4-fluorophenyl)-propylamine17 according to the gene-
ral procedure for amide coupling to give the desired product (31
mg, 34.0%). 1H NMR δ: 8.34 (d, 1H), 7.86 (dd, J=8.6 and 2.69
Hz, 1H), 7.16-7.09 (m, J=5.58 Hz, 4H), 6.9-6.87 (m, J=9.11
Hz, 4H), 6.8 (d, J =8.75 Hz, 1H), 5.9-5.8 (m, 1H), 4.72 (q,
J =8.38 and 8.47 Hz, 2H), 3.92 (t, J =8.1 Hz, 1H), 3.36 (q,
J=6.14 and 7.79 Hz, 2H), 2.28 (q, J=6.87 and 7.60 Hz, 2H).
HRMS: calcd for C23H19F5N2O2 þ Hþ 451.1445; found
451.1418.
N-[3,3-Bis-(4-fluorophenyl)-propyl]-2-(2,2,2-trifluoro-ethoxy)-
isonicotinamide (14). This compound was synthesized on 0.41
mmol scale from 2-(2,2,2-trifluoro-ethoxy)-isonicotinic acid
and 3,3-bis-(4-fluorophenyl)-propylamine17 according to the
general procedure for amide coupling to give the desired product
(61 mg, 34.0%). 1H NMR δ: 8.14 (d, J=5.37 Hz, 1H), 7.17-7.07
(m, 5H), 6.97-6.8 (m, 5H), 6.02-5.09 (m, 1H), 4.71 (q, J=8.45
and 8.55 Hz, 2H), 3.91 (t, J=8.27 Hz, 1H), 3.36 (q, J=6.2 and
7.65 Hz, 2H), 2.27 (q, J=6.77 and 7.67 Hz, 2H). HRMS: calcd
for C23H19F5N2O2 þ Hþ 451.1445; found 451.1420.
4-Cyano-N-[3,3-bis-(4-fluorophenyl)-propyl]-benzamide (15).
This compound was synthesized on 0.20 mmol scale from 4-cya-
nobenzoic acid and 3,3-bis-(4-fluorophenyl)-propylamine17
according to the general procedure for amide coupling to give
1
the desired product (75 mg, 98.0%). H NMR δ: 7.64 (s, 4H),
Pyridine-2-carboxylic Acid (4,4-Diphenyl-propyl)-amide (8).
This compound was synthesized on 0.47 mmol scale from
pyridine-2-carboxylic acid and 4,4-diphenyl-propylamine ac-
cording the general procedure for amide coupling to give the
desired product (17 mg, 11.0%). 1H NMR δ: 8.65 (m, 1H), 8.21
(m, 2H), 7.88 (m, 1H), 7.47 (m, 1H) 7.30-7.26 (m, 7 H), 7.2 (m,
2H), 4.08 (t, J=7.87 Hz, 1H), 3.46 (q, J=8.0 Hz, 2H), 2.45 (m,
2H). HRMS: calcd for C21H20N2O þ Hþ 317.1654; found
317.1650.
N-(3,3-Diphenyl-propyl)-isonicotinamide (9). This compound
was synthesized on 0.47 mmol scale from isonicotinic acid and
3,3-diphenyl-propylamine according to the general procedure
for amide coupling to give the desired product (93.9 mg, 62.7%).
1H NMR δ: 8.69 (m, 2H), 7.44 (m, 2H), 7.30-7.39 (m, 8 H), 7.22
(m, 2H), 6.12 (brs, 1H), 4.06 (t, J=7.72 Hz, 1H), 3.54 (q, J=6.74
Hz, 2H), 2.46 (m, 2H). HRMS: calcd for C21H20N2O þ Hþ
317.1654; found 317.1649.
N-[3,3-Bis-(4-fluorophenyl)-propyl]-benzamide (10). This com-
pound was synthesized on 0.82 mmol scale from benzoic acid and
3,3-diphenyl-propylamine according to the general procedure for
amide coupling to give the desired product (231 mg, 89.5%). 1H
NMR δ: 7.50 (m, 2H), 7.33 (m, 3H), 7.20-7.31 (m, 8 H), 7.12 (m,
2H), 5.92 (brs, 1H), 3.96 (t, J=7.70 Hz, 1H), 3.41 (q, J=6.66 Hz,
2H), 2.35 (m, 2H). HRMS: calcd for C22H21NO þ Hþ 316.1701;
found 316.1706.
N-[3,3-Bis-(4-fluorophenyl)-propyl]-nicotinamide (11). This
compound was synthesized on a 0.48 mmol scale from nicotinic
acid and 3,3-bis-(4-fluorophenyl)-propylamine17 according to
the general procedure for amide coupling to give the desired
compound (25 mg, 14.6%). 1H NMR δ: 8.84 (brs, 1H), 8.61 (brs,
1H), 8.1-7.9 (d, 1H), 7.5-7.22 (m, 1H), 7.00-7.20 (m, 4H),
6.7-6.98 (m, 4H), 6.3-6.55 (m, 1H), 3.92 (t, 1H), 3.36 (q,2H),
2.29 (q, 2H).
7.17-7.09 (m, 4H), 6.96-6.86 (m, 4H), 6.01-5.09 (m, 1H), 3.92
(t, J=7.9 Hz, 1H), 3.39 (q, J=6.02 and 7.93 Hz, 2H), 2.29 (q, J=
6.76 and 7.85 Hz, 2H). HRMS: calcd for C23H18F2N2O þ Hþ
376.1387; found 376.1448.
