Convenient synthesis of quinocetone metabolites: Characterization, theoretical investigation, and cytotoxicity study

Article abstract of DOI:10.1016/j.molstruc.2012.04.071

Quinocetone (3-methyl-2-quinoxalinbenzenevinylketo-1,4-dioxide; QCT) is a new promising antimicrobial growth promoter for quinoxalines. The identification of the major metabolites of QCT has resulted in a number of studies regarding its metabolic pathway.

Full text of DOI:10.1016/j.molstruc.2012.04.071

Journal of Molecular Structure 1022 (2012) 32–36
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Convenient synthesis of quinocetone metabolites: Characterization, theoretical
investigation, and cytotoxicity study
Jiaheng Zhang a, Linxia Li b, Yubo Li a, Bing Peng a, Songqing Li a, Zhiqiang Zhou a, Haixiang Gao a,
,
⇑
Suxia Zhang b,
⇑
a Department of Applied Chemistry, China Agricultural University, Yuanmingyuan West Road 2#, Haidian District, Beijing 100194, China
b Department of Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Yuanmingyuan West Road 2#, Haidian District, Beijing 100194, China
h i g h l i g h t s
"
"
"
Four main metabolites of quinocetone were conveniently synthesized and fully characterized.
Theoretical N–O bond dissociation enthalpies and the octanol-water partition coefficient (Kow) were estimated.
The results from MTT assay have showed that quinocetone and its metabolites have cytotoxicity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Quinocetone (3-methyl-2-quinoxalinbenzenevinylketo-1,4-dioxide; QCT) is a new promising antimicro-
bial growth promoter for quinoxalines. The identification of the major metabolites of QCT has resulted in
a number of studies regarding its metabolic pathway. However, little is known about the systematic syn-
thesis, characterization, and simultaneous determination of its metabolites. To obtain system data for the
four main metabolites of QCT, a convenient synthesis of these compounds was performed. All synthesized
compounds were characterized by infrared spectroscopy, nuclear magnetic resonance, and high-resolu-
tion mass spectroscopy. The theoretical NAO bond dissociation enthalpies (BDEs) and octanol–water par-
tition coefficient (Kow) were estimated. A cytotoxicity assay for these compounds in hepatocytes isolated
from rats was proposed, and the cytotoxicity results were evaluated based on the calculated NAO BDEs.
Ó 2012 Elsevier B.V. All rights reserved.
Received 19 March 2012
Received in revised form 24 April 2012
Accepted 24 April 2012
Available online 1 May 2012
Keywords:
Quinocetone
Metabolites
Synthesis
Bond dissociation enthalpy
Cytotoxicity
1. Introduction
2003 [4]. The majority of pharmacokinetic studies and toxicology
research on veterinary chemicals have focused on the metabolites
of QdNOs. A number of studies have drawn similar conclusion that
Quinocetone
(3-methyl-2-quinoxalinbenzenevinylketo-1,4-
dioxide; QCT) belongs to the family of quinoxaline-1,4-dioxides
(QdNOs), a class of bioactive compounds that exhibit notable anti-
bacterial, antiviral, and antifungal properties [1]. Since the 1960s,
several quinoxaline-1,4-dioxides have been developed as veteri-
nary drugs to promote growth, prevent dysentery, and inhibit bac-
terial enteritis in farm animals [2]. However, two well-known
QdNOs drugs, carbadox (CBX) and olaquindox (OLA), were banned
in 1999 due to toxicity and food safety concerns [3]. As a new
promising synthetic veterinary drug, QCT retains the advantages
of QdNOs and can inhibit bacteria such as Staphylococcus aureus,
Escherichia coli, and Salmonella. The lower toxicity level of QCT than
CBX and OLA has ensured its availability in the Chinese market as a
pharmaceutical agent for swine, poultry, and aquatic animals since
the toxicities of QdNOs are closely associated with their metabo-
lites [5–8]. Our previous study has revealed that QCT is rapidly
metabolized in the liver and kidneys of pigs, thereby producing
several desoxy and reduction metabolites [9].
These compounds are found as residues in edible swine tissue
and can be traced through the gastrointestinal tract of pigs.
