Biomimetic oxidation of denaverine hydrochloride

Article abstract of DOI:10.1002/(sici)1099-0690(199903)1999:3<679::aid-ejoc679>3.0.co;2-w

The behavior of denaverine (1) in various chemical model systems based on metalloporphyrins as catalysts was investigated to determine the possible oxidative metabolism. The resulting derivatives were compared to a biological model system and in vivo metabolism in rat and human.

Full text of DOI:10.1002/(sici)1099-0690(199903)1999:3<679::aid-ejoc679>3.0.co;2-w

FULL PAPER
Biomimetic Oxidation of Denaverine Hydrochloride
Kai Smolinka[a] and Berthold Göber*[a]
Dedicated to Professor Dr. Siegfried Ebel on the occasion of his 65th birthday
Keywords: Biomimetic oxidations / Cytochrome P450 / Metalloporphyrins / Denaverine / Metabolism
The behavior of denaverine (1) in various chemical model
systems based on metalloporphyrins as catalysts was
investigated to determine the possible oxidative metabolism.
The resulting derivatives were compared to a biological
model system and in vivo metabolism in rat and human.
Cytochrome P450 isoenzymes (Cyt P450) play an impor- porphyrin]manganese(III) chloride (MnTPFPPCl) and the
tant role in the metabolism of xenobiotics. Many biological corresponding iron compound FeTPFPPCl are resistant
and chemical models have been studied mimicking such en- towards oxidative degradation and give good yields of aro-
zymatic reactions.[1] In recent years model systems based matic oxygenation products in combination with imidazole
on metalloporphyrins as catalysts have been established suc- or pyridine as co-catalysts.[6] Iodosylbenzene was used to
cessfully mimicking monooxygenations such as alkane hy- compare reaction profile with that of hydrogen peroxide.
droxylations, epoxidations of alkenes, and aromatic hydrox-
ylations.[2] They show similar reactivity and selectivity to
Table 1. Biomimetic systems used
Cyt P450, whereas more simple models, for example Uden-
Catalyst
Co-catalyst O-donor
Medium
friendЈs (Fe2ϩ, ascorbic acid, O2) or FentonЈs reagent
(Fe2ϩ, H2O2) yield more varied results.[3] There are some
reports of application of metalloporphyrin-based model
systems to drug metabolism studies. These systems are com-
posed of a metalloporphyrin as a catalyst, an N-base as
a co-catalyst, and an O-donor such as peroxides, peracids,
iodosylbenzene and derivatives, sodium hypochlorite, and
others.[4] In a previous study we proved the ability of such
biomimetic systems for selective aromatic oxygenations by
comparing the reaction profile of propiverine, a drug not
metabolized by aromatic oxygenation, with that of clemas-
tine, which is favourably metabolized on the aromatic rings.
In both in vivo and aqueous media reactions, these biomi-
metic systems were able to reveal differences in metabolic
pathways.[3b,5]
MnTPFPPCl
imidazole
hydrogen
peroxide
nonaqueous
MnTPFPPCl
MnTPFPPCl
imidazole
pyridine
iodosylbenzene nonaqueous
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
Ϫ
nonaqueous
nonaqueous[a]
nonaqueous
nonaqueous
MnTPFPPCl
FeTPFPPCl
Ϫ
imidazole
pyridine
pyridine
imidazole
imidazole
Ϫ
MnTPFPS4PCl
MnTPFPS4PCl
MnTPFPS4PCl
FeTPFPS4PCl
FeTPFPS4PCl
Ϫ
aqueous,
pH ϭ 1
aqueous,
pH ϭ 6Ϫ8
aqueous,
pH ϭ 1
aqueous,
pH ϭ 1
aqueous,
pH ϭ 6Ϫ8
aqueous,
pH ϭ 1
aqueous,
pH ϭ 6Ϫ8
aqueous,
pH ϭ 6Ϫ8
imidazole
imidazole
imidazole
imidazole
Ϫ
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
hydrogen
peroxide
The spasmolytic drug denaverine hydrochloride (1-HCl)
is used in therapy for more than 25 years. Data is available
on its in vivo metabolism in rat, whilst metabolism in hu-
man is not well documented. The aim of our investigations
was to determine possible oxidative metabolic pathways of
1 by biomimetic reactions. In order to compare the chemi-
cal model systems with a biological model system we exam-
ined the reaction of 1 in a 10,000 g supernatant fraction of
rat liver homogenate, a simple biological model for micro-
somal oxidations.
