Design, synthesis and biological evaluation of 1-hydroxy-2-phenyl-4-pyridyl-1H-imidazole derivatives as xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.01.060

In our previous study, we reported a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic acid derivatives that presented excellent in vitro xanthine oxidase inhibitory potency. As a continuation study, a series of 1-hydroxy-2-phenyl-1H-imidazole derivatives containing a pyridine moiety (4a-g and 5a-g) at the 4-position was designed and synthesized. Evaluation of in vitro xanthine oxidase inhibition demonstrated that the 4a-g series was more potent than the 5a-g series. Compound 4f was the most promising derivative in the series with an IC₅₀ value of 0.64 μM. A Lineweaver-Burk plot revealed that compound 4f acted as a mixed-type xanthine oxidase inhibitor. An iso-pentyloxy group at the 4′-position improved the inhibitory potency. More interestingly, structure-activity relationship analysis indicated that the pyridine para-N atom played a crucial role in the inhibition. Molecular modeling provided a reasonable explanation for the structure-activity relationships observed in this study. In addition, a three dimensional quantitative structure-activity relationships model which possessed reasonable statistics (q² = 0.885 and r² = 0.993) was conducted to further understand the structural basis of these compounds as xanthine oxidase inhibitors. These compounds, especially compound 4f, have good potential for further investigations.

Full text of DOI:10.1016/j.ejmech.2018.01.060

Accepted Manuscript
Design, synthesis and biological evaluation of 1-hydroxy-2-phenyl-4-pyridyl-1H-
imidazole derivatives as xanthine oxidase inhibitors
Tingjian Zhang, Yunying Lv, Yu Lei, Dan Liu, Yao Feng, Jiaxing Zhao, Shaolei Chen,
Fanhao Meng, Shaojie Wang
PII:
S0223-5234(18)30073-4
10.1016/j.ejmech.2018.01.060
EJMECH 10137
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 November 2017
Revised Date: 5 January 2018
Accepted Date: 18 January 2018
Please cite this article as: T. Zhang, Y. Lv, Y. Lei, D. Liu, Y. Feng, J. Zhao, S. Chen, F. Meng, S.
Wang, Design, synthesis and biological evaluation of 1-hydroxy-2-phenyl-4-pyridyl-1H-imidazole
derivatives as xanthine oxidase inhibitors, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.01.060.
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ACCEPTED MANUSCRIPT
and biological
Design,
synthesis
evaluation
of
1-hydroxy-2-phenyl-4-pyridyl-1H-imidazole derivatives as xanthine oxidase
inhibitors
Tingjian Zhanga, b, 1, Yunying Lva, 1, Yu Leia, Dan Liua, Yao Fenga, Jiaxing Zhaoa, Shaolei Chena, Fanhao
Mengb, *, Shaojie Wanga, *
aKey Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang 110016, China
bSchool of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122, China
*Corresponding authors.
Tel/Fax: +86-24-43520230, E-mail: sjwang_99@163.com (S. J. Wang);
Tel.: +86-24-31939448, E-mail: fhmeng@cmu.edu.cn (F. H. Meng).
1 These two authors contributed equally to this work.
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Abstract
In our previous study, we reported a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic acid derivatives that
presented excellent in vitro xanthine oxidase inhibitory potency. As
a continuation study, a series of
1-hydroxy-2-phenyl-1H-imidazole derivatives containing a pyridine moiety (4a-g and 5a-g) at the 4-position was designed
and synthesized. Evaluation of in vitro xanthine oxidase inhibition demonstrated that the 4a-g series was more potent than
the 5a-g series. Compound 4f was the most promising derivative in the series with an IC50 value of 0.64 µM. A
Lineweaver-Burk plot revealed that compound 4f acted as a mixed-type xanthine oxidase inhibitor. An iso-pentyloxy group
at the 4′-position improved the inhibitory potency. More interestingly, structure-activity relationship analysis indicated that
the pyridine para-N atom played a crucial role in the inhibition. Molecular modeling provided a reasonable explanation for
the structure-activity relationships observed in this study. In addition, a three dimensional quantitative structure-activity
relationships model which possessed reasonable statistics (q2 = 0.885 and r2 = 0.993) was conducted to further understand
the structural basis of these compounds as xanthine oxidase inhibitors. These compounds, especially compound 4f, have
good potential for further investigations.
Keywords: 1-Hydroxy-2-phenyl-4-pyridyl-1H-imidazole; Synthesis; Xanthine oxidase inhibitor; Hyperuricemia
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1. Introduction
Xanthine oxidase (XO) is a key enzyme in purine metabolism that catalyzes the hydroxylation of both
hypoxanthine and xanthine in the last two steps of urate biosynthesis in humans [1, 2]. In parallel with the
hydroxylation, reactive oxygen species (ROS) are produced [3]. Therefore, inhibition of XO not only
effectively reduces the production of uric acid for the treatment of hyperuricemia and gout, but also
benefits the pathological conditions caused by the XO-derived ROS such as oxidative damage,
post-ischemic reperfusion injury, diabetes and chronic heart failure [4-6]. Allopurinol (Fig. 1), a prototype
XO inhibitor and a hypoxanthine isomer, has been widely prescribed in the treatment of hyperuricemia and
gout for several decades. However, in some cases, severe life-threatening side effects, including fulminant
hepatitis, renal failure, and Stevens–Johnson syndrome, due to the similar backbone with purine have been
reported [7]. Under these limitations, there is an immense need for non-purine XO inhibitors to be
developed with potent XO inhibitory activity but fewer side effects.
In recent years, a great amount of non-purine XO inhibitors have been reported. Febuxostat [8] (Fig. 1)
is a novel non-purine XO inhibitor that showed a more potent and longer-lasting urate-lowering effect than
allopurinol and was approved in USA in 2009 for the treatment of gout. Topiroxostat (also known as
FYX-051, Fig. 1) is another type of XO inhibitor approved in Japan in 2013. It not only interacts with
amino acid residues of the active pocket but also forms a covalent linkage to molybdenum via oxygen in
the hydroxylation reaction process [9]. In addition, other XO inhibitors have been reported in the literature
including Y-700 [10], selenazoles [11], 1,2,3-triazoles [12], 2-(indol-5-yl)thiazoles [13], phenyl-1,2,4-
triazoles [14], 4-(pyridin-4-yl)-1,2,3-triazoles [15], isonicotinamides [16], isocytosines [17-20], isoxazoles
[21], N-(1,3-diaryl-3-oxo-propyl)amides [22], N-acetyl pyrazolines [23], chalcones [24], fraxamoside [25],
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pyrano[3,2-d]pyrimidines
[26],
benzaldehydes
[27],
2-arylbenzo[b]furans
[28]
and
2-benzamido-4-methylthiazole-5-carboxylic acids [29].
