Metabolic and chemical studies on N-(4-chlorobenzyl)-N'-benzoyhydrazine

Article abstract of DOI:10.1016/S0014-827X(00)00077-X

The in vitro hepatic microsomal metabolism of N-(4-chlorobenzyl)-N'-benzoylhydrazine (CBBAH), a model compound representing N-alkyl substituted hydrazides, was studied using hepatic washed rat microsomal preparations fortified with NADPH to identify the possible N-oxidative, N-dealkylated and hydrolytic metabolites. CBBAH and its potential metabolites were prepared, characterized using spectroscopic techniques and then separated using a reversed phase HPLC system with UV detection at 254 nm. CBBAH was chemically converted to the corresponding hydrazone by m-chloroperbenzoic acid (m-CPBA) oxidation. CBBAH was incubated with rat microsomal preparations in the presence of NADPH, extracted into dichloromethane and evaporated finally under nitrogen. The TLC and HPLC results from the metabolic experiments showed that CBBAH produced the corresponding hydrolytic and N-dealkylated metabolites together with the corresponding hydrazone.

Full text of DOI:10.1016/S0014-827X(00)00077-X

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 624–630
Metabolic and chemical studies on
N-(4-chlorobenzyl)-N%-benzoylhydrazine
:
8
S¸. Gu¨niz Ku¨c¸u¨kgu¨zel, Ilkay Ku¨c¸u¨kgu¨zel, Mert Ulgen *
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara Uni6ersity, 81010 Istanbul, Turkey
Received 2 January 2000; accepted 10 August 2000
Abstract
The in vitro hepatic microsomal metabolism of N-(4-chlorobenzyl)-N%-benzoylhydrazine (CBBAH), a model compound
representing N-alkyl substituted hydrazides, was studied using hepatic washed rat microsomal preparations fortified with NADPH
to identify the possible N-oxidative, N-dealkylated and hydrolytic metabolites. CBBAH and its potential metabolites were
prepared, characterized using spectroscopic techniques and then separated using a reversed phase HPLC system with UV
detection at 254 nm. CBBAH was chemically converted to the corresponding hydrazone by m-chloroperbenzoic acid (m-CPBA)
oxidation. CBBAH was incubated with rat microsomal preparations in the presence of NADPH, extracted into dichloromethane
and evaporated finally under nitrogen. The TLC and HPLC results from the metabolic experiments showed that CBBAH
produced the corresponding hydrolytic and N-dealkylated metabolites together with the corresponding hydrazone. © 2000
Published by Elsevier Science S.A. All rights reserved.
Keywords: N-Alkyl hydrazide; Hydrazone formation; Secondary amines; In vitro metabolism
in vitro hepatic microsomal metabolism of N-(4-
chlorobenzyl)-N%-benzoylhydrazine (CBBAH), a previ-
1. Introduction
ously synthesized and patented insecticide bearing a
nonsubstituted benzoyl and a substituted benzyl moi-
Although N-(substituted benzyl)-N%-substituted ben-
zoylhydrazines have been reported previously to be
eties [6]. Our present aim was not only to investigate
anticonvulsant [1], MAO inhibitor [2] and anti-HIV
the potential N-dealkylation and hydrolytic products of
agents [3], there is no report regarding their in vitro
hepatic microsomal metabolism in the literature. How-
CBBAH, but also to detect whether any corresponding
hydrazone and/or N-oxidative metabolites were formed
ever, there are a few studies on their corresponding
or not. It was also intended to block the possible
hydrazones. For example, the in vitro hepatic microso-
mal metabolism of benzoic acid 4-amino-(4-
fluorophenyl)methylenehydrazide was studied and it
aromatic hydroxylation pathway. In the present work,
the corresponding hydrazone, benzoic acid(4-
chlorophenyl)methylenehydrazide (CBDBAH), which
was reported that this compound produced the corre-
was screened previously for antiepileptic properties [7],
8
sponding hydrolytic metabolites [4]. Ulgen et al. [5] also
was also prepared as a potential metabolic product.
studied the in vitro metabolism of benzoic acid benzyli-
denehydrazide and found that the aromatic hydroxyla-
tion was detected only in the arylidene moiety, but not
in the benzoyl ring. This fact prompted us to study the
2. Experimental
4-Chlorobenzaldehyde (CB) and 4-chlorobenzylchlo-
ride (CBC) and m-chloroperbenzoic acid (m-CPBA)
were purchased from Aldrich. All other chemicals were
purchased from Merck. All melting points were
recorded on a Bu¨chi 530 melting point apparatus and
are uncorrected. UV spectra were recorded on a Shi-
Abbre6iations: CBBAH, N-(4-chlorobenzyl)-N%-benzoylhydrazine;
CB, 4-chlorobenzaldehyde; CBC, 4-chlorobenzylchloride; m-CPBA,
m-chloroperbenzoic acid; BAH, benzoic acid hydrazide; CBDBAH,
benzoic acid(4-chlorophenyl)methylenehydrazide; BA, benzoic acid.
* Corresponding author. Tel.: +90-216-348 4194.
8
E-mail address: mulge@turk.net (M. Ulgen).
0014-827X/00/$ - see front matter © 2000 Published by Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00077-X

