Synthesis and biological evaluation of sorafenib- and regorafenib-like sEH inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.05.011

To reduce the pro-angiogenic effects of sEH inhibition, a structure-activity relationship (SAR) study was performed by incorporating structural features of the anti-angiogenic multi-kinase inhibitor sorafenib into soluble epoxide hydrolase (sEH) inhibitors. The structural modifications of this series of molecules enabled the altering of selectivity towards the pro-angiogenic kinases C-RAF and vascular endothelial growth factor receptor-2 (VEGFR-2), while retaining their sEH inhibition. As a result, sEH inhibitors with greater potency against C-RAF and VEGFR-2 were obtained. Compound 4 (t-CUPM) possesses inhibition potency higher than sorafenib towards sEH but similar against C-RAF and VEGFR-2. Compound 7 (t-CUCB) selectively inhibits sEH, while inhibiting HUVEC cell proliferation, a potential anti-angiogenic property, without liver cancer cell cytotoxicity. The data presented suggest a potential rational approach to control the angiogenic responses stemming from sEH inhibition.

Full text of DOI:10.1016/j.bmcl.2013.05.011

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3732–3737
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of sorafenib- and regorafenib-
like sEH inhibitors
Sung Hee Hwang, Aaron T. Wecksler, Guodong Zhang, Christophe Morisseau, Long V. Nguyen,
⇑
Samuel H. Fu, Bruce D. Hammock
Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To reduce the pro-angiogenic effects of sEH inhibition, a structure–activity relationship (SAR) study was
performed by incorporating structural features of the anti-angiogenic multi-kinase inhibitor sorafenib
into soluble epoxide hydrolase (sEH) inhibitors. The structural modifications of this series of molecules
enabled the altering of selectivity towards the pro-angiogenic kinases C-RAF and vascular endothelial
growth factor receptor-2 (VEGFR-2), while retaining their sEH inhibition. As a result, sEH inhibitors with
greater potency against C-RAF and VEGFR-2 were obtained. Compound 4 (t-CUPM) possesses inhibition
potency higher than sorafenib towards sEH but similar against C-RAF and VEGFR-2. Compound 7 (t-
CUCB) selectively inhibits sEH, while inhibiting HUVEC cell proliferation, a potential anti-angiogenic
property, without liver cancer cell cytotoxicity. The data presented suggest a potential rational approach
to control the angiogenic responses stemming from sEH inhibition.
Received 9 April 2013
Revised 28 April 2013
Accepted 6 May 2013
Available online 15 May 2013
Keywords:
Soluble epoxide hydrolase (sEH)
Sorafenib
Regorafenib
Angiogenesis
C-RAF kinase
VEGFR-2
Ó 2013 Elsevier Ltd. All rights reserved.
Soluble epoxide hydrolase (sEH, EC 3.3.2.10) is an enzyme that
catalyzes the hydrolysis of epoxy fatty acids (EpFAs), including
epoxyeicosatrienoic acids (EETs), to their less bioactive corre-
sponding diols, such as dihydroxyeicosatrienoic acids (DHETs).1
EETs possess anti-inflammatory,2 anti-hypertensive3 and analgesic
properties.4 Therefore, sEH has been a therapeutic target for
numerous indications such as inflammation, pain, hypertension,
atherosclerosis, pulmonary diseases, renal end-organ damage and
diabetes.2,5 EETs have also long been known as a pro-angiogenic
factor particularly in the presence of vascular endothelial growth
factor (VEGF).6–9While this is an attractive property during devel-
opment and in certain cases such as wound healing,10 studies sug-
gested that EETs can promote cancer progression.11 For example,
Panigrahy et al. recently demonstrated their contribution to tumor
growth and metastasis.12
adenine group of ATP which binds to a highly conserved ATP-bind-
ing pocket to inhibit kinase function.14 Sorafenib is a bi-aryl urea
which was originally developed as a therapeutic agent targeting