In our previous report, we employed an imidazole moiety, as an isostere, to replace the thiazole of
Febuxostat and designed a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic acid derivatives [30].
These compounds displayed excellent in vitro potency, especially, compounds 1-3 (Fig. 1) showed higher
activity than the positive control Febuxostat. Structure activity relationship (SAR) analysis and molecular
modeling studies indicated that the 1-hydroxy could form an extra H-bond with Thr1010 and contribute to
the inhibitory potency [30]. The results inspired us to further explore novel XO inhibitors based on the
1-hydroxyimidazole moiety. With compounds 1-3 and Topiroxostat as leading compounds, in this study,
we retained the 1-hydroxy-2-phenylimidazole fragment of 1-3 and hybridized the pyridine moiety of
Topiroxostat (Fig. 2) to obtain a series of 1-hydroxy-2-phenyl-4-pyridyl-1H-imidazole derivatives (4a-g
and 5a-g) by the molecular hybrid method, hoping that, on one hand, the 1-hydroxy-2-phenylimidazole
fragment could maintain its interactions with XO, and on the other hand, the pyridine moiety could pick up
an interaction mode with XO similar to that of Topiroxostat. In addition, molecular modeling studies, three
dimensional quantitative structure-activity relationships (3D-QSAR) and steady-state kinetic analysis were
also performed to investigate the inhibition behaviors of these compounds.
2. Results and discussion
2.1. Chemistry
The synthesis of 2-(4-alkoxy-3-cyanophenyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazoles (4a-f) and
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2-(4-alkoxy-3-cyanophenyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazoles (5a-f) was performed as outlined
in Scheme l. Commercially available 4-hydroxybenzaldehyde was brominated with bromine catalyzed by
iodine in dichloromethane to obtain 3-bromo-4-hydroxybenzaldehyde (6), which was alkylated with
benzyl bromide in DMF in the presence of anhydrous potassium carbonate to provide
3-bromo-4-benzoxybenzaldehyde (7). Compound 7 was cyanided with cuprous cyanide followed by the
removal of the benzyl protecting group under hydrogen atmosphere catalyzed by Pd/C in THF to yield the
key intermediate 9. Compound 9 was alkylated with various alkyl chlorides or alkyl bromides to obtain
4-alkoxy-3-cyanobenzaldehydes (10a-f).
Commercially available 1-(pyridin-4-yl)ethan-1-one and 1-(pyridin-3-yl)ethan-1-one were treated with
tert-butyl nitrite in a sodium ethoxide solution to provide 2-oxo-2-(pyridin-4-yl)acetaldehyde oxime (11)
and 2-oxo-2-(pyridin-3-yl)acetaldehyde oxime (12), respectively. Cyclization of 11 and 12 with
intermediates 10a-f led to 2-(4-alkoxy-3-cyanophenyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazoles (4a-f)
and 2-(4-alkoxy-3-cyanophenyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazoles (5a-f), respectively. By the
same procedure, compounds 11 and 12 cyclized with commercially available 3-cyanobenzaldehyde
provided
2-(3-cyanophenyl)-1-hydroxy-4-(pyridine-4-yl)-1H-imidazole
(4g)
and
2-(3-cyanophenyl)-1-hydroxy-4-(pyridine-3-yl)-1H-imidazole (5g), respectively (Scheme 2).
The HPLC purity of all the synthesized compounds was over 97%. The structures were elucidated by
MS, IR, 1H NMR, and 13C NMR spectra. All spectral data were in accordance with the assumed structures.
In ESI-MS analysis, the target compounds showed [M-OH+2H]+ ion peaks. The IR spectra of the target
compounds displayed cyano stretching vibrations at 2223-2230 cm-1 and hydroxyl stretching vibrations at
3425-3429 cm-1. In 1H NMR spectra, the 1-hydroxy group of imidazole was observed as a broad singlet at
approximately 12.79 ppm in series 4a-g and 13.13 ppm in series 5a-g, respectively.
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2.2. Biological activity
The in vitro bovine XO inhibitory activity of compounds 4a–g and 5a–g was measured
spectrophotometrically by determining uric acid production at 295 nm. Topiroxostat was included as a
reference compound. The testing results are shown in Table 1.
As shown in Table 1, half of the synthesized compounds were effective and possessed micromolar
level potency. Although the effects were much lower than that of the positive control Topiroxostat and
compounds 1-3, some interesting SAR information was obtained. In general, the 4a-g series seemed to be
more potent than the 5a-g series overall. The majority of the compounds in the 4a-g series were effective
with IC50 values ranging from 0.64 to 15.42 µM. Meanwhile, compound 4f, which linked an iso-pentyl for
R1 group, displayed the highest activity with an IC50 value of 0.64 µM, and was 10-fold more effective
than its counterpart 5f (IC50 = 6.73 µM). Additionally, a dose-dependent inhibition of XO by 4f was
exhibited (Fig. 3). Moreover, the ether tail at the 4′-position played a crucial role in the potency; its
removal was accompanied by vanished potency (e.g., 4a, 4c, 4e and 4f versus 4g; 5b, 5d and 5f versus 5g).
Furthermore, it can be found that from 4a to 4c, 4e and 4f, the inhibitory effects were enhanced 3.2-fold,
3.5-fold and 24-fold, respectively, which suggested that as the size of the ether tail at the 4′-position
increased, the potency of the 4a-g series was gradually enhanced (except 4b and 4d).
In contrast, the 5a-g series was less potent. Only three compounds (5b, 5d and 5f) were effective in this
series, which suggested that the pyridine para-N of 4a-g was much welcome than the pyridine meta-N of
5a-g. This result was also supported by the isonicotinamide series in our previous studies [16]. In addition,
compound 5f, bearing an iso-pentyloxy at the 4′-position, exhibited relatively high potency with an IC50
value of 6.73 µM, which indicated that employment of an iso-pentyl as R1 group improved the potency of
both series, 4a-g and 5a-g.
