Discovery of 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase inhibitors and orally active drug candidates for treating of chronic kidney diseases

Article abstract of DOI:10.1016/j.bmcl.2013.12.020

We identified 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted ureas as highly potent soluble epoxide hydrolase (sEH) inhibitors and orally active agents for treating chronic kidney diseases. Compound 19 exhibited excellent sEH inhibitory activity a

Full text of DOI:10.1016/j.bmcl.2013.12.020

Accepted Manuscript
Discovery of 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted urea de-
rivatives as highly potent soluble epoxide hydrolase inhibitors and orally active
drug candidates for treating of chronic kidney diseases
Yuko Kato, Nobuhiro Fuchi, Yutaka Nishimura, Ayano Watanabe, Mai Yagi,
Yasuhito Nakadera, Eriko Higashi, Masateru Yamada, Takumi Aoki, Hideo
Kigoshi
PII:
S0960-894X(13)01392-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.12.020
BMCL 21128
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2013
30 October 2013
4 December 2013
Please cite this article as: Kato, Y., Fuchi, N., Nishimura, Y., Watanabe, A., Yagi, M., Nakadera, Y., Higashi, E.,
Yamada, M., Aoki, T., Kigoshi, H., Discovery of 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted urea
derivatives as highly potent soluble epoxide hydrolase inhibitors and orally active drug candidates for treating of
chronic kidney diseases, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.
2013.12.020
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Discovery of 1-oxa-4,9-
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diazaspiro[5.5]undecane-based trisubstituted
urea derivatives as highly potent soluble
epoxide hydrolase inhibitors and orally
active drug candidates for treating of chronic
kidney diseases
Yuko Kato, Nobuhiro Fuchi, Yutaka Nishimura, Ayano Watanabe, Mai Yagi, Yasuhito Nakadera, Eriko
Higashi, Masateru Yamada, Takumi Aoki, Hideo Kigoshi

Bioorganic & Medicinal Chemistry Letters
journal hom epage: www.elsevier.com
Discovery of 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted urea
derivatives as highly potent soluble epoxide hydrolase inhibitors and orally
active drug candidates for treating of chronic kidney diseases
Yuko Katoa,b,∗, Nobuhiro Fuchia, Yutaka Nishimuraa, Ayano Watanabea, Mai Yagia, Yasuhito Nakaderaa,
Eriko Higashia, Masateru Yamadaa, Takumi Aokia, and Hideo Kigoshib
a Toray Industries, Inc., Pharmaceutical Research Laboratories, 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan
b Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
We identified 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted ureas as highly potent
soluble epoxide hydrolase (sEH) inhibitors and orally active agents for treating chronic kidney
diseases. 19 exhibited excellent sEH inhibitory activity and bioavailability. When administered
orally at 30 mg/kg, 19 lowered serum creatinine in a rat model of anti-glomerular basement
membrane glomerulonephritis but 2,8-diazaspiro[4.5]decane-based trisubstituted ureas did not.
These results suggest that 19 is an orally active drug candidate for treating chronic kidney
diseases.
Revised
Accepted
Available online
Keywords:
sEH inhibitor
2013 Elsevier Ltd. All rights reserved.
Anti-GBM glomerulonephritis rat model
Spirocyclic diamine
Urea
Chronic kidney diseases
Soluble epoxide hydrolase (sEH) is an enzyme that catalyzes
the conversion of epoxyeicosatrienoic acids (EETs) into
dihydroxy eicosatrienoic acids (DHETs) (Figure 1),1 and is
mainly expressed in liver, kidney, and vascular tissue.2 EETs,
which are synthesized by CYP2J- and CYP2C-mediated
epoxidation of arachidonic acid, are associated with
physiologically beneficial effects such as vasodilatation,
vasoprotection, and anti-inflammation.3 Inhibition of sEH
produces effects expected from an increase in EET level.4
We previously reported that 2,8-diazaspiro[4.5]decane-based
trisubstituted ureas are highly potent sEH inhibitors and orally
active drug candidates for treating hypertension (Figure 2, 1).6
Contrary to our expectation, oral administration of these
derivatives failed to reduce serum creatinine in a rat model of
anti-glomerular basement membrane (GBM) glomerulonephritis7.