the pro-angiogenic kinase, C-RAF.15 However, the structural fea-
tures of sorafenib demonstrated multi-kinase inhibitory activities
with potent anti-angiogenic properties via the inhibition of
pro-angiogenic receptor tyrosine kinases (RTKs), such as the VEG-
FR-2.16 As a result, sorafenib displays multi-inhibitory action in the
RAF/MEK/ERK pathway and RTKs to combat tumor angiogenesis. It
is currently used for the treatment of hepatocellular carcinoma
(HCC)17 and renal cell carcinoma (RCC).18
Based on the structural similarity between sorafenib and one
class of sEH inhibitors (Fig. 1A), we tested and found that sorafenib
(NexavarÒ, BAY 43-9006), also displays potent inhibitory activity
against sEH (human sEH IC50 = 12 2 nM).19 As expected, sorafenib
exhibits similar anti-inflammatory responses as conventional sEH
inhibitors in lipopolysaccharide-induced inflammation murine
model.19 In addition, we recently found that regorafenib (Stivarga™,
BAY 73-4506), a second generation derivative of sorafenib for the
treatment of colon or rectal cancer, is a more potent sEH inhibitor
(human sEH IC50 = 0.5 0.1 nM). Data on clinical blood levels from
sorafenib-treated patients suggest that the sEH should be signifi-
cantly inhibited, which may be beneficial during cancer treatment
with sorafenib by reducing renal toxicity, hypertension and pain,2
often associated with pan-kinase anti-angiogenic agents.20
Small-molecule kinase inhibitors13 such as sorafenib and rego-
rafenib, are generally flat, aromatic molecules which mimic the
Abbreviations: HCC, hepatocellular carcinoma; RCC, renal cell carcinoma; GI50
,
concentration required to inhibit cell growth by 50%; EpFAs, epoxygenated fatty
acids; t-CUPM, trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclo-
hexyloxy}-pyridine-2-carboxylic acid methylamide; t-CUCB, trans-3-{4-[3-(4-
chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-benzoic acid; t-AUCB,
trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid; t-TUCB, trans-
4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid.
⇑
Corresponding author. Tel.: +1 530 752 7519; fax: +1 530 752 1537.
E-mail address: bdhammock@ucdavis.edu (B.D. Hammock).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.011

S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3732–3737
3733
A
lipophilic
ring (R1)
proximal
ring
distal
ring
O
O
Cl
O
Cl
O
O
N
H
O
N
H
F
F
N
F
F
N
N
H
N
H
N
H
N
H
F
F
F
sorafenib
regorafenib
IC50 human sEH = 12 2 nM
IC50 human sEH = 0.5 0.1 nM
O
F
F
O
O
O
O
OH
F
OH
N
H
N
H
N
H
N
H
O
O
t-AUCB 11
t-TUCB 12
( )
(
)
IC50 human sEH = 2 1 nM
IC50 human sEH = 0.9 0.1 nM
B
Figure 1. (A) Structures of sorafenib and common sEH inhibitors. (B) Selectivity of sorafenib, t-AUCB (11) and t-TUCB (12) at 10 lM concentration against 10 recombinant
kinases.
On the other hand, urea-based sEH inhibitors {t-AUCB (11) and
t-TUCB (12)} that are structurally related to sorafenib (Fig. 1A), did
not display the cytotoxicity, growth inhibition, or apoptotic effects
of sorafenib in RCC cell lines in our previous study.19 The first
question asked was whether lack of antiproliferative effect in
RCC cells was reflected in their kinase inhibitory activities. We
screened t-AUCB and t-TUCB against a panel of known sorafenib
targets and found that these sEH inhibitors display no significant
maintain sEH inhibition while altering kinase inhibitory activities
(C-RAF and VEGFR-2, the two primary kinase targets of sorafenib
believed to yield its anti-angiogenic properties) and cellular
functions. The cellular responses of the compounds in this small li-
brary of sorafenib-like sEH inhibitors were determined in both
endothelial HUVEC cells as an initial measurement of anti-angio-
genesis, and two epithelial liver cell carcinoma cell lines (HepG2
and Huh-7) as an initial measurement of cytotoxicity.