N-[3,3-Bis-(4-fluorophenyl)-propyl]-4-methanesulfonyl-benza-
mide (16). This compound was synthesized on 0.20 mmol scale
from 4-methanesulfonyl benzoic acid and 3,3-bis-(4-fluoro-
phenyl)-propylamine17 according to the general procedure for
amide coupling to give the desired product (73 mg, 84.1%). 1H
NMR δ: 7.89 (d, J=8.32 Hz, 2H), 7.73 (d, J=8.56 Hz, 2H),
7.17-7.09 (m, 4H), 6.97-6.86 (m, 4H), 6.13-6.00 (m, 1H), 3.93
(t, J=7.9 Hz, 1H), 3.39 (q, J=6.27 and 7.62 Hz, 2H), 2.99 (S, 3
H), 2.3 (q, J = 6.76 and 7.49 Hz, 2H). HRMS: calcd for
C23H21F2NO3S þ Hþ 430.1288; found 430.1261.
3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propylamine
(20). To the t-Boc protected amine 1919 (1.20 g, 2.95 mmol) in
methanol was added and HCl (aq, 6N) (8.0 mL) dropwise. The
mixture was stirred at room temperature for 1 h and evaporated
in vacuo to give a white solid that was triturated twice with
diethyl ether and once with hexanes to give the desired com-
pound as a colorless solid (872 mg, 86.1%). 1H NMR (DMSO-
d6) δ: 7.85 (d, 2H), 7.80 (brs, 3H), 7.65 (d, 2H), 7.50 (m, 2H), 7.36
(m, 2H), 7.14 (m, 4H) 4.25 (m, 1H), 3.19 (s, 3H), 2.69 (m, 2H),
2.31 (m, 2H).
N-[3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl]-
nicotinamide (21). This compound was synthesized on 0.55
mmol scale from nicotinic acid and 3-(4-fluorophenyl)-3-(4-
methanesulfonyl-phenyl)-propylamine (20)18 according to the
general procedure for amide coupling to give the desired product
(140 mg, 58.9%). 1H NMR δ: 9.52 (brs, 1H), 8.64 (m, 1H), 8.50
(m, 1H), 7.88-7.47 (overlapping m, 3H), 7.66 (m, 1H),
7.45-7.41 (m, 2H), 7.24-7.14 (m, 2H), 7.00-6.91 (m, 2H),
4.18 (t, J = 7.30 Hz, 1H), 3.42-3.50 (m, 2H), 2.91 (s, 3H),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5891
2.29-2.48 (m, 2H). HRMS: calcd for C22H21FN2O3S
413.1335 þ Hþ; found 413.1316.
allowed to come to room temperature, and poured into 0.5N aq
hydrochloric acid (80 mL). The aqueous solution was extracted
with ethyl acetate (2 ꢀ 50 mL) and the combined organic
fractions washed with sodium bicarbonate (aq, saturated)
(75 mL), water (50 mL), brine (50 mL), dried over sodium
sulfate, and evaporated in vacuo. The crude product was
purified on silica using heptane/ethyl acetate (3:1) as the eluent.
Fractions containing the product were pooled and evaporated in
N-[3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl]-
6-hydroxy-nicotinamide (22). To 6-hydroxynicotinic acid (139
mg, 1.00 mmol) was added thionyl chloride (1.8 mL) and the
resulting mixture was heated at reflux for 2 h. The mixture was
evaporated in vacuo, redissolved in dichloromethane (3 mL),
and cooled to 0 °C. Triethylamine (0.24 mL) and 3-(4-
fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propylamine (20)
(170 mg, 0.55 mmol) was added, and the mixture was allowed
to come to room temperature and stirred for 2 h. The reaction
was quenched by the addition of water (20 mL) and extracted
twice with diethyl ether (2 ꢀ 10 mL). The combined organic
phase was back-extracted with sodium bicarbonate (saturated,
aq) (10 mL) and brine (10 mL), dried over magnesium sulfate,
evaporated in vacuo, and purified on silica using dichlor-
methane/methanol (95:5) as the eluent. Fractions containing
the product were pooled and evaporated in vacuo to give the
desired product (107 mg, 25.0%). 1H NMR δ: 12.01 (brs, 1H),
11.64 (brs, 1H), 8.06 (s, 1H), 7.76 (m, 1H), 7.71 (m, 2H), 7.37 (m,
2H), 7.13 (m, 2H), 6.93 (m, 1H), 6.83 (m, 1H), 6.35 (m, 1H), 4.04
(t, 1H), 3.29 (q, 2H), 2.98 (s, 3H), 2.32 (q, 2H). HRMS:
C22H21FN2O4S þ Hþ 429.1284; found 429.1259.
1
vacuo to give desired compound (4.10 g, 77.7%). H NMR δ:
7.85 (d,1H), 7.72 (d, 1H) 7.60 (m, 2H), 7.39 (m, 5H), 7.09 (m, 2H)
5.58 (dd, 1 H), 4.76 (m, 3H), 4.35 (dd, 1H).
