Combined theoretical and experimental study on the molecular structure, FT-IR, and NMR spectra of cyadox and 1,4-bisdesoxycyadox

Article abstract of DOI:10.1016/j.molstruc.2012.09.030

1,4-Bisdesoxycyadox, a deoxidized metabolite of cyadox, was synthesised and characterized. Structural and conformational analyses were performed using theoretical calculations employing density functional theory (DFT). The molecular geometry was optimized using B3LYP method with 6-311+G(d,p) basis set and then it was compared with X-ray diffraction data of similar molecular compounds. From the optimized geometry of the molecule, vibrational frequencies of the title compounds were calculated via B3LYP/6-311+G(d,p) approach. The ¹H and ¹³C NMR chemical shift were calculated by gauge-including atomic orbital method with B3LYP/6-311++G(2df,2pd) approach. Comparison of the experimental and calculated 1H and 13C chemical shifts resulted in the reliable assignment of cyadox and 1,4-bisdesoxycyadox. The first, second, total, and mean NO bond dissociation enthalpies were also obtained theoretically.

Full text of DOI:10.1016/j.molstruc.2012.09.030

Journal of Molecular Structure 1035 (2013) 69–75
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Combined theoretical and experimental study on the molecular structure, FT-IR,
and NMR spectra of cyadox and 1,4-bisdesoxycyadox
⇑
Zongyang Li, Jiaheng Zhang, Yubo Li, Haixiang Gao
Department of Applied Chemistry, China Agricultural University, Yuanmingyuan West Road 2#, Haidian District, Beijing 100194, China
h i g h l i g h t s
"
"
"
"
1,4-Bisdesoxycyadox (1,4-BDC) were synthesised as one of cyadox’s main metabolite.
Theoretically optimized structure agree very well with the experimental findings of BDOC.
BDOC was characterized by IR, 1H, 13C NMR spectra.
Bond dissociation enthalpies of NAO in BDOC were estimated theoretically.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2012
Received in revised form 29 August 2012
Accepted 12 September 2012
Available online 21 September 2012
1,4-Bisdesoxycyadox, a deoxidized metabolite of cyadox, was synthesised and characterized. Structural
and conformational analyses were performed using theoretical calculations employing density functional
theory (DFT). The molecular geometry was optimized using B3LYP method with 6-311+G(d,p) basis set and
then it was compared with X-ray diffraction data of similar molecular compounds. From the
optimized geometry of the molecule, vibrational frequencies of the title compounds were calculated via
B3LYP/6-311+G(d,p) approach. The 1H and 13C NMR chemical shift were calculated by gauge-including
atomic orbital method with B3LYP/6-311++G(2df,2pd) approach. Comparison of the experimental and cal-
culated 1H and 13C chemical shifts resulted in the reliable assignment of cyadox and 1,4-bisdesoxycyadox.
The first, second, total, and mean NAO bond dissociation enthalpies were also obtained theoretically.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
Cyadox
1,4-Bisdesoxymequindox
Metabolite
Infrared spectra
NMR
BDE
1. Introduction
of QdNO drugs’ metabolites [4]. Moreover, certain metabolites of
quinoxaline-1,4-dioxide derivatives, especially their desoxy com-
Quinoxaline-1,4-dioxide (QdNO) derivatives, which are benzo-
heterocycles composed of a quinoxaline ring and one or two acy-
clic chains, share the 1,4-di-N-oxide moiety and the accessory
biological properties [1]. Due to their specific antibacterial activity
against gram-negative bacteria, such as Escherichia coli, Salmonella,
and Pasteurella, some QdNOs are widely utilized in the pharmaceu-
tical and veterinary fields [2]. Cyadox, (2-quinoxalinyl-methy-
lee)hydrazine N,N0-dioxide, is one of the notable QdNOs
structurally related to carbadox, olaquindox, mequindox, and quin-
ocetone. As a synthetic growth promoter, cyadox provides better
growth-enhancing functions in food-producing animals, such as
pigs, fish, goats, and poultry. However, problems pertaining to
the toxicity of cyadox limit its application [3].
pounds, are suspected carcinogens and mutagens that cause severe
side effects, thus triggering safety concerns [5]. The cleavage of
NAO bond in QdNO drugs draws extensive attention because of
its toxicological and pharmacological significance. According to
Huang et al.’s research, cyadox is mainly metabolized to its deoxi-
dized metabolites in the microsomes [6]. Therefore, studies on the
mechanism of N-oxide reduction and detailed structure-related
parameters of desoxy metabolites are imperative for the evaluation
of cyadox risk. Currently, the most extensively studied metabolism
profile of cyadox is represented via ultrahigh-performance liquid
chromatography/electrospray ionization quadruple time-of-flight
mass spectrometry (UPLC/ESI-QTOF-MS) combined with an algo-
rithm software [7]. Alternatively, the direct measurement of the
peak area ratio of the standard metabolite synthesis to the internal
standards in an HPLC system is a more practical method of obtain-
ing useful and direct pharmacokinetic information.