multi-kinase inhibition at 10
l
M concentration (Fig. 1B). This con-
The synthetic routes of urea-based sEH inhibitors containing
the cyclohexyl group which are described herein have previously
been disclosed.21 The preparation of urea compounds 4–15, 17,
21 and 22 is depicted in Schemes 1 and 2. Briefly geometric iso-
mers (trans- and cis-) were made starting from the corresponding
alcohols, 1–3 and 16, respectively, using Mitsunobu reaction we
published previously.22 Distal rings in compounds 4–15, and 17
were then installed by nucleophilic aromatic substitution (SNAr)
reaction.23 The compounds 21 and 22 were synthesized starting
from picolinic acid according to the procedure used for the prepa-
ration of sorafenib.24
firmed that there is a distinct structure–activity relationship (SAR)
between sorafenib and structurally related urea-based sEH inhibi-
tors against kinase inhibition, and probably explains the lack of
antiproliferative effects of t-AUCB and t-TUCB in RCC cells. Alterna-
tively, it raises the question whether structural modifications of
urea-based sEH inhibitors could yield altered kinase inhibition
properties towards sorafenib’s primary anti-angiogenic targets, C-
RAF and VEGFR-2, in order to balance the potential adverse effect
stemming from the angiogenic responses of EETs resulting from
high doses of sEH inhibitors.12
Herein, we report SAR study of hybrid compounds between
sorafenib and conventional urea-based sEH inhibitors. To this
end, we investigated whether these structural modifications could
Our previous SAR study with sEH inhibitors such as t-AUCB re-
veal that replacement of the cyclohexyl group, by rigid groups such
as a phenyl or an acetylenyl group, results in equal to or slightly

3734
S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3732–3737
O
O
O
O
R2
O
N
R3
O
O
Me
d
O
OH
O
N
H
R1
N
R1
R1
N
H
N
H
N
H
N
H
N
H
N
H
8
, R1 = 4-Cl, 3-CF3-Ph, R2 = H, R3 = Me
, R1 = 4-Cl, 3-CF3-Ph, R2 = Me, R3 = Me
, R1 = 4-CF3-OPh, R2 = H, R3 = Me
4
5
6
, R1 = 4-Cl, 3-CF3-Ph
, R1 = 4-Cl, 3-CF3-Ph
7
c
b
O
O
N
OH
H
O
e
R1
N
R1
N
H
N
Me
N
H
N
H
H
O
1
f
9
, R = 4-Cl, 3-CF3-Ph
, R1 = 4-Cl, 3-CF3-Ph
1
2, R1 = adamantyl
3, R1 = 4-OCF3-Ph
O
O
O
O
g
H
N
a
R1
OH
R1
N
H
N
H
N
H
N
H
Me
O
O
R1 NCO
h
10, R1 = 4-Cl, 3-CF3-Ph
11, R1 = adamantyl
13, R1 = 4-Cl, 3-CF3-Ph
14, R1 = adamantyl
12, R1 = 4-OCF3-Ph
15, R1 = 4-OCF3-Ph
O
O
Cl
N
OH
O
O
Me
Cl
i
OtBu
OtBu
O
N
H
R1
N
H
N
H
R1
N
N
N
H
N
H
O
18
19
16, R1 = 4-Cl, 3-CF3-Ph
17, R1 = 4-Cl, 3-CF3-Ph
Scheme 1. Reagents and conditions: (a) trans-4-aminocyclohexanol, DMF, rt, 12 h; (b) (i) KOt-Bu, 18, THF, 0 °C to rt, overnight, (ii) (a) 30% TFA in DCM, 6 h, (b) PyBOP,
R2R3NH, DMF, rt, overnight; (c) (i) NaH, 3-fluorobenzonitrile, DMF, rt, 1 d, (ii) 6 N NaOH, EtOH, 90 °C, 1 d; (d) PyBOP, MeNH2, DMF, rt, 12 h; (e) (i) KOt-Bu, 19, THF, 0 °C to rt,
overnight, (ii) (a) 30% TFA in DCM, 6 h, (b) PyBOP, MeNH2, DMF, rt, 12 h; (f) (i) NaH, 4-fluorobenzonitrile, DMF, rt, 12 h, (ii) 6 N NaOH, EtOH, 90 °C, 1 d; (g) PyBOP, MeNH2,
DMF, rt, 12 h; (h) (i) cis-4-nitrobenzoic acid 4-aminocyclohexyl ester,22 4-chloro-3-(trifluoromethyl)phenyl isocyanate, Et3N, DMF, rt, 12 h, (ii) 1 N NaOH, THF, rt, 12 h; (i) (a)
KOt-Bu, 18, THF, 0 °C to rt, overnight, (b) 50% TFA in DCM, 6 h, and (c) PyBOP, MeNH2, DMF, rt, 12 h.
