Synthesis and bioevaluation of 2-phenyl-5-methyl-2H-1,2,3-triazole-4-carboxylic acid/carbohydrazide derivatives as potent xanthine oxidase inhibitors

Article abstract of DOI:10.1039/C6RA24651F

A series of 2-phenyl-5-methyl-2H-1,2,3-triazole-4-carboxylic acids/carbohydrazides as analogues of febuxostat were synthesized and evaluated for their in vitro xanthine oxidase (XO) inhibitory activity. Among these compounds, the carboxylic acid derivatives 7a-h and 8a-h exhibited high potency in the submicromolar/nanomolar range. Steady-state kinetics experiment revealed that 7f was a mixed-type inhibitor of xanthine oxidase. In addition, a molecular docking study of 7f was performed to determine its binding mode at the active site of xanthine oxidase.

Full text of DOI:10.1039/C6RA24651F

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PAPER
Synthesis and bioevaluation of 2-phenyl-5-methyl-
2H-1,2,3-triazole-4-carboxylic acid/
carbohydrazide derivatives as potent xanthine
oxidase inhibitors†
Cite this: RSC Adv., 2016, 6, 114879
a
a
a
a
b
a
Ailong Shi,ab Defa Wang, He Wang, Yue Wu, Haiqiu Tian, Qi Guan, Kai Bao
*
*
a
*
and Weige Zhang
A series of 2-phenyl-5-methyl-2H-1,2,3-triazole-4-carboxylic acids/carbohydrazides as analogues of
febuxostat were synthesized and evaluated for their in vitro xanthine oxidase (XO) inhibitory activity.
Among these compounds, the carboxylic acid derivatives 7a–h and 8a–h exhibited high potency in the
submicromolar/nanomolar range. Steady-state kinetics experiment revealed that 7f was a mixed-type
inhibitor of xanthine oxidase. In addition, a molecular docking study of 7f was performed to determine its
binding mode at the active site of xanthine oxidase.
Received 3rd October 2016
Accepted 30th November 2016
DOI: 10.1039/c6ra24651f
www.rsc.org/advances
bioisosterism is an efficient strategy for the discovery of new
non-purine XO inhibitors, such as 3, 4 and 5 (Fig. 1).11–13 By
Introduction
following this strategy, we designed and synthesized a series of
selenazole derivatives. Among them, 6 displayed potent activity
against XO (Fig. 2).14
Xanthine oxidase (XO) is a ubiquitous molybdo-avoenzyme
existed in organism which catalyzes the oxidation of hypoxan-
thine to xanthine and uric acid (UA).1,2 Normally, UA dissolves
in the blood and passes through the kidneys into urine. Excess
UA accumulating in tissues leads to a hyperuricemic condition
such as gout.3 In addition, it has been demonstrated that high
uric acid level is related to other major diseases including dia-
betes, cardiovascular disease and metabolic syndrome.4–6 XO
inhibitors have been of excellent prospect as a therapeutic
approach of hyperuricemia and its complications by reducing
UA levels.
Allopurinol (1), a structural isomer of hypoxanthine, is the
rst XO inhibitor marketed in 1966 for the treatment of gout.7
However, severe drug-induced hypersensitivity syndrome
impedes its widely application.8 Febuxostat (2), the rst non-
purine XO inhibitor in clinic, is an effective alternative to allo-
purinol and shows greater urate-lowering efficacy and minor
side effects.9,10
In the light of our ongoing efforts to develop new non-purine
XO inhibitors, we herein attempt to replace the thiazole moiety
of febuxostat with an 1,2,3-triazole which is one of the signi-
cant structural fragments of drugs and bioactive molecules.15–17
Furthermore, there have been few reports, to the best of our
knowledge, concerning the structure modication on the
carboxyl group of febuxostat analogous. Hence, a series of 2-
phenyl-5-methyl-2H-1,2,3-triazole-4-carboxylic acids (7a–h and
8a–h) and the hydrazide derivatives (9e–f and 10e–f) were syn-
thesised and evaluated their biological activities (Fig. 2). The
In view of the attractive biological activity exhibited by
febuxostat, numerous analogues of febuxostat have been
designed and synthesized in order to have a better under-
standing of the structure–activity relationships (SAR). Ring
aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China. E-mail: zhangweige@
syphu.edu.cn; baokai@syphu.edu.cn
bSchool of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang 110016, China. E-mail: guanqi@syphu.edu.cn; Fax: +86 2423986393; Tel:
+86 24 23986422
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra24651f
Fig.
inhibitors.
1 Chemical structures of allopurinol and non-purine XO
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Fig. 2 Design of the target compounds.
inhibition type and the interaction of the most potent inhibitor acetate to yield nitriles 14a–h.19 The reduction of the nitro group
with the amino acid residues of the enzyme have also been of 14a–h was performed by using stannous chloride to generate
investigated.
amines 18a–h. Compounds 19a–h were obtained from
commercially available 4-amino-2-nitrophenol (15) via N-Boc
protection, alkylation and deprotection.
The key intermediates 18a–h and 19a–h were diazotized,
respectively, in ice bath followed by condensation with ethyl
acetylacetate in the presence of sodium acetate to afford 20a–h
and 21a–h.20 Treatment of 20a–h and 21a–h with CuBr2 in acetic
acid/N,N-dimethyl formamide mixture afforded esters 22a–h
and 23a–h.21
Carboxylic acids 7a–h and 8a–h were obtained through hydro-
lysis of 22a–h and 23a–h by sodium hydroxide followed by acidi-
cation. On the other hand, hydrazinolysis of esters 22f–g and 23f–
g in ethanol provided carbohydrazides 9f–g and 10f–g, respectively.
Results and discussion
1. Chemistry
The synthesis of the target compounds was performed as
outline in Scheme 1. The commercially available 4-nitrophenol
(11) was applied as starting material and converted into 2-
hydroxy-5-nitrobenzaldehyde (12) via Duff reaction.18 The
alkylations of 12 with appropriate alkyl halides afforded the
corresponding 13a–h. Compounds 13a–h were treated with
hydrochloride hydroxylamine to give aldoximes and subse-
quently underwent dehydration in the presence of copper
Scheme 1 Reagents and conditions: (i) C6H12N4, TFA, 80 ꢀC; (ii) alkyl halide, K2CO3, DMF; (iii) (a) NH2OH$HCl, MeOH, 50 ꢀC; (b) Cu(OAc)2, MeCN,
60 ꢀC; (iv) SnCl2, HCl aq., EtOH, 55 ꢀC; (v) (Boc)2O, THF, 50 ꢀC; (vi) alkyl halide, K2CO3, DMF; (vii) TEA, DCM, rt; (viii) (a) NaNO2, HCl aq., 0 ꢀC; (b)
ethyl acetylacetate, NaOAc, EtOH, H2O; (ix) CuBr2, NH4OAc, DMF, HOAc, 120 ꢀC; (x) NaOH, aq., THF/EtOH; (xi) NH2NH2$H2O, EtOH, 50 ꢀC.
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2. Biological activity
The in vitro bovine XO inhibitory activities of compounds 7a–h,
8a–h and 9e–f, 10e–f were evaluated and compared with the
positive control, febuxostat (Table 1).22,23 As shown in Table 1,
most of the target compounds exhibited moderate XO inhibi-
tory activity. Among them, compounds 7f and 8f bearing iso-
amyl substituent were more potent than the other compounds
(IC50 0.084 and 0.109 mM, respectively). 7a–h carrying the cyano
group at the 3-position of phenyl moiety showed similar or
slightly improved activity with respect to 8a–h carrying the nitro
group. In addition, carboxyl group at the 5-position of the tri-
azole ring was found to be essential for XO inhibition with
respect to carbohydrazides (7e vs. 9e; 7f vs. 9f; 8e vs. 10e; 8f vs.
10f). Increasing the size of the substituent at the 40-position of
the phenyl moiety from n-propyl to n-amyl groups improved XO
inhibitory activity (7e vs. 7a, 7c; 8e vs. 8a, 8c), with the exception
of benzyloxy substituent (7g, 8g). Branched alkoxy substituents
showed more potent inhibitory activity than corresponding
linear chain alkoxy groups (7b vs. 7a; 7d vs. 7c; 7f vs. 7e; 8b vs.