Based on previous in vitro studies of QdNO derivatives, some
correlations exist between the degree of toxicity and the formation
In this study, 1,4-bisdesoxycyadox (1,4-BDC) is synthesised as
the main metabolite of cyadox. A comparison of the theoretical
⇑
Corresponding author. Tel.: +86 1062731991; fax: +86 1062733830.
E-mail address: hxgao@cau.edu.cn (H. Gao).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.09.030

70
Z. Li et al. / Journal of Molecular Structure 1035 (2013) 69–75
and experimental infrared (IR) and nuclear magnetic resonance
(NMR) spectra for the title compounds is reported. This study aims
to determine the use of density functional theory (DFT) in predict-
ing and evaluating spectroscopic properties and to elucidate the
structural differences between cyadox and 1,4-bisdesoxycyadox.
In addition, the NAO bond strength of cyadox measured by the
NAO bond dissociation enthalpies (BDEs) is initially investigated
by DFT calculation.
2.2. Calculations
The molecular structures of the compounds in the ground state
(in vacuo) were optimized using DFT method B3LYP level of theory,
which combines Becke’s three parameter non-local exchange func-
tional with Lee–Yang–Parr’s correlation functional together with
the 6-311+G(d,p) basis set [8–10]. Frequency calculations at the
6-311+G(d,p) level were performed to identify all of the stationary
points as minima and to provide free energies at 298.15 K, which
include entropic contributions by considering the vibrational, rota-
tional, and translational motions of the species. The calculated
chemical shifts of 1H NMR and 13C NMR for the title compounds
were obtained by gauge-including atomic orbital (GIAO) method
using the B3LYP/6-311++G(2df,2dp) level of theory. NMR calcula-
tions were performed using the optimized geometries at the same
level of theory with TMS as reference. The effect of DMSO solvent
on the theoretical NMR parameters was evaluated using the de-
fault integral-equation-formalism polarizable continuum model
(IEF–PCM) [11]. The NAO BDE is the calculated enthalpy change
from the hemolytic bond dissociation reaction:
2. Experimental
2.1. Materials and measurements
Cyadox (98% purity) was provided by the College of Veterinary
Medicine, Huazhong Agricultural University (Wuhan, China). Other
reagents and solvents used in the synthesis were of analytical
grade and purchased from Beijing Chemical Reagent Company.
Melting points were measured with a WRX-4 micro melting point
apparatus (Shanghai Yice Co., China). The infrared spectrum was
recorded on a PerkinElmer FT-IR Spectrum 100 spectrometer in
the spectral range 450 cmꢀ1 to 4000 cmꢀ1 with a scanning speed
of 10 cmꢀ1 minꢀ1 and a resolution of 4.0 cmꢀ1. NMR was recorded
for diluted solutions in DMSO using a BrukerDSX-300 at 300 MHz
for 1H and 75 MHz for 13C. The chemical shifts were expressed in
ppm relative to TMS.
RANAO ðgÞ ! RANÅ ðgÞ þ OÅ ðgÞ
The mean NAO bond dissociation enthalpy of quinocetone is
half of the enthalpy of the following reaction:
ð1Þ
Fig. 1. Synthetic outline of the 1,4-bisdesoxymequindox.
Table 1
Geometrical data of cyadox and 1,4-BDC.
Bond length (Å)
Cyadox
1,4-BDC
Exp.
Bond angle (°)
Cyadox
1,4-BDC
Exp.