O
O
O
O
Me
O
Me
a
b
O
N
H
N
H
OH
R1
N
N
N
N
H
N
H
H2N
21, R1 = adamantyl
20
22, R1 = 4-OCF3-Ph
Scheme 2. Reagents and conditions: (a)24 (i) SOCl2, DMF, 80 °C, 16 h, (ii) MeNH2, MeOH, THF, 0 °C, 4 h, (iii) p-hydroxyaniline, KOt-Bu, K2CO3, DMF, 80 °C, 6 h; (b) R1-NCO,
DMF, rt, 12 h.
less sEH inhibitory activity.22,25 We hypothesized that the cyclo-
hexyl group should be a bioisostere of the phenyl group due to
its size and similar lipophilicity. Therefore we first modified the
‘proximal group’ in sorafenib (Fig. 1A). Two geometric isomers t-
CUPM (4, trans) and 17 (cis), were initially obtained replacing the
phenyl ring of sorafenib by the 1,4-cyclohexyl moiety as the prox-
imal ring.
As seen in Table 1, the high potency of t-CUPM was surprising
because kinase inhibitors utilize a phenyl or planar heterocyclic
ring to maximize lipophilic interaction with residues at ATP-bind-
ing site conserved throughout the kinome. Although the phenyl
group in sorafenib is proposed to interact with three aromatic
residues—Trp530 of the hinge region, Phe582 at the end of the cat-
alytic loop, and Phe594 of the DFG-out ATP-binding region found
in the protein kinase C-RAF26—we observed similar activity
towards C-RAF inhibition with the trans-isomer t-CUPM. Both cis-
and trans-isomers improved sEH inhibition by about 20-fold, but
the cis-isomer 17 had much poorer C-RAF kinase inhibition com-
pared to the trans-isomer t-CUPM, directing our investigation to-
wards only the trans-isomers.