(S)-3-[3-(4-Fluorophenyl)-3-(4-methylsulfanyl-phenyl)-propionyl]-
(R)-4-phenyl-oxazolidin-2-one (28a). To a precooled (-40 °C)
solution of copper(I)bromide methylsulfide complex (4.06 g,
19.8 mmol) in tetrahydrofuran (40 mL) was added dimethylsul-
fide (20 mL), followed by the slow addition of thioanisole
magnesium bromide (0.5 M in tetrahydrofuran) (79.0 mL,
39.5 mmol). The mixture was allowed to warm to -20 °C
over 15 min and a solution of 27a (4.10 g, 13.2 mmol) in
tetrahydrofuran (20 mL) was added dropwise over 1 h. The
resulting solution was stirred at -20 °C for 1 h, allowed to come
to room temperature, stirred overnight, and poured into satu-
rated aq ammonium chloride (200 mL). The mixture was
extracted with ethyl acetate (2 ꢀ 150 mL) and the combined
organic fractions washed with ammonium hydroxide (2 ꢀ 15%
aq) (150 mL), water (150 mL), brine (150 mL), dried over
sodium sulfate, and evaporated in vacuo. The crude product
was purified on silica using heptane/ethyl acetate (3:1) as the
eluent. Fractions containing the product were pooled and
evaporated in vacuo to give desired compound (5.17 g, 90.0%)
N-[3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl]-
6-(2,2,2-trifluoro-ethoxy)-nicotinamide (23). This compound
was synthesized on 0.553 mmol scale from 6-(2,2,2-trifluoro-
ethoxy)-nicotinic acid and 3-(4-fluorophenyl)-3-(4-methanesul-
fonyl-phenyl)-propylamine (20) according to the general proce-
dure for amide coupling to give the desired product (89 mg,
31.5%). 1H NMR δ: 8.36 (d, J=2.62 Hz, 1H), 7.90 (dd, J=8.26
and 2.25 Hz, 1H), 7.79 (d, J=8.39, 2H), 7.38 (d, J=8.39, 2H),
7.18-7.10 (m, 2H), 7.00-6.89 (m, 2H), 6.83 (d, J=8.77 Hz, 2H),
6.03-5.883.93 (m, 1H), 4.73 (q, J=8.43 and 8.43 Hz, 2H), 4.03
(t, J=7.30 Hz, 1 H), 3.37 (q, J=6.20 and 7.81 Hz, 2H), 2.96 (s,
3H), 2.34 (q, J = 6.74 and 7.81 Hz, 2H). HRMS: calcd for
C24H22F4N2O4S 511.1315 þ Hþ; found 511.1320.
1
as a colorless solid. H NMR δ: 6.9-7.3 (m,,13H), 5.33 (dd,
1 H), 4.61 (m, 1H), 4.55 (m, 1H), 4.21 (dd, 1H), 3.78 (dd, 1H),
3.64 (dd, 1H), 2.43 (s, 3H).
(S)-3-(4-Fluorophenyl)-3-(4-methylsulfanyl-phenyl)-propan-
1-ol (29a). To a precooled solution of 28a (5.10 g, 11.7 mmol) in
tetrahydrofuran (60 mL) was added water (10 mL) followed
by the portionwise addition of sodium borohydride (1.17 g,
30.2 mmol), keeping the temperature under 10 °C. The resulting
solution was allowed to come to room temperature, stirred
overnight, tetrahydrofuran was evaporated in vacuo, and water
(50 mL) was added. The solution was extracted with ethyl
acetate (2 ꢀ 50 mL) and the combined organic fractions washed
with brine (50 mL), dried over sodium sulfate, and evaporated in
vacuo. The crude product was purified on silica using heptane/
ethyl acetate (1:1) as the eluent. Fractions containing the
product were pooled and evaporated in vacuo to give desired
compound (2.45 g, 76.0%) as a colorless oil. 1H NMR δ: 7.0-7.3
(m,6H), 6.88 (m, 2H), 4.05 (m, 1H), 3.45 (m, 2H), 2.90 (brs, 1H),
2.35 (s, 3H), 2.18 (m, 2H).
4-Cyano-N-[3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-
propyl]-benzamide (24). This compound was synthesized on 2.90
mmol scale from 4-cyano-benzoic acid and 3-(4-fluorophenyl)-
3-(4-methanesulfonyl-phenyl)-propylamine (20) according to
the general procedure for amide coupling to give the desired
product (1.25 g, 98.5%). 1H NMR δ: 7.43 (d, J=8.37 Hz, 2H),
7.75 (q, J=8.47 and 8.56 Hz, 4H), 7.27-7.21 (m, 2H), 7.04 (t, J=
8.66, 2H), 6.26-6.18 (m, 1H), d, J=8.77 Hz, 2H), 4.13 (t, J=
7.85 Hz, 1H), 3.47 (q, J=6.34 and 8.10 Hz, 2H), 3.047 (s, 3 H),
2.44 (q, J = 7.63 and 7.06 Hz, 2H). HRMS: calcd for
C24H21FN2O3S þ Hþ 437.1335; found 437.1309.
N-[3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl]-
4-methanesulfonyl-benzamide (25). This compound was synthe-
sized on 0.650 mmol scale from 4-methanesulfonyl-benzoic acid
and 3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyla-
mine (20) according to the general procedure for amide coupling
to give the desired product (279 mg, 87.0%). 1H NMR δ: 7.88 (d,
J=8.36 Hz, 2H), 7.76 (t, J=8.05 Hz, 4H), 7.38 (d, 8.33 Hz, 2H),
7.16 (m, 2H), 6.98- 6.90 (m, 2H), 6.28 (m, 1H), 4.05 (t, J=7.97
Hz, 1H), 3.40 (q, J=7.07 and 6.16 Hz, 2H), 3.00 (s, 3 H), 2.94 (s,
3H), 2.45- 2.28 (m, 2H). HRMS: calcd for C24H24FNO5S þ Hþ
490.1158; found 490.1126.