We found that 2,8-diazaspiro[4.5]decane-based trisubstituted
ureas had high sEH inhibitory activity and oral availability. The
solubility and microsomal stability of these compounds can be
conveniently modified by changing the substituent on the amide
group. On the basis of these findings, we hypothesized that
spirocyclic diamine-based trisubstituted ureas could be developed
as sEH inhibitors and we aimed to find other such ureas that
inhibited sEH as strongly as 2,8-diazaspiro[4.5]decane-based
trisubstituted ureas and had different pharmacokinetic profiles.
Here we report that 1-oxa-4,9-diazaspiro[5.5]undecane-based
trisubstituted ureas are highly potent sEH inhibitors and are
available for oral administration in a rat model of anti-GBM
glomerulonephritis.
CO2H
CO2H
CO2
H
CYP2C9
sEH
HO
O
OH
Arachidonic acid
EET
DHET
Figure 1. Role of sEH.
According to a recent report, 5 sEH in proximal tubular cells is
upregulated in chronic proteinuric kidney diseases. According to
that
study,
1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-
trifluoromethoxy-phenyl)-urea reduces long-term elevated serum
creatinine level, interstitial inflammation, fibrosis, and α-smooth
muscle actin expression in adriamycin-induced nephropathic
mice. These findings suggest that sEH inhibitors have potential
use in treating chronic proteinuric kidney diseases.
The hydrolase catalytic pocket of sEH consists of Tyr381,
Tyr465, and Asp333, which are responsible for its enzymatic
activity. Amide or urea derivatives bind to the pocket via
hydrogen bonds between the carbonyl oxygen of the amide or
urea moiety and the hydroxyl group of Tyr381 or Tyr465, and
———
∗ Corresponding author. Tel.: +81-467-32-9632; fax: +81-467-32-9632; e-mail: Yuko_Kato@nts.toray.co.jp

O
F
F
F3CO
F3CO
O
F
O
N
N
H
N
N
H
N
N
F
O
O
1
2
CLogP: 3.33
CLogP: 2.84
Figure 2. Structure of 1 (previously reported) and 2 (newly designed).
Scheme 1. Synthesis of 1-oxa-4,9-diazaspiro[5.5]undecane-based
trisubstituted urea derivatives 2, 19-40 and 8. Reagents and conditions: (a)
R-NCO, DIPEA, CHCl3; (b) H2, Pd(OH)2, MeOH; (c) R’COOH, HATU,
DIPEA, DMF or R’COCl, Et3N, CH2Cl2. (d) 2,6-difluorobenzoyl chloride,
Et3N, CH2Cl2; (e) 4-(trifluoromethoxy)phenyl isocyanate, DIPEA, CHCl3,
37% for 3steps.
Figure 3. Docking studies of human sEH (PDB code: 1VJ5) with 1
(left) and 2 (right).
between the NH of the amide or urea moiety and the carboxylate
of Asp333.8 We previously reported that 1 (Figure 2) exhibits
highly potent sEH inhibitory activity. A docking study of 1 with
human sEH indicated that the oxygen atom of the compound’s
urea moiety binds to Tyr381 and Tyr465 in the hydrolase
catalytic pocket of sEH (Figure 3). We then designed 1-oxa-4,9-
diazaspiro[5.5]undecane-based trisubstituted urea derivative 2.
