Pyrazole and Isoxazole Derivatives as New, Potent, and Selective 20-Hydroxy-5,8,11,14-eicosatetraenoic Acid Synthase Inhibitors

Article abstract of DOI:10.1021/jm020557k

In a previous paper, we reported the N-hydroxyformamidine derivative HET0016 as a potent and selective 20-HETE synthase inhibitor. Despite its attraction as a potential therapeutic agent for cerebral diseases, the preparation of an injectable formulation

Full text of DOI:10.1021/jm020557k

5416
J . Med. Chem. 2003, 46, 5416-5427
P yr a zole a n d Isoxa zole Der iva tives a s New , P oten t, a n d Selective
20-Hyd r oxy-5,8,11,14-eicosa tetr a en oic Acid Syn th a se In h ibitor s
Toshio Nakamura,* Masakazu Sato,* Hiroyuki Kakinuma, Noriyuki Miyata, Kazuo Taniguchi, Kagumi Bando,
Ayumi Koda, and Kazuya Kameo
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 403, Yoshino-Cho 1-Chome, Saitama-Shi,
Saitama, 330-8530, J apan
Received December 10, 2002
In a previous paper, we reported the N-hydroxyformamidine derivative HET0016 as a potent
and selective 20-HETE synthase inhibitor. Despite its attraction as a potential therapeutic
agent for cerebral diseases, the preparation of an injectable formulation of HET0016 was limited
by its poor solubility under neutral conditions and instability under acidic conditions. The
instability of HET0016 in acidic conditions is due to the N-hydroxyformamidine moiety, which
is considered to be essential for the potent and selective activity seen in our previous study.
The activity was maintained when the N-hydroxyformamidine moiety was replaced by an
imidazole ring (3a ; IC50 ) 5.7 ( 1.0 nM), but this was associated with a loss of selectivity for
cytochrome P450s (CYPs). However, other azole derivatives such as isoxazole derivative 23
(IC50 value 38 ( 10 nM) and pyrazole derivative 24 (IC50 value 23 ( 12 nM) showed potent
and selective activities with improved stability.
In tr od u ction
20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE)
is a major metabolite of arachidonic acid (AA) produced
in the kidney,1 and its biological properties have re-
cently been extensively studied. The formation of 20-
HETE from AA is reportedly catalyzed by cytochrome
P450 (CYP) 4A isozymes (CYP4A1, CYP4A2, CYP4A3,
and CYP4A8) in rat kidney,2 and by CYP4A11 and
CYP4F2 in human liver and kidney.3 20-HETE plays
an important role in the regulation of renal vascular
and tubular functions4-6 and contributes to the control
of arterial blood pressure.7 More recent studies have
indicated that 20-HETE is also produced in the brain,
F igu r e 1. Structures of HET0016, 1-ABT, 17-ODYA, DDMS,
DBDD, and 10-SUYS.
where it regulates vascular tone and contributes to the
regulation of cerebral blood flow.8 Therefore, the inhibi-
tion of 20-HETE is now considered a promising new
target for the treatment of renal and cerebral diseases.
Some compounds are known to inhibit the production
of 20-HETE (Figure 1). These compounds are not potent
µM, whereas it does not affect epoxigenase activity at
concentrations up to 50 µM.
In a previous paper, we reported HET0016 (N-
hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) as
or specific inhibitors for 20-HETE formation. 17-Octa-
decynoic acid (17-ODYA)9 inhibits the ω-hydroxylation
of AA; however, its inhibitory effect is not specific for
20-HETE formation, since it also inhibits the formation
of epoxyeicosatrienoic acids (EETs), which are produced
by epoxyganases (CYP1A, CYP2B, CYP2C, and CYP2J
families) from AA.10,11 1-Aminobenzotriazole (1-ABT)12
inhibits the catalytic activity of CYPs and also inhibits
the formation of 20-HETE. N-Methylsulfonyl-12,12-
dibromododec-11-enamide (DDMS)13 and 12,12-dibro-
mododec-11-enoic acid (DBDD)13 inhibit the formation
of 20-HETE with an IC50 value of 2 µM, whereas the
IC50 values for epoxidation of AA were 60 and 51 µM.
Sodium 10-undecynyl sulfate (10-SUYS)14 inhibits the
formation of 20-HETE with an IC50 value of 10.1 ( 2.6
the first potent and selective 20-HETE synthase inhibi-
tor.15 The IC50 value of HET0016 for the formation of
20-HETE by rat renal microsomes was 35.2 ( 4.4 nM,
while its IC50 value for inhibition of the formation of
EETs was 2800 nM.11 HET0016 had very little effect
on the activities of CYP2C9, CYP2D6, CYP3A4, or
cyclooxygenase (COX), even at higher concentrations
(100 µM).11 HET0016 prevented the acute fall in cere-
bral blood flow following subarachnoid hemorrhage
(SAH) in the rat.16 These results indicate that HET0016
is a potent and selective inhibitor of the CYP enzymes
that catalyze the formation of 20-HETE from AA and
possesses attractive properties for the treatment of
cerebrovascular diseases.
Despite its promising pharmacological properties,
HET0016 is not soluble enough for injectable formula-
tions under neutral conditions. The solubility of HET0016
is increased under acidic conditions due to the basicity
* Corresponding authors. TEL: +81 48 669 3029. FAX: +81 48 652
7254. E-mail: toshio.nakamura@po.rd.taisho.co.jp.
10.1021/jm020557k CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/11/2003

Pyrazoles and Isoxazoles as Potent Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5417
Ta ble 1. Inhibition of AA Metabolism Involving Human
20-HETE Synthesizing Enzyme and Inhibitory Activities
against Drug-Metabolizing CYPs by New Heterocyclic
Compounds (1)
F igu r e 2. Comparison of the N-hydroxyformamidine deriva-
tive and the imidazole derivative.
F igu r e 3. Observed NOE enhancements between C5′H and
C3H in 9b, and observed NOE enhancements between C6′H
and C3H in 9c.
F igu r e 4. Observed NOE enhancements between C6′H and
C3H in 25, and observed NOE enhancements between C6′H
and C4′H in 26.
of the N-hydroxyformamidine moiety; however, this is
accompanied by rapid decomposition of the N-hydroxy-
formamidine moiety. Therefore, we have attempted to
replace the N-hydroxyformamidine moiety with some
other acid-stable pharmacologically isostatic moiety.
According to previous X-ray analytic reports on some
N-hydroxyformamidine derivatives,17 the amidine moi-
ety of HET0016 may exist in a cis configuration, which
is structurally similar to a 1,3-azole ring (Figure 2).
Among various 1,3-azoles, imidazole derivatives are
known to be potent CYP inhibitors, and an imidazole
ring may be more stable than an N-hydroxyformamidine
moiety under acidic conditions. Therefore, we examined
replacement of the N-hydroxyformamidine moiety of
HET0016 by an imidazole ring and evaluated its
biological properties in vitro.
a
IC50 value for 20-HETE production from AA by human renal
microsome. Data are calculated from at least triplicate observa-
tions and are presented as a mean ( SE. IC50 was estimated for
each test substance and each enzyme according to the method of
Crespi et al.36 Data are calculated from triplicate observations.
c These compounds were evaluated as a p-toluenesulfonic acid salt.
b
Ch em istr y
Sch em e 1
To begin the synthesis of azole derivatives, the
substituent on the phenyl ring of HET0016 was changed
to a 4-n-butoxy moiety for ease of synthesis. N-(4-n-
Butoxyphenyl)-N′-hydroxyformamidine 1 shows potent
and selective 20-HETE synthase inhibitory activity, like
HET0016 (Table 1), and is suitable as a template
compound instead of HET0016.
Compounds 3a -e were prepared according to the
method shown in Scheme 1. Reaction of 1-(4-hydrox-
yphenyl)imidazole 2a with 1-iodobutane and potassium
carbonate in N,N-dimethylformamide afforded the imi-
dazole 3a , and phenol derivatives 2b, 2c,18 2d ,19 and
2e20 were treated under the same conditions to give the
corresponding 4-n-butoxyphenyl derivatives 3b-e.
Compounds 9a -d were prepared according to the
method shown in Scheme 2. 1-(4-Methoxyphenyl)-2-
methylimidazole 7a was obtained from 2-methylimida-
zole 4 by a diamine-copper complex-catalyzed coupling
reaction with 4-methoxyboronic acid.21 1-(4-Methox-
yphenyl)-4-methylimidazole 7b and 1-(4-methoxyphe-
nyl)-5-methylimidazole 7c were obtained as a mixture
by a reaction similar to that for 7a from 4-methylimi-
dazole 5. 2-(4-Methoxyphenyl)pyrazine 7d was obtained
from 2-chloropyrazine 6 by Suzuki coupling.22 4-Meth-
oxyphenyl derivatives 7a -d were demethylated by
heating with HBr, and subsequent alkylation with
1-iodobutane gave the 4-n-butoxyphenyl derivatives
9a -d . A mixture of 7b and 7c was converted to a
mixture of 8b and 8c, and 8c was isolated by recrys-
tallization from methanol. Alkylation of a mixture of 8b
and 8c gave a mixture of 9b and 9c, and 9b was isolated

5418 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Nakamura et al.
Sch em e 2
a
Reagents: (a) 4-methoxyphenylboronic acid, O2, (Cu(OH)-TMEDA)2Cl2, CH2Cl2; (b) 4-methoxyphenylboronic acid, Pd(OAc)2, PPh3,
K2CO3, DME; (c) HBr, reflux; (d) 1-iodobutane, K2CO3, DMF.
Sch em e 3
a
Reagents: (a) p-tosylmethyl isocyanide, NaCN, EtOH; (b) NH3, MeOH, 110 °C; (c) glyoxal, NH4OH, EtOH/H2O; (d) p-tosylmethyl
isocyanide, NaOMe, MeOH, reflux; (e) NH2OH, MeOH/H2O; (f) tBuOCl, CCl4; (g) acetylene, Et3N, benzene; (h) diethyl cyanomethylphos-
phonate, NaH, toluene; (i) p-tosylmethyl isocyanide, NaH, Et2O/DMSO, reflux; (j) KOH, EtOH/H2O; (k) ethanolamine, 180 °C.
as a p-toluenesulfonic acid salt by recrystallization from
ethyl acetate. The regiochemistries of 9b and 9c were
confirmed by an NOE study (Figure 3).23
Trudell et al.29 The aldehyde 10 was converted into ethyl
3-(4-n-butoxyphenyl)acrylate using the Horner-Em-
mons olefination procedure, and subsequent treatment
with TosMIC afforded 3-ethoxycarbonyl-4-(4-n-butoxy-
phenyl)pyrrole 17. The ester moiety of 17 was hydro-
lyzed with excess potassium hydroxide in 50% methanol
and then decarboxylated by heating in 2-ethanolamine
to give the pyrrole derivative 18.
Compounds 21, 23-26, 29-30, and 32-33 were
prepared according to the method shown in Scheme 4.