Ϫ
Ϫ
[a]
Dichloromethane/methanol (1:1).
Generally, in addition to unchanged 1, nonaqueous bio-
mimetic systems produce five potential metabolites, 2Ϫ6
which are products of cleavage of the ester bond (2), of
N-demethylation (3), of N-oxidation (4), and of C-oxidation
(5, 6) (see Scheme 1). The main products are 5, 2, and 4.
Without a catalyst only cleavage of the ester bond and
N-oxidation take place leading to 2 and 4 in very low
amounts. The use of iodosylbenzene as an O-donor pre-
vents any reaction. The use of methanol instead of aceto-
1. Investigations with (Porphyrin)iron or -manga-
nese Compounds in Nonaqueous Medium
Table 1 shows different chemical model systems used in
our investigations. [5,10,15,20-Tetrakis(pentafluorophenyl)-
[a]
Institut für Pharmazie, Humboldt-Universität zu Berlin,
Goethestr. 54, D-13086 Berlin, Germany
Eur. J. Org. Chem. 1999, 679Ϫ683
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0303Ϫ0679 $ 17.50ϩ.50/0
679

K. Smolinka, B. Göber
FULL PAPER
A likely mechanism of formation of 5 is a further oxi-
dation of the intermediate carbinolamine formed by C-oxi-
dation of one N-methyl group (see Scheme 3).
Scheme 3. Proposed formation of 5
Compound 6, 2-[2-(2-ethylbutoxy)-2,2-diphenylacetoxy]-
acetic acid, results from oxidative deamination followed by
oxidation of the intermediate aldehyde, formed by the hypo-
thetic α-carbinolamine (see Scheme 4).
Scheme 1. Reactions in nonaqueous system
nitrile does not lead to a different reaction profile. No
methyl ester of 2 was found in contrast to the aqueous sys-
tem (see 2.). The use of (porphyrin)iron as catalyst yields
lower levels of products, particulary those produced by ester
bond cleavage and N-demethylation. The main product is 5.
Furthermore, use of pyridine instead of imidazole increases
yields, but due to an easier handling imidazole was prima-
rily used.
Scheme 4. Proposed formation of 6
The formation of α-carbinolamine could be initiated by
hydrogen atom abstraction or electron transfer followed by
proton abstraction.[7]
Compound 5 is the N-formyl derivative of 1. The two
conformational forms (see Scheme 2) occur in a Z/E ratio
of 2:1 up to 1:1 estimated from integrals of formyl and
2. Investigations with (Porphyrin)iron or
-manganese Compounds in Aqueous Solutions
1
Biomimetic oxidations of 1 in aqueous media were per-
formed with (porphyrin)iron or -manganese/imidazole/hy-
drogen peroxide in water (pH ϭ 6Ϫ8) or at pH ϭ 1 by
adding hydrochloric acid (6 mol/L). The influence of higher
pH values on the metabolic spectrum could not be investi-
gated due to the insolubility of 1.
methyl proton signals in the H-NMR spectrum.
Generally, all known derivatives were found in higher
yields in the aqueous system. The most potent system was
[5,10,15,20-tetrakis(pentafluorophenyl)-β-tetrasulfonato-
porphyrin]manganese(III) chloride (MnTPFPS4PCl)/imi-
dazole/hydrogen peroxide at pH ϭ 6Ϫ8. Other products
were detected depending on the catalyst used and/or the pH
value. Various systems are shown in Table 1.
Benzophenone (7) and the methyl esters of benzilic acid
(8) and of O-(2-ethylbutyl)benzilic acid (9) were found in
addition to the known metabolites 2Ϫ6 with (porphy-
Scheme 2. Conformational isomerism of 5
680
Eur. J. Org. Chem. 1999, 679Ϫ683

Biomimetic Oxidation of Denaverine Hydrochloride
FULL PAPER
rin)manganese at pH ϭ 1 or pH ϭ 6Ϫ8 (see Scheme 5). and the absence of any other benzophenone derivative. 2-Hy-
Compounds 8 and 9 are artifacts formed by transesterifi- droxybenzilic acid was not observed. For all detected de-
cation which is caused by working with methanol. Aqueous rivatives see Scheme 5.
solutions of 1 with manganese catalyst and without O-do-
In contrast to the wide metabolic spectrum at pH ϭ 6Ϫ8
nors produced no derivatives. A reaction with molecular reaction of FeTPFPS4PCl/imidazole/H2O2 with 1 generates
oxygen from air could be excluded.
no products in acidic solution.