C1AN12
C1A28C
C2AN12
C2AC3
C2AC5
C3AC6
C3AN13
N13AC28
N12AO14
N13AO15
C5AC8
1.359
1.398
1.399
1.407
1.401
1.399
1.403
1.341
1.273
1.270
1.375
1.406
1.375
1.467
1.281
1.351
1.384
1.206
1.527
1.455
1.148
1.079
1.081
1.081
1.079
1.077
1.082
1.015
1.091
1.091
1.32
1.357
1.406
1.388
1.401
1.404
1.401
1.38
C2AN12AC1
N12AC1AC28
C1AC28AN13
C28AN13AC3
N13AC3AC2
C8AC5AC2
C5AC2AC3
C2AC3AC6
C3AC6AC7
C7AC8AC5
O14AN12AC1
O15AN13AC28
C1AC16AN18
C15AN18AN19
N18AN19AC21
N19AC21AO27
O27AC21AC22
C21AC22AC25
C22AC25AN26
Selected dihedral angles (°)
C2AN12AC1AC16
C1AC15AN18AN19
C18AC19AC21AC22
C19AC21AC22AC25
C21AC22AC25AN26
118.2
120.4
123.2
117.5
120.1
119.3
119.9
120.2
119.3
120.7
121.6
122.0
129.3
121.3
121.6
120.4
124.7
112.6
177.4
117.9
120.7
122.7
117.4
120.4
119.8
119.4
119.5
119.7
120.8
121.3
117.5
125.2
116.8
121.6
120.6
120.9
119
1.426
1.352
1.427
1.415
1.412
1.357
1.308
1.309
1.296
119.9
121.5
1.369
1.415
1.37
1.359
1.405
1.377
C8AC7
C7AC6
131.1
121.6
121.6
120.5
124.5
112.6
177.3
C1AC16
C16AN18
N18AN19
N19AC21
C21AO27
C21AC22
C22AC25
C25AN26
C5AH4
C8AH11
C7AH10
C6AH9
C28AH29
C16AH17
N19AH20
C22AH23
C22AH24
1.477
1.281
1.353
1.381
1.207
1.528
1.455
1.48
1.081
1.081
1.081
1.081
1.084
1.084
1.014
1.091
1.091
A174.8
5.1
6.4
178.6
A179.2
A175.3
5.3
6.5
179
0.95
0.95
0.95
0.95
A179.2

Z. Li et al. / Journal of Molecular Structure 1035 (2013) 69–75
71
OANARANAO ðgÞ ! ÅNARANÅ ðgÞ þ 2OÅ ðgÞ
ð2Þ
crystallized under ꢀ4 °C and filtered. A white solid was obtained,
with a yield of 3 g (95%). The whole synthesised path is illustrated
in Fig. 1.
The bond dissociation energy of the NAO bond is computed
from the heat formation at 298.15 K of the species involved in
the dissociation, i.e.,
EBDE
¼
Df H2ꢁ 98:15;RAN þ Df H2ꢁ 98:15;O ꢀ Df Hꢁ
ð3Þ
298:15;RANAO
All theoretical properties were calculated using the Gaussian 09
program [12]. The obtained theoretical results aided in making de-
tailed assignments of the experimental IR and NMR spectra.
2.3. Synthesis
2.3.1. Synthesis of 2-methylquinoxaline
Sodiumhydrogensulfite (24.5 g) was added to a 40% methylgly-
oxal (21.3 g) solution mixed with 50 ml water and then stirred. O-
phenylenediamine (10.8 g) was dissolved in water (55 ml) with
heating and transferred to a 500 ml round-bottom flask. After stir-
ring for 20 min, the solvent was placed in the dark and stored over-
night. After the addition of sodium metabisulfite (20 g) the mixture
was extracted with 120 mL (3 ꢂ 40 mL) ether. Then the combined
organic phase was washed with 30 mL water (3 ꢂ 10 mL). The
solution was dried over anhydrous MgSO4 and concentrated under
reduced pressure. Oily liquid was obtained, with a yield of 11 g
(51%). 1H-NMR (CDCl3):d = 2.75 (CH3, 3H, s), 7.70 (PhAH, 2H, m),
8.02 (PhAH, 2H, m), 8.72(N@CH, 1H, s); 13C-NMR(CDCl3):d = 22.3,
128.4, 128.6, 128.9, 129.8, 140.7, 141.837, 145.7, 143.5 ppm.
Bp = 245.2–246.9 °C.
2.3.2. Synthesis of quinoxaline-2-carbaldehyde
2-Methyl-quinoxaline (11 g) in a 250 ml round-bottom flask
was dissolved with dioxane and heated at 70 °C. A mixture of sele-
nium dioxide (8.5 g) with dioxane (29 ml) and water (5.9 ml) was
added dropwise into the flask within 90 min. The solution was
then stirred at 85 °C for 4 h, and then filtered and processed with
activated carbon for 30 min. Dioxane was distilled under reduced
pressure and the residue was extracted with 30 mL (3 ꢂ 10 mL)
ether. Then the combined organic phase was washed with 30 mL
wateter (3 ꢂ 10 mL). The ether was dried with MgSO4 at room tem-
perature overnight. The solvent was dried under reduced pressure
and the crude product was isolated as brownish solid (9 g). The so-
lid was refluxed with n-hexane for 3 h and heat-filtered after trea-
ted with activated carbon for 20 min. Then, n-hexane was distilled
to obtain a deep-yellow acicular crystal (48%). 1H-NMR (CDCl3):
d = 7.95 (PhAH, 2H, m), 8.24 (PhAH, 2H, m), 9.43 (N@CH, 1H, s),
10.29 (CHO, 1H, s); 13C-NMR(CDCl3):d = 129.6, 130.4, 131.1,
132.8, 141.9, 142.4, 144.5, 146.0, 192.7; Mp = 106.6–107.2 °C.