8a; 8d vs. 8c; 8f vs. 8e). Consequently, compound 7f represented
the most potent molecule among the series. This nding indi-
cated that the combination of a cyano group and an i-amyl
Fig. 3 Lineweaver–Burk plot of the inhibition of xanthine oxidase by
compound 7f.
group is favourable for the XO inhibitory activity of 2-phenyl-5-
methyl-2H-1,2,3-triazole-4-carboxylic acids/carbohydrazides
derivatives.
3. Steady-state kinetic analysis
A Lineweaver–Burk plot for inhibition of XO by compound 7f
was obtained in the presence or absence of compound 7f with
varying concentration of xanthine as the substrate (20–100 mM).
The interaction of lines indicated that compound 7f was
a mixed-type inhibitor of XO (Fig. 3). Similar ndings have been
reported with febuxostat (2).24
Table 1 In vitro xanthine oxidase inhibitory activity of 2-(3-cyano/
nitro-4-phenyl)-5-methyl-2H-1,2,3-triazole-4-carboxylic acid/car-
bohydrazide derivatives
4. Molecular modeling
To elucidate the interactions of the newly synthesized
compounds with XO and further understand the SARs of the
target compounds, the potential binding mode of compound 7f
at molybdenum-pterin sites was investigated using Discovery
Studio 3.0 (Accelrys, San Diego, CA, USA) soware with the
crystal structure of the xanthine dehydrogenase/febuxostat
complex (PDB: 1N5X).25
The docking study clearly indicated that 7f (yellow) was
perfectly superimposed with febuxostat (green) in the narrow
tunnel towards the molybdenum-pterin center (Fig. 4A).
Hydrogen bonds were found between the carboxylate group of
7f and Thr1010, Arg880, the nitrogen atom at 3-position of tri-
azole and Glu802, the nitrile group and Asn768 (Fig. 4B).25 The
Compound
R
R1
R2
IC50a (mM)
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
8g
8h
n-Propyl
i-Propyl
n-Butyl
i-Butyl
n-Amyl
i-Amyl
Benzyl
Allyl
n-Propyl
i-Propyl
n-Butyl
i-Butyl
n-Amyl
i-Amyl
Benzyl
Allyl
CN
CN
CN
CN
CN
CN
CN
CN
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
CN
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
0.195
0.177
0.135
0.105
0.112
0.084
0.152
0.225
0.162
0.145
0.151
0.123
0.125
0.109
0.180
0.254
NAb
˚
hydrogen bond distances were 2.3, 1.9, 2.0 and 2.3 A, respec-
tively. Moreover, the triazole ring of 7f fostered p–p interaction
˚
with Phe1009 and the interaction distance is 4.2 A. The
CDOCKER interaction energy of 7f was ꢁ43.06 kcal molꢁ1, while
that of febuxostat was ꢁ47.25 kcal molꢁ1. These computational
modeling analyses may help to explain the reason that
compound 7f exhibited potent inhibitory activity, but lower
than that of febuxostat.
9e
9f
10e
10f
n-Amyl
i-Amyl
n-Amyl
i-Amyl
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CN
NO2
NO2
NAb
NAb
NAb
Conclusions
Febuxostat
0.012
In summary, a series of novel XO inhibitors containing 1,2,3-
triazole segments were designed and synthesized. In vitro
activity assay indicated that compounds 7a–h and 8a–h had
a
b
Values are means of the three experiment. NA ¼ not active <50%
inhibition@10.0 mM.
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Fig. 4 (A) Overlapping modes for compound 7f (yellow) and febuxostat (green) binding the active site of xanthine oxidase. (B) The interactions
between the active site of xanthine oxidase and compound 7f on 2D diagram. The surrounding amino acid residues are displayed in ball format
and labelled. The dashed blue arrows represent hydrogen bonds, the solid orange line represents p–p interaction and the interaction distances
(A˚) are shown.
potential inhibitory effects against XO, with IC50 values ranging pressure. The mixture was diluted with H2O (100 mL) and then
from 0.084 to 0.254 mM. Enzyme inhibition kinetic study neutralized with Na2CO3 to pH 5. The precipitate formed was
revealed that the most promising compound 7f acted as collected by ltration, washed with water and dried to afford 12
a mixed-type inhibitor for xanthine oxidase. Furthermore, (6.6 g, 92%) as a yellow solid, mp 146–149 ꢀC.26
molecular docking studies were performed to determine the
probable binding conformation of compound 7f in the XO
3. General procedure for the preparation of compounds 13a–
binding site and offered some insights into the further modi-
h
cation of the target compounds. Our results suggested that 2-
phenyl-5-methyl-2H-1,2,3-triazole-4-carboxylic acids might
serve as new molecular scaffold for the development of non-
purine XO inhibitors. Further research in this area is in prog-
ress in our laboratory.
A mixture of compound 12 (5.0 g, 29.9 mmol), respective alkyl
halide (35.8 mmol), anhydrous K2CO3 (8.2 g, 59.8 mmol) in
DMF (50 mL) was heated at 75 ꢀC for 7 h. The reaction mixture
was diluted with H2O (100 mL) then extracted with EtOAc (100
mL) and the combined organic phases were washed with H2O (3
ꢂ 100 mL) and brine (50 mL), dried over MgSO4 and concen-
trated in vacuo. The residue was recrystallized from EtOH.
3.1. 5-Nitro-2-propoxybenzaldehyde (13a). White solid,
Experimental section
1. Chemistry
(yield: 85%), mp: 64–67 C.27
ꢀ
Unless otherwise noted, all materials were commercial available
and were used without further purication. Thin-layer chro-
matography was performed on FLUKA silica gel aluminum
cards with uorescent indicator 254 nm under UV light. The
column chromatography was performed using silica gel (200–
300 mesh, Qingdao PUKE) with the designated solvents.
Melting points were measured by a YRT-3 melting point
detector without corrected. ESI-HRMS spectra were performed
on Agilent 6530 Accurate-Mass Q-TOF LC/MS (Agilent Tech-
nologies, USA). 1H and 13C NMR spectra were obtained on
Bruker AVANCE 400 (1H, 400 MHz; 13C, 100 MHz) or Bruker
AVANCE 600 (1H, 600 MHz; 13C, 150 MHz), in DMSO-d6 or CDCl3
using TMS as internal standard. Infrared spectra were recorded
using KBr plates on a PE Spectrum-100 instrument.
3.2. 2-Isopropoxy-5-nitrobenzaldehyde (13b). White solid,
(yield: 88%), mp: 77–79 C.28
ꢀ
3.3. 5-Nitro-2-propoxybenzaldehyde (13c). White solid,
(yield: 90%), mp: 67–69 ꢀC. 1H NMR (600 MHz, CDCl3) d 10.47 (s,
1H), 8.68 (d, J ¼ 2.9 Hz, 1H), 8.41 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.12
(d, J ¼ 9.2 Hz, 1H), 4.24 (t, J ¼ 6.4 Hz, 2H), 1.95–1.87 (m, 2H),
1.60–1.50 (m, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H).