However, there are limited characterization data for the four
main metabolites, namely, 1,4-bisdesoxyquinocetone (1,4-BDQ),
1-desoxyquinocetone (1-DQ), 4-desoxyquinocetone (4-DQ), and
3-methyl-2-styrenol-quinoxaline (MSQ). Most available toxicity
studies on QdNOs drugs have focused on the parental drugs rather
than their metabolites. Nevertheless, these drugs are rapidly
metabolized into various kinds of metabolites in vivo, and a few
of the reacting intermediates or final products have been identified
and quantified. To the best of our knowledge, there is no available
information regarding the cytotoxicity of 1,4-BDQ, 1-DQ, 4-DQ, and
MSQ, except for several articles focusing only on QCT [10].
⇑
Corresponding authors.
E-mail addresses: hxgao@cau.edu.cn (H. Gao), suxia@cau.edu.cn (S. Zhang).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.04.071

J. Zhang et al. / Journal of Molecular Structure 1022 (2012) 32–36
33
There are numerous methods for the selective monodeoxygen-
ation and reduction of certain QdNOs [11–13]. However, there are
only a few procedures for the synthesis of the metabolites of QdNO
veterinary drugs. Recently, Li et al. [14] have prepared 1,4-BDQ, 1-
DQ, and 4-DQ from the parent compound QCT using different
selective reagents. Our group has also reported procedures for
the preparation of metabolites from mequindox (MEQ), another
form of QdNO veterinary drug [15,16]. The current study reports
a simple one-step procedure based on the structural similarities
between QCT and MEQ to synthesize 1,4-BDQ, 1-DQ, and 4-DQ
from the corresponding metabolites of MEQ. MSQ was also subse-
quently synthesized from 1,4-BDQ.
1H and 13C NMR spectra were measured on a Bruker DSX-300
instrument at 100.6 and 40.6 MHz, respectively, in CDCl3 with
TMS as the internal standard. The chemical shifts are provided in
d (ppm). The UV and IR spectra were recorded on a Pgeneral T6
new century UV–VIS spectrophotometer and a PerkinElmer FT–IR
Spectrum 100 spectrometer, respectively. High resolution mass
spectrometry (HRMS) were performed on an ACQUITYUPLC™
BEH C18 column (50 mm ꢀ 2.1 mm i.d., 1.7
lm particle size;
Waters Co., Milford, MA, USA) and recorded with a UPLC/ESI-
QTOF-MS system (Waters, Manchester, UK) controlled by Mass-
Lynx™ software.
Oxygenated species, including heterocycles containing one or
more NAO bonds, have been ordered to establish a reactivity scale
based on their abilities to transfer oxygen atoms in several bio-
chemical conversions [17]. A theoretical approach for understand-
ing the toxicity of QdNOs is at an early stage; however, some steps
that depend on NAO bond dissociation enthalpies (BDEs) have
been established. The NAO bond may also lower the octanol–water
partition coefficient (Kow), which can influence the sorption in vivo
or sediments for agricultural applications [8]. Therefore, studies on
the NAO BDEs of QdNOs and Kow are important in the assessment
of drugs. In the present study, calculations of BDEs and Kow using
accurate density functional theory (DFT) and other computational
methods combined with cytotoxicity assay are crucial for deter-
mining pharmacological importance and biologic activity.
In the current study, a convenient synthesis and characteriza-
tion, including the determination of the Kow values of the four main
metabolites of QCT, were described. The first, second, and mean
NAO BDEs for QCT were predicted based on DFT calculations. Tox-
ico-kinetic and cytotoxicity assays in hepatocytes isolated from
rats were proposed. The results were evaluated and associated
with each calculated NAO BDE. This systematic research on QCT
and its metabolites provides useful information on the food safety
evaluation of QCT.
2.3. Methods
Unless otherwise noted, all operations including the synthesis
and analysis were performed in the absence of light or under very
low light conditions because of the lability to light of the target
compounds. The procedures for infrared (IR) spectroscopy, nuclear
magnetic resonance (NMR) imaging, corresponding absorption
spectrums and reverse-phase HPLC analyzes can be found in the
Supplementary Materials.