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2.3. Molecular modeling
To foresee the possible interactions of the synthesized compounds with XO and to rationalize the SARs
observed in this study, molecular modeling simulations of Topiroxostat, 4f and 5f in the binding pocket of
XO were performed. The simulations were carried out with Autodock 4 software package by a similar
protocol to that reported in our previous study [30]. The crystal structure of bovine XO in complex with
Febuxostat (PDB code: 1N5X) was adopted in docking calculations.
As shown in Fig. 4, the 1-hydroxy-2-phenyl-1H-imidazole moiety of 4f and 5f was located at a similar
position in the XO active pocket and presented a set of similar interactions to compound 3 [30] such as the
cyano group forming an H-bond with Asn768, the N-3 atom of imidazole accepting an H-bond from
Glu802 and the ether tail being surrounded by some lipophilic amino acid residues, e.g., Leu648 and
Lys771. In particularly, the 1-hydroxy group formed an extra hydrogen bond with Thr1010 as expected.
Moreover, the pyridine moiety of 4f and 5f could overlap with that of Topiroxostat in the active pocket [9].
However, an obvious difference between the docking models of 4f and 5f was that the pyridine para-N of
4f formed an H-bond with Glu1261, just as Topiroxostat did; in contrast, meta-N of 5f was absent from this
interaction. This difference is consistent with their levels of inhibition potency.
2.4 3D-QSAR analysis
To further understand the structural basis of these compounds for XO inhibitory activity, we
subsequently performed 3D-QSAR study using Topomer CoMFA method. The active compounds (ie, 4a,
4c, 4e, 4f, 5b, 5d and 5f) were used in 3D-QSAR model building. In order to achieve a rational result, we
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combined other published Topiroxostat-like compounds to perform this 3D-QSAR model, including 8 of
1,2,3-triazole derivatives (compounds 13-20) synthesized in our previous studies [15] and 15 of
1,2,4-triazole derivatives (compounds 21-35) reported by Sato T et al [14]. Therefore, The Topomer
CoMFA study was carried out using a total of 30 compounds (Table 2), which randomly divided into
training set and test set (marked with asterisk in Table 2) in the ratio of 5:1. Statistical parameters of the
Topomer CoMFA model showed a reasonable cross-validated correlation coefficient q2 of 0.885, indicating
a good internal prediction ability of the model. The Topomer CoMFA model also exhibited a conventional
correlation coefficient r2 of 0.993 using 5 partial least-squares (PLS) components. The test set was used to
evaluate the predictive ability of our developed model. The experimental and predicted values as well as
their residuals from the training and test set molecules are listed in Table 2. The correlation between the
predicted and experimental values of all compounds was plotted as shown in Fig. 5.
The results were analyzed and visualized using the standard deviation coefficient (StDev*Coeff)
mapping option contoured by steric and electrostatic contributions [31]. The representative steric and
electrostatic contour maps of compound 4f derived from the Topomer CoMFA model are shown in Fig. 6.
In Fig. 6A, one big green contour was found around the ether tail of compound 4f, indicating that a steric
substituent would be favored for R1 group. This result could explain the gradually enhanced potency of
compounds 4a, 4c, 4e and 4f. In Fig. 6A and 6B, a small green contour and a blue contour near the
1-position of imidazole were observed, respectively, which indicated that an electropositive small steric
bulky group would be helpful for activity in this area. Furthermore, a few red contours in Fig. 6B near the
3-position of imidazole suggested that an electronegative group at this position would be welcome;
however, this position can’t tolerate a bulky group according to several yellow contours in Fig. 6A. In Fig.
6C, a yellow contour was observed near the pyridine para-N of 4f, meaning that substituent at this position
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would be disfavored for potency. In Fig. 6D, one blue contour was observed at the meta-position of
pyridine, indicating that the presence of positively charged group at this position may benefit the XO
inhibitory potency.
2.5. Steady-state kinetic analysis
To further investigate the action mode of compounds 4a-g with XO, enzyme kinetics studies of the
representative compound 4f were carried out (Fig. 7). Lineweaver-Burk plot analysis indicated that 4f
acted as a mixed-type inhibitor on XO. This behavior was distinct from Topiroxostat, but similar to
hydroxy-topiroxostat, a metabolite of Topiroxostat with micromolar potency [32]. The different inhibition
type may contribute to the decreased potency of 4f compared with Topiroxostat.
3. Conclusions
In
summary,
we
designed,
synthesized
and
identified
a
series
of
1-hydroxy-2-phenyl-4-pyridyl-1H-imidazole derivatives as novel XO inhibitors. Among them, compound
4f was found to be the most promising derivative in the series with an IC50 value of 0.64 µM. The
Lineweaver-Burk plot revealed that compound 4f acted as a mixed-type XO inhibitor. SAR analysis
indicated that the pyridine para-N atom played a crucial role in the inhibition, and an iso-pentyloxy group
at the 4′-position improved the inhibitory potency. Molecular modeling provided a reasonable explanation
for the SARs observed in this study and 3D-QSAR analysis explored the structural basis of these
compounds. As a result, 1-hydroxy-2-phenyl-4-(pyridin-4-yl)-1H-imidazoles, especially compound 4f,
have good potential for further investigations.
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4. Experimental protocols
4.1. Chemistry
Unless otherwise indicated, reagents and solvents were purchased from commercial sources and used
without further purification. All reactions were monitored by TLC using silica gel aluminum cards (0.2
mm thickness) with fluorescent indicator 254 nm. The column chromatography was performed using silica
gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). The purity was
determined by HPLC (Agilent 1260) by a C18 (250*4.6 mm, 5 µm) column with 0.02 M ammonium
dihydrogen phosphate/acetonitrile (50: 50) as the mobile phase .The UV detection wavelength was 254 nm.
Melting points were recorded on a YRT-3 melting apparatus and were uncorrected. 1H NMR spectra were
recorded on a Bruker 300 MHz spectrometer, a Bruker 400 MHz spectrometer or a Bruker 600 MHz
spectrometer and 13C NMR spectra were recorded on a Bruker 600 MHz spectrometer. Chemical shifts
were expressed in parts per million using tetramethylsilane as an internal reference and CDCl3 or
DMSO-d6 as the solvent. ESI-MS data were gathered using an Agilent 1100 instrument.
4.1.1. Synthesis of 3-bromo-4-hydroxybenzaldehyde (6)
A mixture of 4-hydroxybenzaldehyde (100.0 g, 0.82 mol) and iodine (5.2 g, 21 mmol) in
dichloromethane (500 mL) was stirred at -5 for 15 min, then a solution of bromine (138.0 g, 0.86 mol) in
dichloromethane (300 mL) was slowly added dropwise to maintain the reactive temperature below 0 .