As previously observed in the original SAR study with sorafe-
nib,27 conversion of a methylamide in t-CUPM to a dimethylamide
5 on the distal group (Fig. 1A) causes loss in C-RAF inhibition due to
the loss of H-bonding. Interchange of a methylaminocarbonyl
group and a nitrogen atom in pyridyl group of t-CUPM led only
to 2.3-fold less activity against C-RAF, suggesting that the methyl
amide group in the pyridyl group of the compound 9 still makes
contact with residues that interact with sorafenib. Shifting the

S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3732–3737
3735
Table 1
IC50 values of N-aryl, N0-cyclohexyl ureas 4–15, and 17 for sEH and C-RAF
R1
R2
A
B
sEH
C-Raf
IC50 (nm)
Sorafeniba
12
2
45
5
Regorafeniba
0.5 0.1
>10,00019
0.5 0.1
0.5 0.1
0.5 0.1
8.6 1.0
1.0 0.1
0.5 0.1
0.5 0.1
1.5 0.5
0.9 0.1
0.5 0.1
0.5 0.1
0.5 0.1
0.5 0.1
3.7 1.019
2.9 1.019
NDb
Sunitinib19
t-CUPM (4)
5
6
t-CUCB (7)
8
9
10
t-AUCB (11)
t-TUCB (12)
13
14
>10,000
75 5
4-Cl, 3-CF3-Ph
4-Cl, 3-CF3-Ph
4-OCF3-Ph
4-Cl, 3-CF3-Ph
4-Cl, 3-CF3-Ph
4-Cl, 3-CF3-Ph
4-Cl, 3-CF3-Ph
Adamantyl
4-OCF3-Ph
4-Cl, 3-CF3-Ph
Adamantyl
4-OCF3-Ph
4-Cl, 3-CF3-Ph
3-C(@O)NHMe
3-C(@O)NMe2
3-C(@O)NHMe
3-CO2H
3-C(@O)NHMe
4-C(@O)NHMe
4-CO2H
4-CO2H
4-CO2H
4-C(@O)NHMe
4-C(@O)NHMe
4-C(@O)NHMe
3-C(@O)NHMe
trans
trans
trans
trans
trans
trans
trans
trans
trans
trans
trans
trans
cis
C
C
C
C
C
N
C
C
C
C
C
C
C
N
N
N
C
C
C
C
C
C
C
C
C
N
>10,000
>10,000
>10,000
293 10
175 20
4300 400
>10,000
>10,000
340 40
>10,000
>10,000
1500 200
>10,000
>10,000
15
17
TPPU (1770)19
TUPS (1709)19
a
See the Supplementary data.
ND = not determined.
b
position of the nitrogen atom in the pyridyl group or its removal
has little effect on sEH activity. In contrast to C-RAF inhibition,
the terminal carboxylic acid is not critical for potency on the distal
ring of sEH inhibitors. When present the carboxylic acid is of sim-
ilar activity on sEH to its esters and amides.22,28 Thus avoiding the
methylaminocarbonyl group provides another way to block kinase
activity among sEH inhibitors structurally related to sorafenib.29
We then conversely left the proximal phenyl group intact—
identical to that in sorafenib—and subsequently tested how
replacement of the lipophilic group (R1, (Fig. 1A) in sorafenib by
groups found in t-AUCB and t-TUCB affected C-RAF kinase inhibi-
tion. While the adamantyl group (21, Table 2) is not compatible
for C-RAF inhibition, surprisingly, p-trifluoromethoxyphenyl group
(22) became slightly more potent than sorafenib itself, suggesting
that the lipophilic group (R1, Tables 1 and 2) is not restricted to 4-
Cl, 3-CF3-phenyl moiety for C-RAF inhibition. This indicates that
the lack of inhibition of C-RAF in t-TUCB is more likely due to the
lack of the amide group and the pyridine ring as the distal group
than to the presence of the lipophilic group (R1).
responding methylamide group as in compounds 8, and 13–15,
respectively. Except for compounds 8 and 13 which have the same
lipophilic group (R1) as seen in sorafenib, other compounds 14 and
15 lost C-RAF inhibitory activity.
However replacement of 4-chloro-3-trifluoromethoxy-phenyl
group (R1) in t-CUPM by 4-trifluoromethoxyphenyl group (R1) with
a trans-cyclohexyl proximal ring causes complete loss of C-RAF
inhibition in compound 6, suggesting the importance of the 3,4-
substituents of the R1 group when the proximal phenyl group is re-
placed by the cyclohexyl group. Most sEH inhibitors do not need
large 3,4-substituted aryl group at the lipophilic group (R1) for po-
tent sEH inhibitory acitivity,5 so lacking a single large 3, or 4-aryl
substituent provides a simple way to reduce the potential of sEH
inhibitors with a 1,4-cyclohexyl proximal ring to have inhibitory
activity on the kinases studied here. Therefore we can conclude
that unless the proximal ring is a complete planar group like the
phenyl group as in sorafenib or compounds 21 and 22, the lipo-
philic group (R1) appears to require the 4-chloro,3-trifluorometh-
oxyphenyl moiety to inhibit C-RAF. Otherwise no C-RAF
inhibition was observed in this series. Structurally diverse sEH
inhibitors such as the ones with a piperidyl proximal ring19 (TPPU
and TUPS, Table 1) are not known to be kinase inhibitors.