2.3.1. Preparation of 1,4-BDQ
The synthesis of 1,4-BDQ was performed at 45 °C under contin-
uous stirring of a mixture containing 60 mL of ethanol, 5.83 g
(55.0 mmol) of benzaldehyde, 7.44 g (40.0 mmol) of 1,4-BDM,
and 1.04 g (26 mmol) of sodium hydroxide. The solution was fil-
tered in 95% ethanol after 1 h and afforded 5.95 g (21.7 mmol) of
1,4-BDQ as a yellow solid. m.p. 144.6–147.0 °C. 1H NMR (CDCl3,
300 MHz) d 3.01 (3, s, CH3), 7.45–7.88 (m, ArAH, CH@CH, 9),
8.05–8.17 (m, C8AH, C5AH, 2); 13C NMR (CDCl3, 75 MHz) d
24.029, 123.399, 127.830, 128.232, 128.533, 128.809, 128.945,
129.579, 129.749, 130.807, 131.737, 134.843, 139.737, 142.492,
158.588, 148.640, 153.577; IR (KBr) v: 649, 776, 937, 1123, 1190,
1361, 1482, 1694 cmꢁ1; MS (ESI) m/z (%): 275.11 [M + H]+. HRMS
(ESI) calcd for C18H15N2O [M + H]+ 275.3301, found 275.1159.
2. Materials and methods
2.3.2. Preparation of 1-DQ and 4-DQ
First, 1-DM (8.08 g, 40.0 mmol) or 4-DM (8.08 g, 40.0 mmol)
was dissolved in 100 mL of ethanol and cooled to 0 °C in an ice
bath. This step was followed by the slow addition of 6.36 g
(60.0 mmol) of benzaldehyde and 3.70 g (50.0 mmol) of diethyl-
amine. After 0.5 h, the precipitate was filtered and washed with
95% ethanol to yield 8.73 g (30.1 mmol, yellow solid) of 1-DQ or
7.14 g (24.6 mmol, yellow solid) of 4-DQ. For 1-DQ: m.p. 155.0–
156.3 °C. 1H NMR (CDCl3, 300 MHz) d 2.85 (3, s, CH3), 7.26–7.86
(ArAH, CH@CH, 9), 8.21 (m, C8AH, 1), 8.65 (m, C5AH, 1); 13C
NMR (CDCl3, 75 MHz) d 13.9, 118.9, 123.3, 128.9, 129.0, 130.5,
130.7, 131.0, 131.5, 131.8, 134.5, 137.0, 140.4, 141.9, 150.2,
151.7, 189.9, 200.1; IR (KBr) v: 764, 989, 1343, 1573, 1596,
1670 cmꢁ1; MS (ESI) m/z (%): 291.62 [M + H]+. HRMS (ESI) calcd
for C18H15N2O2 [M + H]+ 291.3291, found 291.6168. For 4-DQ:
m.p. 222.0–224.6 °C. 1H NMR (CDCl3, 300 MHz) d 2.66 (3, s, CH3),
7.13–7.88 (ArAH, CH@CH, 9), 8.09 (m, C8AH, 1), 8.55 (m, C5AH,
1); 13C NMR (CDCl3, 75 MHz) d 22.28, 118.78, 125.06, 128.87,
128.99, 129.39, 129.74, 131.34, 132.28,133.92, 135.39, 137.25,
144.37, 146.14, 153.42, 188.40; IR (KBr) v: 765, 1034, 1128,
1348, 1405, 1483, 1605, 1669 cmꢁ1; MS (ESI) m/z (%): 291.10
[M + H]+. HRMS (ESI) calcd for C18H15N2O2 [M + H]+ 291.3291,
found 291.1026.
2.1. Materials
All reagents and solvents used in the synthesis were analytical
grade and purchased from the Beijing Chemical Reagent Company.
MEQ (purity 98%) and QCT (purity 98%) were provided by the Col-
lege of Veterinary Medicine, Huazhong Agricultural University (Wu-
han, China). 1,4-bisdesoxymequindox (1,4-BDM, purity P 99%), 1-
desoxymaquindox (1-DM, purity P 99%) and 4-desoxymaquindox
(4-DM, purity P 99%) were synthesized according to our published
protocols [15,16]. 3-(4,5-Dimethylthia-zol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT), N-(2-hydroxyethylpi-perazine)-N0-(2-
ethane sulfonic acid) (HEPES), collagenase, Dulbecco’s modified Ea-
gle medium (DMEM), fetal calf serum (FCS), insulin, and collagen
were purchased from Sigma Chemical Company (St. Louis, MO).
