Synthesis and bioevaluation of 1-phenylimidazole-4-carboxylic acid derivatives as novel xanthine oxidoreductase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111883

As part of a continuing study, we designed and synthesized four series of 1-phenylimidazole-4-carboxylic acid derivatives as xanthine oxidoreductase (XOR) inhibitors, evaluated their in vitro inhibitory potencies against XOR and hypouricemic effects in mi

Full text of DOI:10.1016/j.ejmech.2019.111883

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Synthesis and bioevaluation of 1-phenylimidazole-4-carboxylic acid derivatives as
novel xanthine oxidoreductase inhibitors
Haiyan Zhou, Xiaolei Li, Yuanyuan Li, Xinying Zhu, Lei Zhang, Jing Li
PII:
S0223-5234(19)31035-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111883
EJMECH 111883
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2019
Revised Date: 27 October 2019
Accepted Date: 12 November 2019
Please cite this article as: H. Zhou, X. Li, Y. Li, X. Zhu, L. Zhang, J. Li, Synthesis and bioevaluation of
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Synthesis and bioevaluation of 1-phenylimidazole-4-carboxylic acid
derivatives as novel xanthine oxidoreductase inhibitors
#
#
Haiyan Zhou , Xiaolei Li , Yuanyuan Li, Xinying Zhu, Lei Zhang, Jing Li*
School of Biology and Biological Engineering, South China University of Technology,
Guangzhou 510006, P. R. China
*
Address correspondence to this author:
Assoc. Prof. Jing Li, School of Biology and Biological Engineering, South China
University of Technology, Guangzhou 510006, P. R. China
E-mail: lij@scut.edu.cn
Tel./fax: +86 2039380678
#
These authors contributed equally to this work and should be considered as co-first
authors.
1

Abstract
As part of a continuing study, we designed and synthesized four series of 1-
phenylimidazole-4-carboxylic acid derivatives as xanthine oxidoreductase (XOR)
inhibitors, evaluated their in vitro inhibitory potencies against XOR and hypouricemic
effects in mice, and determined their structure-activity relationships (SARs). Most of
the compounds exhibited in vitro XOR inhibition at the nanomolar level. In
comparison to febuxostat (half-maximal inhibitory concentration [IC ] value of 7.0
5
0
nM), compounds Ie and IVa exhibited the most promising XOR inhibitory effects
with IC50 values of 8.0 and 7.2 nM, respectively. In the potassium
oxonate/hypoxanthine-induced acute and long-term hyperuricemia mouse models,
compounds Ie and IVa displayed significant hypouricemic potencies (P < 0.05), that
were slightly weaker than and similar to febuxostat, respectively. More interestingly,
both compounds showed a capacity to improve kidney damage by decreasing
creatinine and urea nitrogen levels compared to the long-term hyperuricemia mouse
group (P < 0.05), while febuxostat showed no significant effect.
Keywords: 1-phenylimidazole-4-carboxylic acid, xanthine oxidoreductase inhibitors,
hypouricemic
1
. Introduction
Hyperuricemia is a common metabolic disorder caused by continuously elevated
serum uric acid, which is produced as the final oxidation product of purine catabolism
in humans and induces the formation of monosodium urate crystal deposits that
eventually cause gout [1–3]. In purine metabolism, xanthine oxidoreductase (XOR) is
2

a key enzyme that catalyzes the oxidation of hypoxanthine and xanthine to generate
the final product uric acid. Therefore, XOR is currently considered the most
promising target for blocking uric acid production in hyperuricemia treatment [4–6].
The representative purine XOR inhibitor allopurinol (Fig. 1) has been the
cornerstone of the treatment of gout and hyperuricemia in the USA for several
decades. Despite its potent urate-lowering properties, allopurinol reportedly induces
several common and severe adverse reactions such as renal failure and allopurinol
hypersensitivity syndrome [7–9]. Hence, it is crucial to develop new XOR inhibitors
with fewer side effects.
Non-purine XOR inhibitors such as febuxostat (Fig. 1) and topiroxostat (FYX-051,
Fig. 1) have been investigated to avoid the side effects of allopurinol. Febuxostat, a
phenylthiazole derivative with outstanding and longer-lasting XOR inhibitory potency
than allopurinol, was approved for the treatment of gout in the USA in 2009 [10, 11].
The triazole derivative topiroxostat was approved for the clinical treatment of
hyperuricemia in Japan in 2013 [12, 13]. Furthermore, other structural XOR inhibitors
are being studied
,
such as imidazoles, pyrazoles (e.g., Y-700, Fig. 1) [14], flavonoids
15], fraxamoside [16], N-(1,3-diaryl-3-oxopropyl)amides [17], N-acetylpyrazolines
18], quinazolines [19], pyrimidines [20], isoxazoles [21], isocytosines [22],
[
[
selenazoles [23], 1,2,3-triazoles [24], and 1,3,4-oxadiazoles [25].
Similarly, we obtained pyrazole derivatives such as WN1703 (Fig. 1) through the
lead optimization of febuxostat via the bioisosterism concept. WN1703 showed
similar XOR inhibitory activity in vitro and similar hypouricemic potency in the
potassium oxonate/hypoxanthine-induced acute and long-term hyperuricemia mouse
models as compared with febuxostat [26 and supplementary material 1]. We also
3