Because of its introduced oxygen atom, 2 has lower cLogP than 1
and thus would have higher aqueous solubility (Figure 2). A
docking study of 2 with human sEH (Figure 3) suggested that the
two carbonyl oxygen atoms of 2 bind to the hydrolase catalytic
pocket in the same manner as 1. Under this hypothesis, we began
Scheme 2. Synthesis of 2,9-diazaspiro[5.5]undecane-based trisubstituted
urea derivatives 15 and 18. Reagents and conditions: (a) Dess-Martin
Periodinane, CH2Cl2, 53%; (b) trimethylbenzylammonium hydroxide 40%
in water, acrylonitrile, 1,4-dioxane, 60%; (c) H2, Pd/C, HCl, EtOH, 29%;
(d) 4-(trifluoromethoxy)phenyl isocyanate, Et3N, CHCl3; (e) TFA, CH2Cl2,
0 °C; (f) 2,6-difluorobenzoyl chloride, Et3N, CH2Cl2.
Replacing the oxygen atom of 8 also led to the lower solubility
(18). Considering that 2 showed higher solubility, we next
focused on derivatives with a 1-oxa-4,9-diazaspiro[5.5]undecane
scaffold.
to
trisubstituted urea derivatives.
explore
1-oxa-4,9-diazaspiro[5.5]undecane-based
The general procedure for the synthesis of 1-oxa-4,9-
diazaspiro[5.5]undecane-based trisubstituted urea derivatives 2,
19-40 and 8 is shown in Scheme 1. Starting material 3 was
synthesized by a reported method.9 Treatment of 3 with
isocyanate afforded 4. Removal of the benzyl protecting group by
The SAR and SPR results for derivatives of 2 with benzamide
moieties are shown in Table 2.10 The removal of the fluorine
atom from 2 improved rat sEH inhibitory activity, and also
improved solubility and microsomal stability (19). Replacing the
trifluoromethoxy group of 2 with a trifluoromethyl group (20)
made little difference in activity or other properties. The effect of
substituent position on the benzamide ring was investigated (21-
24). Removing the substituent reduced human sEH inhibitory
activity (21). Installing a chloro substituent at the ortho (22),
meta (23), and para (24) positions gave the same human sEH
inhibitory activity as 20, and only 22 showed improved rat sEH
inhibitory activity. 22 and 24 exhibited sufficient microsomal
stability. These results suggest that ortho substitution on the
aromatic benzamide ring is particularly favorable for rat sEH
inhibitory activity but meta or para substitution is not. In general,
lower lipophilicity corresponds to better solubility and
microsomal stability.11 To achieve sufficient sEH inhibitory
activity, solubility, and microsomal stability concurrently, we
examined an ortho-substituted derivative with reduced
lipophilicity (25); it had higher sEH inhibitory activity, solubility,
and microsomal stability. Further investigation revealed that only
a cyano group was acceptable at the para position on the
benzamide ring: 26 exhibited good sEH inhibitory activity,
solubility and microsomal stability.
Pd(OH)2-catalyzed
hydrogenation
provided
5.
Then,
condensation with carboxylic acid or acyl chloride led to 2 and
19-40. Treatment of 3 with benzoyl chloride afforded 6. Removal
of the benzyl protecting group by Pd(OH)2-catalyzed
hydrogenation provided 7. Then, treatment with isocyanate led to
8. The synthesis of 2,9-diazaspiro[5.5]undecane-based
trisubstituted urea derivatives 15 and 18 is shown in Scheme 2.
Oxidation of alcohol 9 with Dess-Martin periodinane afforded
10. Michael addition to acrylonitrile provided 11. Then,
sequential reduction of nitrile group and cyclization via reductive
amination gave 2,9-diazaspiro[5.5]undecane 12. Treatment of 12
with isocyanate, removal of the Boc protecting group by TFA,
then, treatment with benzoyl chloride led to 15. Treatment of 12
with benzoyl chloride, removal of the Boc protectiong group by
TFA, then, treatment with isocyanate led to 18.