Fig. 3. Calculated FT-IR (upside) and experimental (downside) spectrums of
cyadox.
2.3.3. Synthesis of 1,4-bisdesoxycyadox
2-Formoxyl-quinoxaline (2 g) was dissolved with 95% ethanol
(25 ml). Neohydrazid (1.2 g) in 5 ml water was added to a 100 ml
round-bottom flask and stirred for 3 h. The solution was
Fig. 4. Calculated FT-IR (upside) and experimental (downside) spectrums of 1,4-
bisdesoxymequindox.
Fig. 2. Optimized molecular structures and atomic numbering of cyadox.

72
Z. Li et al. / Journal of Molecular Structure 1035 (2013) 69–75
Table 2
The observed FT-IR, calculated frequencies using B3LYP/6-31G(d,p) and scaled data with their relative intensities.
Cyadox
1,4-BDC
Unscaled Scaled
Intensity
Observed Proposed assignments
Unscaled Scaled
Intensity
Observed Proposed assignments
644.41
659.23
675.95
699.61
752.45
769.09
795.65
805.64
839.67
875.8
651.9496
651.9496
666.943
631.59
692.63
701.23
781.71
785.07
813.11
829.56
853.24
898.62
905.2
922.81
947.81
967.78
990.02
990.92
998.35
1022.92
1031.22
1110.98
1151.05
1163.07
1224.17
1236.74
1242.51
1253.45
1305.23
638.9796
29.341
19.6106
2.8346
17.601
75.4873
24.0455
6.3874
633.9
x
N17AH18
666.943
683.8586
707.7954
761.2537
778.0884
804.9591
815.066
849.4941
886.0469
896.7506
913.1503
916.7115
959.0916
993.0948
1003.111
1018.681
1041.242
1054.91
1128.389
1131.384
1155.301
1203.741
1213.666
1240.203
1241.842
1261.823
1272.617
1315.514
1383.388
1392.463
1402.54
1415.247
1422.825
1452.073
1459.903
1491.276
1503.973
1547.041
1582.552
1658.925
1662.162
1683.216
1810.417
2398.579
3099.667
3129.795
3221.04
s
CAC(ring)
700.7338
709.4344
790.856
794.2553
822.6234
839.2659
683.8586
707.7954
761.2537
778.0884
804.9591
815.066
849.4941
886.0469
896.7506
913.1503
916.7115
959.0916
993.0948
1003.111
1018.681
1041.242
1054.91
1128.389
1131.384
1155.301
1203.741
1213.666
1240.203
1241.842
1261.823
1272.617
1315.514
1383.388
1392.463
1402.54
1415.247
1422.825
1452.073
1459.903
1491.276
1503.973
1547.041
1582.552
1658.925
1662.162
1683.216
1810.417
2398.579
3099.667
3129.795
3221.04
757.38
dCAC(ring)
CAH(ring)
792.9
x
CAH(ring)
x
863.2229 128.7664
859.21
899.65
mC19AC20
909.1339
915.7908
933.6069
958.8994
979.103
1001.603
1002.514
1010.031
1034.888
1043.285
1123.978
1164.517
1176.678
1238.493
1251.21
9.7762
0.877
866.35
891.6
m
sC21AC22
886.38
902.59
906.11
dC16AH,dC28AH(ring)
dC16AH,dCAH(ring)
dC16AH,dCAH(ring)
13.9699
3.3562
9.1105
1.1405
9.6866
2.846
0.0025 1016.88
4.3696
158.3673
6.9885 1148.57
2.5063
0.0779 1214.57
4.8385
29.6208 1243.79
357.8089
0.216
926.68
945.31
x
qCH2
C14AH15
917.85
941.65
948
q
CH2
m
m
C20AC23
C20-C23
981.61
991.51
1006.9
m
s
s
sC22AC25
(ring)
(ring)
978.19
dsCAH(ring)
dsCAH(ring)
1029.2
m
m
C7AC8(ring)
sN16AN17
1042.71
1115.34
1118.3
1141.94
1189.82
1199.63
1225.86
1227.48
1247.23
1257.9
1300.3
1367.39
1376.36
1386.32
1398.88
1406.37
1435.28
1443.02
1474.03
1486.58
1529.15
1564.25
1639.74
1642.94
1663.75
1789.48
2370.84
3063.82
3093.6
1088.79
1126.98
ds(ring)
q
q
CAH(ring)
CAH(ring)
m
sNAN
sCH2
1257.047
1268.115
1320.501
1330.649
1362.264
1390.47