3-[3-(4-Fluorophenyl-)acryloyl]-(R)-4-phenyl-oxazolidin-2-
one (27a). To a precooled (-10 °C) solution of 4-fluorocinnamic
acid (26) (2.96 g, 17.8 mmol) in tetrahydrofuran (80 mL) was
added trietheylamine (2.98 mmol, 21.4 mmol) followed by the
slow addition of pivaloyl chloride (2.36 g, 19.6 mmol). The
mixture was stirred at -10 °C for 30 min, cooled to -78 °C, and
a solution of the anion of (R)-4-phenyl-2-oxazolidinone
(generated from (R)-4-phenyl-2-oxazolidinone (3.20 g, 19.6
mmol) in tetrahydrofuran (90 mL) at -78 °C by treatment with
lithium diisopropyl amide (2 M in heptane/tetrahydrofuran/
ethylbenzene) (8.47 mL, 16.9 mmol) for 30 min) was added while
at -78 °C. The resulting mixture was stirred at -78 °C for 1 h,
(S)-3-(4-Fluorophenyl)-3-(4-methylsulfanyl-phenyl)-propyl-
azide (30a). To a precooled (-10 °C) solution of 29a (2.45 g, 8.86
mmol) in dichloromethane (40 mL) was added triethylamine
(1.48 mL, 10.6 mmol) followed by the slow addition of toluene-
sulfonyl chloride (2.03 g, 10.6 mmol). The mixture was stirred at
-10 °C for 1 h, stirred overnight at room temperature, and
evaporated in vacuo. The residue was dissolved in N,N-di-
methylformamide (20 mL), sodium azide (1.15 mg, 16.6 mmol)
was added, the mixture was stirred at room temperature over-
night, and water (40 mL) was added. The aqueous phase was
extracted with ethyl acetate (2 ꢀ 20 mL) and the combined
organic fractions washed with water (20 mL), brine (20 mL),
dried over sodium sulfate, and evaporated in vacuo. The crude
product was purified on silica using heptane/ethyl acetate (3:1)
as the eluent. Fractions containing the product were pooled and
evaporated in vacuo to give desired compound (2.52 g, 89.6%)
1
as a colorless oil. H NMR δ: 7.1-7.21 (m,6H), 6.95 (m, 2H),
4.05 (t, 1H), 3.22 (t, 2H), 2.45 (s, 3H), 2.35 (q, 2H).
(R)-3-(4-Fluorophenyl)-3-(4-methylsulfoxide-phenyl)-propyla-
zide (31a). To 30a (2.52 g, 8.36 mmol) was added a mixture of
glacial acetic acid (10 mL) and hydrogen peroxide (30% aq)

5892 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
(8 mL). The resulting solution was stirred at room temperature
for 1 h and water (20 mL) added, the aq phase was extracted
twice with ethyl acetate (2 ꢀ 20 mL), and the organic phase was
washed with brine (15 mL), dried over sodium sulfate, and
evaporated in vacuo to give the desired product (1.27 g 45.0%).
1H NMR δ: 7.62 (d, 1H), 7.39 (d, 1H), 7.20 (m, 1H), 7.00 (m,
1H), 4.13 (m, 1H), 3.25 (t, 2H), 2.70 (s, 3H), 2.30 (q, 2H).
Boc-(S)-3-(4-fluorophenyl)-3-(4-methanesulfoxide-phenyl)-
propylamine (32a). To 31a (1.27 g, 3.76 mmol) was added
methanol (15 mL) and Pd (10% on carbon) (200 mg), and the
mixture was stirred under hydrogen overnight, filtered through
celite (washing with methanol), and evaporated in vacuo to
desired compound as a colorless oil, which was used without
further purification (1.15 g, 47.3%). To the crude amine (1.15 g,
4.00 mmol) in dichloromethane (15 mL) was added di-tert-butyl
dicarbonate (Boc-anhydride) (490 mg, 5.20 mmol) and triethy-
lamine (1.0 mL, 7.20 mmol). The mixture was stirred at room
temperature for 4 h, and the organic phase was washed with aq
HCl (1N) (10 mL) and evaporated to give a colorless oil (1.54 g,
100%). 1H NMR δ: 7.58 (d, 2H), 7.40 (d, 2H), 7.20 (m, 2H), 7.00
(m, 2H), 4.55 (m, 1H), 4.01 (m, 1H) 3.15 (m, 2H), 2.71 (s, 3H), 2.2
(m, 2H) 1.40-1.60 (m, 9 H).
Boc-(S)-3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-pro-
pylamine (33a). To a solution of 32a (1.54 g, 4.00 mmol) in 80 mL
of chloroform is added aluminum oxide (4.19 g, 41.0 mmol)
pretreated with 0.84 mL of water. The resulting suspension was
stirred and treated with 8.37 g of oxone (13.6 mmol) and
refluxed for 23 h. The reaction was cooled to room temperature,
filtered, and evaporated in vacuo to give an amorphous solid.
The crude product was purified on silica using heptane/ethyl
acetate as the eluent (1.62 g, 100%). 1H NMR δ: 7.82 (d, 2H),
7.40 (d, 2H), 7.18 (m, 2H), 7.00 (m, 2H), 4.71 (m, 1H), 4.05 (m,
1H) 3.07 (m, 2H), 3.02 (s, 3H), 2.05 (m, 2H) 1.42 (s, 9 H); 91% ee
chiral HPLC Chiracel Chiralpak AD-H, 4.6 mm ꢀ 250 mm,
85% heptane, 15% ethanol, 1.0 mL/min 25 °C. Retention time
34.5 minor, 37.1 major.
(S)-3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-pro-
pylamine (S-20). To a solution of 33a (1.62 g, 4.00 mmol) in 6 mL
of dichloromethane was added 12 mL of trifluororacetic acid
followed by 0.60 mL of water. The resulting mixture was stirred
for 2 h and 15 min and the solvents evaporated in vacuo. The
resulting oil was taken up in dichloroethane and evaporated in
vacuo. The oil was then taken up in 15 mL of dichloroethane and
para-toulenesulfonic acid was added (760 mg, 4.00 mmol) in one
portion. The reaction was the evaporated in vacuo and the
resulting residue triturated with ether and heptane. The solid
was filtered and dried in vacuo to give a the desired compound
(0.94 g, 49.0%) as a colorless solid. 1H NMR (DMSO-d6) δ: 7.85
(d, 2H), 7.80 (brs, 3H), 7.65 (d, 2H), 7.50 (m, 2H), 7.36 (m, 2H),
7.14 (m, 4H) 4.25 (m, 1H), 3.19(s, 3H), 2.69 (m, 2H), 2.31
(m, 2H).