We were unable to confirm the occurrence of benzilic
acid (10). However, the formation of benzophenones from
benzilic acids is well known, so detection of 7 suggests ear-
lier benzilic acid.
3. Investigations with a 10,000 g Supernatant
Fraction of Rat Liver Homogenate
To compare the chemical system with a biological system
a 10,000 g supernatant fraction of rat liver homogenate was
used. In this fraction many microsomal Cyt P450 occur.
Moreover, this fraction is relatively easy to prepare and to
handle.
2Ϫ4 were isolated from an incubation mixture with
1-HCl. Further products were found in trace amounts. They
do not correspond with products known from our biomi-
metic investigations. Compound 2 occurs in similar amount
in the blank value. It is assumed to be an artifact caused by
the working procedure and not a product of an enzymatic
catalysis.
4. Discussion
Table 2 shows the results of biomimetic studies in com-
parison with the in vitro method and metabolism studies in
rat and in human. Eleven metabolites have been observed
in rat studies as well as intact 1. The identified metabolites
are products of cleavage of the ester and ether bond (10),
of the oxidative N-demethylation (3), of cleavage of the
ether bond and a further ring closure giving 3,3-di-
phenylmorpholin-2-one (13), of reductive cleavage of the
ether bond resulting in 2-(dimethylamino)ethyl 2,2-di-
phenylacetic acid (14) and 2,2-diphenylacetic acid (15), and
of transesterifications generating ethyl benzilate, methyl and
ethyl O-(2-ethylbutyl)benzilate. Three metabolites could not
be identified. The main metabolites are 10 and 13.[8] Prod-
ucts of single ether bond cleavage were not found in either
in the biomimetic way or in the 10,000 g supernatant frac-
tion, or in the metabolism in rat or human. Compound 10
and its methyl ester found in the biomimetic system only in
very low yields. This proves the possibility of O-dealky-
lations with the biomimetic method, but the ether bond
cleavage is not favoured in this study. In contrast to 1 the
[a]
Scheme 5. Reactions in aqueous system: MnTPFPS4PCl/imida-
[b]
zole/H2O2 at pH ϭ 6Ϫ8 or 1; FeTPFPS4PCl/imidazole/H2O2 at
pH ϭ 6Ϫ8
These derivatives are also known from the reaction of 1 biomimetic oxidation of propiverine[5] or clemastine[3b] af-
with the chemical model system FeTPFPS4PCl/imidazole/ fords several products of O-dealkylation in moderate yields.
hydrogen peroxide. In addition, two phenolic compounds
The absence of 14 and 15 in the biomimetic reaction is
were detected in very small amounts. 2-Hydroxybenzo- not surprising, because they are products of reducing reac-
phenone (11) could be elucidated by comparison with an tions. The described biomimetic system mimics oxygen-
authentic sample. The formation of 11 from 7 could be ex- ations, reduced metabolites were never observed. Com-
cluded by biomimetic oxidation of 7 leading to the 3- and pound 3, another metabolite discovered in rat and human,
4-hydroxy derivatives.[6c]
was found in moderate yields. N-Dealkylations are well-
The second phenolic metabolite (12) results from 1 by known reactions mimicked by chemical model systems.
monohydroxylation at one aromatic ring. The 2-position of Furthermore, 2 and its methyl ester 9 were obtained in
the phenolic group is inferable from the occurrence of 11 biomimetic studies. From metabolism in rat only 9 and the
Eur. J. Org. Chem. 1999, 679Ϫ683
681

K. Smolinka, B. Göber
Metabolism in human
FULL PAPER
Table 2. Results in comparison
Metabolite
Biomimetic method
Metabolism in rat
In vitro method
2
ϩ
ϩ
ϩ
ϩ
ϩ[a]
ϩ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ[b]
ϩ
ϩ
Ϫ
ϩ
Ϫ
ϩ
3
4
10
Ϫ
13
14
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
15
further
5, 6, 7, 8, 9, 11, 12
detected,
detected,
not identified
not found[9]
not identified[8]
[a]
[b]
Ethyl and methyl ester of 2. Ϫ No result of enzyme activity.