explored structure–activity relationships (SARs) via the molecular docking and three-
dimensional quantitative structure–activity relationship (3D-QSAR) approaches [27].
Based on the above work, we conducted further optimization experiments (Fig. 2).
Since some simple imidazole analogues are good XOR inhibitors [28], we designed
compounds in which the pyrazole moiety of WN1703 was replaced by imidazole. To
facilitate the discussion of the SARs in the following articles, these compounds are
classified into four series according to functional group substitutions: series I could be
considered as imidazole analogues of WN1703; series II and III were further
modifications of series I that focused on substitution of the C4 of the phenyl moiety
and the coupling mode between the phenyl and imidazole groups, respectively. While
referring to our previous findings on the SARs of the XOR inhibitors [27] and the
three-ring structure of topiroxostat, series IV was designed to determine the
hydrophobic group size that can be accommodated at the entrance of the active site.
Consequently, 22 novel XOR inhibitors were synthesized, some of which were found
to exhibit in vitro XOR inhibition and potent hypouricemic effects in mice.
Fig. 1. Chemical structures of allopurinol, febuxostat, topiroxostat, Y-700, and WN1703.
4

Fig. 2. Design of compounds I–IV.
2
. Results and discussion
2
.1. Chemistry
The series I derivatives were synthesized using the strategy depicted in Scheme 1.
Compound 1 was alkylated using the corresponding alkylamine in the presence of
K CO to produce 2a–e, followed by a coupling reaction with 1H-imidazole-4-ethyl
2
3
formate to obtain 3a–e, subsequent hydrolyzation with NaOH, and then acidification
to yield Ia–e.
5

Scheme 1. Reagents and conditions: (i) alkylamine, K CO , and DMF at 70–80 °C; (ii) CuI,
2
3
K CO , trans-N,Nʹ-dimethyl-1,2-cyclohexanediamine, 1H-imidazole-4-ethyl formate, and DMF at
2
3
1
20 °C; (iii) NaOH aq. (1 M), THF/EtOH (1:1), 65 °C, HCl aq. (1 M), rt.
Similarly, compounds IIa–i were synthesized as presented in Scheme 2. Compound
4
was alkylated with the corresponding alkyl bromide or alkyl iodide in the presence
of K CO to afford 5a–i, followed by a coupling reaction with 1H-imidazole-4-ethyl
2
3
formate to furnish 6a–i, which were hydrolyzed to yield IIa–i.
Scheme 2. Reagents and conditions: (i) K CO , alkyl halide, DMF, 70–80 °C; (ii) CuI, K CO ,
2
3
2
3
trans-N,Nʹ-dimethyl-1,2-cyclohexanediamine, 1H-imidazole-4-ethyl formate, and DMF at 120 °C;
(iii) NaOH aq. (1 M), THF/EtOH (1:1), 65 °C, HCl aq. (1 M), rt.
The synthesis of the target derivatives IIIa–e was accomplished as outlined in
Scheme 3. Commercially available 1-(4-hydroxyphenyl)ethanone (7) was iodinated
with I and KI to generate intermediate 8, which was alkylated with the appropriate
2
C2–C5 alkyl bromide/iodide in dimethylformamide (DMF) in the presence of
6

anhydrous K CO to obtain 9a–d, followed by cyano substitution to afford 10a–d.
2
3
Compounds 10a–d were treated with bromine solution or CuBr to obtain 11a–d. The
2
Delépine reaction of 11a–d was performed through hexamethylenetetramine
treatment, followed by acidic hydrolysis with HCl (conc.) to yield the key
intermediates 12a–d. Cyclization of 12a–d with ethyl thiooxamate in the presence of
trimethyloxonium tetrafluoroborate and CH COONa delivered 13a–d, which were
3
hydrolyzed with NaOH and then acidified to yield IIIa–d. In addition, 13c was
alkylated with CH I and then hydrolyzed to afford IIIe.
3
Scheme 3. Reagents and conditions: (i) KI, I , and NH OH followed by acidification with HCl
2
4
(conc.); (ii) K CO , DMF, and alkyl halide, 70–80 °C; (iii) CuCN and DMF, 140 °C; (iv) Br aq.
2 3 2
(
1 M), or CuBr and dioxane, 120 °C; (v) CHCl , Et O, and HMTA followed by acidic hydrolysis
2
3
2
with HCl (conc.); (vi) ethyl thiooxamate, trimethyloxonium tetrafluoroborate, CH Cl , CH COOH,
2
2
3
CH COONa, and dioxane, 70 °C; (vii) NaOH aq. (1 M) and THF/EtOH (1:1), 65 °C, followed by
3
acidification with HCl aq. (1 M).
7

Compounds IVa–c were synthesized as outlined in Scheme 4. Commercially
available 5-bromo-2-iodobenzonitrile (14) was coupled with the corresponding
alkylboronic acid to produce 15a–c, followed by a coupling reaction with 1H-
imidazole-4-ethyl formate to obtain 16a–c. Subsequently, 16a–c were hydrolyzed
using NaOH in tetrahydrofuran (THF)/ethanol (EtOH) and then acidified to produce
IVa–c.
Scheme 4. Reagents and conditions: (i) alkylboronic acid, K CO , Pd[P(C H ) ] , and DMF,
2
3
6
5 3 4
1
20 °C; (ii) CuI, K CO , trans-N,Nʹ-dimethyl-1,2-cyclohexanediamine, 1H-imidazole-4-ethyl
2 3
formate, and DMF, 120 °C; (iii) NaOH aq. (1 M) and THF/EtOH (1:1), 65 °C, followed by
acidification with HCl aq. (1 M).
2
.2. Biological activity in vitro
The in vitro bovine XOR inhibitory activities of target compounds Ia–e, IIa–i,
IIIa–e, and IVa–c were measured by determining uric acid levels at 295 nm, with
febuxostat as the reference compound [26].
The XOR inhibitory activity of Compounds Ia–e, IIa–i, and IIIa–e are shown in
Table 1. In general, most of compounds I and II (with the exception of IIa and IIb)
displayed certain XOR inhibitory activity with half-maximal inhibitory concentration
(
IC ) values <100 nM, which were significantly superior to the XOR inhibition of
50
8