S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
625
2.1. Preparation of
N-(4-chlorobenzyl)-N%-benzoylhydrazine (CBBAH)
BAH (0.004 mol) was dissolved in absolute ethanol.
To this solution, an absolute ethanolic solution of CBC
(0.002 mol) was added. This mixture was refluxed for 4
h at 110°C. At the end of the reaction, water was added
and the solid product obtained was filtered off, dried
and recrystallized from a mixture of ethanol–petroleum
ether (40–60°C). M.p. 125–127°C. The purity of CB-
BAH was determined by the use of elemental analysis,
TLC and reverse phase HPLC. Analysis for
C14H13ClN2O Calc./Found: 64.49/64.80 (C); 5.02/5.42
(H); 10.74/9.92 (N). The structure of CBBAH was
1
confirmed by the UV, IR, H NMR spectral data. The
UV and IR spectrum of CBBAH showed the character-
istic absorption bands of N-alkyl hydrazide. UV (etha-
nol) umax. (nm): 268, 221 and 205. IR (KBr) cm−1 w:
3269 (CONH); 1641 (CONH); 1600, 1508, 1404 (CꢀC);
Fig. 1. Curve overlay of UV spectra obtained (A) from synthetic
CBDBAH and (B) CBDBAH isolated from the oxidation of CBBAH
by preparative TLC.
1
1089 (CꢁCl). H NMR (CDCl3) l ppm: 4.09 (s, 2H,
ꢁNHꢁCH2ꢁ), 4.30 (s, 1H, ꢁNHꢁCH2), 7.35–7.69 (m,
9H, ArꢁH), 7.71 (b, 1H, ꢁCONHꢁ). The 1H NMR
spectrum of CBBAH in CDCl3 displayed ꢁCONHꢁ,
ꢁNHꢁCH2ꢁ, ꢁNHꢁCH2ꢁ, protons at 7.71, 4.30, 4.09
ppm, respectively. The ꢁCONHꢁ, ꢁNHꢁCH2ꢁNꢁH
protons of iproniazid were observed at 7.58 and 4.31
ppm, respectively [9].
madzu UV-2100S spectrometer (1 mg/100 ml in
methanol). IR spectra were run on a Shimadzu FTIR-
8300 spectrophotometer (1 mg/200 mg in KBr). Ele-
mental analyses were performed on a Carlo Erba 1106
instrument. 1H NMR spectrum was run on Bruker
AVANC-DPX 400 spectrometer.
2.2. Preparation of benzoic
This metabolic study required the synthesis of CB-
BAH together with two of its potential metabolites,
namely benzoic acid hydrazide (BAH) and benzoic
acid(4-chlorophenyl)methylenehydrazide (CBDBAH).
BAH was prepared by the reaction of methyl benzoate
and hydrazine hydrate at 110–130°C. This compound
had an identical melting point of 115°C to its previ-
ously reported value [8]. The attempted oxidation of
CBBAH with m-chloroperbenzoic acid (m-CPBA) was
also carried out.
acid(4-chlorophenyl)methylenehydrazide (CBDBAH)
BAH (0.01 mol) was dissolved in ethanol. To this
solution, an ethanolic solution of CB (0.01 mol) was
added. This mixture was refluxed for 2 h. The solid
product obtained was filtered off, washed with water,
dried and recrystallized from ethanol. The purity of
CBDBAH was confirmed by elemental analysis, TLC
and a reverse phase HPLC. The structure of CBDBAH
was established by its UV and IR spectral data. M.p.
Table 1
Chromatographic properties of the substrate and their potential metabolites
Compound
Abbreviations
HPLC system (S)
Retention time (min)
S a
TLC system (A)
(Rf)
A b
Benzoic acid
Benzoic acid hydrazide
BA
BAH
2.30
3.26
0.07
4-Chlorobenzoic acid
CBA
5.01
4-Chlorobenzaldehyde
N-(4-Chlorobenzyl)-N%-benzoylhydrazine
Benzoic acid(4-chlorophenyl)methylenehydrazide
CB
CBBAH
CBDBAH
17.60
21.25
24.95
0.91
0.61
0.49
a S, acetonitrile: 0.02 M phosphate buffer (pH 7.4) (30:70, v/v) final pH 5.00. Flow rate 0.5 ml/min HPLC column (Novapak C18 5 mm, 15 cm
length×3.9 mm).
b A, diethylether.