3.4. 2-Isobutoxy-5-nitrobenzaldehyde (13d). White solid,
(yield: 91%), mp: 69–71 ꢀC. 1H NMR (600 MHz, CDCl3) d 10.50 (s,
1H), 8.68 (d, J ¼ 2.9 Hz, 1H), 8.41 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.11
(d, J ¼ 9.2 Hz, 1H), 4.00 (d, J ¼ 6.4 Hz, 2H), 2.24 (dt, J ¼ 13.3,
6.6 Hz, 1H), 1.11 (d, J ¼ 6.7 Hz, 7H).
3.5. 5-Nitro-2-(amyloxy)benzaldehyde (13e). White solid,
(yield: 89%), mp: 62–65 ꢀC. 1H NMR (600 MHz, CDCl3) d 10.48 (s,
1H), 8.68 (d, J ¼ 2.9 Hz, 1H), 8.41 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.14–
7.08 (m, 1H), 4.23 (t, J ¼ 6.5 Hz, 2H), 1.93 (dq, J ¼ 13.3, 6.5 Hz,
2. Synthesis of 2-hydroxy-5-nitrobenzaldehyde (12)
A mixture of 4-nitrophenol (6.0 g, 43.2 mmol), hexamethyl- 2H), 1.53–1.48 (m, 2H), 1.43 (dq, J ¼ 14.4, 7.0 Hz, 2H), 0.96 (t, J ¼
enetetramine (7.9 g, 56.2 mmol) in triuoroacetic acid (50 7.3 Hz, 3H).
mL) was heated at 85 ꢀC for 15 h. The reaction mixture was
3.6. 2-(Isoamyloxy)-5-nitrobenzaldehyde (13f). White solid,
cooled to room temperature and concentrated under reduced (yield: 83%), mp: 67–70 ꢀC. 1H NMR (400 MHz, CDCl3) d 10.47 (s,
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1H), 8.68 (d, J ¼ 2.9 Hz, 1H), 8.42 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.13
(d, J ¼ 9.2 Hz, 1H), 4.26 (t, J ¼ 6.4 Hz, 2H), 1.93–1.85 (m, 1H),
1.82 (q, J ¼ 6.5 Hz, 2H), 1.01 (d, J ¼ 6.4 Hz, 6H).
4.7. 2-Benzyloxy-5-nitrobenzonitrile (14g). White solid,
(yield: 69%), mp: 132–134 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.53
(d, J ¼ 2.8 Hz, 1H), 8.41 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.40–7.35 (m,
5H), 7.14 (d, J ¼ 9.3 Hz, 1H), 5.37 (s, 2H).
3.7. 2-(Benzyloxy)-5-nitrobenzaldehyde (13g). White solid,
(yield: 86%), mp: 90–92 C.29
ꢀ
4.8. 2-Allyloxy-5-nitrobenzonitrile (14h). White solid, (yield:
1
66%), mp: 97–100 ꢀC. H NMR (400 MHz, CDCl3) d 8.50 (d, J ¼
3.8. 2-(Allyloxy)-5-nitrobenzaldehyde (13h). White solid,
(yield: 78%), mp: 75–78 C.30
ꢀ
2.8 Hz, 1H), 8.43 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.12 (d, J ¼ 9.3 Hz,
1H), 6.07 (ddt, J ¼ 17.2, 10.4, 5.1 Hz, 1H), 5.59–5.49 (m, 1H), 5.43
(m, 1H), 4.82 (dt, J ¼ 5.1 Hz, 2H).
4. General procedure for the preparation of compounds 14a–
h
5. Synthesis of tert-butyl 4-hydroxy-3-nitrophenylcarbamate
(16)
A mixture of compound 13 (22.4 mmol), hydroxylamine hydro-
chloride (7.7 g, 112 mmol) in methanol (50 mL) was heated at
55 ꢀC for 3 h. The reaction was cooled to room temperature, and
concentrated under reduced pressure. The mixture was diluted
with H2O (200 mL) then extracted with EtOAc (3 ꢂ 150 mL) and
the combined organic phases were washed with brine (50 mL),
dried over MgSO4 and concentrated in vacuo to yield aldoximes.
A mixture of respective aldoxime (12.6 mmol), copper acetate
(0.18 g, 1 mmol) in acetonitrile (50 mL) was heated at 65 ꢀC for
3 h. The reaction mixture was cooled to room temperature, and
concentrated under reduced pressure. The mixture was diluted
A mixture of 4-amino-2-nitrophenol (6.0 g, 43.2 mmol), di-tert-
butyl pyrocarbonate (12.0 g, 55.0 mmol) in THF (60 mL) was
heated at 55 ꢀC for 10 h. The reaction mixture was cooled to
room temperature and concentrated under reduced pressure.
The residue was recrystallized from cyclohexane to yield 16
(10.1 g, 91%) as a yellow solid, mp 59–62 C.31
ꢀ
6. General procedure for the preparation of compounds
17a–h
with H2O (200 mL) then extracted with EtOAc (3 ꢂ 150 mL) and Compounds 17a–h were synthesized in the same way as
the combined organic phases were washed with brine (50 mL), compounds 13a–h.
dried over MgSO4 and concentrated in vacuo. The collected solid
6.1. tert-Butyl 3-nitro-4-propoxyphenylcarbamate (17a).
was puried by ash column chromatography (0–25% EtOAc in Yellow oil, (yield: 82%). 1H NMR (600 MHz, CDCl3) d 7.91 (d, J ¼
hexanes).
2.4 Hz, 1H), 7.49 (s, 1H), 6.99 (d, J ¼ 9.0 Hz, 1H), 6.69 (s, 1H),
4.1. 5-Nitro-2-propoxybenzonitrile (14a). White solid, 4.02 (t, J ¼ 6.4 Hz, 2H), 1.88–1.78 (m, 2H), 1.51 (s, 9H), 1.04 (t, J
(yield: 74%), mp: 75–77 ꢀC. 1H NMR (600 MHz, CDCl3) d 8.48 (d, ¼ 7.4 Hz, 3H).
J ¼ 2.8 Hz, 1H), 8.42 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.08 (d, J ¼ 9.3 Hz,
6.2. tert-Butyl 4-isopropoxy-3-nitrophenylcarbamate (17b).
1H), 4.18 (t, J ¼ 6.4 Hz, 2H), 1.99–1.90 (m, 2H), 1.12 (t, J ¼ 7.4 Hz, Yellow oil, (yield: 88%). 1H NMR (400 MHz, CDCl3) d 7.88 (d, J ¼
3H).
2.6 Hz, 1H), 7.48 (d, 1H), 7.02 (t, J ¼ 9.5 Hz, 1H), 6.74 (s, 1H),
4.2. 2-Isopropoxy-5-nitrobenzonitrile (14b). White solid, 4.57 (dq, 6.1 Hz, 1H), 1.52 (s, 9H), 1.36 (d, J ¼ 6.1 Hz, 6H).
(yield: 70%), mp: 82–84 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.50 (d,
6.3. tert-Butyl 4-butoxy-3-nitrophenylcarbamate (17c).
J ¼ 2.8 Hz, 1H), 8.43 (dd, J ¼ 9.4, 2.8 Hz, 1H), 7.08 (d, J ¼ 9.4 Hz, Yellow oil, (yield: 91%). H NMR (400 MHz, CDCl3) d 7.92 (d,
1
1H), 4.86–4.76 (m, 1H), 1.50 (d, J ¼ 6.1 Hz, 6H).
1H), 7.51 (t, 1H), 6.99 (d, 1H), 6.79 (s, 1H), 4.05 (t, J ¼ 6.4 Hz,
4.3. 2-Butoxy-5-nitrobenzonitrile (14c). White solid, (yield: 2H), 1.78 (m, 2H), 1.51 (s, 9H), 1.47 (m, 2H), 0.96 (t, J ¼ 7.4 Hz,
1
ꢀ
68%), mp: 87–90 C. H NMR (400 MHz, CDCl3) d 8.50 (d, J ¼ 3H).
2.8 Hz, 1H), 8.44 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.09 (d, J ¼ 9.3 Hz,
6.4. tert-Butyl 4-isobutoxy-3-nitrophenylcarbamate (17d).
1
1H), 4.23 (t, J ¼ 6.4 Hz, 2H), 1.95–1.88 (m, 2H), 1.61–1.55 (m, Yellow oil, (yield: 83%), H NMR (400 MHz, CDCl3) d 7.91 (d,
2H), 1.03 (t, J ¼ 7.4 Hz, 3H).
1H), 7.54 (d, 1H), 7.00 (d, 1H), 6.53 (s, 1H), 3.84 (d, J ¼ 6.5 Hz,
4.4. 2-Isobutoxy-5-nitrobenzonitrile (14d). White solid, 2H), 2.14 (dt, J ¼ 13.3, 6.5 Hz, 1H), 1.53 (s, 9H), 1.05 (d, J ¼
(yield: 73%), mp: 77–80 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.51 (d, 6.7 Hz, 6H).
