Peptidyl-urea based inhibitors of soluble epoxide hydrolases

Article abstract of DOI:10.1016/j.bmcl.2006.07.073

We prepared a series of amino acid derived cyclohexyl and adamantyl ureas and tested them as inhibitors of the human soluble epoxide hydrolase, and obtained very potent compounds (K_I = 15 nM) that are >10-fold more soluble than previously descr

Full text of DOI:10.1016/j.bmcl.2006.07.073

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5439–5444
Peptidyl-urea based inhibitors of soluble epoxide hydrolases
Christophe Morisseau,a John W. Newman,a Hsing-Ju Tsai,a
Preston A. Baeckerb and Bruce D. Hammocka,*
aDepartment of Entomology and UCD Cancer Center, University of California, Davis, CA 95616, USA
b
ˆ
Arete Therapeutics, 3912 Trust Way, Hayward, CA 94545, USA
Received 15 June 2006; revised 14 July 2006; accepted 17 July 2006
Abstract—We prepared a series of amino acid derived cyclohexyl and adamantyl ureas and tested them as inhibitors of the human
soluble epoxide hydrolase, and obtained very potent compounds (KI = 15 nM) that are >10-fold more soluble than previously
described sEH inhibitors. While our lead compound 2 showed low apparent bioavailability in dogs and rats, this series of com-
pounds revealed that sEH inhibitor structures could accept large groups that could lead to better orally available drugs.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Soluble epoxide hydrolase (sEH; EC 3.3.3.2) catalyzes
the addition of a water molecule to an epoxide resulting
in the corresponding diol.1 Endogenous substrates for
sEH include epoxyeicosatrienoic acids (EETs), which
are known for their vasodilatory effects as well as for
their anti-inflammatory actions.2 Hydrolysis of the epox-
ides by sEH diminished this activity.3 The inhibition of
sEH led to the accumulation of EETs and other lipid
epoxides in the organism.4 Furthermore, sEH inhibition
in rodent models can successfully treat hypertension,4b,5
and inflammatory diseases,6 as well as protect against
renal damage caused by hypertension.7
properties consistent with successful pharmaceutical
candidates.10 Therefore, there remains a need for novel
sEH inhibitor structures with improved biological
availability and stability for possible future orally
administered in vivo drugs. There is also the need for
synthetic methods amenable to high throughput combi-
natorial or parallel methods.
As shown previously, if placed at an appropriate dis-
tance from the urea moiety polar groups can be incorpo-
rated into one of the alkyl groups of the dialkyl-urea
sEH inhibitors without loss of activity.9 Such modifica-
tions give the new inhibitors better solubility and
availability; however, only straight alkyl chain deriva-
tives such as 12-(3-adamantyl-ureido)-dodecanoic acid
(AUDA; see Fig. 1) were investigated. From the crystal
structure of sEH,8 we observed that while the catalytic
tunnel is restricted around the catalytic residues it
enlarges as one moves away from the site of reaction
to yield relatively large cavities. These structural features
suggest that the enzyme could accommodate branches
on the main alkyl chain. To evaluate a wide variety of
side chains, we used a semi-combinatorial approach.
Because amino acids are simple bifunctional synthons
with a wide variety of side chains, mono- and di-peptidic
derivatives of urea based sEH inhibitors were synthe-
sized (see Fig. 1) and their effects on sEH inhibition
investigated.
The development in our laboratory of novel, stable,
highly potent, and selective inhibitors for sEH4a has
allowed the elucidation of the biology associated with
sEH.1,2d Crystal structure determinations show that
the urea inhibitors establish hydrogen bonding and salt
bridges between the urea function of the inhibitor and
residues of the sEH active site.8 However, these dial-
kyl-ureas have high crystal lattice energies as indicated
by their high melting point, and also have limited sol-
ubility in water,4 likely affecting their in vivo efficacy.