4-(4-n-Butoxyphenyl)oxazole 21 was obtained by heating
2-bromo-4′-n-butoxyacetophenone 20, which was pre-
pared by 2-bromination of commercially available 4′-n-
butoxyacetophenone 19, in formamide.30 2-Formylation
of 19 with ethyl formate and sodium hydride gave 4′-
n-butoxy-2-formylacetophenone 22, and subsequent treat-
ment of 22 with hydroxylamine followed by concentrated
HCl afforded the isoxazole derivative 23. Treatment of
22 with hydrazine afforded 3-(4-n-butoxyphenyl)pyra-
zole 24. Treatment of 22 with N-methylhydrazine gave
5-(4-n-butoxyphenyl)-1-methylpyrazole 25 and 5-(4-n-
butoxyphenyl)-2-methylpyrazole 26, which were easily
separated by silica gel column chromatography. The
regiochemistries of 25 and 26 were determined by an
NOE study (Figure 4).31 5-(4-n-Butoxyphenyl)thiazole
29 was obtained from 1-(4-n-butoxyphenyl)-3-(dimethy-
Compounds 11-13, 16, and 18 were prepared accord-
ing to the method shown in Scheme 3. Treatment of 4-n-
butoxybenzaldehyde 10 with tosylmethylisocyanide
(TosMIC) and a catalytic amount of sodium cyanide in
ethanol afforded 5-(4-n-butoxyphenyl)-4-tosyloxazo-
line,24 successive treatment of which with NH3 in
methanol at 110 °C in a sealed tube without purification
gave 4-(4-n-butoxyphenyl)imidazole 11. Reaction of 10
with glyoxal and ammonium hydroxide solution gave
2-(4-n-butoxyphenyl)imidazole 12.25 Treatment of 10
with TosMIC and sodium methoxide in methanol under
reflux afforded 5-(4-n-butoxyphenyl)oxazole 13.26 3-(4-
n-Butoxyphenyl)isoxazole 16 was synthesized by [3 + 2]
addition of nitrile oxide 15 with acetylene.27 The alde-
hyde 10 was converted into the corresponding 4-(4-n-
butoxyphenyl)benzoxime, and a subsequent reaction
with tert-butyl hypochlorite in carbon tetrachloride gave
the corresponding hydroxamyl chloride 14.28 The hy-
droxamyl chloride 14 was treated with triethylamine
to generate the corresponding nitrile oxide 15 in situ,
and subsequent cycloaddition with acetylene gas af-
forded the isoxazole derivative 16. 3-(4-n-Butoxyphenyl)-
pyrrole 18 was synthesized by the method reported by

Pyrazoles and Isoxazoles as Potent Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5419
Sch em e 4
a
Reagents: (a) Br2, CHCl3; (b) H2NCHO; 180 °C; (c) HCOOMe, NaH, THF; (d) H2NOH, pyridine; (e) concd HCl, THF; (f) H2NNH2,
MeOH; (g) H2NNHMe, MeOH; (h) tBuOCH(NMe2)2, 90 °C; (i) POCl3, NaClO4, CH2Cl2; (j) Na2S‚9H2O, DMF; (k) NH2OSO3H, pyridine,
EtOH; (l) formamidine hydrochloride, tBuOK, tBuOH; (m) AcOEt, NaH, 18-crown-6-ether, THF/EtOH.
Sch em e 5
Sch em e 6
a
Reagents: (a) HCOOEt, NaH, THF; (b) H2NOH, H2O.
lamino)-2-propen-1-thione 28 by the procedure of Lin
et al.32 Treatment of 4′-n-butoxyacetophenone 19 with
tert-butoxybis(dimethylamino)methane afforded 1-(4-n-
butoxyphenyl)-3-(dimethylamino)-2-propen-1-one 27.
Thioenaminone 28 was prepared by the reaction of the
enaminone 27 with phosphorus oxychloride in dichlo-
romethane followed by treatment with sodium perchlo-
rate in water and reaction with sodium sulfide in
aqueous N,N-dimethylformamide. The thioenaminone
28 was converted into the isothiazole derivative 29 by
treatment with hydroxylamine-O-sulfonic acid (HSA).
Reaction of the enaminone 27 with formamidine and
potassium tert-butoxide in tert-butyl alcohol at 50 °C
gave 4-(4-n-butoxyphenyl)pyrimidine 30.33 5-(4-n-Bu-
toxyphenyl)-3-methylisoxazole 32 and 3-(4-n-butoxyphe-
nyl)-5-methylisoxazole 33 were obtained from 2-acetyl-
4′-n-butoxyacetophenone 31 by treatment with hydroxyl-
amine hydrochloride and hydrazine. The 2-acetylac-
etophenone derivative 31 was prepared by acetylation
of the acetophenone derivative 19 with ethyl acetate and
sodium hydride in tetrahydrofuran.34
ride in N,N-dimethylformamide at 120 °C afforded the
tetrazole derivative 35.35
Compounds 37a -d were prepared according to the
method shown in Scheme 6. Acetophenone derivatives
36a -d were formylated with ethyl formate and sodium
hydride to give the corresponding 2-formyl ketone
derivatives, and subsequent treatment with hydroxy-
lamine followed by concentrated HCl afforded the isox-
azole derivatives 37a -d .
4-Methyl-5-(4-n-butoxyphenyl)isoxazole 40 and 4-meth-
yl-3-(4-n-butoxyphenyl)pyrazole 41 were prepared ac-
cording to the method shown in Scheme 7. Formylation
of 38 with ethyl formate and sodium hydride gave 4′-
n-butoxy-2-formylpropiophenone 39, and subsequent
treatment of 39 with hydroxylamine followed by con-
centrated HCl afforded the isoxazole derivative 40.
Reaction of 2-formyl-4′-n-butoxypropiophenone 39 with
hydrazine in methanol afforded the pyrazole derivative
41.
Resu lts a n d Discu ssion
All of the compounds synthesized were evaluated with
regard to their ability to inhibit microsomal synthesis
5-(4-n-Butoxyphenyl)tetrazole 35 was prepared ac-
cording to the method shown in Scheme 5. Reaction of
nitrile 34 with sodium azide and ammonium hydrochlo-

5420 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Nakamura et al.
Sch em e 7a
a
Reagents: (a) HCOOEt, NaH, THF; (b) H2NOH, pyridine; (c) concd HCl, THF; (d) H2NNH2, MeOH.
Ta ble 2. Inhibition of AA Metabolism Involving Human 20-HETE Synthesizing Enzyme and Inhibitory Activities against
Drug-Metabolizing CYPs by New Heterocyclic Compounds (2)
P450 inhibition IC50 (nM)b
compd
R1
X
R2
R3
R4
IC50 (nM)a
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
37a
37b
37c
37d
32
H
O
O
O
O
O
O
O
NMe
NH
NH
NH
NH
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
H
H
H
>1 000
>1 000
>300
NT
NT
120
570
14 200
<46
NT
NT
NT
NT
NT
NT
NT
NT
4-MeO
2-BuO
3-BuO
4-BuO
4-BuO
4-BuO
4-BuO
4-BuO
4-BuO
4-BuO
4-BuO
6 090
7 740
5 220
8 180
8 000
6 480
19 200
17 800
8 700
19 600
14 200
1 630
6 070
480
5 500
1 970
23 500
16 000
3 100
3 160
>100 000
>100 000
>100 000
>100 000
86 400
96 500
>100 000
81 600
21 400
93 500
>100 000
>100 000
>100 000
>100 000
>100 000
93 800
>100 000
>100 000
26 800
>300
>1 000
>1 000
38 ( 10
>1 000
>1 000
>300
40
23
3 230
69 900
8 650
21 100
<46
25c
26
33
41
24
Me
H
H
H
Me
H
44 ( 2.9
23 ( 12
560
70 600
a
IC50 value for 20-HETE production from AA by human renal microsome. Data are calculated from at least triplicate observations,
b
and are presented as a mean ( SE. IC50 was estimated for each test substance and each enzyme, according to the method of Crespi et
al.36 Data are calculated from triplicate observations. c This compound was evaluated as a HCl salt.
of 20-HETE, and those compounds were also evaluated
for their ability to inhibit the major drug-metabolizing
P450 (CYP) enzymes CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4.36 The methods of these assays
are described in the Experimental Section. The obtained
IC50 values are shown in Tables 1 and 2.
inhibitory activity toward either 20-HETE synthase or
CYPs [IC50 value for 9c was 5.9 ( 2.6 nM (n ) 3) and
there was no significant difference in IC50 values
between 3a and 9c (p > 0.05)]. These results suggest
that the range of spatial tolerance around the nitrogen
atom at the 3′-position of imidazole derivatives was
narrow in coordination to heme iron.
Imidazole derivative 3a showed potent inhibitory
activity toward 20-HETE synthase (IC50 value for 3a
was 5.7 ( 1.0nM); however, like other imidazole ana-
logues, 3a also strongly inhibited the drug-metabolizing
CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4; IC50 values of 7, 96, 7, 460, and 348 nM,
respectively). Since some imidazole derivatives show
high affinity to the heme iron atom of CYPs, the loss of
the selective inhibition of CYPs upon replacement of the
N-hydroxyformamidine moiety of 1 with an imidazole
ring may be due to the character of imidazole. To
improve the selectivity, a methyl group was introduced
to the imidazole ring of 3a , based on an observation in
an evaluation of antifungal agents.37 The introduction
of a methyl group at the 2′- or 4′-position of the
imidazole ring of 3a (9a , 9b) decreased the CYP inhibi-
tory activity as expected; however, this was accompa-
nied by a loss of 20-HETE synthase inhibitory activity
[IC50 values for 9a and 9b were >300 nM (n ) 3) and
143 ( 17 nM (n ) 3), respectively, and there was a
significant difference in IC50 values between 3a (n ) 4)
and 9b (p < 0.05)]. Meanwhile, the introduction of a
methyl group at the 5′-position (9c) did not affect the
Next, replacement of the hydroxyformamidine moiety
of 1 with other heteroaromatic rings was examined.
Triazole derivative 3b showed a lower inhibitory activity
toward drug-metabolizing CYPs, but its inhibitory
activity toward 20-HETE synthase was also decreased.
3-Pyridyl derivative 3d showed as potent an inhibitory
activity as 3a to 20-HETE synthase [IC50 value for 3d
was 27 ( 9.6 nM (n ) 3) and there was no significant
difference in IC50 values between 3a and 3d (p > 0.05)],
with elevated selectivity to CYP2C9, CYP2C19, CYP2D6,
and CYP3A4. On the other hand, 4-pyridyl derivative
3e was less active than 3-pyridyl derivative 3d [IC50
value for 3e was 803 ( 100 nM (n ) 5) and there was
a significant difference in IC50 values between 3d and
3e (p < 0.05)], and 2-pyridyl derivative 3c showed
almost no activity (IC50 value for 3c was >1000 nM).
These results suggest that the presence of a nitrogen
atom at the 3′-position of pyridine derivatives is es-
sential for potent inhibitory activity toward 20-HETE
synthase.
