Discovery of a N'-hydroxyphenylformamidine derivative HET0016 as a potent and selective 20-HETE synthase inhibitor.

Article abstract of DOI:10.1016/S0960-894X(01)00614-X

N-(4-Butyl-2-methylphenyl)-N'-hydroxyformamidine (HET0016) was evaluated as the first potent and selective inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthase. The IC(50) value of HET0016 for the production of 20-HETE from arachidonic acid (AA) by human renal microsomes was 8.9+/-2.7 nM, with over 200 times the selectivity of xenobiotic-metabolizing cytochrome P450 enzymes. An examination of the structure-activity relationship revealed that the unsubstituted hydroxyformamidine moiety and the substituent at the para-position of the N-hydroxyformamidine moiety are necessary for the potent activity of HET0016.

Full text of DOI:10.1016/S0960-894X(01)00614-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2993–2995
Discovery of a N0-Hydroxyphenylformamidine Derivative
HET0016y as a Potent and Selective 20-HETE
Synthase Inhibitor
Masakazu Sato, Takaaki Ishii,* Yuko Kobayashi-Matsunaga, Hideaki Amada,
Kazuo Taniguchi, Noriyuki Miyata and Kazuya Kameo
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Saitama, Saitama 330–8530, Japan
Received 10 July 2001; accepted 31 August 2001
Abstract—N-(4-Butyl-2-methylphenyl)-N0-hydroxyformamidine (HET0016) was evaluated as the first potent and selective inhibitor
of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthase. The IC50 value of HET0016 for the production of 20-HETE
from arachidonic acid (AA) by human renal microsomes was 8.9ꢀ2.7 nM, with over 200 times the selectivity of xenobiotic-meta-
bolizing cytochrome P450 enzymes. An examination of the structure–activity relationship revealed that the unsubstituted
hydroxyformamidine moiety and the substituent at the para-position of the N-hydroxyformamidine moiety are necessary for the
potent activity of HET0016. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
and some lack selectivity for xenobiotic-metabolizing
CYPs. HET0016 is the first reported potent and selec-
tive 20-HETE synthase inhibitor. Its IC50 value was
8.9ꢀ2.7 nM for the formation of 20-HETE by human
renal microsomes, while its IC50 values for human
recombinant CYP2C9-, 2D6- and 3A4-catalyzed oxida-
tive metabolism of substrate were 3.3ꢀ0.2, 83.9ꢀ7.0
and 71.0ꢀ21 mM, respectively.1 In comparison, 1-ABT
inhibited CYP2C9-, 2D6- and 3A4 with IC50 values of
42.9ꢀ1.6, 10.5ꢀ0.1 and 0.45ꢀ0.01 mM, these were
similar concentrations to those needed to inhibit the
formation of 20-HETE.1 Epoxyeicosatrienoic acids
(EETs) are produced by epoxyganases (CYP1A, 2B, 2C
and 2J families) from AA. IC50 value for HET0016 for
inhibition of the formation of EETs was 2.8 mM,1 in
contrast, it is known that 17-ODYA is a non-selective
inhibitor of the formation of 20-HETE and EETs.1,11
These results suggest that HET0016 is not a non-selec-
tive inhibitor of CYPs like 1-ABT and 17-ODYA.
HET0016 had a selective inhibitory effect on CYP that
produces 20-HETE from AA without affecting other
CYP isoforms. AA is also metabolized by cyclo-oxyga-
nase (COX), but HET0016 had very little effect on the
activity of COX (IC50 2.3 mM).1 Therefore, we synthe-
sized a series of HET0016 derivatives and elucidated
their ability to inhibit 20-HETE formation. In this
paper, we discuss the structure–activity relationships of
these new potent inhibitors (Fig. 1).
Recently, several studies have been conducted on the
biological properties of 20-hydroxy-5,8,11,14-eicosa-
tetraenoic acid (20-HETE), which is a major metabolite
of arachidonic acid (AA) produced in the kidney.2,3 20-
HETE plays an important role in the regulation of renal
vascular and tubular functions,4ꢁ6 and contributes to
the control of arterial pressure.7 More recent studies
have indicated that 20-HETE is also produced in the
brain, where it regulates vascular tone and contributes
to the autoregulation of cerebral blood flow.8 Therefore,
the regulation of 20-HETE is now considered a pro-
mising new therapeutic target for renal and cerebral
diseases. The formation of 20-HETE from AA is cata-
lyzed by cytochrome P450 (CYP) 4A isozymes
(CYP4A1, 4A2, 4A3 and 4A8) in rat kidney,3 and
CYP4F2 and 4F11 in human liver.9 Some AA analogues
[i.e., 17-octadecynoic acid (17-ODYA)10 and N-methyl-
sulfonyl-12,12-dibromododec-11-enamide (DDMS)11]
and 1-aminobenzotriazole (1-ABT)12 have been reported
to inhibit 20-HETE synthase. However, their IC50 values
for 20-HETE formation are on the order of mM orders,
*Corresponding author. Tel.: +81-48-669-3029; fax: +81-48-652-
7254; e-mail: takaaki.ishii@po.rd.taisho.co.jp
ySee ref 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00614-X