s
CH2
x
CH2
1291.98
dsCAN(ring)
1315.26
1346.51
1374.39
1388.86
N12AO14(ring) 1401.51
1434.7
N13AO15(ring) 1452.28
1468.52
1503.57
0.7127 1320.69
4.5508 1349.55
221.8208
36.6757 1386.95
14.1606 1413.03
19.6322
m
CAN(ring),
mCAC(ring)
1405.11
m
CAC(ring)
1359.17
1384.01
m
m
m
ring,dsCH2,
ring,dsCH2
ring,dsCH2,
m
m
1417.908
1451.486
1469.272
1485.702
1521.162
1545.625
1595.795
1616.241
1669.538
1686.474
1805.52
2397.759
3100.102
3130.321
3192.045
3204.904
3216.336
3227.728
3239.241
3243.52
q
C14AH15
dsCH2
x
x
8.8134
114.8119 1463.61
(ring)
N17AH18
1454.15
dsCH2
21.127
28.0718 1515.94
8.68 1574.2
2.0552 1594.27
7.8883 1611.2
39.8599
450.9498 1778.51
11.507
3.1966 2954.09
0.5998 3077.69
10.005
3.1123
1.7506
3.3602
4.66
10.4197 3203.4
19.0639 3464.17
1494.23
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
s(ring)
s(ring)
s(ring)
s(ring)
1527.75
1577.34
1597.55
1650.23
1666.97
1784.64
2370.03
1503.94
1537.92
1579.22
m
m
m
m
m
ring
ring
ring
ring
ring
as(ring)
sC14AN16
sC19AO25,
sC23AN24
sC20AH
asC20AH
sC26AH15
sC14AH27(ring)
asCAH(ring)
sCAH(ring)
xCH2,xN17AH18
2264.33
3064.25
m
m
m
m
m
m
m
m
m
m
m
sCO,x
CH2,
x
NAH
3094.12
3155.13
3167.84
3179.14
3190.4
3201.78
3206.01
3509
2262
sCN
sCH2
asCH2
sCAH
asCAH(ring)
sCAH(ring)
asCAH(ring)
sCAH(ring)
sCAH(ring)
sNAH
3029.67
3083.1
3150.55
3183.79
3188.84
3202.22
3230.5
3232.55
3242.66
3496.42
3226.149
3239.686
3268.297
3270.371
3280.599
3537.328
3226.149
3239.686
3268.297
3270.371
3280.599
3537.328
asCAH(ring)
sCAH(ring)
sN17AH18
3550.055
3213.72
3440.66
As shown in Table 1, some bond lengths are shorter than the
experimental values. For example, C2AC5, C3AC6, and C6AC7
experimental bond lengths are 1.404, 1.401, and 1.377, respec-
tively, whereas the calculated bond lengths for cyadox are 1.401,
1.399, and 1.375. This difference is caused by two NAO bonds in
the cyadox which exhibit stronger interaction than the experimen-
tal compound. Most of the computed bond lengths are slightly lar-
ger than the experimental data in the crystal. This result is related
to the fact that the experimental data belong to the solid phase,
whereas the theoretical calculations are based on the gaseous
phase. Therefore, the intermolecular coulombic interactions
among neighboring molecules were ignored during calculation.
The differences between cyadox and 1,4-bisdesoxycyadox are also
notable. Without the strong NAO bond, several calculated bond
3. Results and discussion
3.1. Geometrical structure
The initial task for the calculation was to determine the opti-
mized geometries of the title compounds. Due to the unavailability
of the exact crystal structures of these compounds, the optimized
structures can only be compared with 3-methylquinoxaline-2-
carboxilic acid 4-oxide (determined by X-ray studies) [13], which
share highly similar quinoxaline structures with cyadox and 1,4-
bisdesoxycyadox. The optimized parameters (bond lengths, bond
angles) were compared with the experimental data listed in Ta-
ble 1, in accordance with the atom numbering scheme of cyadox
shown in Fig. 2.