J ¼ 2.8 Hz, 1H), 8.44 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.08 (d, J ¼ 9.3 Hz,
1H), 3.99 (d, J ¼ 6.5 Hz, 2H), 2.29–2.18 (m, 1H), 1.12 (d, J ¼ Yellow oil, (yield: 83%). H NMR (400 MHz, CDCl3) d 7.91 (d,
6.7 Hz, 6H).
6.5. tert-Butyl 4-amyloxy-3-nitrophenylcarbamate (17e).
1
1H), 7.52 (t, 1H), 7.01 (d, 1H), 6.61 (s, 1H), 4.07 (t, J ¼ 6.5 Hz,
4.5. 2-Amyloxy-5-nitro-benzonitrile (14e). White solid, 2H), 1.86–1.78 (m, 2H), 1.53 (s, 9H), 1.49–1.44 (m, 2H), 1.42–1.34
(yield: 75%), mp: 69–73 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.50 (d, (m, 2H), 0.94 (t, J ¼ 7.1 Hz, 3H).
J ¼ 2.8 Hz, 1H), 8.44 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.09 (d, J ¼ 9.3 Hz,
6.6. tert-Butyl
4-(isoamyloxy)-3-nitrophenylcarbamate
1H), 4.23 (t, J ¼ 6.5 Hz, 2H), 1.98–1.90 (m, 2H), 1.56–1.49 (m, (17f). Yellow oil, (yield: 90%). 1H NMR (600 MHz, CDCl3)
2H), 1.47–1.38 (m, 2H), 0.97 (t, J ¼ 7.2 Hz, 3H).
d 7.89 (d, J ¼ 2.6 Hz, 1H), 7.51 (s, 1H), 7.00 (d, J ¼ 9.1 Hz, 1H),
4.6. 2-Isoamyloxy-5-nitrobenzonitrile (14f). White solid, 6.59 (s, 1H), 4.08 (t, J ¼ 6.6 Hz, 2H), 1.85 (m, 1H), 1.70 (q, J ¼
(yield: 70%), mp: 87–90 ꢀC. 1H NMR (400 MHz, CDCl3) d 8.50 (d, 6.7 Hz, 2H), 1.51 (s, 9H), 0.95 (d, J ¼ 6.7 Hz, 6H).
J ¼ 2.8 Hz, 1H), 8.45 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.10 (d, J ¼ 9.3 Hz,
6.7. tert-Butyl 4-(benzyloxy)-3-nitrophenylcarbamate (17g).
1H), 4.26 (t, J ¼ 6.5 Hz, 2H), 1.96–1.86 (m, 1H), 1.83 (q, J ¼ Yellow solid, (yield: 89%), mp: 77–79 ꢀC. 1H NMR (600 MHz,
6.5 Hz, 2H), 1.02 (d, J ¼ 6.5 Hz, 6H).
CDCl3) d 7.92 (d, J ¼ 2.7 Hz, 1H), 7.47 (s, 1H), 7.43 (d, J ¼ 7.3 Hz,
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2H), 7.37 (dd, 2H), 7.31 (t, J ¼ 7.3 Hz, 1H), 7.03 (d, J ¼ 9.1 Hz,
1H), 6.51 (s, 1H).
10.1. Ethyl 2-(3-cyano-4-propoxyphenyl)-5-methyl-2H-1,2,3-
triazole-4-carboxylate (22a). Light yellow solid, (yield: 81%), mp:
121–122 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.32 (d, J ¼ 2.7 Hz, 1H),
8.25 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.05 (d, J ¼ 9.2 Hz, 1H), 4.47 (m,
2H), 4.10 (t, J ¼ 6.4 Hz, 2H), 2.61 (s, 3H), 1.96–1.87 (m, 2H), 1.42–
1.48 (m, 3H), 1.11 (t, J ¼ 7.4 Hz, 3H). ESI-MS m/z 315.1 [M + H]+.
10.2. Ethyl 2-(3-cyano-4-isopropoxyphenyl)-5-methyl-2H-
1,2,3-triazole-4-carboxylate (22b). Light yellow, (yield: 84%), mp:
71–75 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.31 (d, J ¼ 2.7 Hz, 1H),
8.25 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.06 (d, J ¼ 9.2 Hz, 1H), 4.72 (m,
1H), 4.47 (m, 2H), 2.61 (s, 3H), 1.47–1.43 (m, 3H), 1.46–1.43 (m,
6H). ESI-MS m/z 315.1 [M + H]+.
10.3. Ethyl 2-(4-butoxy-3-cyanophenyl)-5-methyl-2H-1,2,3-
triazole-4-carboxylate (22c). White solid, (yield: 77%), mp: 67–
68 ꢀC, 1H NMR (600 MHz, CDCl3) d 8.31 (d, J ¼ 2.7 Hz, 1H), 8.25
(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.06 (d, J ¼ 9.2 Hz, 1H), 4.47 (q, J ¼
7.1 Hz, 2H), 4.13 (m, 3H), 2.61 (s, 3H), 1.90–1.85 (m, 2H), 1.59–
1.53 (m, 2H), 1.45 (t, J ¼ 7.1 Hz, 3H), 1.01 (t, J ¼ 7.4 Hz, 3H). ESI-
MS m/z 329.2 [M + H]+.
6.8. tert-Butyl 4-(allyloxy)-3-nitrophenylcarbamate (17h).
Yellow oil, (yield: 87%). 1H NMR (600 MHz, CDCl3) d 7.94 (d, J ¼
2.4 Hz, 1H), 7.52 (s, 1H), 7.02 (d, J ¼ 9.0 Hz, 1H), 6.65 (s, 1H),
6.11–5.98 (m, 1H), 5.49–5.42 (m, 1H), 5.34–5.27 (m, 1H), 4.65 (d,
J ¼ 5.0 Hz, 2H), 1.53 (s, 9H).
7. General procedure for the preparation of compounds
18a–h
A mixture of compound 14 (9.1 mmol), tin(II) chloride dehydrate
(2.3 g, 12 mmol), 2 mL concentrated hydrochloric acid in EtOH
(40 mL) was heated at reux for 5 h. The reaction mixture was
cooled down to room temperature and concentrated under
reduced pressure. The mixture was diluted with H2O (50 mL)
and basied with a 1 M NaOH solution to pH 10. Then the
mixture was extracted with EtOAc (3 ꢂ 100 mL) and the
combined organic phases were washed with brine (50 mL),
dried over MgSO4 and concentrated in vacuo. The collected solid
was used directly in the next step without further purication.
10.4. Ethyl
2-(3-cyano-4-isobutoxyphenyl)-5-methyl-2H-
1,2,3-triazole-4-carboxylate (22d). White solid, (yield: 86%), mp:
77–79 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.32 (d, J ¼ 2.7 Hz, 1H),
8.25 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.04 (d, J ¼ 9.2 Hz, 1H), 4.47 (m,
2H), 3.89 (d, J ¼ 6.5 Hz, 2H), 2.61 (s, 3H), 2.23 (m, 1H), 1.45 (t, J
¼ 7.1 Hz, 3H), 1.10 (d, J ¼ 6.7 Hz, 6H). ESI-MS m/z 329.2 [M +
H]+.
8. General procedure for the preparation of compounds
19a–h
A mixture of compound 17 (1.61 mmol), triuoroacetic acid (2
mL), in DCM (20 mL) was stirred at room temperature for 6 h.
The mixture was quenched with water (20 mL) and neutralized
with sodium bicarbonate to pH 7. The mixture was extracted
with DCM (2 ꢂ 20 mL) and the combined organic phases were
washed with brine (20 mL), dried over MgSO4 and concentrated
in vacuo. The collected solid was used directly in the next step
without further purication.
10.5. Ethyl
2-(3-cyano-4-(amyloxy)phenyl)-5-methyl-2H-
1,2,3-triazole-4-carboxylate (22e). White solid, (yield: 79%), mp:
1
82–83 ꢀC, H NMR (600 MHz, DMSO-d6) d 8.21–8.17 (m, 2H),
7.40 (dd, J ¼ 8.3, 1.4 Hz, 1H), 4.34 (m, 2H), 4.17 (t, J ¼ 6.5 Hz,
2H), 3.32 (s, 2H), 2.50 (s, 3H), 1.79–1.74 (m, 2H), 1.42 (dd, J ¼
9.1, 6.5 Hz, 2H), 1.38–1.35 (m, 2H), 1.33 (t, J ¼ 7.1 Hz, 3H), 0.90
(t, J ¼ 7.2 Hz, 3H). ESI-MS m/z 343.2 [M + H]+.