While medicinal chemistry approaches were used to
increase their inhibitory activity and solubility,9 the lat-
est compounds reported may not possess physical
Keywords: Cardiovascular diseases; Soluble epoxide hydrolase
inhibitors.
We chose to modify (3-cycloalkyl-ureido)-alkanoic acids
because these compounds are poor inhibitors of sEH
when the alkyl chain is less than 6-carbons in length,9
*
Corresponding author. Tel.: +1 530 752 7519; fax: +1 530 752
1537; e-mail: bdhammock@ucdavis.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.073

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C. Morisseau et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5439–5444
We first optimized the distance between the urea func-
tion and the first amino acid using a subset of amino
acids and three (3-cyclohexyl-ureido)-alkanoic acids of
different length (Table 1). For the mouse sEH, good
inhibition is obtained for both inhibitor series with
the 4- and 6-carbon carboxylate tails, suggesting that a
3-carbon spacer (i.e., n = 3) is sufficient for this enzyme.
Compared to the free acid (R = OH), the histidyl deriv-
ative gives the best inhibition potency (smallest IC50)
for both chain length. Interestingly, overall for the
human sEH the 4-carbon chain derivatives (n = 3) gave
better inhibition results than shorter or longer chains.
Similar results were observed for the methyl esters of
the three (3-cyclohexyl-ureido)-alkanoic acids tested
(R = OCH3).13 The crystal structure of the human
sEH with these acids showed that the inhibitor with
the 4-carbon carboxylate tail is oriented the opposite
way in the active site, underlying that structure–activity
relationships are more complex for the human sEH.13
For example, increasing the chain length from 4 to 6
carbons resulted in higher IC50s for all the amino acid
derivatives, except for phenylalanine that is decreased
by an order of magnitude. Furthermore, compared to
the free acid (R = OH), only the phenylalanine deriva-
tive with n = 5 gave a significantly improved inhibition
potency.
Figure 1. General structure of amino acid derived urea based
inhibitors for sEH.
and because we previously showed that ester and amide
derivatives of these compounds could yield potent sEH
inhibitors. Furthermore, to make the peptidic bond,
we select reactants, such as 1-ethyl-3-(3-(dimethylami-
no)-propyl) carbodiimide (EDCI) or N,N-dimethyl-4-
aminopyridine (DMAP), that are not inhibitors of
sEH themselves nor are their reaction products, such
as 1-ethyl-3-(3-dimethylamino)-propyl urea. Therefore,
any inhibition observed was derived from the targeted
peptidic derivatives, allowing us to carry out reactions
on an analytical scale (10 lmol), monitor the reactions
with LC–MS, and test them for inhibition without the
need to purify the products (Scheme 1). The presence
of the desired products was confirmed by positive mode
electrospray LC–MS in all cases reported. The yield of
the reaction was estimated by quantifying the remaining
(3-cycloalkyl-ureido)-alkanoic acid against a 4-point cal-
ibration curve. With the exception of cysteine and lysine,
which formed secondary products and gave erratic
yields and thus are not included herein, we found reac-
tion yields of >95%. Dimethylformamide was added to
the reaction mixtures to give target compound’s concen-
tration of 10 mM. These solutions were used to measure
inhibitor potency directly on purified recombinant
mouse and human sEH,11 using a spectrophotometric
substrate in a 96-well microplate assay.12
Therefore, in a second experiment to optimize the
nature of the amino acid residues, we made amino acid
Table 1. Optimization of the alkyl chain length between the urea
function and the first amino acid for optimal inhibition
O
O
(CH2)n R
N
N
H
H
n
R
Mouse sEH
a
IC50 (lM)
Human sEH
a
IC50 (lM)
1
1
1
1
1
1
1
OH
OCH3
>100
33
>100
70
Arginine
Glutamate
Histidine
Phenylalanine
Valine
>100
>100
36
>100
>100
>100
>100
>100
18
>100
3
3
3
3
3
3
3
OH
OCH3
1.9
36
6.2
25
24
30
21
17
0.33
1.0
Arginine
Glutamate
Histidine
Phenylalanine
Valine
1.7
0.05
1.6
1.4
5
5
5
5
5
5
5
OH
OCH3
24
>100
3.2
0.11
0.23
51
Arginine
Glutamate
Histidine
Phenylalanine
Valine
>100
>100
>100
2.7
0.06
0.15
0.56
41
AUDA
0.05
0.1
Scheme 1. Reagents and conditions: (a) EDCI, DMAP, CH2Cl2,
25 ꢁC, 12 h, 95–99%; (b) KOH, DMF, H2O, 25 ꢁC, 12 h, 100%.