After the completion of the addition, the mixture was reacted at room temperature for 24 h, then diluted
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with 0.9% sodium bisulfite (1000 mL) and stirred for 30 min. The formed precipitate was collected by
filtration, washed with water (100 mL) and dried to give 3-bromo-4-hydroxybenzaldehyde (133.0 g, yield
81.1%) as an off-white solid, which was used directly in the next step. MS (ESI) m/z: 198.9, 200.9 [M-H]-.
1H NMR (600 MHz, DMSO-d6) δ 11.50 (s, 1H, OH), 9.77 (s, 1H, CHO), 8.02 (d, J = 2.0 Hz, 1H, Ar-H),
7.75 (dd, J = 2.0 & 8.4 Hz, 1H, Ar-H), 7.10 (d, J = 8.4 Hz, 1H, Ar-H).
4.1.2. Synthesis of 4-benzoxy-3-bromobenzaldehyde (7)
A mixture of 3-bromo-4-hydroxybenzaldehyde (133.0 g, 0.67 mol), benzyl bromide (125.1 g, 0.73
mol), anhydrous potassium carbonate (183.5 g, 1.33 mol) and potassium iodide (2.2 g, 0.013 mol) in
DMF (700 mL) was reacted at 80 for 8 h under nitrogen atmosphere. After that, the insoluble solid was
filtered out and the filtrate was concentrated to remove near 2/3 of the solvent. The residue was diluted
with ethyl acetate, washed with water and brine, dried over anhydrous Na2SO4 and evaporated in vacuum
to afford 4-benzoxy-3-bromobenzaldehyde (174.4 g, yield 90.5%) as a slight yellow solid, which was
directly used in the next step. MS (ESI) m/z: 291.0, 293.0 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 9.85
(s, 1H, CHO), 8.12 (d, J = 1.9 Hz, 1H, Ar-H), 7.92 (dd, J = 1.9 & 8.5 Hz, 1H, Ar-H), 7.49 (d, J = 7.5 Hz,
2H, Ar-H), 7.45 – 7.39 (m, 3H, Ar-H), 7.36 (t, J = 7.3 Hz, 1H, Ar-H), 5.35 (s, 2H, CH2).
4.1.3. Synthesis of 4-benzoxy-3-cyanobenzaldehyde (8)
A mixture of 4-benzoxy-3-bromobenzaldehyde (93.0 g, 0.32 mol) and cuprous cyanide (37.2 g, 0.45
mol) in DMF (600 mL) was refluxed under nitrogen atmosphere for 6 h. The completed reaction was
determined by TLC. The mixture was cooled to room temperature. Then, dichloromethane (500 mL) and
ammonia (300 mL) were added. The oil layer was collected, successively washed with water and brine,
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dried over anhydrous Na2SO4 and evaporated in vacuum to provide a crude product as a slight yellow
solid, which was treated with methyl tert-butyl ether to yield 4-benzoxy-3-cyanobenzaldehyde as an
off-white solid (56.9 g, yield 75%). MS (ESI) m/z: 238.1 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 9.89
(s, 1H, CHO), 8.32 (d, J = 1.9 Hz, 1H, Ar-H), 8.18 (dd, J = 1.9 & 8.8 Hz, 1H, Ar-H), 7.55 (d, J = 8.8 Hz,
1H, Ar-H), 7.50 (d, J = 7.4 Hz, 2H, Ar-H), 7.44 (t, J = 7.5 Hz, 2H, Ar-H), 7.38 (t, J = 7.3 Hz, 1H, Ar-H),
5.41 (s, 2H, CH2).
4.1.4. Synthesis of 5-formyl-2-hydroxybenzonitrile (9)
A mixture of 4-benzoxy-3-cyanobenzaldehyde (10.0 g) and 10% Pd/C (0.50 g) in THF (40 mL) was
stirred at room temperature for 4 h under hydrogen atmosphere. After the completion of the reaction, the
Pd/C was filtered out and the filtrate was evaporated to give a slight yellow solid (5.0 g, yield 80%). 1H
NMR (600 MHz, DMSO-d6) δ 11.51 (s, 1H, OH), 9.77 (s, 1H, CHO), 8.04 (d, J = 1.8 Hz, 1H, Ar-H), 7.75
(dd, J = 1.8 & 8.4 Hz, 1H, Ar-H), 7.11 (d, J = 8.4 Hz, 1H, Ar-H).
4.1.5. 5-Formyl-2-methoxybenzonitrile (10a).
Into a mixture of 5-formyl-2-hydroxybenzonitrile (8.0 g, 0.04 mol) and anhydrous potassium carbonate
(11.0 g, 0.08 mol) in DMF (20 mL), dimethyl sulfate (5.6 g, 0.044 mol) in DMF (20 mL) was slowly
added dropwise at 0 . The resulting mixture was stirred at room temperature for 5 h, then poured into
water (40 mL) and stirred for 15 min. the resulting solid was filtered, washed with water and dried to give
1
3-cyano-4-methoxybenzonitrile (7.0 g, 79.4%) as a white solid. mp 70.5–72.3 °C. H NMR (300 MHz,
CDCl3) δ 9.88 (s, 1H, CHO), 8.10 (d, 1H, J = 2.0 Hz, Ar-H), 8.06 (dd, 1H, J = 2.1 & 8.8 Hz, Ar-H), 7.10 (d,
1H, J = 8.7 Hz, Ar-H), 3.85 (s, 3H, OCH3).
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4.1.6. General procedure for the synthesis of 2-alkoxy-5-formylbenzonitriles 10b-10f
A mixture of 5-formyl-2-hydroxybenzonitrile (0.04 mol), alkyl chloride or alkyl bromide (0.05 mol)
and anhydrous potassium carbonate (0.08 mol) in DMF (40 mL) was reacted at 80 for 6 h under nitrogen
atmosphere. After that, 2/3 of the solvent was removed by evaporation in vacuum. Then, water (40 mL)
and ethyl acetate (40 mL) were added. The organic layer was collected, washed with brine, dried over
anhydrous Na2SO4 to yield the corresponding 2-alkoxy-5-formylbenzonitriles 10b-10f.