Antibiotics (penicillin–streptomycin mixtures) were purchased
from GIBCO Company (USA). All other chemicals and solvents used
in the extraction and cleanup procedures were analytical grade and
purchased from commercial sources.
Adult male Sprague–Dawley rates (200–250 g) which were fed
with a standard diet and fasted 12 h before the experiment were
provided by the Experimental Animal Research Institute of China
Agricultural University.
2.3.3. Preparation of MSQ
2.2. Instruments
MSQ was prepared from 1,4-BDQ. KBH4 (aq, 0.3 mol/L) was
added dropwise to a solution of 1,4-BDQ (4.14 g, 15 mmol) in
anhydrous alcohol at a temperature of approximately 30 °C. The
reactions were monitored using TLC (thin-layer chromatography,
Melting points were measured with a WRX-4 micro melting
point apparatus (Shanghai Yice Co., China) and were uncorrected.

34
J. Zhang et al. / Journal of Molecular Structure 1022 (2012) 32–36
ethyl acetate/petroleum ether mixtures, 1:1) with pre-coated silica
gel aluminum plates containing a fluorescent indicator. The reac-
tions were stopped when there was only one spot visible by TLC.
Most of the solvent was subsequently removed using a rotary
evaporator. Water (30 ml) was added, and the mixture was washed
three times with chloroform (30 mL at a time). The organic phases
were combined and dried with anhydrous sodium sulfate. Then the
Crude MSQ was obtained by removing the solvent on a rotary
evaporator. The crude product was dissolved in warm diethylether
(80 mL) and kept at 4 °C for 12 h. The resulting white crystals were
separated from the mixture. The recrystallization was repeated
three times using diethyl ether to afford pure MSQ (3.04 g,
11 mol, pale yellow solid). For MSQ: m.p. 84.0–85.1 °C. 1H NMR
(DMSO, 300 MHz) d 2.86 (3, s, CH3), 3.43 (1, d, -OH), 5.71 (1, m,
2-CH), 5.75 (m, C8AH, 1), 6.10 (m, C5AH, 1), 6.88–8.08 (ArAH,
CH@CH, 9); 13C NMR (DMSO, 75 MHz) d 22.459, 73.600, 126.610,
127.739, 128.405, 128.507, 128.655, 128.750, 129.135, 129.625,
129.820, 130.155, 130.379, 136.644, 140.872, 142.150, 153.188,
156.187; IR (KBr) v: 692, 757, 968, 1080, 1449, 1486, 1565,
1596 cmꢁ1; MS (ESI) m/z (%): 276.13. [M + H]+. HRMS (ESI) calcd
for C18H17N2O [M + H]+ 277.1336, found 277.1341.
gree of reduction of the tetrazolium salt MTT to formazan by mito-
chondrial succinic dehydrogenase.
3. Results and discussion
3.1. Synthesis of QCT metabolites
QCT aromatic N-reduced metabolites including 1,4-BDQ, 1-DQ,
and 4-DQ were synthesized by the aldol condensation of the corre-
sponding metabolites of MEQ and benzaldehyde in the presence of
sodium hydroxide in ethanol solutions. Generally, when phospho-
rus trichloride, trimethyl phosphate, and sodium hydrosulfite were
used as the reducing agents for the selective deoxygenation of
quinoxaline 1,4-dioxides, the reaction produced a large amount
of byproducts, and the product was hard to purify. In contrast,
the reactions in the current study proceeded very efficiently,
resulting in good yields and operational simplicity in the separa-
tion of the products. Compared with the selective deoxygenation
of quinocetone, the convenient and specific synthesis of several
QCT metabolites can improve research on the metabolism of the
veterinary drug. For the synthesis of MSQ, the hydroxide ion (eth-
anol potassium hydroxide) reacted rapidly with 1,4-BDM to yield
1-(3-methylquinoxalin-2-yl)-3-phenylprop-2-en-1-ol, which was
easily recrystallized from an ether solution. The synthesized 1,4-
BDQ, 1-DQ, 4-DQ, and MSQ were shown to be P99% pure by
HPLC–UV. The preparation of these compounds is outlined in
Scheme 1.