3-[3-(4-Fluorophenyl-)acryloyl]-(S)-4-phenyl-oxazolidin-2-one
(27b). To a precooled (-10 °C) solution of 4-fluorocinnamic
acid (26) (4.77 g, 28.7 mmol) in tetrahydrofuran (40 mL) was
added trietheylamine (4.80 mmol, 34.4 mmol) followed by the
slow addition of pivaloyl chloride (3.89 g, 31.6 mmol). The
mixture was stirred at -10 °C for 30 min, cooled to -78 °C, and
a solution of the anion of (S)-4-phenyl-2-oxazolidinone
(generated from (S)-4-phenyl-2-oxazolidinone (5.15 g, 31.56
mmol) in tetrahydrofuran (60 mL) at -78 °C by treatment with
lithium diisopropyl amide (2 M in heptane/tetrahydrofuran/
ethylbenzene) (15 mL, 30.00 mmol) for 30 min) was added while
at -78 °C. The resulting mixture was stirred at -78 °C for 1 h,
allowed to come to room temperature, and poured into 0.5N aq
hydrochloric acid (75 mL). The aqueous solution was extracted
with ethyl acetate (2 ꢀ 50 mL) and the combined organic
fractions washed with sodium bicarbonate (aq, saturated) (75
mL), water (50 mL), brine (50 mL), dried over sodium sulfate,
and evaporated in vacuo. The crude product was purified on
silica using heptane/ethyl acetate (3:1) as the eluent. Fractions
containing the product were pooled and evaporated in vacuo to
give desired compound (3.96 g, 43.4%). Spectroscopic data
identical to those of 27a.
(R)-3-[3-(4-Fluorophenyl)-3-(4-methylsulfanyl-phenyl)-propionyl]-
(S)-4-phenyl-oxazolidin-2-one (28b). To a precooled (-40 °C)
solution of copper(I)bromide methylsulfide complex (3.92 g,
19.08 mmol) in tetrahydrofuran (40 mL) was added dimethyl-
sulfide (20 mL), followed by the slow addition of thioanisole
magnesium bromide (0.5 M in tetrahydrofuran) (76.3 mL, 38.16
mmol). The mixture was allowed to warm to -20 °C over
15 min, and a solution of 27b (3.96 g, 12.72 mmol) in tetrahy-
drofuran (20 mL) was added drowise over 1 h. the resulting
solution was stirred at -20 °C for 1 h, allowed to come to room
temperature, stirred overnight, and poured into saturated aq
ammonium chloride (200 mL). The mixture was extracted with
ethyl acetate (2 ꢀ 150 mL), and the combined organic fractions
washed with ammonium hydroxide (2 ꢀ 15% aq) (150 mL),
water (150 mL), brine (150 mL), dried over sodium sulfate, and
evaporated in vacuo. The crude product was purified on silica
using heptane/ethyl acetate (3:1) as the eluent. Fractions con-
taining the product were pooled and evaporated in vacuo to give
desired compound (4.80 g, 87.0%) as a colorless solid. 1H NMR
δ: 6.9-7.3 (m,,13H), 5.35 (dd, 1 H), 4.61 (m, 1H), 4.55 (m, 1H),
4.20 (dd, 1H), 3.80 (dd, 1H), 3,65 (dd, 1H), 2.25 (s, 3H).
(R)-3-(4-Fluorophenyl)-3-(4-methylsulfanyl-phenyl)-propan-1-
ol (29b). To a precooled solution of 28b (4.75 g, 10.91 mmol) in
tetrahydrofuran (60 mL) was added water (10 mL) followed by
the portionwise addition of sodium borohydride (0.89 g, 23.45
mmol), keeping the temperature under 10 °C. The resulting
solution was allowed to come to room temperature, stirred
overnight, tetrahydrofuran was evaporated in vacuo, and water
(40 mL) was added. The solution was extracted with ethyl
acetate (2 ꢀ 50 mL) and the combined organic fractions washed
with water (50 mL), brine (50 mL), dried over sodium sulfate,
and evaporated in vacuo. The crude product was purified on
silica using heptane/ethyl acetate (1:1) as the eluent. Fractions
containing the product were pooled and evaporated in vacuo to
give desired compound (2.11 g, 70.0%) as a colorless oil.
Spectroscopic data identical to those of 28a.
(R)-3-(4-Fluorophenyl)-3-(4-methylsulfanyl-phenyl)-propyl-
azide (30b). To a precooled (-10 °C) solution of 29b (2.10 g,
7.60 mmol) in dichloromethane (30 mL) was added triethyl-
amine (1.27 mL, 9.12 mmol) followed by the slow addtion of
toluenesulfonyl chloride (1.74 g, 9.12 mmol). The mixture was
stirred at -10 °C for 1 h, stirred overnight at room temperature,
and evaporated in vacuo. The residue was dissolved in N,
N-dimethylformamide (15 mL), sodium azide (0.99 g, 15.19
mmol) was added, the mixture was stirred at room temperature
overnight, and water (40 mL) was added. The aqueous phase
was extracted with ethyl acetate (2 ꢀ 20 mL) and the combined
organic fractions washed with water (20 mL), brine (20 mL),
dried over sodium sulfate, and evaporated in vacuo. The crude
product was purified on silica using heptane/ethyl acetate (3:1)
as the eluent. Fractions containing the product were pooled and
evaporated in vacuo to give desired compound (2.25 g, 93.3%)
as a colorless oil. Spectroscopic data identical to those of 29a.