Z. Li et al. / Journal of Molecular Structure 1035 (2013) 69–75
73
Table 3
several molecular properties with good performance. DFT calcula-
tions on harmonic frequencies provide better vibrational frequen-
cies for organic compounds when proper scaling factor is chosen to
correct the calculated frequencies, thus compensating for the
approximate treatment of electron correlation, unharmonious
effects, and basis set deficiencies [15]. Therefore, in this study, the-
oretical harmonic frequencies were scaled by the factor for B3LYP/
6-311+G(d,p) to correct the theoretical error [16]. IR spectrums
from the experiment and B3LYP method are shown in Fig. 3 and
Fig. 4. Assignment of several vibrational frequencies in both theo-
retical and experimental FT-IR are listed in Table 2.
The characteristic CAH stretching vibrations in the quinoxaline
heteroaromatic ring were observed above 3000 cmꢀ1. In the exper-
imental spectrum, the CAH stretching frequencies were
3213.7 cmꢀ1 for cyadox and 3203.4 cmꢀ1 for 1,4-BDC. The theoret-
ical vibrations assigned to the same type of vibration were 3268.3,
3270.4, and 3280.6 cmꢀ1 for cyadox and 3227.7, 3239.2, and
3243.5 cmꢀ1 for 1,4-BDQ. The intense deformation band and the
scissoring CAH bond of the methyl and methylene groups lie in
1386.0–1454.2 cmꢀ1 for cyadox and 1268.1–1451.5 cmꢀ1 for 1,4-
BDC. The CAH vibrations of in-plane were observed at 917.9 and
945.3 cmꢀ1 for the title compounds.
Computed, scaled and experimental NMR data for cyadox and 1,4-BDC.
Cyadox
1,4-BDC
Cal.
Scaled
Exp.
Cal.
Scaled
Exp.
C1
C2
C3
H4
C5
C6
C7
C8
139.17
147.31
147.50
9.04
128.04
128.32
137.97
138.24
8.99
8.08
8.08
134.56
7.76
8.92
135.68
143.60
143.78
9.02
124.84
125.12
134.52
134.78
8.97
8.09
8.08
131.19
7.77
8.90
137.31
137.76
138.00
9.05
119.95
120.13
132.24
132.74
8.54
8.49
8.49
133.74
7.99
7.97
C1
C2
C3
H4
C5
C6
C7
C8
139.49
146.65
146.88
8.98
126.67
126.58
139.30
139.34
8.91
8.30
8.29
135.61
7.82
9.22
135.99
142.96
143.19
8.96
123.51
123.42
135.80
135.84
8.89
8.30
8.29
132.21
7.83
9.20
142.84
143.15
147.99
9.52
129.21
130.92
131.05
130.98
8.19
8.13
8.13
141.30
7.88
7.91
H9
H9
H10
H11
C16
H17
H20
C21
C22
H23
H24
C25
C28
H29
MAE
H10
H11
C16
H17
H20
C21
C22
H23
H24
C25
C28
H29
MAE
171.55
26.96
3.86
167.20
26.46
3.98
165.90
24.50
4.43
173.06
26.77
4.16
168.66
26.27
4.27
165.67
24.58
2.52
3.78
3.90
4.43
4.12
4.23
2.52
115.70
133.69
8.55
112.83
130.34
8.54
116.02
127.70
12.37
120.82
135.58
8.68
117.82
132.18
8.67
116.05
129.23
12.29
3.38
2.2
3.21
2.56
Majority of the CAC and CAN vibrations are bound to the whole
ring, so these frequencies are discussed together with the skeletal
vibrations. Wave numbers occurring within 1503.9–1662.2 cmꢀ1
for cyadox and 1494.2–1686.5 cmꢀ1 for 1,4-BDC were assigned to
MAE: mean absolute error.
the
m CAC (ring) and the m CAN (ring). These specific bands were
found in the experiment IR spectrum of our previous quinoxaline
molecule study [17]. Cyadox shows
a
strong band at
1359.2 cmꢀ1, assigned to the stretching N@O in the experimental
spectrum. The calculated value is predicted to be 17 cmꢀ1 higher
at 1376 cmꢀ1. These vibrational frequencies cannot be seen for
1,4-BDC.
3.3. NMR spectra
The characterization of the compound was further enhanced by
the use of 1H and 13C NMR spectroscopy. The 1H and 13C NMR spec-
tra of the title compounds were recorded using TMS as an internal
standard and dimethylsulfoxide (DMSO-d6) as solvent. GIAO 1H
and 13C chemical shift values (with respect to TMS) were calcu-
lated using the B3LYP method with the 6-311G(d,p) basis set and
compared with the experimental 1H and 13C chemical shift values.