The position of the nitrogen atom on the imidazole
ring also affected the inhibitory activity toward 20-

Pyrazoles and Isoxazoles as Potent Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5421
Ta ble 3. Stability and Water Solubility of HET0016 and
Compounds 23 and 24
heteroaryl derivatives in corresponding formamidine
derivatives strongly suggests that the isoxazole ring of
23 and the pyrazole ring of 24 mimic the N-hydroxy-
formamidine moiety. In addition, the introduction of a
methyl group at the 4′- or 5′-position of the isoxazole
ring of 23 (32, 40) and the 2′-, 3′-, or 4′-position of the
pyrazole ring of 24 (25-26, 33) resulted in a loss of
activity, while introduction of a methyl group at the 5′-
position of a pyrazole ring (41) did not drastically affect
the activity (IC50 value for 41 was 44 ( 2.9 nM). These
results suggest that the spatial tolerance around the
nitrogen atom at the 3′-position of 23 and 24 is narrow
in coordination to the heme iron, as seen in the case of
the imidazole ring.
solubility in
water (µg/mL)
stability (% remaining)
pH 4.0, 50 °C, 1 day
compd
HET0016
23
3.7
5.6
37.2
43.2
100
100
24a
a
MeSO3H salt.
HETE synthase. 4-Imidazolyl derivative 11, which has
a nitrogen atom at the 3′-position, showed as potent an
inhibitory activity toward 20-HETE synthase as 3a (IC50
value for 11 was 23 ( 14 nM), while 2-imidazolyl
derivative 12 showed almost no inhibitory activity (IC50
value for 12 was >1000 nM). These results show that
the nitrogen atom at the 3′-position of imidazole deriva-
tives is also essential for potent 20-HETE synthase
inhibitory activity, as seen with pyridine derivatives.
According to these indications, various heterocyclic
derivatives including a nitrogen atom, namely oxazole,
isoxazole, isothiazole, pyrazole, pyrazine, pyrimidine,
pyrrole, and tetrazole derivatives, were synthesized.
Among these derivatives, compounds with a nitrogen
atom at the 3′-position (pyrazine derivative 9d , oxazole
derivative 13, isoxazole derivative 23, pyrazole deriva-
tive 24, and isothiazole derivative 29) showed inhibitory
activity toward 20-HETE synthase, as expected (IC50
values were 115 ( 35, 32 ( 25, 38 ( 10, 23 ( 12, and
98 ( 24 nM, respectively). In contrast, compounds that
lacked a nitrogen atom at the 3′-position (isoxazole
derivative 16, oxazole derivative 21, and pyrimidine
derivative 30) did not show inhibitory activity toward
20-HETE synthase, even at 1000 nM. These results
strongly support the notion that the nitrogen atom at
the 3′-position of various heterocyclic rings is essential
for the potent inhibitory activity of these derivatives
toward 20-HETE synthase. In contrast to these het-
eroaryl derivatives, pyrrole derivative 18 and tetrazole
derivative 35 showed less inhibitory activity toward 20-
HETE synthase. This difference may be due to the
difference in the coordination ability of the nitrogen
atoms of 18 and 35 to the heme iron.
Con clu sion
Replacement of the N-hydroxyformamidine moiety
of compound 1 by some heterocyclic rings gave the
isoxazole derivative 23 and the pyrazole derivative 24
as potent 20-HETE synthase inhibitors (IC50 values of
38 ( 10 and 23 ( 12 nM, respectively). These com-
pounds showed improved stability under acidic condi-
tions and exhibited much less inhibitory activity toward
drug-metabolizing CYPs CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4. The SAR of these newly syn-
thesized heterocyclic compounds is quite similar to that
of N-hydroxyformamidine derivatives. This result sug-
gests that the structural and electrical properties of
azole rings in heterocyclic compounds such as 23 and
24 are equivalent to those of the N-hydroxyformamidine
moiety of HET0016.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Yanaco
MP-500D melting point apparatus. NMR spectra were re-
corded at 200 or 300 MHz using a Varian Instruments Gemini
2000 or a Varian Instruments INOVA 300 with tetramethyl-
silane as an internal standard. Electron impact (EI) mass
spectra were taken on a Perkin-Elmer Sciex API-300 mass
spectrometer. Electrospray ionization (ESI) mass spectra were
taken on
a Micromass Platform LC mass spectrometer.
Elemental analyses were performed on EA2400 elemental
analyzers, and the results were within 0.4% of calculated
values. Reactions were monitored by TLC analysis using
Merck silica gel 60F-254 thin-layer plates. Column chroma-
tography was carried out on silica gel Wako Pure Chemical
C-200 and NH silica gel Fuji Silicia chromatorex DM1020.
Isoxazole derivative 23 and pyrazole derivative 24
showed potent 20-HETE synthase inhibitory activity
with much less inhibitory activity toward the CYP
enzymes (more than 80-fold to over 10 000-fold selectiv-
ity), and the selectivities of 23 and 24 to CYP 1A2 and
N-(4-n -Bu toxyph en yl)-N′-h ydr oxyfor m am idin e (1). This
1
2C19 were /7 to 1/30 the magnitude of that of HET0016.
compound was synthesized as described in the previous paper:
As expected, 23 and 24 were very stable under acidic
conditions (in both cases 100% remained under pH 4.0,
50 °C, 1 day, Table 3), while HET0016 was unstable
under the same conditions (only 43% remained). Thus,
replacement of the N-hydroxyformamidine moiety of
HET0016 by an isoxazole or pyrazole ring improved the
stability of the compound in acid, and the methane-
sulfonic acid salt of 24 was more soluble than HET0016
in water. The stability and solubility of HET0016 and
compounds 23 and 24 are shown in Table 3.
The SAR of the substituents on the phenyl ring in 23
and 24 is shown in Table 2. Replacement of the 4-n-
butoxy group of 23 by a hydrogen atom (37a ), 4-methoxy
group (37b), or 2- and 3-(4-n-butoxy) group (37c, 37d )
resulted in a loss of activity, as seen in the case of
N-hydroxyformamidine derivatives.14 The similarity of
the SAR of the substituent on the phenyl ring of
15
1
mp 131.5-133.5 °C; H NMR (200 MHz, DMSO-d6) δ 0.92
(t, J ) 7.3 Hz, 3H), 1.35-1.48 (m, 2H), 1.59-1.73 (m, 2H),
3.88 (t, J ) 6.4 Hz, 2H), 6.79 (m, J AB ) 8.8 Hz, 2H), 7.06 (m,
J AB ) 8.8 Hz, 2H), 7.32 (m, J AB ) 10.8 Hz, 1H), 8.33 (m, J AB
) 11.0 Hz, 1H), 9.68 (s, 1H); MS (ESI) m/z 207 (M - H, 70%).
Anal. (C11H16N2O‚2/3H2O) C, H, N.
1-(4-n -Bu toxyp h en yl)im id a zole (3a ). To a mixture of
1-(4-hydroxyphenyl)imidazole 2a (0.5 g, 3.12 mmol) and potas-
sium carbonate (0.52 g, 3.12 mmol) in N,N-dimethylformamide
(6 mL) was added 1-iodobutane (0.57 g, 3.12 mmol), and the
reaction mixture was stirred for 16 h at room temperature.
After stirring, the mixture was diluted with water, and a
colorless precipitate was filtered and purified by silica gel
column chromatography (eluent: hexane/ethyl acetate ) 4/1)
to give a colorless solid. Recrystallization of the solid from
hexane gave 0.57 g (84%) of 3a as a colorless powder: mp 47-
1
50 °C; H NMR (200 MHz, CDCl3) δ 0.99 (t, J ) 7.3 Hz, 3H),
1.41-1.60 (m, 2H), 1.73-1.87 (m, 2H), 4.00 (t, J ) 6.4 Hz, 2H),
6.97 (m, J AB ) 8.8 Hz, 2H), 7.18 (s, 1H), 7.20 (s, 1H), 7.29 (m,

5422 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Nakamura et al.
J AB ) 8.8 Hz, 2H), 7.76 (s, 1H); MS (ESI) m/z 217 (M + H,
62%). Anal. (C13H16N2O) C, H, N.
as described in the procedure for synthesizing 7a to yield 9.23
g (81%) of a mixture of 7b and 7c as a yellow oil: 1H NMR
(200 MHz, CDCl3) δ 2.14 and 2.30 (2s, 3H), 3.85 and 3.88 (2s,
3H), 6.80-7.05 (m, 3H), 7.15-7.34 (m, 2H), 7.53 and 7.66 (2s,
1H).
1-(4-n -Bu toxyp h en yl)tr ia zole (3b). This compound was
prepared from 1-(4-hydroxyphenyl)tridazole 2b (0.52 g, 3.12
mmol) as described in the procedure for synthesizing 3a to
yield 0.49 g (73%) of colorless crystals: mp 59-60.5 °C; 1H
NMR (200 MHz, CDCl3) δ 1.00 (t, J ) 7.4 Hz, 3H), 1.43-1.61
(m, 2H), 1.74-1.88 (m, 2H), 4.02 (t, J ) 6.5 Hz, 2H), 7.01 (m,
J AB ) 9.0 Hz, 2H), 7.56 (m, J AB ) 9.0 Hz, 2H), 8.09 (s, 1H),
8.45 (s,1H); MS (ESI) m/z 218 (M + H, 100%). Anal. (C12H15N3O)
C, H, N.
1-(4-Hyd r oxyp h en yl)-4-m eth ylim id a zole (8b) a n d 1-(4-
Hyd r oxyp h en yl)-5-m eth ylim id a zole (8c). A mixture of
1-(4-methoxyphenyl)-4-methylimidazole 7b and 1-(4-methox-
yphenyl)-5-methylimidazole 7c (4 g, 21.2 mmol) was added to
48% hydrobromic acid (40 mL), and the mixture was stirred
for 54 h at 100 °C. After heating, the reaction mixture was
cooled to room temperature, and 6 M sodium hydroxide (20
mL) and saturated sodium bicarbonate were added. A colorless
solid precipitate was filtered and recrystallized two times from
ethyl acetate/methanol to give 0.26 g (7%) of 8c as light brown
crystals: 1H NMR (200 MHz, DMSO-d6) δ 2.08 (s, 3H), 6.77
(s, 1H), 6.88 (m, J AB ) 9.7 Hz, 2H), 7.20 (m, J AB ) 9.7 Hz,
2H), 7.62 (s, 1H).
The mother liquor was concentrated and recrystallized from
methanol to give 1.97 g (53%) of a 3:2 mixture of 8b and 8c as
a colorless amorphism: 1H NMR (200 MHz, DMSO-d6) δ 2.08
(s, 1.2H), 2.14 (s, 1.8H), 6.77 (s, 0.4H), 6.79-6.95 (m, 2H),
7.13-7.41 (m, 1H), 7.25 (s, 0.6H), 7.62 (s, 0.4H), 7.91 (s, 0.6H),
9.73 (br s, 1H).