a IC50s were determined as described using a spectrophotometric
assay.4a,12
.

C. Morisseau et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5439–5444
5441
derivatives with n = 3 for the murine sEH and n = 5 for
the human sEH. In addition, due to our previous discov-
ery that for the human sEH the replacement of the
cyclohexyl by 1-adamantyl resulted in P10-fold increase
in inhibition, we also prepared derivatives of (3-ada-
mantyl-ureido)-alkanoic acids (Table 2). Other than
the catalytic residues, the active site of both the murine
and human sEHs are quite hydrophobic.1,8 Thus, one
would expect that hydrophobic or aromatic amino acids
would yield good inhibitors. While we observed such
effects for the human sEH, the increase in inhibition
for the murine sEH is not clearly linked to the hydro-
phobicity of the side chain. Furthermore, one would
expect that polar side chains would yield poor sEH
inhibitor as observed for the human sEH. However,
for the murine enzyme, positively charged residues
(i.e., the basic amino acids Arg and His) gave surprising-
ly good inhibition, with the best inhibition obtained with
the histidine conjugate. Because the active site of sEH,
besides being hydrophobic, is also relatively basic,1
one could hypothesize that only uncharged residues
would bind to the enzyme. This hypothesis is supported
by the fact that histidine, which has a 3 pH unit lower
pKa than arginine, yields a far better inhibitor for the
mouse sEH. Alternatively, one could envision p-cation
interactions between the cationic side chain of the inhib-
itors and the aromatic residues that are lining the cata-
lytic cavity of sEH.1 This kind of interaction is very
common in biological systems.14
relationships among inhibitors.4,9 As seen in Table 2, this
is not the case here, indicating that for this structural ser-
ies, it is important to optimize inhibitor structures with
the targeted species (human) enzyme. Furthermore, it
suggests that the series of inhibitors reported herein prob-
ably bind differently for each enzyme studied. While we
obtained a very potent inhibitor for the murine sEH
(the histidine derivative of the 4-carbon carboxylate), less
than optimal inhibition was obtained for the human sEH.
Toward improving inhibition potency for this latter
enzyme, we tested the effect of adding a second amino acid
on the phenylalanine derivatives. Therefore, we prepared
and purified 2-[6-(3-cyclohexyl-ureido)-hexanoylamino]-
3-phenyl-propionic acid 1,15 and following a procedure
similar to the one described in Scheme 1, we prepared a
series of amino acid derivatives of this compound on ana-
lytical scale. We obtained reaction yields >95% and the
products were tested for their ability to inhibit the human
sEH (Table 3). Compared to the free acid (R = OH), the
presence of polar side chains did not enhance the potency
of the inhibitor, while the presence of either aromatic or
hydrophobic side chains reduced the IC50s. These results
agreed with the observed hydrophobicity of the human
sEH catalytic cavity.1,8 The best inhibition was obtained
with isoleucine, phenylalanine, proline, tryptophan or
valine derivatives, suggesting that the human sEH can
fit compounds of various sizes as long as they are hydro-
phobic in nature.