The calculated 1H and 13C isotropic chemical shieldings for TMS at
the B3LYP/6-311++G(2df,2dp) level in DMSO through the IEFPCM
method were 31.71 and 184.55 ppm. The theoretical and experi-
mental chemical shifts, isotropic shielding tensors, and peak
assignments for cyadox and 1,4-BDC are summarized in Table 3.
The linear correlation between the calculated and experimental
values was determined, and the results are presented in Fig. 5. A
good correlation between predicted and observed 13C and 1H
chemical shifts was found. Moreover, the slope and intercept of
the least-square correlation lines were used to scale the GIAO iso-
tropic absolute shielding constants r and to scale chemical shifts in
Fig. 5. The linear regression between the experimental and theoretical 1H and 13C
NMR chemical shifts of title compounds.
lengths, namely, C2AN12, C1AN12, C3AN13, and N13AC28, are
shorter than their parental compounds. In cyadox and 1,4-
bisdesoxycyadox, the theoretical results of the CACAC angles for
the benzene ring fall on the typical hexagonal angle of 120°. This
finding is in contrast to the quinoxaline moiety, in which substitu-
tion leads to some changes in the bond angles. This finding is
consistent with the characteristics of other substituted
quinoxalines[14].
3.2. Vibrational frequency
DFT revolutionalized theoretical modeling. The development of
exchange-correlation functions made possible the calculation of
Fig. 6. Enzimatic reduction of QdNO derivatives drug yielding radical intermediates.

74
Z. Li et al. / Journal of Molecular Structure 1035 (2013) 69–75
Fig. 7. First, second, total and mean NAO BDEs for cyadox were computed at the B3LYP/6-311++G(2df,2dp) level of theory.
the equation d = (dcalc-intercept)/slope. In this study, the intercept
and slope for B3LYP/6-311++G(2df,2dp) level combined with
IEFPCM solvent model are ꢀ0.2271 and 1.0274, respectively.
Results of the comparison with experimental observations are pre-
sented in the correlation graphs based on the calculations. The cor-
relation coefficient is 0.9967. As shown in Table 3, the mean
absolute values for cyadox and 1,4-BDC are 3.38 and 3.21, whereas
the corrected MAE is 2.2 and 2.56 ppm.
The chemical shifts of carbon atoms belonging to the quinoxa-
line moiety observed over 120 ppm are obviously higher than the
carbon on the branched chain C22 and C25, except for C21. This
phenomenon is due to the carbonyl (C@O) atom. The differences
of chemical shifts between cyadox and 1,4-bisdesoxycyadox are
also obvious. In this study, the 13C chemical shifts of quaternary
carbons C2, C3, C1, and C28 of 1,4-BDC are higher than those of
cyadox in terms of the differences in the electronic interactions
with the O atom. Moreover, with the metabolizing of O atom which
belongs to the NAO bond, the inductivities of the N atom to H atom
decrease. The loss of O atom also increases the electron density on
the whole conjugate ring. Based on these observation, the chemical
shifts of H9, H10, H11, and H29 are high and experimentally ob-
served as 8.19, 8.13, 8.13, and 12.29 in 1,4-BDC.
di-N-oxide compound to yield the corresponding N-oxide, whereas
the second NAO BDE is the energy required to break the other
bond. The total and mean NAO BDE are the sum and mean of the
dissociation enthalpies that form the corresponding N-oxide.
The calculated values for the BDE of cyadox are schematically
depicted in Fig. 7. The dissociation of the 12NA14O bond, which
is closer to the branched chain, occurred easily than tꢀh1e
13NA15O bond and yielded a first BDE value of 253.1 kJ mol
.
,
The energy required to remove the 15O atom was 262.1 kJ molꢀ1
almost 10 kJ molꢀ1 higher than the first O atom. The corresponding
value of the total and mean NAO BDE were 525.2 and
525.2 kJ molꢀ1, respectively.
4. Conclusion
In this study, 1,4-Bisdesoxycyadox, a main deoxidized metabo-
lite of cyadox, was synthesised and characterized by spectroscopic
techniques (FT-IR and NMR). To support the solid-state structure,
geometric parameters were optimized using B3LYP method with
6-311+G(d,p) basis set. The results were compared with the X-
ray diffraction data of similar molecular compounds. From the
optimized geometry of the molecule, vibrational frequencies of
the title compounds were calculated via B3LYP/6-311+G(d,p) ap-
proach and compared with the experimental data. The magnetic
isotropic shielding constants were calculated using GIAO/B3LYP/
6-311++G(2df,2pd) method. Linear correlations with the experi-
mental 1H and 13C chemical shifts were obtained. The computed
chemical shifts, scaled by the linear correlation equation, produced
the experimental results with MAE lower than 2.2 ppm. Moreover,
the first, second, total, and mean NAO bond dissociation enthalpies
(BDEs) were obtained theoretically. The predicted values based on
3.4. NAO BDE calculation
With the development of QdNO derivatives and progress in
drug safety, several studies which feature experimental validation
or theoretical calculation resulted in remarkable achievements.