compounds III. SARs were revealed with reference to their XOR inhibitory activities.
1) The XOR inhibitory activity of the alkylamine-containing derivatives was superior
to that of the alkoxy-containing derivatives, which reconfirmed our previous study
26]. (2) Comparison of IIa–g revealed that inhibitory activity gradually increased
(
[
with carbon chain length. Generally, when substituents at the phenyl 4-position (R)
possessed the same number of carbon atoms, the inhibitory activity of chain-alkane-
substituted derivatives was superior to that of cyclic-alkane-substituted derivatives,
such as Ia versus Ic and IIf versus IIh. (3) Compound Ie, which contains a
cycloheptylamine substituent at the phenyl 4-position (R), exhibited the strongest
inhibitory activity, which was equivalent to that of febuxostat (t test, P > 0.05),
suggesting that the most appropriate molecular volume could maximize inhibitory
activity. (4) The coupling mode between the phenyl and imidazole groups affected
XOR inhibitory activity. Obviously, when XR was the same, compounds III showed
lower XOR inhibition than compounds II, for example, IIa and IIIa, IIb and IIIb, IIc
and IIIc, and IIh and IIId. (5) A methyl group introduced at the R position led to a
1
fourfold reduction in XOR inhibitory activity as compared IIIe with IIIc, further
indicating that although those compounds can be considered as structural
modifications of febuxostat, methyl is not necessary for their XOR inhibitory activity
after the bioisosterism of five-membered heterocycles. Taken together, the results in
Table 1 suggest that imidazole analogues were generally tolerated for the structural
optimization of WN1703; however, we did not obtain compounds with significantly
higher XOR inhibitory activity, except Ie with XOR inhibition similar to febuxostat.
9

Table 1. In vitro XOR inhibitory activities of designed compounds Ia–e, IIa–i and IIIa–e (n = 3).
XR
CN
N
N
HOOC
R1
III
-
R1
IC (nM)
Compd.
-XR
)CH CH(CH
IC (nM) Compd.
-XR
IC (nM)
Compd.
IIIa
IIIb
IIIc
IIId
-XR
50
50
50
Ia
Ib
Ic
Id
Ie
-N(CH
3
2
3
)
2
27.1
34.1
55.6
30.5
8.0
IIa
IIb
IIc
IId
IIe
IIf
-OCH
CH
2
3
226.3
197.9
94.5
73.9
67.9
40.6
29.8
63.0
47.4
-OCH
CH
2
3
-H
-H
-H
-H
-CH
3
948.0
750.0
510.0
387.0
2158.0
-N(CH
-N(CH
)
)
-OCH(CH
)
-OCH(CH
3
)
2
2
4
3
2
-OCH
-OCH(CH
-OCH C(CH
-OCH(CH
-OCH CH(CH
-OCH(CH
CH(CH
2
)
-OCH
-OCH(CH
-OCH CH(CH
CH(CH
2
3
)
2
2
2
5
3
2
-N(CH
) CHCH
2 4
)CH
3
CH
2
)
4
3
2
3
-N(CH
)
)
IIIe
2
)
3
2
7
2
3
3
CH )
3 2
CH )
3 2
2
IIg
IIh
IIi
2
2
)
2
4
Febuxostat
一
7.0
-OCH(CH2)5
10

As shown in Table 2, IVa was the most potent XOR inhibitor with an IC value of 7.2 nM,
5
0
and its inhibitory activity was equivalent to that of febuxostat (t test, P > 0.05). The inhibitory
activity of IVb was approximately twofold higher than that of IVc, indicating that para
substitution of the benzene ring was superior to ortho substitution. Although compounds
IVa–c displayed potent inhibitory activity against XOR, more derivatives of series IV are
needed for future discussions regarding SARs.
Table 2. In vitro XOR inhibitory activities of designed compounds IVa–c (n = 3).
Compd.
R
IC (nM)
5
0
IVa
7.2
IVb
IVc
34.5
74.4
O
O
Febuxostat
-
7.0
2
.3. Molecular modeling
To further study the binding mode, molecular docking was performed between several
compounds (Ia, Ie, IVa, WN1703, and febuxostat) and XOR using Schrodinger Glide 10.1
software [29–31]. As shown in Fig. 3A, the binding model of the compounds and XOR had
three main features: (1) the carboxyl group on the heterocyclic ring interacted with Arg880
(
O–H–N: 2.33 Å, O–H–N: 1.97 Å) and Thr1010 (O–H–N: 2.22 Å) through hydrogen bonds;
2) hydrogen bonds were also formed between the cyano groups and Asn768 (N–H–N: 2.44
(
Å); (3) the five-membered heterocyclic rings formed a slanted sandwich between Phe914 and
11