MS, Autospect, Micromass, magic bullet as matrix. For CI MS,
MAT Finnigan 95 ST, isobutane as reactant gas. Ϫ HPLC: Hewlett
Packard 1090, using a LiChroCart column (125 ϫ 4 mm) with pre-
column LiChroCart (4 ϫ 4 mm) containing LiChroSpher RP se-
lect-B (5 µm), Merck, diode-array detector, monitoring at 230 nm,
flow 1 mL/min, linear gradient: 60Ϫ98% or 70Ϫ98% methanol in
disodium hydrogen phosphate solution 0.002 mol/L, 0Ϫ20 min. Ϫ
TLC: Analytical TLC; HPTLC plates, silica gel 60F254, 5 ϫ 7.5 cm,
Merck, saturation time 15 min, front 6 cm. Preparative TLC; PSC
plates, silica gel 60F254, 2 mm, 20 ϫ 20 cm, Merck. Eluents used;
ethyl ester of 2 are known. They are artifacts like ethyl ben-
zilate formed by isolating procedures using methanol or
ethanol.[8] The possible primary metabolite is 2. The forma-
tion of 2 shows conformity with our biomimetic investi-
gations.
Less than 1% of the applied dose of 1-HCl could be de-
tected in metabolism studies in human.[9] Besides un-
changed 1, compounds 3 and 10 were quantified. They are
generated in chemical model systems, too. Thus, all meta-
bolites formed by oxidations and known from metabolism dichloromethane/methanol (9:1), dichloromethane/cyclohexane/
methanol/25% ammonia solution (5:3.5:1.5:0.05), cyclohexane/di-
oxane/ethyl acetate/ethanol/25% ammonia solution (1.5:1.5:4:2:1),
toluen/dioxane/glacial acetic acid (7:2.7:0.3), and toluene/ethyl
acetate (8:2). Detection reagents; concentrated sulphuric acid, Dra-
gendorff reagent by AB-DDR 87, 3-methyl-2-benzothiazolinone
hydrazone (MBTH) reagent, after saturation of plates with am-
monia vapour, coupling with MBTH solution (2% in methanol)
and following oxidation with potassium hexacyanoferrate(III) solu-
tion (8% in water).
in rat or human were found in biomimetic studies, with the
exception of 13. A comparison of quantities of different
methods was not indicated. Elaboration of validated
quantification does not seem beneficial because of the wide
product spectrum in chemical model system, no published
quantities of metabolites in rat, and the recovery of less
than 1% of applied dose in metabolism studies in human.
Total 1 is oxidized to 10Ϫ20% in the biomimetic system as
estimated from isolated derivatives.
With the incubation of the 10,000 g supernatant fraction
as an in vitro method only 3 was identified corresponding
with metabolism in rat and human, whilst 2 is not a product
of enzymatic activity. The main in vivo metabolites 10 and
13 were not found, cleavage of the ether bond was not ob-
served. Most derivatives were obtained with the described
biomimetic method. Mainly, metabolic reactions are sup-
posed to occur on the ester side chain. Aromatic oxygen-
ations are not expected due to their minor role in biomi-
metic studies. The minor role of the ether bond cleveage is
noteworthy. The importance of this fact will be seen in
further metabolism studies. The new derivatives 5, 6 could
be useful as reference substances for additional screening in
future studies on the metabolism of 1 in human.
Materials: Commercial reagents were purchased from standard
chemical suppliers and were used without further purification.
