Biomimetic oxidation of 2-methylimidazole derivative with a chemical model system for cytochrome P-450

Article abstract of DOI:10.1248/cpb.50.1137

A chemical model system for cytochrome P-450, consisting of tetraphenylporphyrin manganese chloride (TPPMnCl) and iodosylbenzene, efficiently oxidized 2-methylimidazole to 2-methylimidazolone. This system next applied to 4-(2-methyl-1-imidazolyl)-2,2-diph

Full text of DOI:10.1248/cpb.50.1137

August 2002
Notes
Chem. Pharm. Bull. 50(8) 1137—1140 (2002)
1137
Biomimetic Oxidation of 2-Methylimidazole Derivative with a Chemical
Model System for Cytochrome P-450
Hiroyuki MIYACHI* and Yoshio NAGATSU
Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd.; 2399–1 Mitarai, Nogi-machi, Shimotsuga-gun,
Tochigi 329–0114, Japan. Received April 18, 2002; accepted May 28, 2002
A chemical model system for cytochrome P-450, consisting of tetraphenylporphyrin manganese chloride
(TPPMnCl) and iodosylbenzene, efficiently oxidized 2-methylimidazole to 2-methylimidazolone. This system was
next applied to 4-(2-methyl-1-imidazolyl)-2,2-diphenylbutyramide, a muscarinic acetylcholine receptor antago-
nist under clinical trial, affording the previously unisolated imidazole ring 5-mono-oxidized derivative that is
considered to be the precursor of the main metabolites. This system, which is superior to the copper-ascorbate
system, should be applicable to in vitro studies of various drugs containing the 2-methylimidazole moiety.
Key words 2-methylimidazole derivative; 2-methylimidazolone derivative; metabolic intermediate; drug metabolism; P-450
chemical model; metalloporphyrin
Cytochrome P-450 (designated as P-450) is a family of tem, but found that reproducibility was poor (data not
heme-containing metallo-enzymes named after the absorp- shown).
tion band at 450 nm of their carbon monoxide form. P-450
In this paper, we present a novel mono-oxidation of 2-
metabolizes various kinds of xenobiotics, including drugs, methylimidazole by using metalloporphyrin–iodosylbenzene
oxidatively or reductively, playing an important role in drug as a chemical model system for cytochrome P-450.10—12) Ap-
metabolism.1) Many chemical models for P-450 have been plication of this system to a 2-methylimidazole-containing
developed to elucidate and/or mimic the function of the en- candidate drug is also described.
zymes. Several models function as efficient oxidation sys-
tems for simple substrates, but there have been only a few re-
Experimental
General Melting points were determined on a Yanagimoto micro melt-
ports on the application of these models to the in vitro meta-
bolic study of heterocycles, which are often used as an im-
portant structural backbone for drugs and candidate drugs.
For example, in the case of the oxidation of 3-isobutyryl-2-
isopropylpyrazolo[1,5-a]pyridine (Ibudilast; IBPP), which
has been developed as an antiasthma drug and cerebral va-
sodilator, ring hydroxylation of the pyrazolo[1,5-a]pyridine
nucleus occurred smoothly to give the monoepoxide deriva-
tive along with the diepoxide derivative.2) Those compounds
are unstable precursors of the final metabolites of IBPP.
2-Methylimidazole is an important pharmacophore, e.g., in
thromboxane A2 synthase inhibitors, aromatase inhibitors,
5-hydroxytryptamine (5HT) inhibitors, muscarinic acetyl-
choline receptor antagonists, and so on.3) In general, the imi-
dazole ring is rather metabolically labile, affording ring poly-
oxygenated and related metabolites.4) However, there has
ing point apparatus and are uncorrected. 1H-NMR spectra were measured in
CDCl3 with tetramethylsilane (TMS) and the solvent peak as internal stan-
dards, on a JEOL JMN-A400 spectrometer. Mass spectra (MS) were ob-
tained on a JEOL JMS-HX110 spectrometer. Column chromatography was
carried out on Merck silica gel 60. Analytical thin-layer chromatography
(TLC) was performed on Merck precoated silica gel 60F254 plates, and the
compounds were visualized by UV illumination (254 nm) or by heating after
spraying with phosphomolybdic acid in ethanol. HPLC analysis was per-
formed on a TOSOH ODS-80TM column (4.6ϫ250 mm). Chromatographic
conditions were as follows: samples were eluted at a flow-rate of 0.5 ml/min
with 0.05 mol/l ammonium acetate–methanol solution (25 : 1 v/v). The sam-
ples were monitored by measuring the absorbance at 220 nm. The areas of
the chromatographic peaks were calculated by use of a Hitachi chromato-in-
tegrator (D-2000). Elemental analysis was performed in the microanalytical
laboratory of Kyorin Pharmaceutical Co., Ltd.
Chemicals 2-Methylimidazole, tetraphenylporphyrin manganese chlo-
ride (TPPMnCl), tetraphenylporphyrin iron chloride (TPPFeCl), manganese
chloride (MnCl2), iodosylbenzene (PhIO), dichloromethane, and acetonitrile
were of reagent grade and were used without further purification. 4-(2-
been no report on the isolation and structural determination Methyl-1-imidazolyl)-2,2-diphenylbutyramide13—16) (KRP-197) and its
tetradeuterio derivative (D4-KRP-197) were synthesized by the Discovery
Research Laboratory of Kyorin Pharmaceutical Co. Ltd.
of imidazole ring mono-oxygenated metabolites (designated
as imidazolones), which are thought to be precursors of the
final metabolites of various drugs and candidate drugs.
It has been reported for several imidazole-containing
drugs that trace amounts of drug equivalents are retained in
connective tissue after administration to laboratory ani-
mals.5—7) Although the mechanism(s) of the retention in con-
nective tissue is not yet known, it may involve the reaction of
a reactive intermediate (such as imidazolone), formed by me-
Chart 1. Reaction of 2-Methylimidazole with the TPPMnCl–PhIO System
tabolism of the imidazole moiety, with tissue macromole-
cules.8) Therefore, it is important to develop an efficient
method for the preparation of imidazolone derivatives under
biomimetic reaction conditions. Direct transformation of 2-
methylimidazole to 2-methylimidazolone using a cupro-
ascorbate system (cupric chloride and L-ascorbic acid) was
reported, but the yield was very poor.9) We retested this sys- Chart 2. Reaction of 2-Methylimidazolone with Benzaldehyde
To whom correspondence should be addressed. e-mail: hiroyuki.miyachi@mb2.kyorin-pharm.co.jp
© 2002 Pharmaceutical Society of Japan