Phe1009 by π–π stacking. However, there were still subtle differences in their binding
patterns. (1) As shown in Fig. 3B, to match the XOR binding cavity, the dihedral angle
between the benzene ring and five-membered heterocyclic ring of these compounds was not
exactly the same, which might have altered the π–π interaction between the five-membered
heterocyclic ringsand Phe914 and Phe1009. Although the dihedral angle data of these
compounds cannot show clear rules, it further suggests that bioisosterism substitution of
different five-membered heterocycles would affect the spatial conformation of the whole
molecule, thus affecting its affinity for XOR. (2) The substituent at the C4 position of the
phenyl moiety mainly provided hydrophobic interactions at the initial part of the XOR
hydrophobic channel, so the size of the substituent affected compound inhibitory activity to
some extent. For example, it may be that Ie fitted more neatly into the hydrophobic pocket
than Ia, which gave it stronger inhibitory potency. (3) IVa with a hydrophobic benzene ring
substituted at the C4 position of the phenyl moiety exhibited an extra π–π interaction between
the benzene ring and Phe649, which might explain its superior XOR inhibitory activity
among these compounds. The above results of virtual molecular docking essentially agree
with the in vitro XOR inhibitory activity data, which may help clarify the SARs of these
compounds.
12

13

A
B
Fig. 3. A: 3D (left) and 2D (right) docking modes of XOR with compounds Ia, Ie, IVa, WN1703, and
febuxostat. B: The overlays of five compounds (a: Ia (yellow), WN1703 (green) and febuxostat (blue); b:
Ie (red), WN1703, and febuxostat; c: IVa (orange), WN1703, and febuxostat). The hydrogen bonds are
represented by yellow and purple in 3D and 2D maps, respectively. Salt bridges are represented by red-
violet solid lines in 2D maps. π–π bonds are represented by black and green dotted arrows in 3D and 2D
maps, respectively. CCMo: catalytic center containing Mo; EAS: entrance of the active site; HC:
hydrophobic channel.
2
.4. Hypouricemic effect in vivo
14

The in vivo hypouricemic effects of compounds Ie and IVa were further verified by
measuring serum uric acid levels in an acute hyperuricemia mouse model (Fig. 4). The acute
hyperuricemia mouse model was based on our previous research methods. Compared with
the normal group (data not shown as they were below the detection limit), serum uric acid
levels in the model group effectively increased and were maintained throughout the
experiment [26, 32]. Compared with the hyperuricemia model group, compounds Ie and IVa
significantly reduced serum uric acid levels at a dosage of 5 mg/kg during the entire test
period after drug administration (P < 0.05). Although the uric-acid-lowering effects of these
two compounds were slightly weaker than that of febuxostat, this experiment confirmed that
both have uric-acid-lowering effects.
a
b
c
Fig. 4. Hypouricemic effects of compounds Ie and IVa at a dosage of 5 mg/kg in the in vivo potassium
oxonate/hypoxanthine-induced acute hyperuricemia mouse model (n = 8). (a) Time-dependent changes in
serum uric acid levels after oral administration of Ie, IVa, or febuxostat. (b) Serum uric acid levels 4 h
after oral administration of Ie, IVa, or febuxostat. (c) Areas under the curve (uric acid, 2–8 h, AUC) after
oral administration of Ie, IVa, or febuxostat. Data are expressed as mean ± S.D. *P < 0.05, **P < 0.01 vs
#
model group, P < 0.05 vs febuxostat group.
Furthermore, we explored the hypouricemic effects of compounds Ie and IVa compared
with febuxostat in a long-term hyperuricemia mouse model. To prevent mouse death caused
by intolerance during model establishment, the dose used (250 mg/kg potassium oxonate +
1
50 mg/kg hypoxanthine) was lower than that in the acute hyperuricemia model (250 mg/kg
15

potassium oxonate + 400 mg/kg hypoxanthine). As expected, Ie and IVa displayed
continuous and potent hypouricemic effects compared with the hyperuricemia group at a
dosage of 5 mg/kg (P < 0.05), and the hypouricemic effects of both compounds were similar
to that of febuxostat. In addition, some studies suggest that hyperuricemia may lead to renal
injury [33–36], but another group did not find evidence of a direct relationship [37]. In the
current experiment, creatinine and urea nitrogen levels in the hyperuricemia group were
significantly higher than those in the vehicle group, suggesting that renal injury occurred.
More interestingly, Ie and IVa decreased creatinine and urea nitrogen levels compared to the
hyperuricemia group (P < 0.05). This effect was not observed for febuxostat or WN1703
[
supplementary material 1], suggesting that Ie and IVa could alleviate hyperuricemia-
induced renal injury to some extent.
a
b
c
Fig. 5. Levels of uric acid (a), creatinine (b), and urea nitrogen (c) after oral administration of Ie, IVa, or
febuxostat at a dosage of 5 mg/kg in the potassium oxonate/hypoxanthine-induced long-term
#
hyperuricemia mouse model (n = 8, *P < 0.05 vs model group, P < 0.05 vs vehicle group).
3
. Conclusions
Based on our previous me-too optimization with febuxostat as the lead compound, a series
of 1-phenylimidazole-4-carboxylic acid derivatives were designed as novel XOR inhibitors.
Among these compounds, Ie and IVa emerged as the most potent XOR inhibitors, with IC50
16