1-(4-n -Bu toxyp h en yl)-5-m eth ylim id a zole (9b). To a 3:2
mixture of 1-(4-hydroxyphenyl)-4-methylimidazole 8b and 1-(4-
hydroxyphenyl)-5-methylimidazole 8c (0.2 g, 1.15 mmol) in
N,N-dimethylformamide (2 mL) was added potassium carbon-
ate (0.19 g, 1.38 mmol) and 1-iodobutane (0.21 g, 1.38 mmol).
The reaction mixture was stirred for 18 h at room temperature.
After stirring, the mixture was diluted with water and
extracted with ethyl acetate. The organic layer was dried over
magnesium sulfate and concentrated, and the residue was
purified by NH silica gel column chromatography (eluent:
hexane/ethyl acetate ) 1/1) to give a mixture of 0.15 g (57%)
of 9b and 9c as a colorless oil. Compound 9b was isolated as
a salt with p-toluenesulfonic acid by recrystallization from
ethyl acetate: (p-toluenesulfonic acid salt) mp 163-164 °C;
1H NMR (200 MHz, DMSO-d6) δ 0.95 (t, J ) 7.3 Hz, 3H), 1.38-
1.56 (m, 2H), 1.67-1.80 (m, 2H), 2.17 (d, J ) 0.9 Hz, 3H), 2.29
(s, 3H), 4.07 (t, J ) 6.5 Hz, 2H), 7.09-7.21 (m, 4H), 7.48 (m,
J AB ) 8.1 Hz, 2H), 7.55 (m, J AB ) 8.8 Hz, 2H), 9.25 (d, J ) 1.5
Hz, 1H); MS (ESI) m/z 231 (M + H, 90%). Anal. (C21H26N2O4S-
C7H8O3S) C, H, N.
2-(4-n -Bu toxyp h en yl)p yr id in e (3c). This compound was
prepared from 2-(4-hydroxyphenyl)pyridine 2c (0.46 g, 2.69
mmol) as described in the procedure for synthesizing 3a to
1
yield 0.449 g (73%) of colorless crystals: mp 47-48.5 °C; H
NMR (200 MHz, CDCl3) δ 0.99 (t, J ) 7.2 Hz, 3H), 1.42-1.61
(m, 2H), 1.73-1.88 (m, 2H), 4.03 (t, J ) 6.5 Hz, 2H), 7.00 (m,
J AB ) 8.8 Hz, 2H), 7.13-7.21 (m, 1H), 7.64-7.78 (m, 2H), 7.95
(m, J AB ) 8.8 Hz, 2H), 8.62-8.68 (m, 1H); MS (ESI) m/z 228
(M + H, 53%). Anal. (C15H17NO) C, H, N.
3-(4-n -Bu toxyp h en yl)p yr id in e (3d ). This compound was
prepared from 3-(4-hydroxyphenyl)pyridine 2d (0.2 g, 1.17
mmol) as described in the procedure for synthesizing 3a to
yield 0.21 g (79%) of a colorless oil: (p-toluenesulfonic acid
salt) mp 132-133.5 °C; 1H NMR (200 MHz, DMSO-d6) δ 0.95
(t, J ) 7.3 Hz, 3H), 1.38-1.56 (m, 2H), 1.64-1.80 (m, 2H),
2.29 (s, 3H), 4.06 (t, J ) 6.5 Hz, 2H), 7.08-7.18 (m, 4H), 7.48
(m, J AB ) 8.0 Hz, 2H), 7.88 (m, J AB ) 9.0 Hz, 2H), 7.93-8.02
(m, 1H), 8.67-8.80 (m, 2H), 9.15 (d, J ) 2.2 Hz, 1H); MS (ESI)
m/z 228 (M + H, 100%). Anal. (C22H25NO4S‚C7H8O3S) C, H,
N.
4-(4-n -Bu toxyp h en yl)p yr id in e (3e). This compound was
prepared from 4-(4-hydroxyphenyl)pyridine 2e (0.2 g, 1.17
mmol) as described in the procedure for synthesizing 3a to
yield 0.230 g (86%) of a light brown powder: mp 58.5-59.5
°C; 1H NMR (200 MHz, CDCl3) δ 1.00 (t, J ) 7.3 Hz, 3H), 1.42-
1.61 (m, 2H), 1.73-1.88 (m, 2H), 4.03 (t, J ) 6.5 Hz, 2H), 7.01
(m, J AB ) 8.8 Hz, 2H), 7.48 (m, J AB ) 6.4 Hz, 2H), 7.60 (m,
J AB ) 8.8 Hz, 2H), 8.62 (m, J AB ) 6.4 Hz, 2H); MS (ESI) m/z
228 (M + H, 100%). Anal. (C15H17NO) C, H, N.
1-(4-Meth oxyp h en yl)-2-m eth ylim id a zole (7a ). A mix-
ture of 2-methylimidazole (1 g, 12.2 mmol), 4-methoxyphenyl-
boronic acid (3.7 g, 24.4 mmol), and [Cu(OH)-TMEDA]2Cl2
(0.57 g, 1.22 mmol) in dichloromethane (48 mL) was stirred
for 18 h at room temperature under an atmosphere of O2. The
mixture was then filtered through Celite and the filtrate was
concentrated to give a crude oil, which was purified by NH
silica gel column chromatography (eluent: hexane/ethyl ac-
etate ) 4/1 to ethyl acetate) to give 2.15 g (94%) of 7a as a
yellow oil: 1H NMR (200 MHz, CDCl3) δ 2.45 (s, 3H), 3.88 (s,
3H), 6.93-7.05 (m, 4H), 7.20 (m, J AB ) 9.9 Hz, 2H).
1-(4-Hydr oxyph en yl)-2-m eth ylim idazole (8a). 1-(4-Meth-
oxyphenyl)-2-methylimidazole 7a (2 g, 10.6 mmol) was added
to 48% hydrobromic acid (20 mL) and the mixture was stirred
for 16 h at 100 °C. After heating, the reaction mixture was
cooled to room temperature. To the reaction mixture was added
6 M sodium hydroxide (10 mL) and an aqueous solution of
sodium bicarbonate, and a precipitated colorless solid was
filtered and dried to give 0.745 g (40%) of 8a as a colorless
powder: 1H NMR (200 MHz, DMSO-d6) δ 2.21 (s, 3H), 6.83-
6.93 (m, 3H), 7.13-7.26 (m, 3H), 9.80 (s, 1H).
1-(4-n -Bu toxyph en yl)-4-m eth ylim idazole (9c). This com-
pound was prepared from 1-(4-hydroxyphenyl)-4-methylimi-
dazole 8c (0.15 g, 0.86 mmol) as described in the procedure
for synthesizing 3a to yield 0.11 g (56%) of 8c as a brown oil:
1
(p-toluenesulfonic acid salt) mp 130-130.5 °C; H NMR (200
MHz, DMSO-d6) δ 0.95 (t, J ) 7.2 Hz, 3H), 1.35-1.56 (m, 2H),
1.64-1.80 (m, 2H), 2.30 (s, 1H), 2.35 (d, J ) 0.9 Hz, 3H), 4.05
(t, J ) 5.5 Hz, 2H), 7.07-7.22 (m, 4H), 7.48 (m, J AB ) 8.5 Hz,
2H), 7.67 (m, J AB ) 8.5 Hz, 2H), 7.93 (t, J ) 1.5 Hz, 1H), 9.46
(d, J ) 1.5 Hz, 2H); MS (ESI) m/z 231 (M + H, 100%). Anal.
(C21H26N2O4S‚C7H8O3S) C, H, N.
2-(4-Meth oxyp h en yl)p yr a zin e (7d ). To a mixture of
2-chloropyrazine (1 g, 8.7 mmol), 4-methoxyphenylboronic acid
(1.6 g, 10.56 mmol), triphenylphosphine (0.23 g, 0.88 mmol),
and palladium(II) acetate (0.05 g, 0.22 mmol) in 1,2-dimethox-
yethane (10 mL) was added a 2 M aqueous solution of
potassium carbonate (12 mL, 24 mmol), and the mixture was
stirred for 5 h at 100 °C under an atmosphere of N2. The
mixture was then diluted with water and extracted with ethyl
acetate, and the extract was dried over magnesium sulfate and
concentrated to give a crude oil, which was purified by silica
gel column chromatography (eluent: hexane/ethyl acetate )
3/1 to 1/1) to give 1.57 g (97%) of 7d as a colorless solid: 1H
NMR (200 MHz, CDCl3) δ 3.89 (s, 3H), 7.05 (m, J AB ) 9.0 Hz,
2H), 8.00 (m, J AB ) 9.0 Hz, 2H), 8.45 (d, J ) 2.5 Hz, 1H), 8.60
(t, J ) 1.5 Hz, 1H), 9.00 (d, J ) 1.5 Hz, 1H).
1-(4-n -Bu toxyph en yl)-2-m eth ylim idazole (9a). This com-
pound was prepared from 1-(4-hydroxyphenyl)-2-methylimi-
dazole 8a (0.2 g, 1.15 mmol) as described in the procedure for
synthesizing 3a to yield 0.17 g (64%) of a light brown oil: (p-
toluenesulfonic acid salt) mp 148-149 °C; 1H NMR (200 MHz,
DMSO-d6) δ 1.00 (t, J ) 7.3 Hz, 3H), 1.41-1.60 (m, 2H), 1.65-
1.84 (m, 2H), 2.37 (s, 3H), 2.68 (s, 3H), 4.04 (t, J ) 6.4 Hz,
2H), 7.18-7.27 (m, 4H), 7.42 (s, 1H), 7.88 (m, J AB ) 8.1 Hz,
2H); MS (ESI) m/z 231 (M + H, 100%). Anal. (C21H26N2O4S‚
C7H8O3S) C, H, N.
2-(4-Hyd r oxyp h en yl)p yr a zin e (8d ). This compound was
prepared from 1-(4-hydroxyphenyl)-4-methylimidazole 7b (1.5
g, 8.06 mmol) as described in the procedure for synthesizing
8a to yield 1.06 g (77%) of an orange powder: mp 130-130.5
4-Met h yl-1-(4-m et h oxyp h en yl)im id a zole (7b ) a n d 5-
Meth yl-1-(4-m eth oxyp h en yl)im id a zole (7c). These com-
pounds were prepared from 4-methylimidazole (5 g, 60.9 mmol)

Pyrazoles and Isoxazoles as Potent Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5423
1
°C; H NMR (200 MHz, CDCl3) δ 6.98 (m, J AB ) 9.7 Hz, 2H),
hexane/ethyl acetate ) 9/ 1) to give 2.48 g (98%) of 14 as a
yellow oil: 1H NMR (300 MHz, CDCl3) δ 0.99 (t, J ) 7.5 Hz,
3H), 1.43-1.58 (m, 2H), 1.73-1.88 (m, 2H), 4.00 (t, J ) 6.2
Hz, 2H), 6.99 (m, J AB ) 9.0 Hz, 2H), 7.76 (m, J AB ) 9.0 Hz,
2H).