Based on the results obtained at analytical scale (Tables
1–3), we prepared N-[6-(3-adamantyl-ureido)-hexanoyl]-
phenylalanyl-tryptophan 2.16 We then determined the
Previously, while investigating largely hydrophobic
inhibitors, we observed reasonable agreement between
the two enzymes with regard to the structure–activity
Table 3. Inhibition of human sEH by mono-amino acid derivatives
of 1
Table 2. Inhibition of mouse and human sEH by mono-amino acid
derivatives of n-(3-adamantyl-ureido)-alkanoic acid
O
O
H
N
N
N
R
O
H
H
O
(CH2)n
O
N
H
N
R
H
R
Mouse sEH
a
n = 3 IC50 (lM)
Human sEH
a
n = 5 IC50 (lM)
a
R
IC50 (lM)
OH
OCH3
Alanine
>100
0.10
>100
16
>100
1.8
OH (1)
Alanine
1.0
0.26
2.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.7
Arginine
Aspartate
Glutamate
Glycine
Arginine
Aspartate
Glutamate
Glycine
>100
>100
>100
0.05
1.3
>100
>100
1.5
Histidine
Isoleucine
Leucine
Histidine
Isoleucine
Leucine
5.5
0.13
0.88
0.87
0.10
0.10
2.4
15
Methionine
Phenylalanine
Proline
4.8
0.8
Methionine
Phenylalanine
Proline
82
47
>100
>100
>100
41
Serine
Serine
Threonine
Tryptophan
Tyrosine
Valine
33
Threonine
Tryptophan
Tyrosine
Valine
1.3
8.4
0.10
0.23
0.10
18
30
80
55
a IC50s were determined as described using a spectrophotometric
a IC50s were determined as described using a spectrophotometric
assay.4a,12
4a,12
assay.
.

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C. Morisseau et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5439–5444
Table 4. Pharmacokinetic profile data for compounds 2 and AUDA as
obtained via oral dosing in a canine model
inhibition constant of compound 2 for the human sEH
(Fig. 2) and found a KI of 15 3 nM (n = 3). This is
in the same order of magnitude as previously reported
sEH inhibitors,4,9 confirming that 2 is a very potent
human sEH inhibitor in vitro, as suggested by analytical
scale results. Our ultimate objective is to obtain novel
sEH inhibitors with improved solubility, higher biologi-
cal availability, and in vivo stability. Therefore, we next
tested the peptidyl-urea sEH inhibitor solubility in sodi-
um phosphate buffer (100 mM, pH 7.4) as previously
described.9a All prepared amino acid derivatives had sol-
ubilities >500 lM. This is P10-fold the solubility of the
corresponding compounds with a straight carboxylate
chain, such as AUDA,9 suggesting that the peptidyl-
urea based inhibitors could be given in drinking water,
and should dissolve readily from tablets. Finally, we
tested the oral bioavailability of 2 in a canine model
(Table 4). The presence of amino acids in some com-
pounds can greatly increase their bioavailability due to
the presence of di/tri-peptide specific transporters in
the gut.18 Thus, one would expect that compounds like
2 might be readily bioavailable. Surprisingly, as shown
in Table 4, small amounts of 2 were detected in dog
Time (min)
2
AUDA
Plasma concentration (nM)
0
15
0.00
0.62
0.82
0.33
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
6.97
30
60
20.43
30.69
26.31
13.39
6.12
120
180
240
300
360
480
1440
1.61
1.17
0.86
0.00
AUCa (·104 nM min)
<0.01
0.31
a Area under the curve, estimated from a plot of inhibitor plasma
concentration (nM) versus time (minutes) following an oral dose of
0.3 mg/Kg of the indicated compounds in 6 mL of tristerate.17
plasma. Furthermore, expected metabolites for 2, 6-(3-
adamantyl-ureido)-hexanoic acid and its phenylalanine
derivatives, were not detected, suggesting that 2 is poorly
absorbed from the gut or rapidly metabolized by an
Figure 2. Determination of the KI of 2 for the human sEH. The enzyme ([E]final = 3 nM) diluted in sodium phosphate buffer (100 mM, pH 7.4) was
incubated for 5 min at 30 ꢁC with the inhibitor ([I]final = 0–50 nM), before the addition of substrate (tDPPO; [S]final = 3.6–50 lM). The inhibition
constant (KI) was determined as described.4a,9a

C. Morisseau et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5439–5444
5443
Hammock, B. D.; Busse, R.; Fleming, I. Hypertension
2005, 45, 759; (c) Loch, D.; Hoey, A.; Morisseau, C.;
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alternate route, either within the gut or the body.