values similar to those of febuxostat. Molecular modeling studies of selected compounds
provided some explanations for their binding model to XOR and inhibitory activity in vitro;
the results also clarified their SARs. Notably, while Ie and IVa exhibited weaker
hypouricemic effects than febuxostat in the acute hyperuricemia mouse model, both exhibited
considerable hypouricemic effects similar to febuxostat in the long-term hyperuricemia
mouse model. Moreover, the creatinine- and urea nitrogen-lowering effects suggested that Ie
and IVa could mitigate kidney injury; this point needs further examination and confirmation
in our future research.
4
. Experimental protocols
4
.1. Chemistry
Unless otherwise indicated, reagents and solvents were purchased from commercial
suppliers and used without further purification. All of the reactions were monitored via thin-
layer chromatography using silica gel glass plates (Qingdao Ocean Chemical, Qingdao,
Shandong, P. R. China) with an ultraviolet indicator at 254 nm.
Column chromatography was performed using silica gel (200–300 mesh) from Qingdao
1
13
Ocean Chemical. H NMR and C NMR spectra were obtained on Bruker Avance III HD
00 or 600 MHZ spectrometers (Bruker, Germany) using tetramethylsilane as the internal
standard. High-resolution mass spectrometry (HRMS) spectra were determined on an Agilent
290 instrument operating in electrospray ionization (ESI) mode (Agilent Technologies,
USA).
4
1
4
.1.1. General procedure for the preparation of compounds 2a–e
17

A mixture of compound 1 (5.0 mmol), alkylamine (15.0 mmol), and K CO (15.0 mmol) in
2
3
DMF (10 mL) was reacted at 70–80 °C for 3–5 h. After the reaction was complete, the
mixture was poured into H O (100 mL) and extracted with ethyl acetate (100 mL × 2). The
2
organic layer was collected, washed with brine (100 mL × 3), dried over anhydrous Na SO ,
2
4
and concentrated under vacuum to yield the crude product, which was purified by flash
column chromatography (0–15% ethyl acetate in petroleum ether).
1
4
.1.1.1. 5-bromo-2-(isobutyl(methyl)amino)-benzonitrile (2a) Yellow oil (yield: 93%); H
NMR (400 MHz, CDCl ) δ 7.54 (d, 1H, J = 1.3 Hz, ArH), 7.42 (dd, 1H, J = 9.1, 3.3 Hz, ArH),
3
6
.75 (d, 1H, J = 9.1 Hz, ArH), 3.19 (d, 2H, J = 7.5 Hz, -NCH CH CH(CH ) ), 3.05 (s, 3H, -
3 2 3 2
NCH CH CH(CH ) ), 2.01 (m, 1H, -NCH CH CH(CH ) ), 0.89 (d, 6H, J = 6.6 Hz, -
3
2
3 2
3
2
3 2
NCH CH CH(CH ) ).
3
2
3 2
1
4
.1.1.2. 5-bromo-2-pyrrolidin-benzonitrile (2b) Yellow solid (yield: 90%); H NMR (400
MHz, CDCl ) δ 7.51 (d, J = 2.5 Hz, 1H, ArH), 7.36 (dd, J = 9.2, 2.5 Hz, 1H, ArH), 6.51 (d, J
3
=
9.2 Hz, 1H, ArH), 3.62-3.54 (m, 4H, -N(CH ) (CH ) ), 2.04-1.98 (m, 4H, -N(CH ) (CH ) ).
2 2 2 2 2 2 2 2
1
4
.1.1.3. 5-bromo-2-(piperidin-1-yl)-benzonitrile (2c) White solid (yield: 92%); H NMR (600
MHz, CDCl ) δ 7.62 (dd, J = 2.3, 0.7 Hz, 1H, ArH), 7.52 (ddd, J = 8.8, 2.3, 0.9 Hz, 1H, ArH),
3
6
.86 (d, J = 8.9 Hz, 1H, ArH), 3.18-3.12 (m, 4H, -N(CH ) ), 1.76 (m, 4H, -N(CH ) ), 1.63-
2 5 2 5
1
.56 (m, 2H, -N(CH ) ).
2
5
1
4
.1.1.4. 5-bromo-2-(4-methylpiperidin-1-yl)-benzonitrile (2d) White solid (yield: 90%); H
NMR (600 MHz, CDCl ) δ 7.62 (d, J = 2.4 Hz, 1H, ArH), 7.52 (dd, J = 8.9, 2.4 Hz, 1H, ArH),
3
6
1
1
=
.86 (d, J = 8.9 Hz, 1H, ArH), 3.54 (d, J = 12.3 Hz, 2H, -N(CH ) (CH ) CHCH ), 2.78 (t, J =
2 2 2 2 3
3.0 Hz, 2H, -N(CH ) (CH ) CHCH ), 1.76 (d, J = 12.6 Hz, 2H, -N(CH ) (CH ) CHCH ),
2
2
2 2
3
2 2
2 2
3
.53 (m, 1H, -N(CH ) (CH ) CHCH ), 1.48-1.41 (m, 2H, -N(CH ) (CH ) CHCH ), 1.00 (d, J
2
2
2 2
3
2 2
2 2
3
6.4 Hz, 3H, -N(CH ) (CH ) CHCH ).
2
2
2 2
3
18