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Vol. 50, No. 8
Reaction with the Chemical Model In accordance with the method re-
ported by Cook et al.,17) 1 equivalent of substrate, 0.01—0.05 molar equiva-
lent(s) of metalloporphyrin and 1—3 molar equivalent(s) of oxidant were
dissolved in 10 ml of mixed solvent of dichloromethane and acetonitrile
(1 : 1 v/v), and the mixture was stirred at room temperature (see Chart 1).
The reaction was monitored by HPLC and/or TLC.
Results and Discussion
Figure 1 shows HPLC chromatograms indicating the for-
mation of 2-methylimidazolone from 2-methylimidazole in
the TPPMnCl–PhIO system. When an equimolar amount of
PhIO was used as the oxidant, the reaction was incomplete,
and a considerable amount of the starting material remained,
even after 5 h. However, the reaction went to completion
when 2 molar-equivalents of PhIO was used, and 2-
methylimidazolone was obtained almost quantitatively. With-
out metalloporphyrin manganese chloride, little reaction took
place. Furthermore, little reaction took place when MnCl2
was used instead of TPPMnCl. These results clearly indi-
cated that 2-methylimidazole was oxidized not by PhIO it-
self, but by an oxidant formed by the reaction between
TPPMnCl and PhIO, presumably a manganese-oxenoid
species.18) The effect of the central metal is small, because
similar efficiency was obtained by the use of TPPFeCl as a
catalyst. The structural determination of 2-methylimida-
Fig. 1. HPLC Chromatograms of the Reaction Products of 2-Methylimi-
dazole with the TPPMnCl-PhIO System
(A) 2-methylimidazole. (B) Synthetic 2-methylimidazolone.29) (C) After 5 h reaction
(1 eq of PhIO was used). (D) After 4 h reaction (2 eq of PhIO was used). ᮀ and ᭿ indi-
cate 2-methylimidazole and 2-methylimidazolone respectively.
zolone was done by 1H-NMR, and high-resolution mass Fig. 2. Chemical Structure of KRP-197
spectrometry.19) Furthermore, the structure was confirmed by
derivatization of 2-methylimidazolone to the aldol adduct
with benzaldehyde.
2-Methylimidazolone reacted smoothly with benzaldehyde
at room temperature in the absence of base to afford the (Z)-
a,b-unsaturated ketone derivative20) in good yield, indicating
that the acidity of the methylene moiety of 2-methylimida-
zolone is high.
Based on these observations, we applied this biomimetic
oxidation reaction system to 4-(2-methyl-1-imidazolyl)-2,2-
diphenyl butyramide13—16) (KRP-197; Figure 2), which was
developed as a dual antagonist of muscarinic acetylcholine
a) BrCD2CD2Br, DB-18-crown-6, 50% NaOH; b) 2-methylimidazole, TEA, DMF;
c) KOH, 2-PrOH.
receptors M1 and M3. KRP-197 is a candidate drug for the
treatment of urinary incontinence associated with bladder Chart 3. Synthesis of D4-KRP-197
muscle instability, and is currently under clinical study. The
imidazole ring mono-oxidation product of KRP-197 (desig-
nated as KRP-197-imidazolone) is thought to be an unstable
precursor of the final metabolites of KRP-197, but has not
yet been isolated.21) To make structural analysis easier, a
mixture of KRP-197 and its tetradeuterio analog (designated
as D4-KRP-197 (Chart 3)) was used as a substrate (desig-
nated as (H4, D4-KRP-197)).
When a mixture of (H4, D4)-KRP-197, 0.01 molar-equiva-
Chart 4. Oxydation of (H4, D4)-KRP-197 with the P-450 Chemical Model
lent of TPPMnCl, and 1.5 molar-equivalent of PhIO in a
mixed solvent of dichloromethane–acetonitrile (1 : 1 v/v; 10
ml) was stirred for 6 h at room temperature, a product which
showed molecular-ion peaks at 336 (MϩH)ϩ and 340
(MϩH)ϩ was isolated in about 30% yield, together with con-
siderable recovery of the starting material (Chart 4).22) In the
mass spectrum (see Fig. 3), (MϩH)ϩϭ336 was 16 m/z larger
than KRP-197, and (MϩH)ϩϭ340 was 16 m/z larger than
D4-KRP-197. These mass unit gains correspond to one oxy-
gen atom. These results indicated that the product is a mono-
oxygenated derivative of (H4, D4)-KRP-197. The mass frag-
ments of 238 (MϩH)ϩ and 242 (MϩH)ϩ corresponded to
the (C6H5)2C(CONH2)CX2CX2 (XϭH, or D) moiety, and no
System
fragment with a mass 16 units higher was seen in the spec-
trum. These results indicated that mono-oxygenation had oc-
curred not on the phenyl ring, but on the imidazole ring of
KRP-197. The oxygenated position of the imidazole nucleus
was determined by heteronuclear multiple bond connectivity
(HMBC) analysis to be the 5-position (see Chart 4). Further-
more, the 5-imidazolone structure was confirmed by compar-
ison of the NMR spectra of the isolated product and an au-
thentic sample, which was prepared from 2,2-diphenylace-
tonitrile.23) These compounds exhibited identical 1H- and