The most important highlights of these breakthroughs are the rad-
ical intermediates that they enzymatically reduced in vivo, causing
cytotoxic DNA strand breaks and pharmaceutical effect. This mech-
anism (Fig. 6) is widely accepted by the formation of these inter-
mediates including drug radical and hydroxyl radical, which is
directly dependent on the NAO bond [18]. Therefore, the measure
and evaluation of NAO bond strength is important for systematic
research of QdNOs drugs. For example, the high NAO BDE of tira-
pazamine (TPZ) yields corresponding activated TPZ, which causes
high DNA damage. However, limited NAO BDE data are available
for QdNO veterinary drug. However, studying this structure-
related parameter for cyadox is necessary. In this study, the first,
second, total, and mean NAO BDE of cyadox are calculated in
B3LYP/6-311++G(2df,2dp) level of theory. Typically, the first NAO
BDE is the energy required to break the weaker bond in the
6-311++G(2df,2pd) were 253.1, 262.1, 525.2, and 525.2 kJ molꢀ1
respectively.
,
Acknowledgements
This work was supported by 973 Fund, the Ministry of Science
and Technology, P.R. China (Grant No. 2009CB118801), the
National Natural Science Foundation of China (Project No.
20977112), Program for New Century Excellent Talents in Univer-
sity and the Specialized Research Fund for the Doctoral Program of
Higher Education of China New Teachers’ Fund for Doctor Stations,
Ministry of Education (Grant No. 20090008120015).

Z. Li et al. / Journal of Molecular Structure 1035 (2013) 69–75
[11] E. Cancès, B. Mennucci, J. Tomasi, J. Chem. Phys. 107 (1997) 3032.
75
References
[12] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M.
Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y.
Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery, J.E. Peralta, F. Ogliaro, M.
Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J.
Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi,
Gaussian 09, ReVision A.02, Gaussian, Inc., Wallingford, CT, 2009.
[1] D. Zhao, L. He, C. Pu, A. Deng, Anal. Bioanal. Chem. 391 (2008) 2653.
[2] Q. Chen, S. Tang, X. Jin, J. Zou, K. Chen, T. Zhang, X. Xiao, Food Chem. Toxicol. 47
(2009) 328.
[3] X. Wang, Q. He, Y. Wang, A. Ihsan, L. Huang, W. Zhou, S. Su, Zhen Liu, Z. Yuan,
Regul. Toxicol. Pharm. 59 (2011) 324.
[4] X. Huang, H. Zhang, X. Wang, L. Huang, L. Zhang, C. Yan, Y. Liu, Z. Yuan, Chem-
Biol. Interact. 185 (2010) 227.
[5] H.C. Zhang, C. Huang, Environ. Sci. Technol. 39 (2005) 593.
[6] L. Huang, Y. Wang, Y. Tao, D. Chen, Z. Yuan, J. Chromatogr. B 874 (2008) 7.
[7] Z. Liu, L. Huang, M. Dai, D. Chen, Y. Wang, Y. Tao, Z. Yuan, Rapid Commun. Mass
Spectrom. 22 (2008) 1009.
[13] Y.B. Li, W.F. Zhou, J.L. Wang, H.X. Gao, Z.Q. Zhou, Acta Cryst. E 66 (2010) o1801.
[14] Sß. Yurdakul, T. Polat, J. Mol. Struct. 963 (2010) 194.
_
}
[15] E. Inkaya, M. Dinçer, O. Ekici, A. Cukurovali, J. Mol. Struct. 1026 (2012)
117.
[16] J.P. Merrick, D. Moran, L. Radom, J. Phys. Chem. A 111 (2007) 11683.
[17] J.H. Zhang, X. He, H.X. Gao, J. Mol. Struct. 1004 (2011) 109.
[18] J.R.B. Gomes, M.D.M.C. Ribeiro da Silva, M.A.V. Ribeiro da Silva, Chem. Phys.
Lett. 429 (2006) 18–22.
[8] P.J. Stephens, F.J. Devlin, C.F. Chabalowski, M.J. Frisch, J. Phys. Chem. 98 (1994)
11623.
[9] A.D. Becke, J. Chem. Phys. 98 (1993) 5648.
[10] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785.