1
4
.1.1.5. 5-bromo-2-(azocan-1-yl)- benzonitrile (2e) White solid (yield: 93%); H NMR (400
MHz, CDCl ) δ 7.53 (s, 1H, ArH), 7.37 (d, J = 9.2 Hz, 1H, ArH), 6.68 (d, J = 9.3 Hz, 1H,
3
ArH), 3.69 (s, 4H, -N(CH ) ), 1.80 (s, 2H, -N(CH ) ), 1.60 (d, J = 10.1 Hz, 8H, -N(CH ) ).
2
7
2 7
2 7
4
.1.2. General procedure for the preparation of compounds 3a–e
A mixture of compound 2a–e (2.4 mmol), CuI (0.2 mmol), 1H-imidazole-4-ethyl
carboxylate (2.0 mmol), trans-N,N-dimethyl-1,2-cyclohexanediamine (0.4 mmol), and K CO3
2
(4.2 mmol) in DMF (5 mL) was stirred at 120 °C for 24 h under a nitrogen atmosphere.
Subsequently, the reaction was diluted with H O (30 mL) and extracted with ethyl acetate (20
2
mL × 2). The organic phases were combined, washed with brine (30 mL × 3), dried over
anhydrous Na SO , and concentrated under vacuum. The residue was purified by flash
2
4
column chromatography (12–30% ethyl acetate in petroleum ether).
4
.1.2.1. ethyl 1-(3-cyano-4-(isobutyl(methyl)amino)phenyl)-1H-imidazole-4- carboxylate (3a).
1
White solid (yield: 42%); H NMR (600 MHz, CDCl ) δ 7.84 (d, J = 1.3 Hz, 1H, -NCH),
3
7
.74 (d, J = 1.2 Hz, 1H, -NCH), 7.50 (d, J = 2.7 Hz, 1H, ArH), 7.39 (dd, J = 9.1, 2.8 Hz, 1H,
ArH), 6.96 (d, J = 9.2 Hz, 1H, ArH), 4.40 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.33 (d, J = 7.5
2
3
Hz, 2H, -NCH CH CH(CH ) ), 3.18 (s, 3H, -NCH CH CH(CH ) ), 2.12-2.02 (m, 1H, -
3
2
3 2
3
2
3 2
NCH CH CH(CH ) ), 1.41 (t, J = 7.1 Hz, 3H, -COOCH CH ), 0.95 (d, J = 6.6 Hz, 6H, -
3
2
3 2
2
3
NCH CH CH(CH ) ).
3
2
3 2
4
.1.2.2. ethyl 1-(3-cyano-4-(pyrrolidin-1-yl)phenyl)-1H-imidazole-4-carboxylate (3b). White
1
solid (yield: 43%); H NMR (600 MHz, CDCl ) δ 7.82 (s, 1H, -NCH), 7.72 (s, 1H, -NCH),
3
7
.46 (d, J = 2.7 Hz, 1H, ArH), 7.34 (dd, J = 9.2, 2.6 Hz, 1H, ArH), 6.73 (d, J = 9.2 Hz, 1H,
19

ArH), 4.40 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.67 (t, J = 6.5 Hz, 4H, -N(CH ) (CH ) ),
2
3
2 2
2 2
2
.09-2.03 (m, 4H, -N(CH ) (CH ) ), 1.41 (t, J = 7.1 Hz, 3H, -COOCH CH ).
2 2 2 2 2 3
4
.1.2.3. ethyl 1-(3-cyano-4-(piperidin-1-yl)phenyl)-1H-imidazole-4-carboxylate (3c). White
1
solid (yield: 40%); H NMR (600 MHz, CDCl ) δ 7.86 (d, J = 1.3 Hz, 1H, -NCH), 7.77 (d, J
3
=
1.3 Hz, 1H, -NCH), 7.57 (d, J = 2.7 Hz, 1H, ArH), 7.48 (dd, J = 8.9, 2.7 Hz, 1H, ArH),
7
.09 (d, J = 8.9 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.28-3.23 (m, 4H, -
2
3
N(CH ) (CH ) CH ), 1.83-1.77 (m, 4H, -N(CH ) (CH ) CH ), 1.65 (t, J = 11.8 Hz, 2H, -
2
2
2 2
2
2 2
2 2
2
N(CH ) (CH ) CH ), 1.41 (t, J = 7.1 Hz, 3H, -COOCH CH ).
2
2
2 2
2
2
3
4
.1.2.4. ethyl 1-(3-cyano-4-(4-methylpiperidin-1-yl)phenyl)-1H-imidazole-4-carboxylate (3d).
1
White solid (yield: 47%); H NMR (600 MHz, CDCl ) δ 7.86 (d, J = 1.4 Hz, 1H, -NCH),
3
7
.77 (d, J = 1.4 Hz, 1H, -NCH), 7.57 (d, J = 2.7 Hz, 1H, ArH), 7.47 (dd, J = 8.9, 2.7 Hz, 1H,
ArH), 7.09 (d, J = 8.9 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.66 (d, J =
2
3
1
2.3 Hz, 2H, -N(CH ) CHCH ), 2.89 (m, -N(CH ) CHCH ), 1.81 (dd, J = 12.7, 2.0 Hz, 2H, -
2 4 3 2 4 3
N(CH ) CHCH ), 1.62-1.54 (m, 1H, -N(CH ) CHCH ), 1.51-1.44 (m, 2H, -N(CH ) CHCH ),
2
4
3
2 4
3
2 4
3
1
.41 (t, J = 7.1 Hz, 3H, -COOCH CH ), 1.02 (d, J = 6.5 Hz, 3H, -N(CH ) CHCH ).
2 3 2 4 3
4
.1.2.5. ethyl 1-(4-(azocan-1-yl)-3-cyanophenyl)-1H-imidazole-4-carboxylate (3e). White
1
solid (yield: 40%); H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 1.3 Hz, 1H, -NCH), 7.73 (d, J
3
=
1.3 Hz, 1H, -NCH), 7.48 (d, J = 2.8 Hz, 1H, ArH), 7.35 (dd, J = 9.3, 2.8 Hz, 1H, ArH),
6
.89 (d, J = 9.4 Hz, 1H, ArH), 4.40 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.82-3.75 (m, 4H, -
2
3
N(CH ) ), 1.91-1.81 (m, 4H, -N(CH ) ), 1.68-1.55 (m, 6H, -N(CH ) ), 1.41 (t, J = 7.1 Hz, 3H,
2
7
2 7
2 7
-
COOCH CH ).
2 3
4
.1.3. General procedure for the preparation of compounds Ia–e
20