With these data in hand, we then converted the free carboxylic
acid of sEH inhibitors t-CUCB (7), 10, t-AUCB and t-TUCB to the cor-
We then evaluated all of our compounds against VEGFR-2 to
determine which of these compounds have potentially anti-angio-
genic property. As seen in Figure 2, only four compounds (t-CUPM,
Table 2
IC50 values of ureas 21 and 22 for sEH and C-RAF
9, 21 and 22) displayed potency towards VEGFR-2 at 10 lM con-
centration. These data yielded crucial evidence on the structural
features which appear to be most import for VEGFR-2 inhibition.
(1) There is some flexibility in the lipophilic group (R1) and the
proximal ring, and (2) compound 8 which is structurally similar
to compound t-CUPM, shows little inhibitory activity suggesting
the pyridine ring in R2 is critical for VEGFR-2 inhibition.
To determine if potency against VEGFR-2 correlated with anti-
angiogenic properties, we tested the ability of these compounds
to halt the growth of HUVEC cells, which is used as preliminary
model for angiogenesis.30 We observed that compounds t-CUPM
and 9 displayed similar effects on HUVEC cells compared to
R1
sEH
C-RAF
45
1000 100
30
IC50 (nM)
Sorafenib
21
22
4-Cl, 3-CF3-Ph
Adamantyl
4-OCF3-Ph
12
0.5 0.1
0.5 0.1
2
5
5

3736
S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3732–3737
Table 4
IC50 values of compounds t-CUPM, 17, 21, and 22 for B-RAFV600E
B-RAFV600E
IC50 (nM)
Sorafenib
Regorafenib
t-CUPM
17
13 2 (3818
)
1937
570 30
>5000
21
>5000
22
>5000
This study reveals that structural modification of sorafenib
slightly alters sEH inhibitory activity, but affects C-RAF kinase
and VEGFR-2 inhibition dramatically. Compounds possessing both
a left side lipophilic group (R1) and a right side distal group with a
methylamide, or the identical right side groups as in sorafenib, re-
tain C-RAF inhibition and/or VEGF receptor kinase inhibition. Such
kinase inhibition is easy to remove from potent selective sEH
inhibitors by altering the distal ring and thus avoid adverse effects
associated with kinase inhibition. In addition, current study also
demonstrated that a 1,4-cyclohexyl group, especially, as a trans-
form as in the compound t-CUPM could be act as a bioisostere
for the phenyl ring in sorafenib, which give an advantage over
sorafenib by having narrower spectrum of kinase inhibition. This
is illustrated by the selective inhibition of C-RAF over B-RAFV600E
(Table 4). This selectivity should reduce B-RAF driven side effects
for example when treating tumors initiated by oncogenic Kras
where B-RAF is not required but C-RAF is critical for downstream
MEK signaling.34 In addition, reducing the aromatic character of
the sorafenib improved its physical characteristics.35 Therefore
the compound t-CUPM might have reduced side effects compared
to sorafenib.
Figure 2. Percent inhibition against VEGFR-2 at 10 lM concentration of test
compounds.
sorafenib, whereas 21 and 22 displayed over two-fold loss in
growth inhibition (Table 3). Interestingly, the compounds t-CUCB,
8, and 17 also inhibited HUVEC growth with similar to the com-
pound t-CUPM and 9 despite their lack of VEGFR-2 inhibition. This
suggested that some of these compounds may be cytotoxic rather
than anti-angiogenic.