10.6. Ethyl 2-(3-cyano-4-isoamyloxyphenyl)-5-methyl-2H-
1,2,3-triazole-4-carboxylate(22f). White solid, (yield: 93%), mp:
75–77 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.31 (d, J ¼ 2.7 Hz, 1H),
8.25 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.06 (d, J ¼ 9.2 Hz, 1H), 4.47 (q, J ¼
7.1 Hz, 2H), 4.16 (t, J ¼ 6.6 Hz, 2H), 2.61 (s, 3H), 1.98–1.88 (m,
1H), 1.78 (q, J ¼ 6.6 Hz, 2H), 1.45 (t, J ¼ 7.1 Hz, 3H), 1.00 (d, J ¼
6.6 Hz, 6H). ESI-MS m/z 343.2 [M + H]+.
9. General procedure for the preparation of compounds
20a–h and 21a–h
To a mixture of substituted phenylamine (2.6 mmol) and 15%
HCl (5 mL) was added drop-wise NaNO2 (0.27 g, 3.0 mmol) in
ꢀ
H2O (5 mL) at 0 C. Aer the completion of addition, the reac-
tion mixture was stirred at this temperature for 30 min. Ethyl
acetylacetate (0.39 g, 3 mmol), anhydrous sodium acetate (1.6 g,
20 mmol), and EtOH (10 mL) was added to the reaction mixture
at 0 ꢀC. Then, the mixture was ltered, and the residue was
dried to afford yellow solids (20d) in 90–95% yields. The
collected solid was used directly in the next step without further
purication.
10.7. Ethyl
2-(4-benzyloxy-3-cyanophenyl)-5-methyl-2H-
1,2,3-triazole-4-carboxylate (22g). White solid, (yield: 67%), mp:
150–152 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.35 (d, J ¼ 2.7 Hz, 1H),
8.24 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.50–7.32 (m, 5H), 7.11 (d, J ¼
9.2 Hz, 1H), 5.28 (s, 2H), 4.46 (m, 2H), 2.60 (s, 3H), 1.44 (m, 3H).
ESI-MS m/z 363.1 [M + H]+.
10.8. Ethyl 2-(4-allyloxy-3-cyanophenyl)-5-methyl-2H-1,2,3-
triazole-4-carboxylate (22h). White solid, (yield: 73%), mp: 102–
104 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.34 (d, J ¼ 2.7 Hz, 1H), 8.26
(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.06 (d, J ¼ 9.2 Hz, 1H), 6.07 (ddd, J ¼
10.6, 5.1 Hz, 1H), 5.51 (dd, J ¼ 1.2 Hz, 1H), 5.38 (dd, J ¼ 10.6,
1.2 Hz, 1H), 4.73 (d, J ¼ 5.1 Hz, 2H), 4.47 (q, J ¼ 7.1 Hz, 2H), 2.61
(s, 3H), 1.45 (t, J ¼ 7.1 Hz, 3H). ESI-MS m/z 313.1 [M + H]+.
10.9. Ethyl 5-methyl-2-(3-nitro-4-propoxyphenyl)-2H-1,2,3-
triazole-4-carboxylate (23a). Yellow solid, (yield: 86%), mp: 119–
120 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.59 (d, J ¼ 2.7 Hz, 1H), 8.26
10. General procedure for the preparation of compounds
22a–h and 23a–h
A mixture of corresponding compound 20 or 21 (0.63 mmol),
ammonium acetate (0.092 g, 1.2 mmol), CuBr2 (0.011 g, 0.05
mmol) in DMF (5 mL) and HOAc (5 mL) was heated at 110 ꢀC for
5 h. The reaction mixture was diluted with H2O (20 mL) then
extracted with EtOAc (3 ꢂ 20 mL) and the combined organic
phases were washed with brine (20 mL), dried over MgSO4 and
concentrated in vacuo. The residue was puried by ash column
chromatography (0–25% EtOAc in hexanes).
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(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.17 (d, J ¼ 9.2 Hz, 1H), 4.47 (q, J ¼ dissolved in water (20 mL) and acidied with a 1 M HCl solution
7.1 Hz, 2H), 4.13 (t, J ¼ 6.6 Hz, 2H), 2.61 (s, 3H), 1.90 (m, 2H), to pH 3. The solid was collected by ltration, washed with water
1.45 (t, J ¼ 7.1 Hz, 3H), 1.09 (m, 3H). ESI-MS m/z 335.1 [M + H]+. and puried by ash column chromatography (0–35% EtOAc in
10.10. Ethyl 2-(4-isopropoxy-3-nitrophenyl)-5-methyl-2H- hexanes) to yield the desired product.
1,2,3-triazole-4-carboxylate (23b). Yellow solid, (yield: 78%), mp:
11.1. 2-(3-Cyano-4-propoxyphenyl)-5-methyl-2H-1,2,3-
87–89 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.54 (d, J ¼ 2.7 Hz, 1H), triazole-4-carboxylic acid (7a). White solid, (yield: 88%), mp:
8.23 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.17 (d, J ¼ 9.2 Hz, 1H), 4.74 (m, 166–169 ꢀC, 1H NMR (600 MHz, DMSO-d6) d 13.52 (s, 1H), 8.27–
1H), 4.49–4.43 (m, 2H), 2.61 (s, 3H), 1.46–1.42 (m, 3H), 1.44–1.42 8.19 (m, 2H), 7.44 (d, J ¼ 8.9 Hz, 1H), 4.17 (t, J ¼ 6.4 Hz, 2H), 2.52
(m, 6H). ESI-MS m/z 335.1 [M + H]+.
(s, 3H), 1.83–1.77 (m, 2H), 1.03 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100
10.11. Ethyl 2-(4-butoxy-3-nitrophenyl)-5-methyl-2H-1,2,3- MHz, DMSO-d6) d 161.99, 159.91, 148.47, 138.80, 131.92, 125.34,
triazole-4-carboxylate (23c). Yellow solid, (yield: 79%), mp: 77– 123.56, 115.20, 114.27, 101.32, 70.79, 21.76, 11.30, 10.15. HRMS:
79 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.59 (d, J ¼ 2.7 Hz, 1H), 8.26 calcd for C14H14N4O3 [M ꢁ H]ꢁ 285.0993, found 285.0990.
(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.17 (d, J ¼ 9.2 Hz, 1H), 4.47 (q, J ¼
11.2. 2-(3-Cyano-4-isopropoxyphenyl)-5-methyl-2H-1,2,3-
7.1 Hz, 2H), 4.17 (m, 2H), 2.61 (s, 3H), 1.88–1.81 (m, 2H), 1.54 triazole-4-carboxylic acid (7b). White solid, (yield: 90%), mp:
(dq, J ¼ 14.7, 7.4 Hz, 2H), 1.45 (t, J ¼ 7.1 Hz, 3H), 0.99 (t, J ¼ 180–182 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.35 (d, J ¼ 2.7 Hz, 1H),
7.4 Hz, 3H). ESI-MS m/z 349.1 [M + H]+.
8.29 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.10 (d, J ¼ 9.2 Hz, 1H), 4.74 (dq, J
10.12. Ethyl
2-(4-isobutoxy-3-nitrophenyl)-5-methyl-2H- ¼ 6.2 Hz, 1H), 2.67 (s, 3H), 1.48 (d, J ¼ 6.2 Hz, 6H). 13C NMR (100
1,2,3-triazole-4-carboxylate (23d). Yellow solid, (yield: 84%), mp: MHz, CDCl3) d 164.33, 158.75, 148.99, 136.36, 131.11, 123.97,
104–106 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.60 (d, J ¼ 2.7 Hz, 1H), 123.66, 114.31, 113.07, 102.68, 71.69, 20.78, 10.57. HRMS: calcd
8.26 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.16 (d, J ¼ 9.2 Hz, 1H), 4.47 (q, J ¼ for C14H14N4O3 [M ꢁ H]ꢁ 285.0993, found 285.0975.