We performed structure–activity relationship (SAR) and
structure–property relationship (SPR) studies on the diazaspiro
scaffolds (Table 1).10 4-(Trifluoromethoxy)phenyl and 2,6-
difluorobenzoyl groups were selected as the left- and right-hand
substituents, respectively. As we expected, 2 had moderate
inhibitory activity against human sEH and better solubility than
1. However, the microsomal stability of 2 was lower than that of
1. Lower sEH inhibitory activity was found in 8, whose
diazaspiro scaffold was constructed with the left- and right-hand
substituents swapped. In 15, which had a carbon atom in place of
the oxygen atom in the 1-oxa-4,9-diazaspiro[5.5]undecane
scaffold of 2, sEH inhibitory activity was improved but
microsomal stability and solubility became problematically low.
Derivatives with
a heteroaromatic ring as the amide
substituent were explored in order to evaluate the effectiveness of
reduced lipophilicity for improving solubility and microsomal
10
stability. As expected, almost all compounds in Table 3
exhibited improved solubility and microsomal stability.
However, we observed decreased human sEH inhibitory activity
in 27 which contained an unsubstituted five-membered ring. The
derivatives with five-membered rings bearing a methyl or

cyclopropyl substituent (28-30) exhibited increased human sEH
inhibitory activity. Remarkably, pyrazole derivative 30, which
contained an NH moiety, was tolerated in humans and exhibited
sEH inhibitory activity, but was not tolerated in rats. 31 with an
unsubstituted pyridine ring showed lower activity, and 32 with a
chloro substituent on the pyridine ring effectively inhibited sEH
in humans and rats. A pyrazine ring was also an applicable
substituent (33). Although 34 exhibited moderate human sEH
inhibitory activity, its rat sEH inhibitory activity was low.
10
Table 4 shows the results for derivatives with an aliphatic
amide. 35 with a pivalamide moiety had high sEH inhibitory
activity, and 36 also showed increased human sEH inhibitory
activity, but its rat sEH inhibitory activity was 0.4-fold that of 35.
37 exhibited potent sEH inhibitory activity but was labile to
CYP-mediated metabolism. The derivatives with bulky
substituents (38-40) had moderate human sEH inhibitory activity,
but showed decreased rat sEH inhibitory activity.
We selected compounds 19, 28, and 33 and evaluated their
pharmacokinetic profiles (Table 5). 19 had good bioavailability,
whereas 33 had modest bioavailability, which was attributed to
low permeability resulting from low lipophilicity (cLogP: 0.90).
We next investigated the effect of 19 on serum creatinine level
in a rat model of anti-GBM glomerulonephritis7 (Figure 4). The
serum creatinine level was significantly higher than that of the
normal rat and increased in a time-dependent manner. Oral
administration of 19 at 30 mg/kg significantly reduced serum
creatinine in the rat model. The result indicate that 19 prevented
the progression of glomerulonephritis. We do not yet know why
2,8-diazaspiro[4.5]decane-based trisubstituted ureas failed to
reduce serum creatinine in the rat model, but we speculate that
these derivatives did not sufficiently penetrate into renal tissue.
Currently, we are investigating the exposure of renal tissue to
these ureas and 19 in order to elucidate the reason behind the
difference in renal protective effect.
3
2
1
0
Normal
Vehicle
Compound 19 30 mg/kg
2
3
4
5
Week
Figure 4 Effect of 19 on serum creatinine (sCre) in a rat model of anti-
GBM glomerulonephritis. 19 (30 mg/kg) was orally administered once
daily for 3 weeks from 2 weeks after injection of anti-GBM antibody.
In
conclusion,
we
identified
1-oxa-4,9-
diazaspiro[5.5]undecane-based trisubstituted ureas as highly
potent sEH inhibitors and orally active agents for treating chronic
kidney diseases. 19 exhibited excellent sEH inhibitory activity
and bioavailability, as well as a renal protective effect in a rat
model of anti-GBM glomerulonephritis. These results suggest
that 19 is an orally active drug candidate for treating chronic
kidney diseases. We are currently investigating further
derivatization of 19.