(R)-3-(4-Fluorophenyl)-3-(4-methylsulfoxide-phenyl)-propyl-
azide (31b). To 30b (2.25 g, 7.09 mmol) was added a mixture of
glacial acetic acid (10 mL) and hydrogen peroxide (30% aq)
(8 mL). The resulting solution was stirred at room temperature
for 1 h and water (20 mL) added, the aqueous phase was
extracted twice with ethyl acetate (2 ꢀ 20 mL), the organic
phase washed with brine (15 mL), dried over sodium sulfate, and
evaporated in vacuo to give the desired product (1.27 g, 57.0%).
Spectroscopic data identical to those of 31a.
(R)-3-(4-Fluorophenyl)-3-(4-methanesulfoxide-phenyl)-propy-
lamine (32b). To 31b (1.00 g) was added methanol (15 mL) and
Pd (10% on carbon) (167 mg), the mixture was stirred under
hydrogen overnight, filtered through celite (washing with
methanol), and evaporated in vacuo to desired compound as a

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5893
colorless oil (0.50 g, 24.0%). To this amine (0.500 g, 1.72 mmol)
in dichloromethane (10 mL) was added di-tert-butyl dicarbo-
nate (Boc-anhydride) (490 mg, 2.23 mmol) and triethylamine
(0.430 mL, 3.00 mmol). The mixture was stirred at room
temperature for 4 h and the organic phase washed with aq
HCl (1N) (10 mL) and evaporated to give the desired compound
(672 mg, 100%) as a colorless oil. Spectroscopic data identical to
those of 32a.
Boc-(R)-3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-pro-
pylamine (33b). To a solution of the above sulfoxide (32b)
(885 mg, 2.26 mmol) in 35 mL of chloroform was added 1.8
(17.6 mmol) g of aluminum oxide pretreated with 360 μL of
water. The resulting suspension was treated with 3.6 g of oxone
(5.8 mmol) and refluxed for 24 h. The reaction was cooled to
room temperature, filtered, and the solvents evaporated in
vacuo. The crude product was purified on silica using hep-
tane/ethyl acetate as the eluent 0.94 g (2.26 mmol, 100%).
Spectroscopic data same as 33a; 82% ee chiral HPLC Chiracel
Chiralpak AD-H, 4.6 mm ꢀ 250 mm, 85% heptane, 15%
ethanol, 1.0 mL/min 25 °C. Retention time 34.4 major, 37.2
minor).
(R)-3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyl-
amine (R-20). To a solution of 33b 0.94 g (2.30 mmol) in 5 mL of
dichloromethane was added 10 mL of trifluororacetic acid
followed by 0.500 mL of water. The resulting mixture was
stirred for 1 h and solvents removed in vacuo. The oil was taken
up in 15 mL of dichloroethane and para-toluenesulfonic acid
(396 mg, 0.94 mmol) was added in one portion. The reaction was
the evaporated in vacuo, and the resulting residue triturated
with ether and heptane. The solid was filtered and dried in vacuo
to give the desired compound (1.01 g, 91.0%) as colorless solid.
1H NMR (DMSO-d6) δ: 7.85 (d, 2H), 7.80 (brs, 3H), 7.65 (d,
2H), 7.50 (m, 2H), 7.36 (m, 2H), 7.14 (m, 4H) 4.25 (m, 1H),
3.19(s, 3H), 2.69 (m, 2H), 2.31 (m, 5H).
(S)-4-Cyano-N-[3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-
propyl]-benzamide (S-24). This compound was synthesized on
0.21 mmol scale from 4-cyano-benzoic acid and (S)-3-(4-
fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propylamine (S-20)
according to the general procedure for amide coupling to give
the desired compound (50 mg, 54.9%). 1H NMR δ: 7.69-7.77 (m,
6H), 7.38 (m, 2H), 7.15 (m, 2H), 7.69 (m, 2H), 6.36 (brt, 1H), 4.04
(t, J=7.42 Hz, 1H), 3.36 (q J=6.14 Hz, 2H), 3.94 (s, 3H), 2.34 (m,
2H). HRMS: calcd for C24H21FN2O3S 437.1335; found 437.1325.
(R)-4-Cyano-N-[3-(4-fluorophenyl)-3-(4-methanesulfonyl-phe-
nyl)-propyl]-benzamide (R-24). This compound was synthesized
on 0.21 mmol scale from 4-cyano-benzoic acid and (R)-3-
(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propylamine
(R-20) (according to the general procedure for amide coupling
to give the desired compound (51 mg, 55.0%). 1H NMR δ:
7.69-7.77 (m, 6H), 7.38 (m, 2H), 7.15 (m, 2H), 7.69 (m, 2H),
6.36 (brt, 1H), 4.04 (t, J=7.42 Hz, 1H), 3.36 (q J=6.14 Hz, 2H),
3.94 (s, 3H), 2.34 (m, 2H). HRMS: calcd for C24H21FN2O3S
437.1335; found 437.1318.
7.19 (m, 2H), 6.96 (m, 2H), 6.45 (m, 1H), 4.10 (brt, J=8.5 Hz,
1H), 3.33 (m, 2H), 3.10 (s, 3H), 2.37 (m, 2H). HRMS: calcd for
C22H21FN2O4S þ Hþ 429.1284; found 429.1282.
In Vitro Molecular Assay for Inhibition of Human or Rat sEH.