THF/ethanol (1:1, 10 mL) was added to 3a–e (1.0 mmol), and the resulting mixture was
maintained at 70 °C for 2 h. After completion of the reaction, the mixture was diluted with
H O (100 mL), acidified to pH 2 with 1 M HCl solution, and stirred in an ice bath for 30 min.
2
The resulting solid was filtered, washed with water, and then purified by recrystallization
from methanol to yield the desired product.
4
.1.3.1. 1-(3-cyano-4-(isobutyl(methyl)amino)phenyl)-1H-imidazole-4-carboxylic acid (Ia).
1
White solid (yield: 95%); H NMR (600 MHz, DMSO-d ) δ 8.35 (d, J = 1.3 Hz, 1H, -NCH),
6
8
.28 (d, J = 1.3 Hz, 1H, -NCH), 8.02 (d, J = 2.8 Hz, 1H, ArH), 7.83 (dd, J = 9.2, 2.8 Hz, 1H,
ArH), 7.17 (d, J = 9.3 Hz, 1H, ArH), 3.28 (d, J = 7.5 Hz, 2H, -NCH CH CH(CH ) ), 3.06 (s,
3
2
3 2
3
H, -NCH CH CH(CH ) ), 1.99 (m, 1H, -NCH CH CH(CH ) ), 0.87 (d, J = 6.6 Hz, 6H, -
3 2 3 2 3 2 3 2
13
NCH CH CH(CH ) ). C NMR (151 MHz, DMSO-d ) δ 163.90, 153.28, 137.13, 134.80,
3
2
3 2
6
1
27.77, 127.47, 127.26, 124.51, 119.33, 119.22, 99.64, 61.50, 41.91, 27.01, 20.21.
+
HRMS(ESI): Calcd for C H N O [M+H] 299.1508, Found 299.1494.
1
6
18
4
2
4
.1.3.2. 1-(3-cyano-4-(pyrrolidin-1-yl)phenyl)-1H-imidazole-4-carboxylic acid (Ib). White
1
solid (yield: 97%); H NMR (600 MHz, DMSO-d ) δ 8.31 (d, J = 1.3 Hz, 1H, -NCH), 8.25 (d,
6
J = 1.3 Hz, 1H, -NCH), 7.92 (d, J = 2.8 Hz, 1H, ArH), 7.75 (dd, J = 9.3, 2.8 Hz, 1H, ArH),
6
.87 (d, J = 9.3 Hz, 1H, ArH), 3.56 (t, J = 6.5 Hz, 4H, -N(CH ) (CH ) ), 1.98-1.94 (m, 4H, -
2 2 2 2
13
N(CH ) (CH ) ). C NMR (151 MHz, DMSO-d ) δ 163.92, 149.39, 137.04, 134.65, 127.64,
2
2
2 2
6
1
27.44, 125.73, 124.50, 120.32, 116.30, 93.79, 50.28, 25.70. HRMS(ESI): Calcd for
+
C H N O [M+H] 283.1195, Found 283.1179.
15
14
4
2
4
.1.3.3. 1-(3-cyano-4-(piperidin-1-yl)phenyl)-1H-imidazole-4-carboxylic acid (Ic). White
1
solid (yield: 95%); H NMR (600 MHz, DMSO-d ) δ 8.39 (s, 1H, -NCH), 8.33 (s, 1H, -NCH),
6
8
.16 (d, J = 2.7 Hz, 1H, ArH), 7.93 (dd, J = 8.9, 2.7 Hz, 1H, ArH), 7.26 (d, J = 9.0 Hz, 1H,
ArH), 3.17-3.15 (m, 4H, -N(CH ) (CH ) CH ), 1.72 -1.67 (m, 4H, -N(CH ) (CH ) CH ), 1.57
2
2
2 2
2
2 2
2 2
2
21