Table 1. SAR and SPR of diazaspiro scaffolds.
sEH IC50(nM)
Solubility (µg/mL)
Human liver
microsomal
stabilitya
Structure
Compound
cLogP
3.33
Human
0.4
Rat
4.5
JP1b
JP2c
1
24
21
0
2
0.7
18.5
N.D.d
3.3
44
52
9
39
46
9
0.086
2.84
2.86
3.89
8
5.4
N.D.
15
0.125
0.602

18
2.96
N.D.
0
0
N.D.
3.89
a Units: mL/min/mg protein.
b Simulated gastric fluid compliant with Japanese Pharmacopeia.
c Simulated intestinal fluid compliant with Japanese Pharmacopeia.
d N.D.: Not determined.
Table 2. SAR and SPR of 1-oxa-4,9-diazaspiro[5.5]undecane-based urea derivatives.
O
R
O
R'
N
N
N
H
O
sEH IC50(nM)
Solubility (µg/mL)
Human liver
microsomal
stabilitya
R
R'
Compound
cLogP
2.84
Human
Rat
JP1b
JP2c
F
F
F
OCF3
OCF3
CF3
2
0.7
18.5
9.3
44
39
0.086
0.03
F
19
20
1.1
1.3
3.2
1.4
1.2
1.0
0.8
1.9
71
43
62
10
26
29
92
85
62
38
63
12
21
23
93
82
2.67
19.1
N.D.d
13
0.093
N.D.
0.048
0.1
3.04
F
CF3
CF3
CF3
CF3
CF3
CF3
21
22
23
24
25
26
2.66
3.44
3.44
3.44
2.14
2.26
Cl
Cl
36.9
24.9
6.8
0.021
0.039
0.003
Cl
O
HN
15
CN
a Units: mL/min/mg protein.
b Simulated gastric fluid compliant with Japanese Pharmacopeia.
c Simulated intestinal fluid compliant with Japanese Pharmacopeia.
d N.D.: Not determined.
Table 3. SARs and SPRs of 1-oxa-4,9-diazaspiro[5.5]undecane-based urea derivatives.
O
R
O
R'
N
N
N
H
O
sEH IC50(nM)
Human
Solubility (µg/mL)
Human liver
microsomal
stabilitya
R
R'
Compound
cLogP
Rat
JP1b
JP2c
O
CF3
27
5.4
N.D.
75
75
N.D.d
1.83

N
O
OCF3
OCF3
OCF3
CF3
28
29
30
31
32
33
34
1.4
0.6
1.1
4.2
0.6
0.9
1.8
25.7
22.2
96.9
N.D.
11.6
8.8
84
86
93
85
90
91
96
83
89
92
83
89
92
93
0.047
0.087
0.035
N.D.
1.22
1.67
1.91
1.56
2.10
0.90
1.49
N
O
NH
N
N
N
OCF3
OCF3
OCF3
0.062
0.012
0.029
Cl
N
N
O
N
H
>20
a Units: mL/min/mg protein.
b Simulated gastric fluid compliant with Japanese Pharmacopeia.
c Simulated intestinal fluid compliant with Japanese Pharmacopeia.
d N.D.: Not determined.
Table 4. SARs and SPRs of 1-oxa-4,9-diazaspiro[5.5]undecane-based urea derivatives.
O
R
O
R'
N
N
H
N
O
sEH IC50(nM)
Human
Solubility (µg/mL)
Human liver
microsomal
stabilitya
R
R'
Compound
cLogP
Rat
JP1b
JP2c
CF3
CF3
35
36
2.9
2.1
12
76
73
0.098
0.074
2.07
1.81
31.4
79
69
79
63
OCF3
OCF3
37
38
0.6
0.4
14.5
28
0.357
0.228
2.67
3.25
37
33
OCF3
OCF3
39
40
0.5
0.7
71.6
>20
66
92
59
90
0.461
0.132
2.82
1.41
OMe
N
a Units: mL/min/mg protein.
b Simulated gastric fluid compliant with Japanese Pharmacopeia.
c Simulated intestinal fluid compliant with Japanese Pharmacopeia.