Finally, since C-RAF is overexpressed in a high number of HCC
tumors,31 and sEH is highly expressed in the liver,32 we treated
two epithelial liver carcinoma cells, HepG2 and Huh-7, to correlate
sEH, C-RAF and VEGFR-2 inhibition to cytotoxicity and potential
anti-tumorigenic properties. Consistent with our previous observa-
tions in RCC cell lines,19 sEH inhibitors (TUPS and TPPU) that are
structurally dissimilar to sorafenib displayed no observable effects
Lastly, while conventional sEH inhibitors t-AUCB and t-TUCB
display little cytotoxicity, the corresponding methylamide deriva-
tives (14 and 15, respectively) significantly affected HCC cell viabil-
ities. These data reinforce the notion that inhibition of sEH does
not contribute to cytotoxicity and suggest that the carboxylic acid
derivative confers selectivity towards sEH. In addition, compound
t-CUCB displays an ability to inhibit the growth of HUVEC stimu-
lated by growth factors such as VEGF, but does not suppress cancer
cell growth, suggesting that general cytotoxicity is not the mecha-
nism for inhibition of HUVEC growth. Therefore, such compounds
might possess a potential to reduce adverse effect associated with
angiogenesis related to sEH inhibition.
on cell viability up to 100 lM (data only shown for 25 lM) (Ta-
ble 3). Initial observation with compounds t-CUPM, 8, 9, and 13
suggested there may be a direct correlation between C-RAF inhibi-
tion and cytotoxicity; however, compounds 8 and 17 display sim-
ilar ability to halt cellular proliferation despite significant
differences in C-RAF inhibition. Moreover, among these com-
pounds only t-CUPM and 9 inhibit both C-RAF and VEGFR-2. Lastly,
22 displayed similar cytotoxicity as t-CUPM, 9 and 22, yet pos-
sessed significant differences in C-RAF inhibition. These data are
consistent with previous work with sorafenib derivatives,33 sug-
gesting that C-RAF kinase inhibition may not be critical for the
cytotoxic responses of sorafenib in HCC cells.
In summary, we have performed a SAR study against sEH and
kinases, such as C-RAF and VEGFR-2, with a series of compounds
that resemble both sorafenib and conventional urea-based sEH
inhibitors. Sorafenib is such a potent inhibitor of the sEH that this
activity is thought to stabilize endogenous EpFAs including anti-
hypertensive, anti-inflammatory, and analgesic EETs. Surprisingly
regorafenib is 24-fold more potent than sorafenib as a sEH inhibi-
Table 3
GI50 concentrations of test compounds for HCC cells (HepG2 and Huh-7) and HUVEC
GI50 (lM)
HepG2
Huh-7
HUVEC
Sorafenib
4
5
6
7
8
9
10
11
12
13
14
15
17
21
22
TUPS
TPPU
4.5
7.0
10
7.0
>25
6.5
7.0
>25
>25
15
>25
7.0
7.0
7.5
10
7.0
>25
>25
4.0
8.0
8
7.0
>25
7.5
8.0
>25
>25
20
6
11
tor. A recent study showed that epoxides of the
x-3 fatty acid DHA,
ND
>25
10.5
11
10.5
25
>25
>25
>25
>25
>25
10
when stabilized against hydrolysis, inhibit angiogenesis, tumor
growth and metastasis by a non-VEGF dependent pathway.36
Acknowledgments
This work was supported in part by NIEHS grant ES02710, NIE-
HS Superfund grant P42 ES04699, NIHLB grant HL059699 and the
CounterACT Program, National Institutes of Health Office of the
Director, and the National Institute of Neurological Disorders and
Stroke, Grant Number U54 NS079202. A.T. Wecksler was supported
by Award Number T32CA108459 from the National Institutes of
Health. B.D. Hammock is a George and Judy Senior Fellow of the
American Asthma Society.
16
5.5
5.5
8.5
7.0
7.0
>25
>25
25
>25
>25
>25

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