7.1 Hz, 2H), 3.92 (m, 2H), 2.61 (s, 3H), 2.18 (m, 1H), 1.45 (t, J ¼
11.3. 2-(4-Butoxy-3-cyanophenyl)-5-methyl-2H-1,2,3-
7.1 Hz, 3H), 1.08 (d, J ¼ 6.7 Hz, 6H). ESI-MS m/z 349.1 [M + H]+. triazole-4-carboxylic acid (7c). White solid, (yield: 83%), mp:
10.13. Ethyl
5-methyl-2-(4-amyloxy-3-nitrophenyl)-2H- 157–158 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.34 (d, J ¼ 2.6 Hz, 1H),
1,2,3-triazole-4-carboxylate (23e). Yellow solid, (yield: 84%), mp: 8.29 (dd, J ¼ 9.2, 2.6 Hz, 1H), 7.09 (d, J ¼ 9.2 Hz, 1H), 4.17 (t, J ¼
84–85 ꢀC, 1H NMR (600 MHz, CDCl3) d 8.58 (d, J ¼ 2.7 Hz, 1H), 6.4 Hz, 2H), 2.66 (s, 3H), 1.93–1.85 (m, 2H), 1.58 (dq, J ¼ 14.7,
8.25 (dd, J ¼ 9.1, 2.7 Hz, 1H), 7.17 (d, J ¼ 9.2 Hz, 1H), 4.47 (q, J ¼ 7.4 Hz, 2H), 1.03 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3)
7.2 Hz, 2H), 4.16 (t, J ¼ 6.5 Hz, 2H), 2.60 (d, J ¼ 4.6 Hz, 3H), 1.89– d 166.03, 160.90, 150.26, 137.63, 132.49, 125.29, 124.72, 115.34,
1.84 (m, 2H), 1.51–1.44 (m, 5H), 1.40 (dt, J ¼ 14.8, 7.2 Hz, 2H), 113.06, 103.01, 69.74, 31.09, 19.32, 13.99, 11.81. HRMS: calcd
0.94 (t, J ¼ 7.3 Hz, 3H). ESI-MS m/z 363.2 [M + H]+.
for C15H16N4O3 [M ꢁ H]ꢁ 299.1150, found 299.1146. IRmax/cmꢁ1
10.14. Ethyl 2-(4-isoamyloxy-3-nitrophenyl)-5-methyl-2H- 3426, 1700, 1287 (COOH), 2232 (CN), 2962, 2928, 2875, 1468,
1,2,3-triazole-4-carboxylate (23f). Yellow solid, (yield: 80%), mp: 1437, 1384 (CH3, CH2), 1614, 1531, 1505 (Ar), 1259 (C–O–C)
1
ꢀ
85–86 C, H NMR (400 MHz, CDCl3) d 8.64–8.56 (m, 1H), 8.28 (KBr).
(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.20 (d, J ¼ 9.2 Hz, 1H), 4.48 (q, J ¼
11.4. 2-(3-Cyano-4-isobutoxyphenyl)-5-methyl-2H-1,2,3-
7.1 Hz, 2H), 4.21 (t, J ¼ 6.6 Hz, 2H), 2.63 (s, 3H), 1.96–1.85 (m, triazole-4-carboxylic acid (7d). White solid, (yield: 77%), mp:
1H), 1.78 (q, J ¼ 6.6 Hz, 2H), 1.47 (t, J ¼ 7.1 Hz, 3H), 1.00 (d, J ¼ 184–186 ꢀC, 1H NMR (600 MHz, CDCl3) d 8.34 (d, J ¼ 2.7 Hz, 1H),
6.6 Hz, 6H). ESI-MS m/z 363.2 [M + H]+.
8.28 (dd, J ¼ 9.1, 2.7 Hz, 1H), 7.07 (d, J ¼ 9.2 Hz, 1H), 3.91 (d, J ¼
10.15. Ethyl
2-(4-benzyloxy-3-nitrophenyl)-5-methyl-2H- 6.6 Hz, 2H), 2.65 (s, 3H), 2.22 (dt, 6.6 Hz, 1H), 1.10 (d, J ¼ 2.9 Hz,
1,2,3-triazole-4-carboxylate (23g). Yellow solid, (yield: 75%), mp: 6H). 13C NMR (100 MHz, DMSO-d6) d 161.98, 160.01, 148.49,
90–91 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.64 (d, J ¼ 2.7 Hz, 1H), 138.86, 131.97, 125.40, 123.58, 115.14, 114.36, 101.37, 75.23,
8.27 (dd, J ¼ 9.1, 2.7 Hz, 1H), 7.51–7.35 (m, 5H), 7.25 (d, J ¼ 28.61, 18.72, 11.30. HRMS: calcd for C15H16N4O3 [M ꢁ H]ꢁ
9.1 Hz, 1H), 5.33 (s, 2H), 4.49 (q, J ¼ 7.1 Hz, 2H), 2.63 (s, 3H), 299.1150, found 299.1134. IRmax/cmꢁ1 3430, 1704, 1288
1.47 (t, J ¼ 7.1 Hz, 3H). ESI-MS m/z 383.1 [M + H]+.
(COOH), 2232 (CN), 2961, 2929, 2877, 1469, 1438, 1384 (CH3,
10.16. Ethyl 2-(4-allyloxy-3-nitrophenyl)-5-methyl-2H-1,2,3- CH2), 1614, 1533, 1506 (Ar), 1259 (C–O–C) (KBr).
triazole-4-carboxylate (23h). Yellow solid, (yield: 68%), mp: 153–
11.5. 2-(4-Amyloxy-3-cyanophenyl)-5-methyl-2H-1,2,3-
155 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.64 (d, J ¼ 2.7 Hz, 1H), 8.28 triazole-4-carboxylic acid (7e). White solid, (yield: 91%), mp:
(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.20 (d, J ¼ 9.2 Hz, 1H), 6.13–6.03 (m, 155–157 ꢀC, 1H NMR (600 MHz, DMSO-d6) d 8.23–8.17 (m, 1H),
1H), 5.54 (dd, J ¼ 17.3, 1.2 Hz, 1H), 5.40 (dd, J ¼ 10.6, 1.2 Hz, 7.42 (d, J ¼ 9.3 Hz, 1H), 4.18 (t, J ¼ 6.5 Hz, 2H), 2.50 (s, 3H), 1.80–
1H), 4.79–4.74 (m, 2H), 4.49 (q, J ¼ 7.1 Hz, 2H), 2.63 (s, 3H), 1.46 1.72 (m, 2H), 1.47–1.39 (m, 2H), 1.39–1.31 (m, 2H), 0.90 (t, J ¼
(t, J ¼ 7.1 Hz, 3H). ESI-MS m/z 333.1 [M + H]+.
7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) d 161.95, 159.95,
148.50, 138.74, 131.91, 125.37, 123.61, 115.20, 114.29, 101.32,
69.42, 28.97, 27.46, 21.78, 13.89, 11.29. HRMS: calcd for
C
16H18N4O3 [M ꢁ H]ꢁ 313.1306, found 313.1305. IRmax/cmꢁ1
11. General procedure for the preparation of compounds 7a–
h and 8a–h
3426, 1708, 1289 (COOH), 2232 (CN), 2958, 2929, 2860, 1467,
A mixture of corresponding compound 22 or 23 (0.3 mmol), 1 M 1436, 1384 (CH3, CH2), 1614, 1529, 1506 (Ar), 1258 (C–O–C)
NaOH solution (3.0 mL) in THF/EtOH (1 : 1) (5.0 mL) was heated (KBr).
at 45 ꢀC for 1.5 h. Aer the reaction was completed, the solvent
11.6. 2-(3-Cyano-4-isoamyloxyphenyl)-5-methyl-2H-1,2,3-
was removed under reduced pressure. The residue was triazole-4-carboxylic acid (7f). White solid, (yield: 92%), mp:
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185–186 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.36 (d, J ¼ 2.7 Hz, 1H), 8.30 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.20 (d, J ¼ 9.2 Hz, 1H), 3.96 (d, J ¼
8.30 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.10 (d, J ¼ 9.2 Hz, 1H), 4.19 (t, J ¼ 6.4 Hz, 2H), 2.70 (s, 3H), 2.26–2.15 (m, 1H), 1.10 (d, J ¼ 6.7 Hz,
6.6 Hz, 2H), 2.67 (s, 3H), 1.95 (m, 1H), 1.81 (q, J ¼ 6.6 Hz, 2H), 6H). 13C NMR (100 MHz, DMSO-d6) d 160.91, 150.24, 147.64,
1.02 (d, J ¼ 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) d 161.97, 137.89, 130.03, 123.29, 115.38, 114.56, 74.57, 26.65, 17.71,
159.92, 148.47, 138.77, 131.91, 125.31, 123.55, 115.19, 114.26, 10.29. HRMS: calcd for C14H16N4O5 [M ꢁ H]ꢁ 319.1048, found
101.30, 67.99, 36.99, 24.62, 22.38, 11.29. HRMS: calcd for 319.1029.