4.1.6.1. 2-(Allyloxy)-5-formylbenzonitrile (10b)
A slight yellow oil, yield 90.4%. 1H NMR (600 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H),
8.15 (dd, J = 8.8
& 2.1 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 6.07 (m, 1H), 5.48 (dd, J = 17.3 & 1.6 Hz, 1H), 5.35
(dd, J = 10.6
& 1.4 Hz, 1H), 4.96 – 4.79 (m, 2H).
4.1.6.2. 5-Formyl-2-isopropoxybenzonitrile (10c)
A slight yellow solid, yield 82.4%. Mp 51.5–52.6 °C. 1H NMR (300 MHz, CDCl3) δ 9.88 (s, 1H, CHO),
8.09 (d, 1H, J = 2.0 Hz, Ar-H), 8.05 (dd, 1H, J = 2.1 & 8.7 Hz, Ar-H), 7.10 (d, 1H, J = 8.8 Hz, Ar-H), 4.80
(m, 1H, OCH), 1.46 (d, 6H, J = 6.1 Hz, 2CH3).
4.1.6.3. 5-Formyl-2-isobutoxybenzonitrile (10d)
A yellow oil, yield 93.2%. 1H NMR (300 MHz, CDCl3) δ 9.89 (s, 1H, CHO), 8.10 (d, 1H, J = 2.0 Hz,
Ar-H), 8.06 (dd, 1H, J = 2.1 & 8.7 Hz, Ar-H), 7.09 (d, 1H, J = 8.7 Hz, Ar-H), 3.95 (t, 2H, J = 6.5 Hz,
OCH2), 2.22 (m, 1H, CH), 1.01 (d, 6H, J = 6.8, 2CH3).
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4.1.6.4. 2-(sec-Butoxy)-5-formylbenzonitrile (10e)
A yellow oil, yield 90.5%. 1H NMR (300 MHz, CDCl3) δ 9.88 (s, 1H, CHO), 8.09 (d, 1H, J = 1.8 Hz,
Ar-H), 8.04 (dd, 1H, J = 1.8 & 8.8 Hz, Ar-H), 7.09 (d, 1H, J = 8.8 Hz, Ar-H), 4.56 (m, 1H, OCH), 1.79 (m,
2H, CH2), 1.41 (d, 3H, J = 6.1 Hz, CH3), 1.01 (t, 3H, J = 7.4, CH3).
4.1.6.5. 5-Formyl-2-isopentyloxybenzonitrile (10f)
A yellow solid, yield 87.8%., mp 47.1–48.5°C. 1H NMR (300 MHz, CDCl3) δ 9.90 (s, 1H, CHO), 8.09
(s, 1H, Ar-H), 8.06 (d, 1H, J = 8.8 Hz, Ar-H), 7.11 (d, 1H, J = 8.8 Hz, Ar-H), 4.21 (t, 2H, J = 6.5 Hz,
OCH2), 1.91 (m, 1H, CH), 1.80 (m, 2H, CH2), 1.00 (d, 6H, J = 6.6, 2CH3).
4.1.7. Synthesis of 2-oxo-2-(pyridin-4-yl)acetaldehyde oxime (11)
Into a sodium ethoxide solution prepared by dissolving the metal sodium (4.0 g, 0.18 mol) in
anhydrous ethanol (100 mL), 4-acetylpyridine (20.0 g, 0.17 mol) was added. The solution was stirred at -5
for 15 min, and then tert-butyl nitrite (21.9 g, 0.22 mol) was added dropwise. The reactive solution was
stirred at 0-10 for 24 h then frozen in the fridge for 24 h. The generated precipitate was collected by
filtration to provide a red solid (4.1 g, yield 82.0%). MS (ESI) m/z: 149.1 [M-H]-.
4.1.8. Synthesis of 2-oxo-2-(pyridin-3-yl)acetaldehyde oxime (12)
2-Oxo-2-(pyridin-3-yl)acetaldehyde oxime was synthesized by a procedure similar to that for the
preparation of 2-oxo-2-(pyridin-4-yl)acetaldehyde oxime with 3-acetylpyridine instead of 4-acetylpyridine.
A slight red solid (yield 78.0%). MS (ESI) m/z: 149.1 [M-H]-.
14

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4.1.9. General
2-(4-alkoxy-3-cyanobenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazoles
2-(4-alkoxy-3-cyanobenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazoles 5a-5g
procedure
for
the
synthesis
of
4a-4g
and
A mixture of 4-alkoxy-3-cyanobenzaldehyde (0.015 mol) and ammonium acetate (0.15 mol) in
methanol (30 mL) was added 2-oxo-2-(pyridin-4-yl)acetaldehyde oxime (0.018 mol) or
2-oxo-2-(pyridin-3-yl)acetaldehyde oxime (0.018 mol) for the synthesis of 4a-4g and 5a-5g, respectively.
The mixture was refluxed for 24 h under nitrogen atmosphere. The solvent was evaporated in vacuum and
the residue was treated with ethyl acetate (50 mL) and water (50 mL). The oil layer was separated and
washed with brine, dried over anhydrous Na2SO4 and evaporated in vacuum to obtain a crude product,
which was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the
corresponding 4a-4g and 5a-5g.
4.1.9.1. 2-(3-Cyano-4-methoxybenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4a)
A yellow solid (0.9 g, yield 21.4%). HPLC 98.2%. Mp 206.0 -208.6 . MS (ESI) m/z: 277.1
[M-OH+2H]+; 1H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H, NOH), 8.53 (s, 2H, Py-H), 8.27 (m, 2H,
Ar-H), 8.07 (s, 1H, Imidazole-H),7.79 (s, 2H, Py-H), 7.40 (s, 1H, Ar-H), 3.97 (s, 3H, CH3); 13C NMR (150
MHz, DMSO-d6) δ 160.73, 149.84, 144.86, 141.43, 138.58, 131.66, 129.89, 123.53, 118.79, 117.53,
116.09, 112.92, 100.71, 56.60; IR (KBr, cm-1) 3426.6, 2921.9, 2851.5, 2224.5, 1608.4.