Regardless, as measured by the AUCs of the parent
molecules (Table 4), compound 2 appears far less
bioavailable than AUDA, our reference inhibitor.9a
Similar results were obtained in rats.
6. (a) Schmelzer, K. R.; Kubala, L.; Newman, J. W.; Kim, I.
H.; Eiserich, J. P.; Hammock, B. D. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 9772; (b) Smith, K. R.; Pinkerton, K. E.;
Watanabe, T.; Pedersen, T. L.; Ma, S. J.; Hammock, B. D.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2186.
7. (a) Zhao, X.; Yamamoto, T.; Newman, J. W.; Kim, I. H.;
Watanabe, T.; Hammock, B. D.; Stewart, J.; Pollock, J.
S.; Pollock, D. M.; Imig, J. D. J. Am. Soc. Nephrol. 2004,
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46, 975.
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In conclusion, we have reported a new series of potent
peptidyl-urea based sEH inhibitors which are substan-
tially more water soluble than previously described
sEH inhibitors. While our lead compound, 2, showed
low apparent bioavailability, this series of compounds
revealed that sEH inhibitor structures could accept large
groups if placed at an appropriate distance from the cen-
tral pharmacophore. Further exploration within this
area of the molecular structure could yield inhibitors
with higher biological availability and stability, leading
the way to orally available drugs. Furthermore, these
findings suggest that refinements of the sEH inhibitor
structure that depart from the simple, flexible aliphatic
backbone characteristic of the AUDA-like compounds
should be performed using human sEH. Moreover, it
is clear that the procedure described here offers a rapid
route to synthesize a variety of sEHI with both natural
and non-natural amino acids by both liquid- and solid-
phase procedures. One also can reverse the polar group
on the urea side of the molecule using an amine linker to
have the peptide chain serve as the N- rather than C-ter-
minal equivalent. The structure and polarity of the pep-
tide chain can be altered with amides and esters of free
amino and carboxylic acid functionalities or by deriva-
tives commonly used to alter the structure and polarity
of the NH bond.
10. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Deliv. Rev. 2001, 46, 3.
11. (a) Grant, D. E.; Storms, D. H.; Hammock, B. D. J. Biol.
Chem. 1993, 268, 17628; (b) Beetham, J. K.; Tian, T.;
Hammock, B. D. Arch. Biochem. Biophys. 1993, 305, 197;
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Acknowledgments
14. Meyer, E. A.; Castellano, R. K.; Diederich, F. Angew.
Chem. Int. Ed. 2003, 42, 1210.
This study was supported in part by NIEHS Grant
ES02710, NIEHS Superfund Grant P42 ES04699, NIE-
HS Center Grant P30 ES05707, and NHLBI STTR
Grant R41 HL078016.
15. 2-[6-(3-cyclohexyl-ureido)-Hexanoylamino]-3-phenyl-pro-
pionic acid 1 was prepared following the method described
on Scheme 1, starting with 0.2 mmol of reagents. The
target product was purified by reverse phase (C-18)
chromatography with a 60:40 methanol/water solvent
mixture. We obtained 70 mg (Yield: 93%) of 1 as a white
1
solid; mp 141–142 ꢁC. ESIMS m/z = 404.26 (M+H)+; H
References and notes
NMR (300 MHz, DMSO/TMS): d 12.62 (s, 1H, COOH),
8.07 (d, J = 8.4 Hz, 1H, NH amide), 7.24–7.13 (br m, 5H,
phenyl), 5.61 (m, 2H, NH urea), 4.38 (m, 1H, C2) 3.30 (m,
1. Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol.
Toxicol. 2005, 45, 311.