2.4. Theoretical study
NAO BDEs were calculated using the Gaussian 09 program [18].
The geometries of the molecules and related radical species were
optimized at the B3LYP/6-31 + g(d,p) level, which has been used
in numerous structure optimizations. This method is feasible be-
cause of the high accuracy requirements and practical, acceptable
computational cost [19,20]. Subsequent frequency calculations at
the same level verified that these optimized structures were the
global minimum without imaginary frequencies and provided ther-
mal data. Single-point energies for the optimized structures of the
molecules and radicals were refined and computed with the more
flexible B3LYP/6-311 + g(2df,2pd) level. The energies obtained
from the extended basis set calculations were used to estimate
the NAO BDEs. The gas-phase mean NAO molar BDEs for MEQ were
used as a reference to validate the theoretical method. The theoret-
ical Kow values for QCT, 1,4-BDQ, 1-DQ, 4-DQ, and MSQ were calcu-
lated based on the constants assigned to the various functional
groups using the ChemAxon computer program [21].
3.2. Theoretical calculation results
The NAO BDEs were calculated from the enthalpy change of the
following hemolytic bond dissociation reaction:
RANAOðgÞ ! RANÅðgÞ þ OÅðgÞ
ð1Þ
The mean NAO BDE of QCT was half of the enthalpy of the following
reaction:
OANARANAOðgÞ ! NARANÅðgÞ þ 2OÅðgÞ
ð2Þ
The bond dissociation energy of the NAO bond was computed from
the heats of formation at 298.15 K of the species involved in the dis-
sociation, i.e.:
2.5. Cytotoxicity study
EBDE
¼
Df H2ꢂ 98:15;RAN þ Df H2ꢂ98:15;O ꢁ Df Hꢂ
ð3Þ
298:15;RANAO
The hepatocytes were isolated by the two-step collagenase per-
fusion method. In the first step, about 100 g of liver was washed in
500 mL of 20 mM HEPES solution without calcium to remove blood
and weaken the cell–cell junctions. The hepatocytes were released
by collagenase (50 mg/100 mL) perfusion for 20 min (recirculation
system), centrifugation, and resuspension thrice in the second step.
After isolation, the hepatocytes were suspended in DMEM contain-
ing 10% FCS, 100.0 g/mL streptomycin, 100 U/mL penicillin, and
10.0 g/mL insulin at pH 7.65. The hepatocytes were then placed
into 100-mm diameter plastic dishes, pre-coated with collagen,
and maintained in a 5% CO2 incubator at 37 °C. To determine cyto-
toxicity, the hepatocytes were plated onto 96-well plates (104 cells
per well). After the monolayers of cells became confluent in the 96-
well plate, the rat hepatocytes were treated with different concen-
tration ranges of QCT, 1,4-BDQ, 1-DQ, 4-DQ, and MSQ in DMEM
without FCS, with no drug (control), and/or with DMSO alone
(vehicle control) for 4 h. The vehicle controls were specified with
a volume of DMSO corresponding to the largest volume of test
compound added to the treatment dishes. Various toxicity end
points were assayed in the control and drug-exposed cells after
4 h. Mitochondrial function was evaluated by measuring the de-
To evaluate the accuracy of the B3LYP/6-311+g(2df,2pd) level,
the experimental data for the mean NAO BDE of MEQ
(251.6 4.2 kJ molꢁ1) was used as a Ref. [22]. In our calculation,
the theoretical value for the mean NAO BDE of MEQ was
253.4 kJ molꢁ1, and the difference between the theoretical and
experimental values was very small. Therefore, the proposed
method was suitable for calculating the NAO BDEs of QdNO deriv-
atives. For QCT, which also has two different NAO bonds due to dif-
ferent chemical environments, these bonds in QCT were expected
to exhibit different dissociation energies. Consequently, the NAO
BDE can be described according to the first, second, total, and mean
NAO BDEs. The first NAO BDE was the energy required to break the
weakest bond in the di-N-oxide compound to yield the corre-
sponding N-oxide. The second NAO BDE was the energy required
to break the bond in the N-oxide compound to yield the parent
quinoxaline. The total and mean NAO BDEs were the sum and
mean of the former two dissociation enthalpies, respectively. The
full results for the computed BDEs for QCT are schematically
shown in Fig. 1. The dissociation of the 4-NAO bond, which is clo-
ser to the methyl group, occurred more easily and yielded a BDE

J. Zhang et al. / Journal of Molecular Structure 1022 (2012) 32–36
35
Scheme 1. Synthesis of 1,4-BDQ, 1-DQ, 4-DQ and MSQ.