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S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
Fig. 2. RP-HPLC chromatogram obtained (A) from authentic CBBAH and its potential metabolites; (B) following extraction of male rat
microsomal incubation mixture with CBBAH as substrate; (C) from incubation with denatured microsomes; (D) from incubation with microsomes
in which co-factors were omitted; (E) from incubation of microsomes in which substrate and co-factors were omitted; (F) from co-incubation of
BAH and CB with denatured microsomes.

S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
627
Fig. 2. (Continued)
164–165°C (lit. 170–175°C) [10]. Analysis for
C14H11ClN2O Calc./Found: 64.99/64.89 (C); 4.28/4.56
(H); 10.83/10.85 (N). The UV spectrum exhibited a
characteristic E band for aromatic group at 202 nm and
a characteristic K band for chromophoric ꢁCHꢀNꢁ
group at 306 nm [4]. UV (ethanol) umax. (nm): 306, 221
and 204. IR spectrum of compound CBDBAH showed
the characteristic absorption bands of hydrazone (NꢁH
3288; CꢀO 1664; ꢁCHꢀNꢁ 1620 cm−1). IR (KBr)
cm−1w: 3288 (CONH); 1664 (CONH); 1620 (CHꢀN);
1602, 1543, 1487 (CꢀC); 1086 (CꢁCl).
Na2SO4 and concentrated under reduced pressure to
leave a mixture of a pale yellow and white solid. This
mixture was dissolved in acetone and chromatographed
on preparative silica gel plates (0.5 mm thickness) in
conjunction with diethylether. The corresponding hy-
drazone (CBDBAH) and 4-chlorobenzaldehyde (CB)
were obtained from the bottom and top bands, respec-
tively. The silica-layers corresponded to these bands
were scraped off and extracted into dichloromethane
and the solvent was evaporated. Two solid products
were obtained. The solid corresponding to the bottom
band gave a m.p. at 160–165°C, UV (ethanol) umax.
(nm): 305, 221 and 204 (Fig. 1). HPLC Rt=24.90, TLC
Rf=0.48 whereas the solid corresponding to the top
band gave a HPLC Rt=17.50, and a TLC Rf=0.90.
2.3. Attempted oxidation of
N-(4-chlorobenzyl)-N%-benzoylhydrazine
The oxidation products of CBBAH were synthesized
by the oxidation reaction with m-CPBA [11]. CBBAH
(0.001 mol, 0.261 g) was dissolved in dry acetone and
the flask kept in the dark. To this stirred and cooled
solution, m-CPBA (0.002 mol, 0.43 g 80%, in dry
acetone) was added dropwise over 30 min. The reaction
was continued for further 5 h and terminated by re-
moval of the solvent under reduced pressure. The resid-
ual solid was dissolved in dichloromethane (25 ml) and
this solution was subsequently washed with aqueous
K2CO3 (0.5 M, 3×25 ml); dried with anhydrous
2.4. Analytical separation
The separation techniques were based on TLC and
an isocratic HPLC system. TLC was carried out using
prepared silica-gel GF254 0.25 mm on glass plates (E.
Merck) with diethylether as solvent system A. Com-
pounds were identified by their absorbances at 254 nm
(Table 1). A mixture of 4-dimethylaminocinnamalde-
hyde (0.2% w/v) in hydrochloric acid (1 M)–ethanol
(1:1, v/v) reagent was sprayed on to the plates after