4.1.9.2. 2-(4-Allyl-3-cyanobenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4b)
A yellow solid, yield 26.7%. HPLC 98.2%. Mp 216.0 -218.6 . MS (ESI) m/z: 303.1 [M-OH+2H]+;
15

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1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H, NOH), 8.54 (d, 2H, J = 4.0 Hz, Py -H), 8.29 (m, 2H, Ar-H),
8.08 (s, 1H, Imidazole-H), 7.79 (d, 2H, J = 4.0 Hz, Py-H), 7.41 (d, 1H, J = 8.0 Hz, Ar-H), 6.10 (m, 1H,
—CH=CH2), 5.51 (m, 1H,
—CH=CH2), 5.46 (m, 1H,
—
CH=CH2), 4.48 (d, 2H, J = 4.0 Hz, OCH2); 13C NMR
(150 MHz, DMSO-d6) δ 159.71, 149.99, 145.47, 141.16, 137.52, 133.66, 132.59, 131.89, 130.27, 123.97,
118.36, 116.26, 115.02, 113.96, 101.17, 69.47; IR (KBr, cm-1) 3426.7, 3078.9, 2920.0, 2230.5, 1607.1.
4.1.9.3. 2-(3-Cyano-4-isopropoxybenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4c)
A yellow solid, yield 19.4%. HPLC 98.6%. Mp 202.3 -205.2 . MS (ESI) m/z: 305.1 [M-OH+2H]+; 1
H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H, NOH), 8.56 (d, 2H, J = 5.1 Hz, Py-H), 8.27 (m, 2H, Ar-H),
8.09 (s, 1H, Imidazole-H),7.84 (d, 2H, J = 5.3 Hz, Py-H), 7.43 (d, 1H, J = 8.8 Hz, Ar-H), 4.89 (m, 1H, CH),
1.37 (d, 6H, J = 6.0 Hz, 2CH3); 13C NMR (150 MHz, DMSO-d6) δ 159.22, 149.85, 144.91, 141.44, 138.56,
131.56, 130.02, 123.26, 118.78, 117.51, 116.21, 114.73, 101.71, 71.73, 21.61; IR (KBr, cm-1) 3429.1,
3046.4, 2923.7, 2224.3, 1607.1.
4.1.9.4. 2-(3-Cyano-4-isobutoxybenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4d)
A yellow solid, yield 24.6%. HPLC 97.6%. Mp 243.0 -244.7 . MS (ESI) m/z: 319.2 [M-OH+2H]+;
1H NMR (400 MHz,DMSO-d6) δ 12.89 (s, 1H, NOH), 8.54 (d, 2H, J = 4.0 Hz, Py-H), 8.27 (m, 2H, Ar-H),
8.06 (s, 1H, Imidazole-H), 7.79 (d, 2H, J = 4.0 Hz, Py-H), 7.39 (d, 1H, J = 8.0 Hz, Ar-H), 3.98 (d, 2H,
CH2), 2.12 (m, 1H, CH), 1.03 (d, 6H, J = 8.0 Hz, 2CH3); 13C NMR (150 MHz, DMSO-d6) δ 160.22,
149.88, 144.94, 141.40, 138.56, 131.65, 129.83, 123.40, 118.76, 117.52, 115.97, 113.69, 100.95, 74.89,
27.62, 18.75; IR (KBr, cm-1) 3425.4, 2956.3, 2852.6, 2227.9, 1607.2.
16

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4.1.9.5. 2-(4-(sec-Butoxy) 3-cyanobenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4e)
A yellow solid, yield 21.4%. HPLC 98.2%. Mp 184.7 -191.0 . MS (ESI) m/z: 319.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H, NOH), 8.54 (d, 2H, J = 4.0 Hz, Py-H), 8.26 (m, 2H, Ar-H),
8.07 (s, 1H, Imidazole-H), 7.79 (d, 2H, J = 4.0 Hz, Py-H), 7.43 (d, 1H, J = 8.0 Hz, Ar-H), 4.69 (m, 1H,
CH), 1.73 (m, 2H, CH2), 1.32 (d, 3H, J = 8.0 Hz, CH3), 0.98 (t, 3H, J = 8.0 Hz, CH3); 13C NMR (150 MHz,
DMSO-d6) δ 159.54, 149.84, 145.02, 141.46, 138.56, 131.61, 130.04, 128.64, 123.24, 118.73, 116.16,
114.71, 101.71, 76.39, 28.40, 18.82, 9.20; IR (KBr, cm-1) 3426.7, 3094.7, 2924.0, 2227.5, 1605.9.
4.1.9.6. 2-(3-Cyano-4-isopentyloxybenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4f)
A yellow solid, yield 19.4%. HPLC 98.3%. Mp 148.6 -152.0 . MS (ESI) m/z: 333.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H, NOH), 8.53 (s, 2H, Py-H), 8.27 (s, 2H, Ar-H), 8.07 (s, 1H,
Imidazole-H), 7.79 (s, 2H, Py-H), 7.41 (d, 1H, J = 8.0 Hz, Ar-H), 4.21 (m, 2H, CH2), 1.82 (m, 1H, CH),
1.68 (m, 2H, CH2), 0.96 (d, 6H, J = 8.0 Hz, 2CH3); 13C NMR (150 MHz, DMSO-d6) δ 160.13, 149.83,
144.91, 141.45, 138.57, 131.57, 129.86, 123.38, 118.78, 117.48, 116.04, 113.58, 100.92, 67.60, 37.02,
24.61, 22.36; IR (KBr, cm-1) 3425.4, 2956.3, 2923.1, 2227.9, 1607.2.
4.1.9.7. 2-(3-Cyanobenzyl)-1-hydroxy-4-(pyridin-4-yl)-1H-imidazole (4g)
A slight yellow solid, yield 15.4%, HPLC 97.2%. Mp: 208.6 . -209.8 . MS (ESI) m/z: 246.9
[M-OH+2H]+; 1H NMR (400 MHz,DMSO-d6) δ 13.13 (s,1H, NOH), 8.55 (d, 2H, J = 4.4 Hz, Py-H), 8.39
(s, 1H, Ar-H), 8.34 (d, 1H, J = 7.9 Hz Ar-H), 8.16 (s, 1H, Imidazole-H), 7.87 (d, 1H, J = 7.6 Hz, Ar-H),
7.82 (d, 2H, J = 5.1 Hz, Py-H), 7.73 (t, 1H, J = 7.8 Hz, Ar-H); 13C NMR (150 MHz, DMSO-d6) δ 149.93,
144.75, 141.28, 139.04, 131.76, 131.24, 130.20, 129.53, 128.32, 118.85, 118.53, 118.23, 112.02; IR (KBr,
17

ACCEPTED MANUSCRIPT
cm-1) 3429.2, 3073.9, 3041.8, 2231.5, 1605.0.