2. (a) Harder, D. R.; Campbell, W. B.; Roman, R. J. J. Vasc.
Res. 1995, 32, 79; (b) Campbell, W. B.; Gebremedhin, D.;
Pratt, P. F.; Harder, D. R. Circ. Res. 1996, 78, 415; (c)
Node, K.; Huo, Y.; Ruan, X.; Yang, B.; Spiecker, M.;
Ley, K.; Zeldin, D. C.; Liao, J. K. Science 1999, 285, 1276;
(d) Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog.
Lipid Res. 2005, 44, 1.
0
1H, CH cyclohexyl), 3.04–2.75 (br m, 4H, C3 and C6 ),
0
1.99 (t, J = 7.4 Hz, 2H, C2 ), 1.70–1.00 (br m, 16H, CH2s
0
from cyclohexyl and C3 –5 ) ppm.
0
16. N-[6-(3-adamantyl-ureido)-hexanoyl]-phenylalanyl-tryp-
tophan 2 was obtained by reacting 6-(3-adamantyl-
ureido)-hexanoic acid with the commercially available
phenylalanyl-tryptophan dipeptide following the method
described on Scheme 1, starting with 0.2 mmol of
reagents. The target product was purified by reverse
phase (C-18) chromatography with a 70:30 methanol/
water solvent mixture. We obtained 45 mg (Yield: 35%)
of 2 as a light tan colored solid; mp 115–120 ꢁC (dec.);
ESIMS m/z = 642.60 (M+H)+; 1H NMR (300 MHz,
DMSO/TMS): d 12.70 (br s, 1H, COOH), 10.86 (d,
J = 8.8 Hz, 1H, NH indol), 8.25 (dd, J = 7.6 Hz, 1H, NH
amide), 7.90 (dd, J = 7.6 Hz, 1H, NH amide), 7.55 (t,
J = 7.1 Hz, 1H, CH indol), 7.34–6.92 (br m, 9H, phenyl
and indol), 5.56 (m, 1H, NH urea), 5.42 (m, 1H, NH
3. Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub, N. L.
Prog. Lipid Res. 2004, 43, 55.
4. (a) Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng,
J.; Greene, J. F.; Sanborn, J. R.; Hammock, B. D. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 8849; (b) Yu, Z.; Xu, F.;
Huse, L. M.; Morisseau, C.; Draper, A. J.; Newman, J.
W.; Parker, C.; Graham, L.; Engler, M. M.; Hammock, B.
D.; Zeldin, D. C.; Kroetz, D. L. Circ. Res. 2000, 87, 992.
5. (a) Imig, J. D.; Zhao, X.; Capdevila, J. H.; Morisseau, C.;
Hammock, B. D. Hypertension 2002, 39, 690; (b) Jung, O.;
Brandes, R. P.; Kim, I.; Schweda, F.; Schmidt, R.;

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C. Morisseau et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5439–5444
0
urea), 4.62–4.38 (br m, 2H, C2 and C2 ), 3.23–2.68 (br m,
collected at 0, 15, 30, 60, 120, 180, 240, 300, 360, 480, and
1440 min, and analyzed by LC/MS/MS (Quattro Premier;
Micromass, MA). The pharmacokinetic parameters were
calculated with WinNonlin 5.0 (Pharsight, CA).
18. Oh, D. M.; Han, H. K.; Williamson, R. M.; Bigge, C. F.;
Amidon, G. L.; Stewart, B. H.; Surendran, N. J. Pharm.
Sci. 2002, 12, 2579.
0
6H, C3, C3 and C6 ), 2.06–1.45 (br m, 17H, C2 and
00
00
00
adamantyl), 1.40–0.93 (br m, 6H, C3 –5 ) ppm.
00
17. Selected soluble epoxide hydrolase inhibitors were pre-
pared at 6 mg/mL in triglycerides. Each dog was dosed
orally with three different inhibitors per experiment
(dose = 0.3 mg/kg per inhibitor). Plasma samples were