August 2002
1139
Fig. 3. Mass Spectrum of the Product of the Reaction of (H4, D4)-KRP-197 with the TPPMnCl-PhIO System
13C-NMR spectra.
References and Notes
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In this paper, we have shown that 2-methylimidazole and a
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model system for cytochrome P-450, TPPMnCl–PhIO, to af-
ford the 2-methylimidazolone mono-oxidation product. The
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P450-independent pathway,24) but this suggestion was based
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P-450-dependent. Further study is under way.
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P-450 could oxidize 2-methylimidazole to 2-methylimida-
zolone. A 2-methylimidazole-containing drug was similarly
mono-oxidized to afford a putative precursor of the main
metabolites. The efficiency of the reaction was superior to
that of a reported method using a copper-ascorbate system.
Further application of this chemical model system for in
vitro metabolic studies of various drugs and/or drug candi-
dates containing a heterocyclic structure is in progress.
19) 1H-NMR (400 MHz, CDCl3) d 1.99 (3H, s), 3.90 (2H, s), 10.76 (1H,
br s). FAB-MS m/z: 98.0505 (Calcd for C4H6N2O: 98.0480).
20) (Z)-5-Benzylidene-2-methyl-3,5-dihydro-imidazol-4-one; mp. 171.0—
1
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7.37—7.46 (3H, m), 8.13 (2H, dd, Jϭ8.3, 1.7 Hz), 8.82 (1H, br s).
FAB-MS m/z: 186.0772 (Calcd for C11H10N2O: 186.0793). Anal.
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5.38; N, 14.99.
21) The metabolic study of KRP-197 will be published elsewhere.
22) 1H-NMR (400 MHz, CDCl3) d 2.14 (3H, t, Jϭ2.0 Hz), 2.61—2.65
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15.71, 37.77, 38.41, 58.23, 58.92, 128.44—128.80, 142.26, 163.57,
176.24, 181.42. FAB-MS m/z: 336.1728 (Calcd for C20H22N3O2:
336.1712), 340.1977 (Calcd for C20H18D4N3O2: 340.1963).
Acknowledgments The authors wish to thank to Professor Tsunehiko
Higuchi, Faculty of Pharmaceutical Sciences, Nagoya City University, for
fruitful discussions. Thanks are also due to H. Saito and H. Furuta of Kyorin
Pharmaceutical Co., Ltd., for their analytical work.

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Vol. 50, No. 8
23) Synthesis of authentic KRP-197-5-imidazolone will be published else-
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