2994
M. Sato et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2993–2995
Chemistry
N-methylation of 1a via trifluoroacetamide for 7 (Scheme
1). Introduction of a hydroxyformamidine group for
3a–3m via an iminoether intermediate like 2b gave lower
yields due to the formation of N,N0-diarylamidine by-
products.
HET0016 (3a) was synthesized from commercially
available 4-butyl-2-methylaniline (1a) by successive
treatment with a slight excess of dimethylformamide
dimethyl-acetal in refluxing toluene and hydroxylamine
hydrochloride in MeOH at room temperature.13 Com-
pounds 3b–3m were synthesized from the corresponding
anilines in the same way. The introduction of a methyl
group to each atom of the hydroxyformamidine moiety
of 3a was accomplished by treating the intermediate 2a
with O-methyl and N-methylhydroxylamine for 4 and 5,
respectively, in refluxing MeOH, by treating 1a with
triethylorthoacetate (neat at 60 ꢂC) for 6, and by the
Results and Discussion
Compounds 3a–3m and 4–7 were evaluated for their
ability to inhibit the catalytic activity of human cyto-
chrome enzymes; their IC50 values are shown in Table 1.
Compounds 4–7, which have a methyl group on the
N-hydroxyformamidine moiety, showed lower inhibi-
tory activity than 3a. This suggests that the N-hydroxy-
formamidine moiety might be essential for the potent
inhibitory activity of 3a. Next, modification of the sub-
stituents on the phenyl ring of 3a was examined. Dis-
placement of the para-Bu group (3b) resulted in
dramatic decrease in activity (about 1/400), whereas
displacement of the ortho-Me group (3g) did not affect
the activity. These observations suggest that the butyl
group at the para-position of the N-hydroxy-
formamidine moiety is also essential for the potent
activity of 3a. Indeed, moving the para-Bu group to the
meta-position (3c) also reduced the activity (about 1/80).
Introduction of various alkyl groups (3e–3k) at the
para-position of N-hydroxyformamidine indicated that
an alkyl group larger than a methyl group is sufficient
for potent activity (3f–3k), and branched alkyl groups
such as isopropyl (3i), sec-butyl (3j) and tert-butyl (3k)
also give tolerable results. Introduction of a benzyl
group (3l) somewhat decreased the activity (about 1/6),
but replacing the Bu group of 3g with a propoxy group
(3m) did not affect the activity.
Figure 1.
Scheme 1.

M. Sato et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2993–2995
2995
Table 1.
Conclusions
N-Phenyl-N0-hydroxyformamidine derivatives exhibited
potent inhibitory activity against 20-HETE synthase.
Unsubstituted N-hydroxyformamidine was essential for
the potent activity, and the substituent at the para-
position of N-hydroxyformamidine had large effect on
the potent inhibitory activity, while those at the ortho-
and meta-position did not.
IC50 for CYPs (mM)1
2C9 2D6 3A4
Compd R1
R2
R3 IC50 (nM)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
4
Me
Me
Me
Bu
H
H
H
H
H
H
H
H
H
Bu
H
H
H
Me
Et
Bu
hexyl
i-Pr
s-Bu
t-Bu
PhCH2
PrO
H
H
Bu
H
H
H
H
H
H
H
H
H
H
8.9ꢀ2.7
3.3
83.9
71
3625
720
77
24
42
79
17
8.8
0.3
30
>100
98
32
>100
94
51
>100
>100
65
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20% at 1 mM
669
52
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We also examined the effects of compounds 3a–3m on
CYP isoforms (CYP 2C9, CYP 2D6 and CYP 3A4) that
could be important enzymes for drug metabolism. The
results are shown in Table 1. Compound 3a and its
derivatives inhibited CYP 2C9-, CYP 2D6- and CYP
3A4-catalyzed oxidative metabolism of substrate with
IC50 values on the order of mM. The inhibitory effects
on CYP 2C9, CYP 2D6 and CYP 3A4 were 1000 times
weaker than those on CYPs that catalyzed o-hydroxy-
lation of AA. Compounds 3a–3m were not non-selective
inhibitors of CYPs like 1-ABT. This means that N-
hydroxyformamidine derivatives can be the selective
inhibitors of CYPs that produce 20-hydroxy-5,8,11,14-
eicosatetraenoic acid from AA.
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