Table 5. Pharmacokinetic profiles of 19, 28, and 33 in rat.
i.v. (0.2 mg/kg)
Compound
p.o. (1 mg/kg)

C5min
(ng/mL)
71
AUC0-
t1/2
CLtot
Vdss
Cmax
AUC0-
tmax
(hr)
1
F
∞
∞
(ng·hr/mL)
173
(hr)
2.63
1.17
1.53
(mL/hr/kg)
1157
(mL/kg)
3562
3315
1825
(ng/mL)
20.4
(ng·hr/mL)
306
(%)
35.4
24.1
11.1
19
28
33
77.7
134
90.2
2216
9.58
123
2
111
1808
8.64
61.3
1
a
i.v.: Intravenous. p.o.: Oral administration. C: Concentration. AUC: Area under the blood concentration time curve. t1/2: Half-life. CLtot: Total body
clearance. Vdss: Volume of distribution. F: Oral bioavailability.
purchased from Cayman Chemical Company) or rat (the enzyme
was expressed in Sf9 insect cells using baculovirus) in buffer
(BisTris–HCl, 25 mM, pH 7.0, containing 0.1 mg/ml BSA) was
incubated with a inhibitor at room temperture for 30 min. To the
resultant solution cyano(6-methoxy-naphthalen-2-yl)methyl trans-
[(3-phenyloxiran-2-yl)methyl] carbonate (purchased from Cayman
Chemical Company) was added and incubated at room temperture
for 20-45 min. ZnSO4 was added and the resultant solution of
fluorescence intensity (excitation filter 330 nm, emission filter 465
nm) was measured. The reduction rate of enzyme activity by
inhibitors were calculated using the fluorescence intensity, and
IC50 values were determined. In these assays IC50 values of a
representative sEH inhibitor 12-(3-adamantan-1-ylureido)-
dodecanoic acid (AUDA) were 3 nM (human), 10 nM (murine),
10 nM (rat).
References and notes
1. Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog. Lipid Res.
2005, 44, 1.
2. Pacifici, G. M.; Temellini, A.; Giuliani, L.; Rane, A.; Thomas, H.;
Oesch, F. Arch. Toxicol. 1988, 62, 254.
3. For a review: Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub,
N. L. Prog. Lipid Res. 2004, 43, 55.
4. For a recent review of sEH inhibitors: Shen, H. C.; Hammock, B.
D. J. Med. Chem. 2012, 55, 1789.
5. Zhao, X. Y, Yamamoto, T.; Newman, J. W.; Kim, I.-H.; Watanabe,
T.; Hammock, B. D.; Pollock, J. S.; Pollock, D. M.; Imig, J. D. J.
Am. Soc. Nephrol. 2004, 15, 1244.
6. Kato, Y.; Fuchi, N.; Saburi, H.; Nishimura, Y.; Watanabe, A.;
Yagi, M.; Nakadera, Y.; Higashi, E.; Yamada, M.; Aoki, T.
Bioorg. Med. Chem. Lett. 2013, 23,5975.
11. Nassar, A. E. F.; Kamel, A. M.; Clarimont, C. Drug Discov.
Today 2004, 9, 1020.
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Exp. Biol. Med. 1978, 159, 324.
8. Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D.
W. Protein Sci. 2006, 15, 58.
Supplementary Material
9. Connors, R.V.; Dai, K.; Eksterowicz, J.; Fan, P.; Fisher, B.; Fu, J.;
Li, K.; Li, Z.; McGee, L.R.; Sharma, R.; Wang, X.; McMinn, D.;
Mihalic, J.; Deignan, J. PCT Int. Appl., WO 2009085185, 2009.
10. The sEH inhibition assays were performed as described by Jones,
P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.;
Hammock, B. D.( Anal. Biochem. 2005, 343, 66.). A solution of
recombinant sEH from human or mouse (the enzymes were
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