Fig. 1. First, second, total, and mean NAO BDEs for QCT were computed at the B3LYP/6-311 + g(2df,2pd) level of theory.
value of 243.4 kJ molꢁ1. This BDE was determined to be the first
NAO BDE of QCT. The metabolic pathway producing the desoxy
metabolites were presumed to be the same.
Given the growing interest in using computational techniques
for regulatory purposes, the prediction of Kow, a well-studied
chemical property, has been developed rapidly with numerous the-
oretical models. ChemAxon is one of the popular desktop chemical
software programs that provides accurately predicted Kow values
based on a published database. Strock et al. [8] have examined
the Kow values of another QdNO derivative, CBX, and its associated
Table 1
Selected properties of quinocetone and its metabolites.
Compound
Molecular weight (g molꢁ1
)
MP (°C)
logKow
LD50 (rat mg/kg b.w)
QCT
306.32
274.32
290.32
290.32
276.33
186.5–187.5
144.6–147.0
155.0–156.3
222.0–224.6
84.0–85.1
1.29
3.55
2.76
2.08
2.89
8687.32a
1,4-BDQ
1-DQ
4-DQ
MSQ
–
–
–
–
a
Ref. [23].

36
J. Zhang et al. / Journal of Molecular Structure 1022 (2012) 32–36
cytes isolated from rats was also proposed. QCT and 1-DQ both
displayed the most potent cytotoxicities, and the cleavage of the
4-NAO bond may be ultimately responsible for the toxicity of
QdNOs. This systematic study can improve pharmacokinetic and
residue studies on veterinary drugs.
Acknowledgments
This work was supported by 973 Fund, the Ministry of Science
and Technology, PR China (Grant No. 2009CB118801), National
Natural Science Foundation of China (Project No. 20977112), the
Specialized Research Fund for the Doctoral Program of Higher Edu-
cation of China (Grant No. 20090008120015) and Program for New
Century Excellent Talents in University (NCET-10-0777).
Appendix A. Supplementary data
Fig. 2. Cytotoxicity results using MTT assay for each of the five chemicals. Cell
cultures were exposed to the drugs for 4 h. Control value was taken as 100%. The
data were presented as means SE from three independent experiments.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2012.04.
071.
N-oxide reduced metabolites by this software, and found trends
consistent with the experimental data. In addition to the selected
properties of QCT and its metabolites, the Kow values determined
by ChemAxon are summarized in Table 1. From the logP plugin
of ChemAxon, no significant difference at the 95% confidence level
was observed in the Kow measurements as a function of pH for QCT
and its metabolites. The Kow values were averaged for each system.
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243.4 kJ molꢁ1
, the toxicity can be mainly attributed to the
cleavage of the 4-NAO bond rather than that of the 1-NAO bond.
Notably, the complete elucidation of the cytotoxic mechanism of
QCT is outside the scope of our current work. However, based on
the MTT assay results, careful monitoring procedures are impera-
tive for the safe use of these compounds as animal feed, and aware-
ness of their toxic effects to animals and general consumers should
be raised.
4. Conclusions
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This study reported the convenient synthesis of the four main
metabolites of QCT, namely, 1,4-BDQ, 1-DQ, 4-DQ, and MSQ. All
synthesized compounds were characterized by IR, NMR, and
HRMS. The theoretical NAO BDEs and Kow were calculated by the
B3LYP/6-311 + g(2df,2pd) DFT method and ChemAxon software,
respectively. A cytotoxicity assay for these compounds in hepato-