4.1.9.8. 2-(3-Cyano-4-methoxybenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5a)
A yellow solid, yield 15.4%. HPLC 97.2%. Mp 199.9 -204.2 . MS (ESI) m/z: 277.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H, NOH), 9.06 (s, 1H, Py-H), 8.42 (d, 1H, J = 3.1 Hz, Py -H),
8.31 (m, 2H, Ar-H), 8.19 (m, 1H, Py-H), 7.88(s, 1H, Imidazole-H), 7.40 (m, 2H, Ar-H & Py-H), 3.97 (s,
3H, CH3); 13C NMR (150 MHz, DMSO-d6) δ 160.62, 147.30, 145.84, 144.73, 137.54, 131.61, 131.35,
130.09, 129.79, 123.70, 123.66, 116.12, 114.53, 112.90, 100.68, 56.58; IR (KBr, cm-1) 3420.4, 3104.4,
2922.9, 2227.9, 1602.2.
4.1.9.9. 2-(4-Allyloxy-3-cyanobenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5b)
A yellow solid, yield 19.4%. HPLC 97.8%. Mp 212.3 -215.2 . MS (ESI) m/z: 303.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.81 (s, 1H, NOH), 9.06 (d, 1H, J = 2.2 Hz, Py-H), 8.43 (dd, 1H, J =
1.7 Hz & 4.8 Hz, Py-H), 8.29 (d, 1H, J = 2.2 Hz, Ar-H), 8.25 (dd, 1H, J = 2.3 Hz & 8.8 Hz, Ar-H), 8.19 (m,
1H, Py-H), 7.88 (s, 1H, Imidazole-H), 7.40 (m, 2H, Ar-H & Py-H), 6.09 (m, 1H, CH=CH2), 5.51 (dd, 1H, J
= 1.7 Hz &17.2 Hz, -CH=CH2), 5.36 (dd, 1H, J = 1.6 Hz & 14.1 Hz, -CH=CH2), 4.80 (d, 2H, J = 5.2 Hz,
OCH2); 13C NMR (150 MHz, DMSO-d6) δ 159.50, 147.30, 145.84, 144.71, 137.30, 132.49, 131.46, 131.35,
129.82, 129.61, 123.81, 123.66, 118.21, 116.07, 114.51, 113.92, 101.04, 69.33; IR (KBr, cm-1) 3428.5,
3082.0, 2964.0, 2227.4, 1601.3.
4.1.9.10. 2-(3-Cyano-4-isopropoxybenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5c)
A yellow solid, yield 12.4%. HPLC 98.5%. Mp 202.3 -205.2 . MS (ESI) m/z: 305.0 [M-OH+2H]+;
18

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, NOH), 9.07 (s, 1H, Py-H),8.43 (d, 1H, J = 3.8 Hz, Py-H),
8.27 (m, 2H, Ar-H), 8.19 (d, 1H, J = 7.7 Hz, Py-H), 7.92 (s, 1H, Imidazole-H), 7.41 (d, 2 Hz, J = 8.0 Hz,
Ar-H), 7.43 (m, 2H, Ar-H & Py-H), 4.80 (m, 1H, CH), 1.36 (d, 6H, J = 6.0 Hz, 2CH3); 13C NMR (150
MHz, DMSO-d6) δ 159.12, 147.28, 145.82, 144.80, 137.27, 131.52, 131.38, 129.94, 129.72, 123.68,
123.42, 117.42, 116.26, 114.71, 101.71, 71.72, 21.62; IR (KBr, cm-1) 3425.9, 3098.4, 2922.3, 2227.9,
1617.4.
4.1.9.11. 2-(3-Cyano-4-isobutoxybenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5d).
A yellow solid, yield 20.1%. HPLC 97.3%. Mp 191.6 -195.8 . MS (ESI) m/z: 319.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, NOH), 9.06 (s, 1H, Py-H), 8.42 (d, 1H, J = 4.7 Hz, Py-H),
8.27 (m, 2H, Ar-H), 8.19 (d, 1H, J = 8.0 Hz, Py-H), 7.90 (s, 1H, Imidazole-H), 7.42 (m, 2H, Ar-H & Py-H),
3.98 (d, 2H, J = 6.4 Hz, CH2), 2.10 (m, 1H, CH), 1.03 (d, 6H, J = 6.6 Hz, 2CH3); 13C NMR (150 MHz,
DMSO-d6) δ 160.09, 147.30, 145.85, 144.70, 138.13, 131.55, 131.33, 130.09, 129.70, 123.65, 123.58,
116.01, 115.43, 113.64, 100.92, 74.87, 27.62, 18.74; IR (KBr, cm-1) 3427.1, 2960.7, 2921.7, 2229.4,
1621.0.
4.1.9.12. 2-(4-(sec-Butoxy)-3-cyanobenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5e)
A yellow solid, yield 16.7%. HPLC 98.4%. Mp 195.1 -197.9 . MS (ESI) m/z: 319.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, NOH), 9.06 (s, 1H, Py-H), 8.42 (s, 1H,Py -H), 8.27 (m, 2H,
Ar-H), 8.19 (d, 1H, J = 8.0 Hz, P y-H), 7.91 (s, 1H, Imidazole-H), 7.39 (m, 2H, Ar-H & Py-H), 4.69 (m,
1H, CH), 1.74 (m, 2H, CH2), 1.32 (d, 3H, J = 6.0 Hz, CH3), 0.97 (t, 3H, J = 6.0 Hz, CH3); 13C NMR (150
MHz, DMSO-d6) δ 159.47, 147.05, 145.63, 144.85, 136.54, 131.76, 131.72, 130.22, 129.62, 123.79,
19

ACCEPTED MANUSCRIPT
123.34, 117.28, 116.23, 114.66, 101.70, 76.40, 28.44, 18.86, 9.23; IR (KBr, cm-1) 3425.3, 2963.2, 2922.2,
2227.1, 1620.0.
4.1.9.13. 2-(3-Cyano-4-isopentyloxybenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5f)
A yellow solid, yield 12.1%. HPLC 97.9%. Mp 197.3 -203.9 . MS (ESI) m/z: 333.1 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, NOH), 9.07 (s, 1H, Py-H), 8.42 (s, 1H, Py–H), 8.26 (m,
2H, Ar-H), 8.19 (d, 1H, J = 7.9 Hz, Py-H), 7.89 (s, 1H, Imidazole-H), 7.39 (m, 2H, Ar-H & Py-H), 4.19 (t,
2H, J = 6.5 Hz, CH2), 1.83 (m, 1H, CH), 1.67 (q, 2H, J = 6.5 Hz, CH3), 0.95 (d, 6H, J = 6.6 Hz, 2CH3); 13C
NMR (150 MHz, DMSO-d6) δ 160.02, 147.24, 145.79, 144.77, 137.42, 131.53, 131.37, 129.76, 129.58,
123.66, 123.53, 117.07, 116.07, 113.58, 100.90, 67.58, 37.03, 24.61, 22.37; IR (KBr, cm-1) 3425.5, 2956.4,
2922.0, 2228.4, 1620.7.