C
16H18N4O3 [M ꢁ H]ꢁ 313.1306, found 313.1288. IRmax/cmꢁ1
11.13. 2-(4-Isoamyloxy-3-nitrophenyl)-5-methyl-2H-1,2,3-
3432, 1704, 1289 (COOH), 2231 (CN), 2957, 2927, 1466, 1437, triazole-4-carboxylic acid (8e). Light yellow, (yield: 89%), mp:
1384 (CH3, CH2), 1615, 1532, 1506 (Ar), 1259 (C–O–C) (KBr).
183–184 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.63 (d, J ¼ 2.6 Hz, 1H),
11.7. 2-(4-Benzyloxy-3-cyanophenyl)-5-methyl-2H-1,2,3- 8.30 (dd, J ¼ 9.2, 2.6 Hz, 1H), 7.21 (d, J ¼ 9.2 Hz, 1H), 4.19 (t, J ¼
triazole-4-carboxylic acid (7g). White solid, (yield: 86%), mp: 6.4 Hz, 2H), 2.67 (s, 3H), 1.94–1.84 (m, 2H), 1.55–1.47 (m, 2H),
199–201 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.39 (d, J ¼ 2.6 Hz, 1H), 1.41 (dt, J ¼ 14.3, 7.2 Hz, 2H), 0.97 (t, J ¼ 7.2 Hz, 3H). 13C NMR
8.28 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.51–7.40 (m, 5H), 7.15 (d, J ¼ (100 MHz, CDCl3) d 165.73, 152.41, 150.30, 139.86, 137.64,
9.2 Hz, 1H), 5.32 (s, 2H), 2.67 (s, 3H). 13C NMR (100 MHz, CDCl3) 131.73, 124.43, 116.98, 115.19, 100.12, 70.42, 28.71, 28.07,
d 163.20, 163.19, 150.55, 149.12, 139.14, 136.56, 133.92, 131.10, 22.45, 14.08, 11.73. HRMS: calcd for C15H18N4O5 [M ꢁ H]ꢁ
127.83, 127.51, 126.06, 123.22, 115.93, 114.89, 70.70, 10.53. 333.1204, found 333.1187. IRmax/cmꢁ1 3425, 1702, 1279
HRMS: calcd for C18H14N4O3 [M ꢁ H]ꢁ 333.0933, found (COOH), 1540 (NO2), 2959, 2933, 2873, 1469, 1433, 1383 (CH3,
333.1100.
11.8. 2-(4-Allyloxy-3-cyanophenyl)-5-methyl-2H-1,2,3-
CH2), 1625, 1590, 1497 (Ar), 1255 (C–O–C) (KBr).
11.14. 2-(4-Isoamyloxy-3-nitrophenyl)-5-methyl-2H-1,2,3-
triazole-4-carboxylic acid (7h). White solid, (yield: 84%), mp: triazole-4-carboxylic acid (8f). Light yellow, (yield: 90%), mp:
212–215 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.38 (d, J ¼ 2.7 Hz, 1H), 200–201 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.62 (d, J ¼ 2.7 Hz, 1H),
8.30 (dd, J ¼ 9.3, 2.7 Hz, 1H), 7.10 (d, J ¼ 9.3 Hz, 1H), 6.09 (ddd, J 8.30 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.22 (d, J ¼ 9.2 Hz, 1H), 4.22 (t, J ¼
¼ 22.3, 10.6, 5.1 Hz, 1H), 5.54 (dd, J ¼ 17.3, 1.1 Hz, 1H), 5.41 (d, J 6.5 Hz, 2H), 2.67 (s, 3H), 1.90 (td, J ¼ 13.3, 6.6 Hz, 1H), 1.79 (q, J
¼ 10.6 Hz, 1H), 4.77 (d, J ¼ 5.1 Hz, 2H), 2.67 (s, 3H). 13C NMR ¼ 6.5 Hz, 2H), 1.01 (d, J ¼ 6.6 Hz, 6H). 13C NMR (100 MHz,
(100 MHz, CDCl3) d 164.16, 160.05, 150.05, 137.35, 132.60, CDCl3) d 164.63, 152.24, 150.13, 139.82, 137.45, 131.65, 124.27,
131.33, 124.98, 124.59, 118.92, 115.05, 113.35, 103.18, 70.13, 116.87, 115.05, 68.74, 37.52, 24.90, 22.47, 11.56. HRMS: calcd
10.54. HRMS: calcd for C14H12N4O3 [M ꢁ H]ꢁ 283.0837, found for C15H18N4O5 [M ꢁ H]ꢁ 333.1204, found 333.1205. IRmax/cmꢁ1
283.0832.
3428, 1704, 1281 (COOH), 1540 (NO2), 2959, 2873, 1472, 1432,
11.9. 5-Methyl-2-(3-nitro-4-propoxyphenyl)-2H-1,2,3- 1385 (CH3, CH2), 1625, 1591, 1497 (Ar), 1256 (C–O–C) (KBr).
triazole-4-carboxylic acid (8a). White solid, (yield: 80%), mp:
11.15. 2-(4-Benzyloxy-3-nitrophenyl)-5-methyl-2H-1,2,3-
1
ꢀ
204–205 C, H NMR (600 MHz, DMSO-d6) 8.42 (d, J ¼ 2.7 Hz, triazole-4-carboxylic acid (8g). Light yellow, (yield: 93%), mp:
1H), 8.21 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.55 (d, J ¼ 9.2 Hz, 1H), 4.18 (t, 204–206 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.66 (d, J ¼ 2.7 Hz, 1H),
J ¼ 6.3 Hz, 2H), 2.52 (s, 3H), 1.80–1.72 (m, 2H), 0.99 (t, J ¼ 8.29 (dd, J ¼ 9.1, 2.6 Hz, 1H), 7.51–7.38 (m, 5H), J ¼ 9.1, 7.26 (d,
7.4 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) d 162.93, 151.12, 1H) 5.33 (s, 2H), 2.66 (s, 3H). 13C NMR (100 MHz, CDCl3)
148.65, 139.09, 138.96, 131.09, 124.29, 116.48, 115.58, 71.19, d 163.19, 150.55, 149.12, 139.14, 136.56, 133.92, 131.10, 127.83,
21.78, 11.30, 10.19. HRMS: calcd for C13H14N4O5 [M ꢁ H]ꢁ 127.51, 126.06, 123.22, 115.93, 114.89, 70.70, 28.69, 10.53.
305.0891, found 305.0874.