This assay identifies compounds that inhibit the binding inter-
action of either human or rat soluble epoxide hydrolase with a
tetramethyl rhodamine-labeled probe. Test compounds were
dissolved and serially diluted in DMSO, with final dilution in
assay buffer (20 mM TES, 200 mM NaCl, 0.05% w/v CHAPS, 1
mM TCEP, pH=7.0) to achieve 1% DMSO in the assay. Test
compounds were dispensed into a black, flat-bottom 96-well
plate. Positive controls were reaction mixtures containing no
test compound; negative controls (blanks) were reaction mix-
tures containing a reference inhibitor. The reaction was started
with the addition of a mixture of either human or rat sEH (final
assay concentration is 10 nM) and probe (final assay concentra-
tion is 2.5 nM). The reaction was mixed by briefly shaking the
plate on an orbital shaker, and the plates were incubated in the
dark for 30 min at room temperature. Fluorescence polarization
is measured on an LJL Analyst using a 530 nm excitation filter, a
580 nm emission filter, and a 561 nm dichroic mirror. Concen-
tration-response data were fitted to a 4-parameter equation to
determine IC50 values.
In Vitro Cellular Assay for Inhibition of sEH in Human Hep G2
Cells. This assay identifies compounds that inhibit the ability of
soluble epoxide hydrolase to convert 14,15-epoxy-eicosatienoic
acid to 14,15-dihydroxyeicosatrienoic acid in human HepG2
cells. HepG2 cells, grown in low glucose DMEM supplemented
with 10% fetal bovine serum, were seeded at 20000 cells/well in
96-well poly D-lysine-coated plates 18 h prior to the assay. On
the day of the assay, test compounds were dissolved and serially
diluted in DMSO, with final dilution in assay medium (low
glucose DMEM containing 6.6 μg/mL BSA) to achieve a 0.3%
DMSO in the assay. Positive controls were wells containing no
test compound; negative controls (blanks) were reaction mix-
tures containing a reference inhibitor. The growth medium was
aspirated from the cells, they were washed once with phosphate
buffered saline containing calcium and magnesium, and assay
medium was added. Test compounds were dispensed into the
plates, which were then placed in a humidified incubator at 37 °C
with 5% CO2 for 30 min. After addition of 14,15-epoxy-
eicosatienoic acid to a final concentration of 330 nM, the plates
were placed in a humidified incubator at 37 °C with 5% CO2 for
2 h. Quantitation of 14,15-dihydroxyeicosatrienoic acid in the
supernatants was performed using a commercially available
ELISA kit (cat. no. DH 21 from Detroit R&D, Inc.). Concen-
tration-response data were fitted to a 4-parameter equation to
determine IC50 values.
Assessment of Stability in Rat and Human Liver Microsomes.
The stability of the compounds in rat and human liver micro-
somes were determined by calculating the half-lives of com-
pounds from two time points (0 and 30 min). The incubation
consisted of a microsomal content was 1 mg/mL, a NADPH
(nicotinamide adenine dinucleotide phosphate-oxidase) concen-
tration of 2.5 mM, and a compound concentration of 1 μM. All
compounds were preincubated at 37 °C for 5 min on a Tecan
Genesis. Following preincubation, NADPH was added and
samples were incubated at 37 °C for 30 min on a Tecan Genesis.
Reactions were stopped when 80 uL of each sample was
collected at 0 and 30 min and crashed with 160 μL of cold
acetonitrile. The crashed samples were filtered and analyzed by
LC/MS/MS. The half-life of disappearance of parent molecule
(t1/2) was calculated by dividing 0.693 by the slope of parent loss.
Methods for Computational Analyses of Compounds 7 and 8.
Glide 4.0 docking26 in standard precision mode was used to
predict putative binding modes of 7 and 8 in the catalytic site of
human sEH represented by an in-house crystal structure. The
protein was prepared for docking using 500 steps of Polak-Rib-
ier conjugant gradient minimization in the presence of com-
pound 1 constraining heavy atoms with a force constant of
(S)-N-[3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-pro-
pyl]-6-hydroxy-nicotinamide (S-22). This compound was synthe-
sized on 0.42 mmol scale from 6-hydroxynicotinic acid and (S)-
3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propylamine
(S-20) according to the general procedure for amide coupling to
give the desired compound (100 mg, 56.0%). 1H NMR δ: 8.20
(brs, 1H), 7.80 (m, 1H), 7.79 (m, 2H), 7.42 (m, 3H), 7.19 (m, 2H),
6.96 (m, 2H), 6.45 (m, 1H), 4.10 (brt, J=8.5 Hz, 1H), 3.33 (m,
2H), 3.10 (s, 3H), 2.37 (m, 2H). HRMS: calcd for
C22H21FN2O4S þ Hþ 429.1284; found 429.1259.
(R)-N-[3-(4-Fluorophenyl)-3-(4-methanesulfonyl-phenyl)-pro-
pyl]-6-hydroxy-nicotinamide (R-22). This compound was syn-
thesized on 0.42 mmol scale from 6-hydroxynicotinic acid and
(R)-3-(4-fluorophenyl)-3-(4-methanesulfonyl-phenyl)-propyla-
mine (R-20) according to the general procedure for amide
coupling to give the desired compound (95 mg, 53.2%). 1H
NMR δ: 8.20 (brs, 1H), 7.80 (m, 1H), 7.79 (m, 2H), 7.42 (m, 3H),

5894 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Eldrup et al.
2
100 kcal/mol/A . Two hydrogen bonding constraints to Asp 335
˚
Table 7. Refinement Statistics
and to either Tyr 383 or Tyr 466 were required for accepted
docking solutions. Standard conditions were chosen for all
other docking parameters. Force field calculations were per-
formed using the OPLS2005 force field as implemented by
Schrodinger Inc. Geometry optimization calculations were per-
formed using DFT/B3LYP with a 6-31G** basis set using
Jaguar6.5.27
compd
1
24
˚
resolution range, A
˚
unit cell, A P6(5)22
38.1-2.5
a = b = 92.609
c = 244.046
1321310/23208
99(100)
36.25-1.95
a = b = 92.410
243.983
3243810/183207
98(96)
(R = β = 90; γ = 120)
reflections measured/unique
completeness (outer shell)
reflections work/test
I/σ (outer shell)
21903/1115
4.7(0.9)
4378
42950/2271
11.7(1.6)
4500
Method for Identification of in Vitro Metabolites. Additional
incubations were optimized to identify the phase I metabolites of
1 and 15. The incubations consisted of a microsomal content of
1 mg/mL, a NADPH concentration of 2.5 mM, and a com-
pound concentration of 10 μM. The mixtures were preincubated
at 37 °C for 4 min. The reactions were initiated by the addition of
NADPH. Control samples were prepared in the absence of
NADPH, where additional buffer was added to equal the
volume of NADPH added to the test compound mixtures.