4.1.9.14. 2-(3-Cyanobenzyl)-1-hydroxy-4-(pyridin-3-yl)-1H-imidazole (5g)
A yellow solid, yield 21.3%. HPLC 97.1%. Mp 222.2 -226.9 . MS (ESI) m/z: 246.9 [M-OH+2H]+;
1H NMR (400 MHz, DMSO-d6) δ 13.04 (s, 1H, NOH), 9.09 (s, 1H, Py-H), 8.44 (d, 1H, J = 4.7 Hz, Py-H),
8.38 (s, 1H, Ar-H), 8.33 (d, 1H, J = 8.0 Hz, Py-H), 8.21 (d, 1H, J = 8.0 Hz, Ar-H), 8.01 (s, 1H,
Imidazole-H), 7.86 (m, 1H, Py -H), 7.73 (t, 1H, Ar-H), 7.43 (m, 1H, Py -H); 13C NMR (150 MHz,
DMSO-d6) δ 147.48, 145.91, 144.52, 131.59, 131.47, 131.38, 130.20, 129.43, 128.19, 127.25, 123.70,
118.55, 116.36, 114.52, 111.98; IR (KBr, cm-1) 3428.8, 2923.4, 2853.7, 2226.6, 1632.6.
4.2. Assay of in vitro XO inhibitory activity
20

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Bovine XO inhibitory potency in vitro was assayed spectrophotometrically by measuring uric acid
formation at 295 nm at 25°C. The testing method was based on the procedure reported by Matsumoto et al.
[32] with modification. The assay mixture contained 0.1 M sodium pyrophosphate buffer (pH 8.3), 0.3 mM
Na2EDTA, 64 µM xanthine, 15 U/L XO (Sigma, X4875), and the test compound. The XO inhibition by
various compounds was measured by the reduction of the uric acid concentration. The enzyme needed
pre-incubation for 15 min with the test compound, and the reaction was started by addition of xanthine.
Topiroxostat was used as a positive control. All the tests were performed in triplicate. Compounds
presenting inhibitory effects over 50% at the concentration of 10 µg/mL were further tested at a wide range
of concentrations to calculate their IC50 value by using SPSS 20.0 software.
4.3. Molecular modeling
AutoDock 4 [33] was used to perform docking calculations. The crystal structure of bovine XO in
complex with Febuxostat (PDB code: 1N5X) [8] was adopted in docking simulations. An 80 × 80 × 80 Å
grid box with a grid spacing of 0.375 Å was generated to define the binding pocket. Affinity grid fields
were generated using the auxiliary program AutoGrid 4. Ligand structures were built and minimized with
Accelrys Discovery Studio 3.0 software package. Flexible torsions in the ligands were assigned, and all
dihedral angles were allowed to freely rotate.
The Lamarckian genetic algorithm was used to determine the appropriate binding positions,
orientations, and conformations of ligands [33]. All other parameters were maintained as default. In each
group, the lowest binding energy configuration with the highest percentage frequency was selected as the
group representative.
21

ACCEPTED MANUSCRIPT
4.4. 3D-QSAR analysis
In this study, 30 of Topiroxostat-like compounds were used to conduct 3D-QSAR model with Topomer
CoMFA method, including 7 of imidazole derivatives synthesized in this work, 8 of 1,2,3-triazole
derivatives reported in our previous studies [15] and 15 of 1,2,4-triazole derivatives reported by Sato T et
al [14]. The structures of compounds and their biological data in the form of pIC50 are listed in Table 2.
The 3D structures were prepared using Sybyl X-1.1. The Gasteiger-Hückel charge was applied to each
molecule and energy minimization was performed using the Tripos force field [34, 35]. The 30 compounds
were divided into the training set (25 compounds) and the test set (5 compounds) in the ratio of 5:1. The
training set was used to build the 3D-QSAR model, and the test set was used to test the predictions of the
model [36]. Each molecule in training set was divided into two sets of fragments shown as S1 and S2
groups (Fig. 6). A carbon sp3 probe was applied for calculating steric and electrostatic parameters. PLS
regression was used to generate the Topomer CoMFA model.
Acknowledgments
This work was supported by Program for Liaoning Excellent Talents in University (LR2012035) and
the Scientific Research Program of Hainan Province (ZDYF2016143).
References
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1. Legends for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Scheme 1 and Scheme 2.
Fig. 1 Chemical structures of Allopurinol, Febuxostat, Topiroxostat and compounds 1-3.
Fig. 2 Design of compounds 4a-g and 5a-g.
Fig. 3 The inhibition of XO by compound 4f. Values are means ± SD, n = 3.
Fig. 4 Binding modes of Topiroxostat (A), 4f (B) and 5f (C) within the XO binding pocket.
Fig. 5 Contour maps of Topomer CoMFA. (A) Steric contour map depicted around S1; (B) electrostatic contour map
depicted around S1; (C) steric contour map depicted around S2; (D) electrostatic contour map depicted around S2.
Fig. 6 The correlation chart of experimental versus predicted pIC50 values for the training set and test set compounds.
Fig. 7 Lineweaver-Burk plot analysis of XO inhibition by compound 4f.
Scheme 1 Reagents and conditions: (i) Br2, I2, CH2Cl2, 5 °C, 24 h; (ii) BnBr, KI, K2CO3, DMF, N2, 80 °C, 8 h; (iii) CuCN,
DMF, 150 °C, 8 h; (iv) H2, Pd/C, THF, 25 °C, 5 h; (v) RCl or RBr, KI, K2CO3, N2, DMF, 80 °C, 8 h; (vi) t-BuONO,
C2H5ONa, EtOH, 0~10 °C, 24 h; (vii) NH4OAc, CH3OH, reflux, 24 h.
Scheme 2 Reagents and conditions: NH4OAc, CH3OH, reflux, 24 h.
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2. Graphics for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig.6, Fig. 7, Scheme 1 and Scheme 2
Fig. 1
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Fig. 2
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Fig. 3
29