HRMS: calcd for C17H14N4O5 [M ꢁ H]ꢁ 353.0891, found
11.10. 2-(4-Isopropoxy-3-nitrophenyl)-5-methyl-2H-1,2,3- 353.0889.
triazole-4-carboxylic acid (8b). White solid, (yield: 94%), mp:
11.16. 2-(4-Allyloxy-3-nitrophenyl)-5-methyl-2H-1,2,3-
201–203 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.58 (d, J ¼ 2.7 Hz, 1H), triazole-4-carboxylic acid (8h). Light yellow, (yield: 91%), mp:
8.28 (dd, J ¼ 9.3, 2.7 Hz, 1H), 7.22 (d, J ¼ 9.3 Hz, 1H), 4.78 (m, 220–222 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.65 (d, J ¼ 2.7 Hz, 1H),
1H), 2.67 (s, 3H), 1.46 (d, J ¼ 6.1 Hz, 6H). 13C NMR (100 MHz, 8.30 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.22 (d, J ¼ 9.2 Hz, 1H), 6.08 (ddd, J
DMSO-d6) d 162.93, 149.51, 148.64, 140.12, 131.02, 123.99, ¼ 22.2, 10.4, 5.0 Hz, 1H), 5.58–5.51 (m, 1H), 5.41 (dd, J ¼ 10.4,
117.63, 115.52, 72.80, 21.57, 11.31. HRMS: calcd for C13H14N4O5 1.2 Hz, 1H), 4.79 (d, J ¼ 5.0 Hz, 2H), 2.67 (s, 3H). 13C NMR (100
[M ꢁ H]ꢁ 305.0891, found 305.0878.
MHz, DMSO-d6) d 161.89, 150.55, 148.69, 139.19, 138.95, 132.22,
11.11. 2-(4-Butoxy-3-nitrophenyl)-5-methyl-2H-1,2,3- 131.33, 124.23, 118.17, 116.84, 115.67, 69.95, 11.29. HRMS:
triazole-4-carboxylic acid (8c). White solid, (yield: 91%), mp: calcd for C13H12N4O5 [M ꢁ H]ꢁ 303.0736, found 303.0716.
183–185 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.62 (d, J ¼ 2.7 Hz, 1H),
8.30 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.21 (d, J ¼ 9.2 Hz, 1H), 4.20 (t, J ¼
12. General procedure for the preparation of compounds 9e–
6.4 Hz, 2H), 2.67 (s, 3H), 1.88 (tt, J ¼ 12.5, 6.4 Hz, 2H), 1.61–1.51
(m, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
f and 10e–f
d 161.86, 151.13, 148.60, 138.98, 138.88, 131.03, 124.21, 116.42, A mixture of the corresponding compound 22 or 23 (0.3 mmol),
115.49, 69.50, 30.37, 18.52, 13.55, 11.26. HRMS: calcd for hydrazine hydrate (1.0 mL) in EtOH (10.0 mL) was heated at
C
14H16N4O5 [M ꢁ H]ꢁ 319.1048, found 319.1041.
55 ꢀC for 5 h. Aer the reaction was completed, the solvent was
11.12. 2-(4-Isobutoxy-3-nitrophenyl)-5-methyl-2H-1,2,3- then removed under reduced pressure. The residue was puried
triazole-4-carboxylic acid (8d). White solid, (yield: 86%), mp: by ash column chromatography (0–5% methanol in DCM) to
203–205 ꢀC, 1H NMR (400 MHz, CDCl3) d 8.64 (d, J ¼ 2.7 Hz, 1H), yield the desired product.
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12.1. 2-(4-Amyloxy-3-cyanophenyl)-5-methyl-2H-1,2,3-
RSC Advances
All the experiments were repeated three to four times and values
were expressed as means of several experiments.
triazole-4-carbohydrazide (9e). White solid, (yield: 77%), mp:
113–115 ꢀC, 1H NMR (600 MHz, DMSO-d6) d 8.30 (d, J ¼ 2.7 Hz,
1H), 8.23 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.45 (d, J ¼ 9.2 Hz, 1H), 4.19 (t,
J ¼ 6.5 Hz, 2H), 2.50 (s, 3H), 1.80–1.74 (m, 2H), 1.46–1.40 (m,
14. Molecular modeling
2H), 1.36 (dt, J ¼ 14.0, 7.2 Hz, 2H), 0.90 (t, J ¼ 7.2 Hz, 3H). 13
C
Discovery Studio 3.0 was used to perform molecular docking.
The crystal structure of XO (PDB code: 1N5X) with the corre-
sponding entry was adopted as a target protein. In the docking
process, the protein was prepared via several operations, which
was illustrated in detail in our previous study.14 Then, the
compound 7f was drawn with Chemdraw and the energy fully
minimized using the CHARMm force eld. Finally, it was
docked into the binding site using the CDOCKER protocol with
the default settings. The schematic diagrams of interactions
between the active site of XO and compound 7f were analyzed by
Discovery Studio soware package.
NMR (150 MHz, DMSO-d6) d 159.71, 159.60, 146.41, 139.50,
132.05, 124.97, 123.41, 115.39, 114.33, 101.27, 69.42, 27.98,
27.47, 21.78, 13.90, 10.81.
12.2 2-(3-Cyano-4-isopentyloxyphenyl)-5-methyl-2H-1,2,3-
triazole-4-carbohydrazide (9f). White solid, (yield: 68%), mp:
124–126 ꢀC, 1H NMR (600 MHz, DMSO-d6) d 8.30 (d, J ¼ 2.7 Hz,
1H), 8.23 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.47 (d, J ¼ 9.2 Hz, 1H), 4.22 (t,
J ¼ 6.6 Hz, 2H), 2.50 (s, 3H), 1.85–1.78 (m, 1H), 1.67 (q, J ¼
6.6 Hz, 2H), 0.95 (d, J ¼ 6.6 Hz, 6H). 13C NMR (150 MHz, DMSO-
d6) d 159.75, 159.63, 146.46, 139.54, 132.09, 125.03, 123.45,
115.44, 114.38, 101.29, 68.04, 24.66, 22.43, 20.52, 10.85. HRMS:
calcd for C16H20N6O2 [M + H]+ 329.1721, found 329.1721. IRmax
/
cmꢁ1 3423, 1669 (CONHNH2), 2230 (CN), 2958, 2929, 1468,
1433, 1384 (CH3, CH2), 1625, 1543, 1508 (Ar), 1256 (C–O–C)
(KBr).
Acknowledgements
We gratefully acknowledge the National Natural Science Foun-
dation of China (81502932, 81673293, 81602969). This work was
also supported by Program for Innovative Research Team of the
Ministry of Education and Program for Liaoning Innovative
Research Team in University.
12.3. 5-Methyl-2-(3-nitro-4-pentyloxyphenyl)-2H-1,2,3-
triazole-4-carbohydrazide (10e). Light yellow, (yield: 60%), mp:
117–118 ꢀC, 1H NMR (600 MHz, DMSO-d6) d 8.47 (d, J ¼ 2.7 Hz,
1H), 8.23 (dd, J ¼ 9.2, 2.7 Hz, 1H), 7.57 (d, J ¼ 9.2 Hz, 1H), 4.24–
4.18 (m, 2H), 2.51 (s, 3H), 1.76–1.71 (m, 2H), 1.43–1.37 (m, 2H),
1.37–1.30 (m, 2H), 0.88 (t, J ¼ 7.2 Hz, 3H). 13C NMR (150 MHz,
DMSO-d6) d 159.55, 150.92, 146.62, 139.75, 139.16, 131.25,
124.01, 116.54, 115.33, 69.82, 28.03, 27.48, 21.76, 13.92, 10.85.
HRMS: calcd for C15H20N6O4 [M + H]+ 349.1619, found
349.1616.
12.4. 2-(4-Isopentyloxy-3-nitrophenyl)-5-methyl-2H-1,2,3-
triazole-4-carbohydrazide (10f). Light yellow, (yield: 66%), mp:
120–122 ꢀC, 1H NMR (600 MHz, DMSO-d6) d 9.83 (s, 1H), 8.46 (d,
J ¼ 2.6 Hz, 1H), 8.22 (dd, J ¼ 9.2, 2.6 Hz, 1H), 7.59 (d, J ¼ 9.2 Hz,
1H), 4.24 (t, J ¼ 6.5 Hz, 2H), 2.51 (s, 3H), 1.83–1.75 (m, 1H), 1.64
(q, J ¼ 6.5 Hz, 2H), 0.92 (d, J ¼ 6.6 Hz, 6H). 13C NMR (150 MHz,
DMSO-d6) d 159.89, 151.25, 146.94, 140.08, 139.48, 131.59,
124.30, 116.87, 115.63, 68.71, 37.40, 24.87, 22.68, 11.19.
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114888 | RSC Adv., 2016, 6, 114879–114888
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