7.95 (m, J AB ) 9.7 Hz, 2H), 8.45 (d, J ) 2.5 Hz, 1H), 8.59 (t, J
) 1.4 Hz, 1H), 8.98 (d, J ) 1.4 Hz, 1H).
2-(4-n -Bu toxyp h en yl)p yr a zin e (9d ). This compound was
prepared from 1-(4-hydroxyphenyl)-4-methylimidazole 8b (0.2
g, 1.16 mmol) as described in the procedure for synthesizing
3a to yield 0.21 g (79%) of a colorless powder: mp 72-74.5
°C; 1H NMR (200 MHz, CDCl3) δ 1.00 (t, J ) 7.3 Hz, 3H), 1.43-
1.61 (m, 2H), 1.72-1.88 (m, 2H), 4.05 (t, J ) 6.5 Hz, 2H), 7.03
(m, J AB ) 8.6 Hz, 2H), 7.98 (m, J AB ) 8.6 Hz, 2H), 8.45 (d, J
) 2.4 Hz, 1H), 8.57-8.61 (m, 1H), 8.99 (d, J ) 1.4 Hz, 1H);
MS (ESI) m/z 229 (M + H, 65%). Anal. (C14H16N2O) C, H, N.
4-(4-n -Bu toxyp h en yl)im id a zole (11). To a stirred sus-
pension of tosylmethylisocyanide (TosMIC) (1.07 g, 5.5 mmol)
and 4-n-butoxybenzaldehyde 10 (1 g, 5.6 mmol) in dry ethanol
(16.5 mL) was added finely powdered sodium cyanide (0.027
g, 0.55 mmol). The reaction mixture was stirred for 20 min
and the resulting clear solution was concentrated in vacuo.
The residue was dissolved in a saturated solution of ammonia
in dry methanol (44 mL) and heated at 110 °C for 18 h in a
sealed tube. After heating, the reaction mixture was cooled to
room temperature and concentrated to give a crude oil, which
was purified by silica gel column chromatography (eluent:
ethyl acetate) to give a colorless solid. Recrystallization of the
solid from hexane/ethyl acetate gave 0.188 g (16%) of 11 as a
colorless powder: mp 120-122 °C; 1H NMR (200 MHz, CDCl3)
δ 0.98 (t, J ) 7.3 Hz, 3H), 1.41-1.60 (m, 2H), 1.71-1.86 (m,
2H), 3.99 (t, J ) 6.5 Hz, 2H), 6.94 (m, J AB ) 8.6 Hz, 2H), 7.25
(s, 1H), 7.64 (m, J AB ) 8.6 Hz, 2H), 7.69 (s, 1H); MS (ESI) m/z
217 (M + H, 100%). Anal. (C13H16N2O) C, H, N.
2-(4-n -Bu toxyp h en yl)im id a zole (12). To a solution of 4-n-
butoxybenzaldehyde 10 (1 g, 5.61 mmol) in ethanol (7.5 mL)
at 0 °C was added a solution of 40% glyoxal in water (1.3 mL)
and 20 M ammonium hydroxide (1.9 mL). The reaction mixture
was stirred for 30 min at 0 °C and then at room temperature
overnight. The reaction mixture was concentrated and the
residue was purified by NH silica gel column chromatography
(eluent: ethyl acetate) to give a colorless solid. Recrystalliza-
tion of the solid from hexane/ethyl acetate gave 0.69 g (57%)
of 12 as colorless crystals: mp 172-173 °C; 1H NMR (200 MHz,
CDCl3) δ 0.99 (t, J ) 7.4 Hz, 3H), 1.41-1.60 (m, 2H), 1.72-
1.86 (m, 2H), 4.01 (t, J ) 6.6 Hz, 2H), 6.96 (m, J AB ) 8.7 Hz,
2H), 7.13 (s, 2H), 7.75 (m, J AB ) 8.7 Hz, 2H); MS (ESI) m/z
217 (M + H, 86%). Anal. (C13H16N2O) C, H, N.
5-(4-n -Bu toxyp h en yl)oxa zole (13). To a mixture of 4-n-
butoxybenzaldehyde 10 (1 g, 5.61 mmol) and sodium methoxide
(0.91 g, 16.8 mmol) in methanol (5 mL) was added TosMIC
(1.30 g, 6.73 mmol). The reaction mixture was refluxed for 16
h with stirring. The mixture was diluted with water and
extracted with chloroform, and the extract was dried over
magnesium sulfate and concentrated to give a crude oil, which
was purified by silica gel column chromatography (eluent:
hexane/ethyl acetate ) 7/3) to give a colorless solid. Recrys-
tallization of the solid from hexane/ethyl acetate gave 0.63 g
(43%) of 13 as colorless crystals: mp 57.5-59 °C; 1H NMR
(200 MHz, CDCl3) δ 0.98 (t, J ) 7.2 Hz, 3H), 1.40-1.60 (m,
2H), 1.71-1.86 (m, 2H), 4.00 (t, J ) 6.4 Hz, 2H), 6.95 (m, J AB
) 8.6 Hz, 2H), 7.67 (m, J AB ) 8.6 Hz, 2H), 7.86 (s, 1H), 7.92
(s, 1H); MS (EI) m/z 217 (M+, 27%). Anal. (C13H15NO2) C, H,
N.
3-(4-n -Bu toxyp h en yl)isoxa zole (16). A solution of 4-n-
butoxybenzohydroxamyl chloride 14 (2.4 g, 10.5 mmol) in
benzene (105 mL) was bubbled with acetylene gas for 10 min.
After bubbling, triethylamine (2.12 g, 21.0 mmol) was added
dropwise at 0 °C, and the mixture was stirred for 1 h at 60
°C. The reaction mixture was then cooled, diluted with brine,
and extracted with ethyl acetate, and the extract was dried
over magnesium sulfate and concentrated to give a light yellow
residue. The crude oil was purified by silica gel column
chromatography (eluent: hexane/ethyl acetate ) 13/1) to give
1
1.29 g (57%) of 16 as a colorless solid: mp 34.5-35.5 °C; H
NMR (300 MHz, CDCl3) δ 0.99 (t, J ) 7.4 Hz, 3H), 1.44-1.60
(m, 2H), 1.74-1.85 (m, 2H), 4.02 (t, J ) 6.6 Hz, 2H), 6.60 (d,
J ) 1.5 Hz, 1H), 6.97 (m, J AB ) 9.0 Hz, 2H), 7.75 (m, J AB
)
9.0 Hz, 2H), 8.41 (d, J ) 1.5 Hz, 1H); MS (ESI) m/z 240 (M +
Na, 73%). Anal. (C13H15NO2) C, H, N.
4-(4-n -Bu t oxyp h en yl)-3-et h oxyca r b on ylp yr r ole (17).
To a stirred suspension of sodium hydride (60% in oil, 0.90 g,
22.4 mmol) in toluene (23 mL) was added diethylphospho-
noacetic acid ethyl ester (5.02 g 22.4 mmol) at 0 °C, and the
mixture was stirred for 1 h at room temperature. 4-n-
Butoxybenzaldehyde 10 (4 g, 22.4 mmol) was then added and
the reaction mixture was stirred for 30 min at room temper-
ature. After stirring, the reaction mixture was diluted with
water and extracted with ethyl acetate, and the extract was
dried over magnesium sulfate and concentrated to give a
colorless oil which was used without further purification. A
mixture of the oil, TosMIC (4.44 g, 22.8 mmol), diethyl ether
(16 mL), and dimethyl sulfoxide (8 mL) was added dropwise
to a stirred suspension of sodium hydride (60% in oil, 1.15 g,
28.7 mmol) in diethyl ether (29 mL). The mixture was stirred
for 16 h at 60 °C and then cooled to room temperature. The
reaction mixture was diluted with water and extracted with
ethyl acetate, and the extract was dried over magnesium
sulfate and concentrated to give a brown oil. The crude oil was
purified by silica gel column chromatography (eluent: hexane/
ethyl acetate ) 4/1 to 1/1) to give 0.96 g (15%) of 14 as a
colorless powder:1H NMR (200 MHz, CDCl3) δ 0.99 (t, J ) 6.5
Hz, 3H), 1.26 (t, J ) 6.5 Hz, 3H), 1.38-1.61 (m, 2H), 1.58-
1.86 (m, 2H), 3.99 (t, J ) 6.5 Hz, 2H), 4.22 (q, J ) 6.5 Hz,
2H), 6.76 (t, J ) 1.5 Hz, 1H), 6.90 (m, J AB ) 8.2 Hz, 2H), 7.41
(m, J AB ) 8.2 Hz, 2H), 7.49 (t, J ) 1.5 Hz, 1H); MS (ESI)
m/z 310 (M + Na, 100%). Anal. (C17H21NO3) C, H, N.
3-(4-n -Bu toxyp h en yl)p yr r ole (18). To a mixture of 4-(4-
n-butoxyphenyl)-3-ethoxycarbonylpyrrole 17 (0.2 g, 0.694 mmol)
dissolved in ethanol (1 mL) was added potassium hydroxide
(0.39 g, 6.94 mmol), and the mixture was stirred for 4 h at 80
°C. After stirring, the reaction mixture was cooled to room
temperature, diluted with water, acidified with 12 M HCl, and
extracted with ethyl acetate. The organic layer was dried over
magnesium sulfate and concentrated to give a brown oil, which
was dissolved in 2-ethanolamine (3 mL) and refluxed for 3 h
with stirring. After heating, the reaction mixture was cooled
to room temperature and purified by silica gel column chro-
matography (eluent: hexane/ethyl acetate ) 1/1) to give 0.05
g (33%) of 18 as a brown amorphism: 1H NMR (300 MHz,
CDCl3) δ 0.98 (t, J ) 7.4 Hz, 3H), 1.43-1.57 (m, 2H), 1.72-
1.82 (m, 2H), 3.97 (t, J ) 6.6 Hz, 2H), 6.48 (dd, J ) 2.7 Hz, 4.2
Hz, 1H), 6.82 (dd, J ) 2.7 Hz, 4.8 Hz, 1H), 6.88 (m, J AB ) 8.9
Hz, 2H), 7.01 (dd, J ) 2.0 Hz, 4.2 Hz, 1H), 7.44 (m, J AB ) 8.9
Hz, 2H); MS (EI) m/z 217 (M+, 48%). Anal. (C14H17NO‚1/4H2O)
C, H, N.