1-HCl and metabolites 2Ϫ4, and 10 were gifts from Apogepha Arz-
neimittel GmbH, Dresden (Germany). The methyl esters 8 and 9
were prepared according to ref.[8] MnTPFPPCl and FeTPFPPCl
were prepared according to ref.[6]
FeTPFPS4PCl according to ref.[10]
,
MnTPFPS4PCl and
2-(N-Formyl-N-methylamino)ethyl O-(2-Ethylbutyl)benzilate (5): 1H
NMR: δ ϭ 0.7 (6 H, CH3), 1.27 (5 H, CHϪCH2), 2.5 and 2.6 [1
H and 1 H, (Z)- and (E)-NϪCH3], 3.0 (2 H, OϪCH2), 3.3 and 3.4
(2 H and 2 H, NϪCH2), 4.19 and 4.25 (2 H and 2 H, COϪ
OϪCH2), 7.2Ϫ7.4 (10 H, aromatic H), 7.6 and 7.8 (1 H and 1 H,
CHO). Ϫ 13C NMR: δ ϭ 11.54 and 11.56, 23.84 and 23.86, 30.0
and 35.6, 42.1, 43.6 and 48.5, 62.0 and 63.2, 86.5, 128.19, 128.29,
128.41, 128.80, 128.83, 141.2 and 141.5, 163.0 and 163.1, 172.1. Ϫ
EI MS; m/z (%): 297 (< 1), 267 (17), 183 (100), 165 (17), 105 (77),
86 (23), 77 (22), 72 (8). Ϫ FAB MS; m/z: 420 [Mϩ ϩ Na]. Ϫ ESI
MS; m/z: 398 [Mϩ ϩ H], 420 [Mϩ ϩ Na].
2-[2-(2-Ethylbutoxy)-2,2-diphenylacetoxy]acetic Acid (6): 1H NMR:
δ ϭ 0.8 (6 H, CH3), 1.2 (5 H, CHϪCH2), 3.1 (2 H, OϪCH2), 4.2
(2 H, COϪOϪCH2), 7.1Ϫ7.4 (10 H, aromatic H). Ϫ 13C NMR:
δ ϭ 11.0, 23.1, 41.2, 64.9, 65.2, 85.2, 127.42, 127.46, 128.4, 142.1,
168.9, 170.9. Ϫ EI MS; m/z (%): 267 (24), 183 (100), 165 (23), 105
(75), 77 (25). Ϫ FAB MS; m/z: 393 [Mϩ ϩ Na], 357 [Mϩ ϩ H Ϫ
H2O]. Ϫ CI MS; m/z: 357 [Mϩ ϩ H Ϫ H2O].
Now, the application of such biomimetic systems to the
evaluation of analytical procedures in order to isolate and
elucidate derivatives of new drug substances for metabolism
studies is in progress.
Experimental Section
Equipment: NMR: Bruker DPX 300 (300 MHz and 75 MHz, for
1H and 13C, respectively), CDCl3 as solvent, TMS as internal stand-
ard. Ϫ MS: Hewlett Packard 5995 A (EIMS), 70 eV. For ESI MS,
Triple-Quadrupol-MS TSQ 700, Finnigan MAT Bremen. For FAB
2-(Dimethylamino)ethyl O-(2-Ethylbutyl)-2-hydroxybenzilate (12):
EI MS; m/z (%): 299 (1), 283 (< 1), 199 (16), 181 (4), 121 (9), 105
682
Eur. J. Org. Chem. 1999, 679Ϫ683

Biomimetic Oxidation of Denaverine Hydrochloride
FULL PAPER
(26), 93 (3), 77 (9), 71 (50), 58 (100). Ϫ FAB MS; m/z: 400 [Mϩ
H], 422 [Mϩ ϩ Na].
ϩ
Acknowledgments
We would like to thank the Apogepha Arzneimittel GmbH,
Dresden (Germany) for financial support and Dr. Bartoszek from
the Institut für Angewandte Chemie Adlershof e.V., Berlin (Ger-
many) for FAB MS.
Methods: Chemical model system: Ϫ (i) Nonaqueous system:
1 mmol of (porphyrin)iron or -manganese, 10 mmol of N-base, and
700 mmol of 1-HCl were dissolved in 10 mL of dichloromethane/
acetonitrile (1:1). After addition of 1500 mmol of oxidant, the mix-
ture was stirred at room temperature for 24 h. Blank values were
carried out without catalyst or oxidant. Ϫ (ii) Aqueous system:
Compounds were dissolved in the same way and ratio in 10 mL of
water, if necessary hydrochloric acid (6 mol/L) was added. Ϫ (iii)
Analytical procedure: Products were separated by preparative layer
chromatography and purified by TLC. Aqueous systems were
freeze-dried initially and then dissolved in 10 mL of methanol. Iso-
lated derivatives were characterized by TLC, HPLC, MS, and
NMR data, if possible they were compared with authentic samples.
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in ice-cold phosphate buffer (0.01 mol/L, pH ϭ 7.6) with 1.15%
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[10]
Received September 10, 1998
[O98412]
Eur. J. Org. Chem. 1999, 679Ϫ683
683