628
S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
development. A dark red color was obtained. The
HPLC chromatograph consisted of an isocratic system,
a Rheodyne syringe loading sample injector valve
(model 7725) fitted with a 20 ml sample loop, a model
481 Waters wavelength UV detector and a Unicam
4880 Chromatography Data Handling System integra-
tor. The HPLC column (Spherisorb, m-Bondapak C18
5
mm, 25 cm length×4.6 mm and Novapak C18 5 mm, 15
cm length×3.9 mm) was purchased from Phase Sepa-
rations Limited, UK. The guard column packing mate-
rial (Whatman Pellicular ODS) was purchased from
Whatman International Ltd., Maidstore, Kent, UK.
The composition of the mobile phases, flow rates and
the retention times of the CBBAH and its potential
metabolites were given in Table 1. All these compounds
were detected by their absorbance at 254 nm.
2.4.1. Biological studies
Glucose-6-phosphate dehydrogenase (G6PH) was
purchased from the Boehringer Mannheim Corporation
(London) Ltd. Nicotinamide adenine dinucleotide
phosphate mono sodium salt (NADP) and glucose-6-
phosphate (G6P) disodium salt were obtained from
Sigma Ltd. The animals used in this investigation were
male rats. Hepatic washed male rat microsomes were
prepared at 0°C using the calcium chloride precipita-
tion method [12]. Incubation was carried out in a
shaking water-bath at 37°C using a standard co-factor
solution consisting of NADP+ (disodium salt, 1 mmol),
G6P (disodium salt, 5 mmol), G6PDH (0.5 unit) and
MgCl2 (50% w/w, 10 mmol) in 1 ml of phosphate buffer
(0.2 M, pH 7.4) for each flask. Co-factor solutions were
pre-incubated for 5 min before the addition of substrate
(2 mmol) in methanol (50 ml) and microsomes equiva-
lent to 0.25 g original liver. Incubation was continued
for 30 min, terminated and the incubates were extracted
with dichloromethane (2×5 ml). Extracts were evapo-
rated to dryness under a stream of nitrogen at 20°C.
Dry organic residues were reconstituted in 200 ml of
methanol for HPLC and 50 ml of methanol for TLC
analysis.
Fig. 3. TLC chromatogram obtained following extraction from male
rat microsomal incubation mixture with CBBAH as substrate. 1,
BAH; 2, CBDBAH; 3, CBBAH; 4, CB; 5, test; 6, incubation of CB
and BAH in the presence of denatured microsomes; 7, control
(denatured microsomes); 8, control (omission of co-factors); 9, con-
trol (omission of the substrate).
accordance with the findings reported by Low et al.
[13]. Since the chemical oxidation of CBBAH using
m-CPBA yielded CBDBAH as the major product, it
could be proposed that this hydrazone would also arise
from a direct oxidation of CBBAH via dehydratation
of an unstable N-hydroxy intermediate (Fig. 4) [14]. CB
was further oxidized to 4-chlorobenzoic acid (CBA).
BA was found as a metabolite which may arise either
from the hydrolysis of CBBAH or alternatively from
the hydrolysis of BAH. All these metabolites were not
observed with control incubates with denatured micro-
somes or incubates lacking co-factors. This indicates
that these metabolites formed via enzymatic reactions.
Metabolic and chemical reactions possibly involved in
the biotransformation of CBBAH are shown in Fig. 4.
N-Dealkylated and hydrolytic metabolites were de-
tected together with a hydrazone function formed. Ben-
zoic acid hydrazide produced by the N-dealkylation
reaction is possible to form hydrazone with reactive
carbonyl groups, i.e. pyruvic acid and a-ketoglutaric
acid. If this occurs, all a-keto acid resources needed for
the pyruvate oxidation by tricarboxylic acid cycle
would be depleted. This may interrupt the carbohydrate
3. Results and discussion
Following the metabolic experiments using CBBAH
as a substrate, the N-dealkylation products, i.e. BAH
and CB were observed by RP-HPLC (Fig. 2) and TLC
(Fig. 3). The formation of the corresponding hydrazone
(CBDBAH) as a major in vitro metabolic product was
also demonstrated. The control experiments were per-
formed to investigate the mechanism of formation of
this metabolite by co-incubation of BAH with CB in
the presence of denatured microsomes. The result indi-
cated that the formation of CBDBAH was most proba-
bly via condensation of CB and BAH. This was in

S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
629
Fig. 4. In vitro microsomal metabolic reactions of CBBAH.

630
S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
8
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Evaluation of some arylhydrazones of p-aminobenzoic acid hy-
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and the amino acid metabolisms. However, in our
structure, the hydrazone formation from the benzoic
acid hydrazide and 4-chlorobenzaldehyde produced by
N-dealkylation may prevent, to some extent, these po-
tential toxic responses. In addition, it may be possible
that BAH produced by N-dealkylation from CBBAH
may result in hepatic necrosis by a similar mechanism
as in isoniazid and isopropylhydrazine. Isoniazid is
known to produce a hydrazone with vitamin B6 and a
deficiency for this vitamine occurs. BAH may similarly
have this undesired effect.
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Acknowledgements
This work was supported by the Research Fund of
Marmara University Project number SBDYD-102.
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