4-n -Bu toxyben zoh yd r oxa m yl Ch lor id e (14). To a solu-
tion of 4-n-butoxybenzaldehyde 10 (2 g, 11.2 mmol) in metha-
nol (33 mL) was added a mixture of hydroxylamine hydro-
chloride (0.93 g, 13.5 mmol) and sodium acetate trihydrate
(1.83 g, 13.5 mmol) in water (17 mL). The reaction mixture
was stirred for 16 h at room temperature. The mixture was
then diluted with water and extracted with ethyl acetate, and
the extract was dried over magnesium sulfate and concen-
trated to give a colorless oil. Without further purification, the
oil was dissolved in carbon tetrachloride (30 mL) and added
to a solution of tert-butyl hypochlorite (1.37 g, 12.6 mmol) in
carbon tetrachloride (10 mL). The reaction mixture was stirred
for 1 h at room temperature and concentrated, and the residue
was purified by silica gel column chromatography (eluent:
2-Br om o-4′-n -bu toxya cetop h en on e (20). To a solution of
4′-n-butoxyacetophenone 19 (2.5 g, 13.0 mmol) in chloroform
(75 mL) was added bromine (2.75 g, 14.3 mmol) dropwise at 0
°C. The reaction mixture was stirred for 2 h at room temper-
ature and then diluted with an aqueous solution of sodium
thiosulfate and extracted with chloroform. The organic layer
was dried over magnesium sulfate and concentrated to give a

5424 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Nakamura et al.
yellow oil, which was purified by silica gel column chroma-
tography (eluent: hexane/ethyl acetate ) 19/1) to give 1.85 g
(52%) of 20 as a light yellow oil: 1H NMR (200 MHz, CDCl3)
δ 0.99 (t, J ) 7.2 Hz, 3H), 1.41-1.60 (m, 2H), 1.72-1.87 (m,
2H), 4.05 (t, J ) 6.5 Hz, 2H), 4.40 (s, 2H), 6.95 (m, J AB ) 8.8
Hz, 2H), 7.97 (m, J AB ) 8.8 Hz, 2H).
3-(4-n -Bu toxyp h en yl)-2-m eth ylp yr a zole (25) a n d 3-(4-
n -Bu toxyp h en yl)-1-m eth ylp yr a zole (26). To a solution of
4′-n-butoxy-2-hydroxyacrylophenone 22 (0.5 g, 2.27 mmol) in
tetrahydrofuran (5 mL) was added a solution of N-methylhy-
drazine (0.115 g, 2.50 mmol) in tetrahydrofuran (5 mL). The
reaction mixture was stirred for 2 h at room temperature and
then diluted with brine and extracted with ethyl acetate. The
organic layer was dried over magnesium sulfate and concen-
trated, and the residue was purified by NH silica gel column
chromatography (eluent: hexane/ethyl acetate ) 8/1) to give
0.100 g (19%) of 25 as a colorless oil and 0.170 g (33%) of 26
as a colorless powder. 25 as the hydrochloride salt: mp 151.5-
4-(4-n -Bu toxyp h en yl)oxa zole (21). 2-Bromo-4′-n-butoxy-
acetophenone 20 (0.2 g, 0.74 mmol) was dissolved in formamide
(2.5 mL) and stirred for 1 h at 180 °C. After stirring, the
reaction mixture was cooled to room temperature, diluted with
water, and extracted with ethyl acetate. The extract was dried
over magnesium sulfate and concentrated to give a brown oil.
The crude oil was purified by silica gel column chromatography
(eluent: hexane) to give 0.091 g (57%) of 21 as a colorless
1
154 °C; H NMR (200 MHz, DMSO-d6) δ 0.95 (t, J ) 7.2 Hz,
3H), 1.37-1.55 (m, 2H), 1.66-1.80 (m, 2H), 3.82 (s, 3H), 4.03
(t, J ) 6.4 Hz, 2H), 6.32 (d, J ) 2.0 Hz, 1H), 7.04 (m, J AB
)
1
powder: mp 52-52.5 °C; H NMR (200 MHz, CDCl3) δ 0.99
8.8 Hz, 2H), 7.43 (m, J AB ) 8.8 Hz, 2H), 7.44 (d, J ) 2.0 Hz,
1H); MS (ESI) m/z 231 (M + H, 100%). Anal. (C14H18ClN2O)
C, H, N. 26: mp 57-59.5 °C; 1H NMR (200 MHz, CDCl3) δ
0.98 (t, J ) 7.3 Hz, 3H), 1.41-1.60 (m, 2H), 1.70-1.84 (m, 2H),
3.94 (s, 3H), 3.99 (t, J ) 6.5 Hz, 2H), 6.46 (d, J ) 2.2 Hz, 1H),
6.92 (m, J AB ) 8.7 Hz, 2H), 7.35 (d, J ) 2.2 Hz, 1H), 7.71 (m,
J AB ) 8.7 Hz, 2H); MS (ESI) m/z 253 (M + Na, 100%). Anal.
(C13H16N2O-HCl) C, H, N.
(t, J ) 7.3 Hz, 3H), 1.41-1.62 (m, 2H), 1.72-1.87 (m, 2H),
4.01 (t, J ) 6.5 Hz, 2H), 6.96 (m, J AB ) 8.8 Hz, 2H), 7.23 (s,
1H), 7.58 (m, J AB ) 8.8 Hz, 2H), 7.88 (s, 1H); MS (EI) m/z 217
(M+, 51%). Anal. (C13H15NO2) C, H, N.
4′-n -Bu toxy-2-h yd r oxya cr ylop h en on e (22). To a stirred
suspension of sodium hydride (60% in oil, 0.51 g, 14.3 mmol)
in tetrahydrofuran (13 mL) and methyl formate (0.859 g, 14.3
mmol) was added 4′-n-butoxyacetophenone 19 (2.5 g, 13.0
mmol) in tetrahydrofuran (2.5 mL). The reaction mixture was
stirred for 2 h at room temperature and then diluted with 0.5
M HCl and extracted with ethyl acetate. The organic layer was
dried over magnesium sulfate and concentrated, and the
residue was purified by silica gel column chromatography
(eluent: hexane/ethyl acetate ) 8/ 1) to give 2.05 g (72%) of
22 as a yellow oil:1H NMR (200 MHz, CDCl3) δ 0.99 (t, J )
7.4 Hz, 3H), 1.40-1.61 (m, 2H), 1.72-1.87 (m, 2H), 4.04 (t, J
) 6.5 Hz, 2H), 6.16 (d, J ) 4.8 Hz, 1H), 6.94 (m, J AB ) 8.8 Hz,
2H), 7.89 (m, J AB ) 9.0 Hz, 2H), 8.14 (d, J ) 4.4 Hz, 1H).
4′-n -Bu toxy-3-(d im eth yla m in o)a cr ylop h en on e (27). 4′-
n-Butoxyacetophenone 19 (0.5 g, 2.6 mmol) was dissolved in
tert-butoxybis(dimethylamino)methane (1.36 g, 7.8 mmol) and
stirred for 1 h at 90 °C. After stirring, the reaction mixture
was diluted with a mixture of ethyl acetate/hexane ) 2/1, and
a solid yellow precipitate was collected by filtration and dried
to give 0.52 g (81%) of 27 as a light yellow powder: 1H NMR
(200 MHz, CDCl3) δ 0.98 (t, J ) 7.2 Hz, 3H), 1.40-1.61 (m,
2H), 1.67-1.88 (m, 2H), 3.02 (br s, 6H), 4.01 (t, J ) 6.5 Hz,
2H), 5.72 (d, J ) 12 Hz, 1H), 6.91 (m, J AB ) 8.8 Hz, 2H), 7.80
(d, J ) 12 Hz, 1H), 7.90 (m, J AB ) 8.8 Hz, 2H).
5-(4-n -Bu toxyp h en yl)isoxa zole (23). To a mixture of 4′-
n-butoxy-2-hydroxyacrylophenone 22 (1 g, 4.54 mmol) dis-
solved in pyridine (4.5 mL) was added hydroxylamine hydro-
chloride (0.379 g, 5.45 mmol). The reaction mixture was stirred
for 1 h at 80 °C and then poured into water, acidified with 6
M HCl, and extracted with ethyl acetate. The organic layer
was dried over magnesium sulfate and concentrated to give a
colorless solid. Without further purification, the solid was
dissolved in tetrahydrofuran (1.6 mL) and 12 M HCl (0.8 mL)
was added. The reaction mixture was stirred for 1 h at room
temperature, and then diluted with water and extracted with
ethyl acetate. The extract was dried over magnesium sulfate
and concentrated to give a light yellow oil. The crude oil was
purified by silica gel column chromatography (eluent: hexane/
ethyl acetate ) 19/1) to give 0.168 g (17%) of 23 as a colorless
5-(4-n -Bu toxyp h en yl)isoth ia zole (29). To a mixture of 4′-
n-butoxy-3-(dimethylamino)acrylophenone 27 (1 g, 4.04 mmol)
dissolved in anhydrous dichloromethane (4 mL) was added a
solution of phosphorus oxychloride (0.62 g, 4.04 mmol) in
anhydrous dichloromethane (1 mL) at 0 °C. After the reaction
mixture was stirred at room temperature for 30 min, the
reaction mixture was concentrated to give a yellow solid. The
solid was added to a stirred solution of sodium perchlorate
monohydrate (1.7 g, 12.1 mmol) in water (3 mL). The reaction
mixture was vigorously stirred at 0 °C for 30 min, and a solid
yellow precipitate was collected by filtration and added to a
stirred ice-cold mixture of N,N-dimethylformamide (8.3 mL),
sodium sulfide nonahydrate (1.02 g, 4.24 mmol), and water (1
mL). The reaction mixture was stirred at room temperature
for 2 h, diluted with water and extracted with chloroform, and
the extract was washed with brine, dried over magnesium
sulfate, and concentrated to give 4′-n-butoxy-3-(dimethylami-
no)thioacrylophenone 28 as a red-brown oil.
1
powder: mp 48-49.5 °C; H NMR (200 MHz, CDCl3) δ 0.99
(t, J ) 7.3 Hz, 3H), 1.46-1.56 (m, 2H), 1.73-1.87 (m, 2H),
4.02 (t, J ) 6.6 Hz, 2H), 6.39 (d, J ) 2.0 Hz, 1H), 6.97 (m, J AB
) 8.6 Hz, 2H), 7.72 (m, J AB ) 8.6 Hz, 2H), 8.26 (d, J ) 2.0 Hz,
1H); MS (ESI) m/z 240 (M + Na, 39%). Anal. (C13H15NO2) C,
H, N.