The reactions were continued for 30 min and stopped by the
addition of 1 mL of ice cold acetonitrile. The resulting mixtures
were vortexed for 10 s and centrifuged to remove particulates.
The control and test compound supernatants (1 mL each) were
dried under nitrogen at 40 °C. The dried supernatants were
reconstituted in H2O/acetonitrile/acetic acid (70/30/0.1, v/v/v),
vortexed, and centrifuged. A LC/MS/MS system consisting of a
Hewlett-Packard Series 1100 system (auto injector, pump, and
diode array detector) and a Micromass Ultima triple quadru-
pole mass spectrometer was used for the metabolite ID experi-
ments. LC/MS/MS experiments were conducted to obtain full
scan spectra and product ion spectra for the protonated parent
ions of the compounds and the phase I metabolites.
Assessment of Compound Exposure in Rats. Male Spra-
gue-Dawley rats (Charles River, Co.) were purchased precan-
nulated (jugular catheter) in order to facilitate precise blood
sampling times to increase throughput and to reduce the stress
on the animals caused by serial bleedings. Three fed rats were
dosed orally with one compound at a dose of 5 mg/kg. Blood
was collected into heparin-containing tubes serially from each
animal at 1, 2, 4, and 6 h postdosing and centrifuged to generate
plasma. The plasma samples were stored at -20 °C until
analysis. The plasma concentrations were obtained by extrac-
tion of the compound from an aliquot of rat plasma by
precipitation of proteins with acetonitrile and quantitation by
LC/MS/MS. The LC/MS/MS system consisted of a Hewlett-
Packard Series 1100 pump, Leap Technologies injector, and an
Applied Biosystems API4000 mass spectrometer. The LC/MS/
MS system was operated in multireaction monitoring (MRM)
mode.
no. of atoms
no. of waters
37
152
R/Rfree
rmsd (A) bonds
rmsd (deg) angles
22.6/29.1
0.004
0.8
21.4/25.5
0.008
1.1
˚
12 mg/mL with 5 μL of a well solution containing: 0.2 M lithium
sulfate, 36% PEG 3350, and 0.1 M Tris pH 8.4 and 0.5 μL of
0.006 mM β-D-hexadecyl maltoside. Crystals were observed to
grow within 1 week at 4 °C. A 100 mM stock solution of
compounds 1 and 24 were prepared in DMSO. Prior to addition
of compound, crystals were transferred to a stabilizing solution
containing 0.2 M lithium sulfate, 40% PEG 3350, and 0.1 M Tris
pH 8.4. The stock compound solution was added to the drop
containing the protein to give a final concentration of 10 mM.
The crystals were then allowed to soak for 1 week at 4 °C.
Cryoprotection of the crystals was achieved by gradually adding
sucrose to the stabilizing solution to a final concentration of
20%. The crystals were then flash frozen in liquid nitrogen.
Diffraction data for the cocrystal containing 1 was collected on
the SGX-CAT beamline at the Advanced Photon Source using a
˚
wavelength of 0.929 A and a MAR165 CCD 165. Diffraction
data for the cocrystal containing 24 was collected on the
PXI beamline Swiss Light Source using a MAR 225 CCD. Data
reduction was performed using Mosflm. Molecular replacement
was performed using the program AMORE in conjunction with
the apo human soluble epoxide structure 1S8O.pdb. Refinement
and model building was performed using CNX and Phenix.
Initial refinement of the model was performed with no waters
present in the model. Following the addition of water molecules
to the model and subsequent rounds of refinement, the ligand was
added to the model. Table 7 contains the data reduction and
refinement statistics of the final models.
Acknowledgment. We thank Michael August and Lori
Patnaude for performing the high-throughput screen of our
compound collection against sEH.
Expression and Purification of Human sEH. Human soluble
epoxide hydrolase was obtained using a baculovirus expression
system. The cells were lysed using sonication in a buffer contain-
ing 20 mM MOPS (3-(N-morpholino)propanesulfonic acid)
pH 7.4, 75 mM NaCl, 10% glycerol 1 mM EGTA (ethylene
glycol tetraacetic acid), 1 mM EDTA (ethylenediaminetetra-
acetic acid), 3 mM DTT (dithiothreitol), 1 mM PMSF (phenyl-
methylsulfonyl fluoride), μg/mL pepstatin, and 4 μg/mL leu-
peptin. The lysate was clarified by centrifugation and loaded
onto a benzyl-thio sepharose column that had been equilibrated
with 20 mM MOPS pH 7.4, 75 mM NaCl, 10% glycerol, 1 mM
EDTA, 3 mM DTT. The protein was eluted using the same
buffer as used for equilibration plus 1 mM trans-1,3-diphenyl-
2,3-epoxypropan-1-one (chalcone oxide). The eluted protein
was then concentrated and dialyzed against a buffer containing
100 mM sodium phosphate pH 7.4, 50 mM NaCl and 3 mM
DTT. Plasma levels of 1 and 13 were determined using a 1%
CMC/0.2% Tween80/98.8% water formulation, whereas a 80%
PEG 400/20% pH2 30 mM citric acid formulation was used for
the determination of plasma levels of 24.
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