Without further purification, the oil was dissolved in ethanol
(30 mL), and pyridine (0.626 g, 8.08 mmol) and a solution of
hydroxylamine-O-sulfonic acid (0.53 g, 4.65 mmol) in ethanol
(5 mL) were added at 0 °C. The reaction mixture was stirred
at room temperature for 30 min, and the solvents were then
removed under reduced pressure to give a residue. Water was
added to the residue, the mixture was extracted with ethyl
acetate, and the extract was washed with saturated aqueous
solution of sodium bicarbonate, dried over magnesium sulfate,
and concentrated to give a brown oil. The crude oil was purified
by silica gel column chromatography (eluent: hexane/ethyl
acetate ) 9/1) to give a yellow solid. Recrystallization of the
solid from hexane gave 0.255 g (27%) of 29 as a yellow
3-(4-n -Bu toxyp h en yl)p yr a zole (24). To a solution of 4′-
n-butoxy-2-hydroxyacrylophenone 22 (1 g, 4.54 mmol) in
methanol (27 mL) was added hydrazine monohydrate (0.273
g, 5.45 mmol). The reaction mixture was stirred for 1 h at room
temperature and then diluted with water. The solid yellow
precipitate was filtered and purified by silica gel column
chromatography (eluent: hexane/ethyl acetate ) 2/1) to give
0.740 g (75%) of 24 as a light yellow powder: mp 72-76 °C;
1H NMR (200 MHz, CDCl3) δ 0.99 (t, J ) 7.2 Hz, 3H), 1.41-
1.61 (m, 2H), 1.72-1.87 (m, 2H), 4.00 (t, J ) 6.2 Hz, 2H), 6.54
(d, J ) 2.2 Hz, 1H), 6.95 (m, J AB ) 9.0 Hz, 2H), 7.60 (d, J )
2.2 Hz, 1H), 7.65 (m, J AB ) 9.0 Hz, 2H); MS (ESI) m/z 240 (M
+ Na, 38%). Anal. (C13H16N2O‚1/2H2O) C, H, N. Methane-
sulfonic acid salt: mp 130.5-131.5 °C; 1H NMR (200 MHz,
DMSO-d6) δ 0.94 (t, J ) 11.0 Hz, 3H), 1.38-1.52 (m, 2H),
1.63-1.80 (m, 2H), 2.38 (s, 3H), 4.00 (t, J ) 9.8 Hz, 2H), 6.69
(d, J ) 3.3 Hz, 1H), 6.98 (m, J AB ) 12.9 Hz, 2H), 7.71 (m, J AB
) 12.9 Hz, 2H), 7.77 (d, J ) 3.3 Hz, 1H). Anal. (C13H16N2O‚
CH4O3S) C, H, N.
1
powder: mp 75-76.5 °C; H NMR (200 MHz, CDCl3) δ 0.99
(t, J ) 7.2 Hz, 3H), 1.40-1.61 (m, 2H), 1.72-1.87 (m, 2H),
4.01 (t, J ) 6.3 Hz, 2H), 6.95 (m, J AB ) 8.8 Hz, 2H), 7.31 (d, J
) 1.6 Hz, 1H), 7.54 (m, J AB ) 8.8 Hz, 2H), 8.43 (d, J ) 1.6 Hz,
1H); MS (ESI) m/z 234 (M + H, 100%). Anal. (C13H15NOS) C,
H, N.
4-(4-n -Bu toxyp h en yl)p yr im id in e (30). To a mixture of
formamidine hydrochloride (029 g, 3.60 mmol) suspended in
tert-butyl alcohol (11 mL) was added a warm solution of

Pyrazoles and Isoxazoles as Potent Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5425
potassium tert-butoxide (0.63 g, 7.20 mmol) in tert-butyl alcohol
(11 mL). The mixture was stirred vigorously for 45 min at room
temperature. To this mixture was then added 4′-n-butoxy-3-
(dimethylamino)acrylophenone 27 (0.5 g, 2.02 mmol) in tert-
butyl alcohol (11 mL). The reaction mixture was stirred for
16 h at 50 °C. The mixture was cooled, poured into ice water,
and extracted with ethyl acetate. The organic layer was dried
over magnesium sulfate and concentrated, and the residue was
purified by NH silica gel column chromatography (eluent:
hexane/ethyl acetate ) 8/1 to 1/1) to give a colorless solid.
Recrystallization of the solid from hexane gave 0.24 g (52%)
2′-n -Bu toxya cetop h en on e (36c). This compound was
prepared from 2′-hydroxyacetophenone (3 g, 22.0 mmol) as
described in the procedure for synthesizing 3a to yield 3.07 g
(73%) of a colorless oil: 1H NMR (200 MHz, CDCl3) δ 1.00 (t,
J ) 7.0 Hz, 3H), 1.42-1.63 (m, 2H), 1.76-1.94 (m, 2H), 2.64
(s, 3H), 4.08 (t, J ) 6.0 Hz, 2H), 6.90-7.04 (m, 2H), 7.38-7.51
(m, 1H), 7.70-7.79 (m, 1H).
3′-n -Bu toxya cetop h en on e (36d ). This compound was
prepared from 3′-hydroxyacetophenone (3 g, 22.0 mmol) as
described in the procedure for synthesizing 3a to yield 3.99 g
(94%) of a colorless oil: 1H NMR (200 MHz, CDCl3) δ 1.00 (t,
J ) 7.2 Hz, 3H), 1.40-1.63 (m, 2H), 1.70-1.89 (m, 2H), 2.60
(s, 3H), 4.03 (t, J ) 6.2 Hz, 2H), 7.06-7.16 (m, 1H), 7.37 (t, J
) 8.5 Hz, 1H), 7.46-7.59 (m, 2H).
5-(4-Meth oxyp h en yl)isoxa zole (37b). To a stirred mix-
ture of sodium hydride (60% in oil, 14.6 g, 385.4 mmol), ethyl
formate (43.8 g, 730.8 mmol), and tetrahydrofuran (300 mL)
was added 4′-methoxyacetophenone 36b (30 g, 182.7 mmol)
in tetrahydrofuran (100 mL) at 0 °C. The reaction mixture was
stirred for 2.5 h at room temperature, and then diluted with
water (200 mL) and washed with ethyl acetate. The aqueous
layer was separated and hydroxylamine hydrochloride (12.7
g, 182.7 mmol) was added. The mixture was stirred for 17 h
at room temperature, and then diluted with 1 M HCl and
extracted with ethyl acetate. The organic layer was dried over
magnesium sulfate and concentrated to give a yellow solid.
The crude solid was recrystallized from diethyl ether to give
15.8 g (49%) of 37b as a light yellow powder: mp 62-64 °C;
1H NMR (200 MHz, CDCl3) δ 3.87 (s, 3H), 6.40 (d, J ) 2.0 Hz,
1H), 6.99 (m, J AB ) 9.0 Hz, 2H), 7.74 (m, J AB ) 8.8 Hz, 2H),
8.25 (d, J ) 1.6 Hz, 1H); MS (ESI) m/z 198 (M + Na, 70%).
Anal. (C10H9NO2) C, H, N.
1
of 30 as a colorless powder: mp 81.5-82.5 °C; H NMR (200
MHz, CDCl3) δ 1.00 (t, J ) 7.4 Hz, 3H), 1.42-1.62 (m, 2H),
1.74-1.89 (m, 2H), 4.05 (t, J ) 6.5 Hz, 2H), 7.02 (m, J AB ) 8.8
Hz, 2H), 7.65 (m, J AB ) 5.4 Hz, 1H), 8.07 (m, J AB ) 8.8 Hz,
2H), 8.70 (m, J AB ) 5.4 Hz, 1H), 9.21 (d, J ) 1.0 Hz, 3H); MS
(ESI) m/z 251 (M + Na, 100%). Anal. (C14H16N2O) C, H, N.
2-Acetyl-4′-n -bu toxya cetop h en on e (31). A mixture of two
drops of dry ethanol, 4′-n-butoxyacetophenone 19 (2 g, 10.4
mmol), and dibenzo-18-crown-6 (0.062 g, 0.166 mmol) in
tetrahydrofuran (13 mL) was added to a stirred mixture of
sodium hydride (60% in oil, 0.853 g, 21.8 mmol) and ethyl
acetate (1.83 g, 20.8 mmol) in tetrahydrofuran (13 mL) at room
temperature. The mixture was stirred for 3 h at 80 °C and
then cooled to room temperature and diluted with 1 M HCl.
The mixture was extracted with ethyl acetate and the extract
was dried over magnesium sulfate and concentrated, and the
residue was purified by silica gel column chromatography
(eluent: hexane/ethyl acetate ) 8/1) to give 2.17 g (89%) of 31
as yellow crystals:1H NMR (200 MHz, CDCl3) δ 0.99 (t, J )
7.4 Hz, 3H), 1.40-1.60 (m, 2H), 1.72-1.84 (m, 2H), 2.17 (s,
3H), 4.03 (t, J ) 6.5 Hz, 2H), 6.11 (s, 1H), 6.93 (m, J AB ) 9.0
Hz, 2H), 7.86 (m, J AB ) 9.0 Hz, 2H).
5-P h en ylisoxa zole (37a ). This compound was prepared
from acetophenone (2.47 g, 20.5 mmol) as described in the
procedure for synthesizing 37b to yield 1.70 g (57%) of a yellow
oil: 1H NMR (300 MHz, CDCl3) δ 6.52 (d, J ) 1.2 Hz, 1H),
7.43-7.52 (m, 3H), 7.76-7.83 (m, 2H), 8.29 (d, J ) 1.2 Hz,
1H); MS (EI) m/z 145 (M+, 100%). Anal. (C9H7NO) C, H, N.
5-(2-n -Bu toxyp h en yl)isoxa zole (37c). This compound
was prepared from 2′-n-butoxyacetophenone 36c (1.75 g, 9.10
mmol) as described in the procedure for synthesizing 37b to
yield 0.930 g (35%) of a colorless powder: mp 33-34.5 °C; 1H
NMR (200 MHz, CDCl3) δ 1.01 (t, J ) 7.2 Hz, 3H), 1.43-1.63
(m, 2H), 1.82-1.94 (m, 2H), 4.12 (t, J ) 6.3 Hz, 2H), 6.82 (d,
J ) 1.8 Hz, 1H), 6.97-7.12 (m, 2H), 7.34-7.44 (m, 1H), 8.01
(dd, J ) 1.8 Hz, 7.8 Hz, 1H), 8.31 (d, J ) 1.8 Hz, 1H); MS
(ESI) m/z 240 (M + Na, 100%). Anal. (C13H15NO2) C, H, N.
5-(3-n -Bu toxyp h en yl)isoxa zole (37d ). This compound
was prepared from 3′-n-butoxyacetophenone 36d (3 g, 15.6
mmol) as described in the procedure for synthesizing 37b to
yield 0.910 g (27%) of a colorless oil: 1H NMR (200 MHz,
CDCl3) δ 1.00 (t, J ) 7.4 Hz, 3H), 1.45-1.62 (m, 2H), 1.73-
1.85 (m, 2H), 4.03 (t, J ) 6.3 Hz, 2H), 6.51 (d, J ) 2.0 Hz,
1H), 6.94-7.02 (m, 1H), 7.30-7.42 (m, 3H), 8.29 (d, J ) 2.0
Hz, 1H); MS (EI) m/z 217 (M+, 36%). Anal. (C13H15NO2) C, H,
N.
4′-n -Bu toxyp r op iop h en on e (38). This compound was
prepared from 4′-hydroxypropiophenone (3.3 g, 22.0 mmol) as
described in the procedure for synthesizing 3a to yield 4.20 g
(93%) of a colorless oil: 1H NMR (200 MHz, CDCl3) δ 0.99 (t,
J ) 7.2 Hz, 3H), 1.21 (t, J ) 7.2 Hz, 3H), 1.40-1.62 (m, 2H),
1.70-1.90 (m, 2H), 2.96 (q, J ) 7.2 Hz, 2H), 4.03 (t, J ) 5.6
Hz, 2H), 6.91 (m, J AB ) 8.8 Hz, 2H), 7.95 (m, J AB ) 8.8 Hz,
2H).
4′-n -Bu toxy-2-h yd r oxy-1-m eth yla cr ylop h en on e (39). To
a stirred suspension of sodium hydride (60% in oil, 1.94 g, 48.4
mmol) in ethyl formate (19.26 g, 260 mmol) was added 4′-n-
butoxypropiophenone 38 (3.8 g, 18.4 mmol) in tetrahydrofuran
(24 mL). The reaction mixture was stirred for 20 min at room
temperature, and then diluted with 0.5 M HCl and extracted
with ethyl acetate. The organic layer was dried over magne-
sium sulfate and concentrated, and the residue was purified
by silica gel column chromatography (eluent: hexane/ethyl
acetate ) 9/ 1) to give 1.12 g (26%) of 39 as a colorless
5-(4-n -Bu toxyp h en yl)-3-m eth ylisoxa zole (32). To a solu-
tion of 2-acetyl-4′-n-butoxyacetophenone 31 (0.3 g, 1.28 mmol)
in pyridine (3 mL) was added hydroxylamine hydrochloride
(0.098 g, 1.41 mmol). The mixture was stirred for 30 min at
room temperature. To this mixture was then added 6 M HCl
(7 mL), and the result was extracted with ethyl acetate. The
organic layer was washed with brine, dried over magnesium
sulfate, and concentrated to give a light yellow solid. The crude
solid was recrystallized from hexane/ethyl acetate to give 0.100
g (34%) of 32 as a colorless powder: mp 77.5-79 °C; 1H NMR
(200 MHz, CDCl3) δ 0.99 (t, J ) 7.3 Hz, 3H), 1.40-1.61 (m,
2H), 1.72-1.84 (m, 2H), 2.33 (s, 3H), 4.01 (t, J ) 6.4 Hz, 2H),
6.23 (s, 1H), 6.95 (m, J AB ) 8.8 Hz, 2H), 7.68 (m, J AB ) 8.8
Hz, 2H); MS (ESI) m/z 254 (M + Na, 73%). Anal. (C14H17NO2)
C, H, N.
3-(4-n -Bu toxyp h en yl)-5-m eth ylp yr a zole (33). To a solu-
tion of 2-acetyl-4′-n-butoxyacetophenone 31 (0.2 g, 0.85 mmol)
in methanol (5 mL) was added hydrazine monohydrate (0.047
g, 0.94 mmol). The mixture was stirred for 1 h at room
temperature. This mixture was then concentrated and the
residue was purified by silica gel column chromatography
(eluent: hexane/ethyl acetate ) 3/1) to give 0.175 g (89%) of
33 as a colorless powder: mp 100-101 °C; 1H NMR (200 MHz,
CDCl3) δ 0.98 (t, J ) 7.4 Hz, 3H), 1.41-1.61 (m, 2H), 1.70-
1.83 (m, 2H), 2.35 (s, 3H), 4.00 (t, J ) 6.5 Hz, 2H), 6.28 (s,
1H), 6.93 (m, J AB ) 8.7 Hz, 2H), 7.61 (m, J AB ) 8.7 Hz, 2H);
MS (ESI) m/z 231 (M + H, 100%). Anal. (C14H18N2O) C, H, N.
5-(4-n -Bu toxyp h en yl)tetr a zole (35). To a mixture of 4-n-
butoxybenzonitrile 34 (5 g, 28.5 mmol) and ammonium chloride
(1.68 g, 31.4 mmol) in N,N-dimethylformamide (57 mL) was
added sodium azide (2.04 g, 31.4 mmol). The reaction mixture
was stirred for 30 h at 120 °C. After stirring, the mixture was
cooled to room temperature and the solvent was removed
under reduced pressure. The mixture was diluted with 1 M
HCl and a colorless solid precipitate was filtered and recrys-
tallized from water/ethanol to give 4.60 g (74%) of 35 as
colorless crystals: mp 198.5-200 °C; 1H NMR (200 MHz,
DMSO-d6) δ 0.95 (t, J ) 7.3 Hz, 3H), 1.36-1.57 (m, 2H), 1.66-
1.81 (m, 2H), 4.07 (t, J ) 6.3 Hz, 2H), 7.15 (m, J AB ) 8.9 Hz,
2H), 7.97 (m, J AB ) 8.9 Hz, 2H); MS (ESI) m/z 217 (M - H,
100%). Anal. (C11H14N4O) C, H, N.

5426 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Nakamura et al.
powder: 1H NMR (200 MHz, CDCl3) δ 1.00 (t, J ) 7.2 Hz, 3H),
1.40-1.62 (m, 2H), 1.70-1.89 (m, 2H), 2.02 (s, 3H), 4.03 (t, J
) 6.5 Hz, 2H), 6.95 (m, J AB ) 9.2 Hz, 2H), 7.67 (m, J AB ) 9.2
Hz, 2H), 8.53 (d, J ) 4.8 Hz, 1H).
45 min for CYP2C9 and CYP3A4, and metabolite concentra-
tions were measured.
Fluorescence per well was measured using a fluorescent
plate scanner (ARVO 1420 multilabel counter, Wallac, Turku,
Finland). Metabolite concentrations were measured using the
excitation and emission wavelength (CYP1A2, 405 and 460 nm;
CYP2C9, 405 and 535 nm; CYP2C19, 405 and 460 nm;
CYP2D6, 390 and 460 nm; CYP3A4, 405 and 535 nm,
respectively). Detection of the products of either assay was
linear over the range used for these assays.
IC50 values were estimated for each test compounds and
each enzyme, according to the method of Crespi et al. (1997).36
Sta tistica l An a lysis. Data are reported as the mean ( SE.
Significance was evaluated by the unpaired t-test. The level
of significance was p<0.05.
Mea su r em en t of Solu bility. About 2 mg of each compound
was added to 2 mL of water, and the mixture was shaken by
using a shaker (model SA31, Yamato Kagaku) at room
temperature. Then the suspension was centrifuged for 10 min
at 25 °C, 11 000 rpm by using a centrifugal separator (model
CF 15R, Hitachi). The supernatant was diluted with a mixed
solvent of water/acetonitrile ) 1/1. The concentration was
measured by HPLC.
5-(4-n -Bu toxyp h en yl)-4-m eth ylisoxa zole (40). This com-
pound was prepared from 4′-n-butoxy-2-hydroxy-1-methylacry-
lophenone 39 (0.7 g, 3.0 mmol) as described in the procedure
for synthesizing 23 to yield 0.38 g (55%) of a light yellow
1
powder: mp 71-72 °C; H NMR (200 MHz, CDCl3) ? 0.99 (t,
J ) 7.2 Hz, 3H), 1.45-1.61 (m, 2H), 1.73-1.84 (m, 2H), 2.24
(s, 3H), 4.02 (t, J ) 6.5 Hz, 2H), 7.00 (m, J AB ) 8.8 Hz, 2H),
7.66 (m, J AB ) 8.8 Hz, 2H), 8.12 (s, 1H); MS (ESI) m/z 254 (M
+ Na, 100%). Anal. (C14H17NO2) C, H, N.
3-(4-n -Bu toxyp h en yl)-4-m eth ylisoxa zole (41). This com-
pound was prepared from 4′-n-butoxy-2-hydroxy-1-methylacry-
lophenone 39 (0.2 g, 0.85 mmol) as described in the procedure
for synthesizing 24 to yield 0.105 g (54%) of a light yellow
1
powder: mp 71.5-72.5 °C; H NMR (200 MHz, CDCl3) δ 0.99
(t, J ) 7.3 Hz, 3H), 1.42-1.60 (m, 2H), 1.72-1.85 (m, 2H),
2.21 (s, 3H), 4.01 (t, J ) 6.5 Hz, 2H), 6.97 (m, J AB ) 8.8 Hz,
2H), 7.43 (s, 1H), 7.48 (m, J AB ) 8.8 Hz, 2H); MS (ESI) m/z
253 (M + Na, 100%). Anal. (C14H18N2O) C, H, N.
Biologica l Eva lu a tion : Ar a ch id on ic Acid (AA) Me-
ta bolism . The compounds were dissolved in 100% DMSO and
diluted with reaction buffer. The final concentration of DMSO
was 1% and did not affect 20-HETE synthesis enzyme activity.
To examine the effect of the compounds on the activity of 20-
HETE synthesizing enzyme, the compounds (10-10-10-6 mol/
L, five concentrations) were incubated for 6 h at 37 °C with
human renal microsome (HCCC, Laurel, MD) (100 µg/mL),
tritiated arachidonic acid (2 µCi/mL), and NADPH (1 mM) in
reaction buffer (50 mM MOPS/5 mM MgCl2/1 mM EDTA, pH
7.4). Reaction was terminated by addition of formic acid (pH
3.5). Reaction mixtures were separated on Unifilter2000
(Whatman J apan KK, Tokyo, J apan) filled with ODS-SS
(Sensyu Scientific Co., Ltd., Tokyo, J apan). Tritiated 20-HETE
was eluted with 70% acetonitrile and the radioactivity of the
eluate was measured by liquid scintillation counting. Before
starting the above-mentioned experiments, we have completed
the validation of this assay system by using the radio HPLC
and have confirmed that we could exactly measure the
production of 20-HETE. In the present study, we found that
human renal microsomes produced only tritiated 20-HETE
peak from tritiated arachidonic acid under our present ex-
perimental conditions by the use of HPLC according to the
procedure of Miyata et al.11 The retention time of this peak
was corresponded to that of the cold 20-HETE (Sigma Chemi-
cal Co., St. Louis, MO), and any peaks of other metabolites
including EETs and DHETs were not detected. Curve-fitting
and parameter estimation were carried out by using Origin
6.0J (OriginLab Corp., MA).
The HPLC analysis was performed with a Shimadzu HPLC
system composed of a LC-10AD, SPD-10AV, and SIL-10A. The
condition for HPLC was as follows: mobile phase, 10 mmol/L
aqueous ammonium acetate solution/acetonitrile ) 45/55; flow
rate, 1.0 mL/min; column, reverse-phase (Capcell Pak UG120,
4.6 mm i.d. + 150 mm; Shiseido) at 40 °C; and detection
wavelength, 255 nm.
Mea su r em en t of Sta bility. About 0.5 mg of each com-
pound was added to 5 mL of pH 4.0 Briton-Robinson buffer,
and the mixture was shaken by using a shaker (model SA31,
Yamato Kagaku) at room temperature for 2 h. Then the
suspension was filtrated with membrane filter (0.45 µm,
PALL). The filtrate was stored at 50 °C in an oven (model DS-
44, Yamato Kagaku). After 24 h the sample solution was
diluted with a mixed solvent of water/acetonitrile ) 1/1 and
analyzed by HPLC. The HPLC method was same as that for
solubility measurement.
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(Wirburn, MA). The CYP-specific substrates used were as
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J M020557K