1
3
(
dt, J = 11.4, 5.8 Hz, 2H, -N(CH ) (CH ) CH ). C NMR (151 MHz, DMSO-d ) δ 163.85,
2 2 2 2 2 6
1
2
55.69, 137.17, 134.95, 130.08, 127.21, 126.82, 124.50, 120.69, 117.91, 105.57, 52.93, 26.08,
+
3.85. HRMS(ESI): Calcd for C H N O [M+H] 297.1352, Found 297.1337.
1
6
16
4
2
4
.1.3.4. 1-(3-cyano-4-(4-methylpiperidin-1-yl)phenyl)-1H-imidazole-4-carboxylic acid (Id).
1
White solid (yield: 95%); H NMR (600 MHz, DMSO-d ) δ 8.39 (d, J = 1.2 Hz, 1H, -NCH),
6
8
.32 (d, J = 1.2 Hz, 1H, -NCH), 8.15 (d, J = 2.3 Hz, 1H, ArH), 7.93 (dd, J = 9.0, 2.7 Hz, 1H,
ArH), 7.26 (d, J = 9.0 Hz, 1H, ArH), 3.52 (d, J = 12.2 Hz, 2H, -N(CH ) CHCH ), 2.84 (dd, J
2
4
3
=
11.9, 10.3 Hz, 2H, -N(CH ) CHCH , 1.76 (d, J = 10.9 Hz, 2H, -N(CH ) CHCH ), 1.58-
2 4 3 2 4 3
1
.50 (m, 1H, -N(CH ) CHCH ), 1.32 (m, 2H, -N(CH ) CHCH , 0.97 (d, J = 6.5 Hz, 3H, -
2 4 3 2 4 3
1
3
N(CH ) CHCH ). C NMR (151 MHz, DMSO-d ) δ 163.86, 155.46, 137.16, 134.96, 130.05,
2
4
3
6
1
27.19, 126.80, 124.48, 120.73, 117.92, 105.49, 52.21, 34.35, 30.23, 22.10. HRMS(ESI):
+
Calcd for C H N O [M+Na] 333.1327, Found 333.1316.
1
7
18
4
2
4
.1.3.5. 1-(4-(azocan-1-yl)-3-cyanophenyl)-1H-imidazole-4-carboxylic acid (Ie). White solid
1
(
yield: 98%); H NMR (600 MHz, DMSO-d ) δ 8.32 (d, J = 1.1 Hz, 1H, -NCH), 8.26 (d, J =
6
1
.2 Hz, 1H, -NCH), 7.93 (d, J = 2.9 Hz, 1H, ArH), 7.75 (dd, J = 9.4, 2.9 Hz, 1H, ArH), 7.07
(
d, J = 9.5 Hz, 1H, ArH), 3.76-3.71 (m, 4H, -N(CH ) (CH ) (CH ) ), 1.77-1.73 (m, 4H, -
2
2
2 2
2 3
13
N(CH ) (CH ) (CH ) ), 1.59-1.46 (m, 6H,-N(CH ) (CH ) (CH ) ). C NMR (151 MHz,
2
2
2 2
2 3
2 2
2 2
2 3
DMSO-d ) δ 164.00, 149.60, 137.08, 134.75, 128.38, 127.63, 125.90, 124.46, 120.16, 117.23,
6
+
9
4.44, 52.54, 27.33, 26.81, 24.82. HRMS(ESI): Calcd for C H N O [M+H] 325.1665,
1
8
20
4
2
Found 325.1661.
4
.1.4. General procedure for the preparation of compounds 5a–i
22

K CO (15.3 mmol) was added to compound 4 (5.1 mmol) in DMF and stirred at room
2
3
temperature (rt) for 1 h. Alkyl halide (15.3 mmol) was added to the mixture and heated at 60–
00 °C for 3–10 h. Subsequently, the mixture was cooled to rt, diluted with H O (150 mL),
1
2
and extracted with ethyl acetate (150 mL × 2). The organic phases were combined, washed
with H O (200 mL × 2) and brine (200 mL × 2), dried over anhydrous Na SO , evaporated
2
2
4
under vacuum, and purified by flash column chromatography (0–10% ethyl acetate in
petroleum ether).
1
4
.1.4.1. 5-bromo-2-ethoxy-benzonitrile (5a). White solid (yield: 97%); H NMR (400 MHz,
CDCl ) δ 7.65 (d, J = 2.4 Hz, 1H, ArH), 7.60 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 6.85 (d, J = 9.0
3
Hz, 1H, ArH), 4.14 (q, J = 7.0 Hz, 2H, -OCH CH ), 1.48 (t, J = 7.0 Hz, 3H, -OCH CH ).
2
3
2
3
1
4
.1.4.2. 5-bromo-2-isopropoxy-benzonitrile (5b). White solid (yield: 85%); H NMR (400
MHz, CDCl ) δ 7.66-7.56 (m, 2H, ArH), 6.85 (d, J = 9.0 Hz, 1H, ArH), 4.67-4.57 (m, 1H, -
3
OCH(CH ) ), 1.40 (d, J = 6.1 Hz, 6H, -OCH(CH ) ).
3
2
3 2
1
4
.1.4.3. 5-bromo-2-isobutoxy-benzonitrile (5c). Colourless oil (yield: 92%); H NMR (400
MHz, CDCl ) δ 7.64 (d, J = 2.4 Hz, 1H, ArH), 7.59 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 6.84 (d, J
3
=
9.0 Hz, 1H, ArH), 3.81 (d, J = 6.5 Hz, 2H, -OCH CH(CH ) ), 2.23-2.09 (m, 1H, -
2 3 2
OCH CH(CH ) ), 1.06 (d, J = 6.7 Hz, 6H, -OCH CH(CH ) ).
2
3 2
2
3 2
1
4
.1.4.4. 5-bromo-2-(sec-butoxy)-benzonitrile (5d). Colourless oil (yield: 87%); H NMR (400
MHz, CDCl ) δ 7.66-7.56 (m, 2H, ArH), 6.84 (d, J = 9.0 Hz, 1H, ArH), 4.43-4.34 (m, 1H, -
3
OCHCH CH CH ), 1.88-1.64 (m, 1H, -OCHCH CH CH ), 1.35 (d, J = 6.1 Hz, 2H, -
3
2
3
3
2
3
OCHCH CH CH ), 1.00 (t, J = 7.4 Hz, 2H, -OCHCH CH CH ).
3
2
3
3
2
3
23

1
4
.1.4.5. 5-bromo-2-(neopentyloxy)-benzonitrile (5e). White solid (yield: 90%); H NMR (400
MHz, CDCl ): δ 7.62 (d, 1H, J = 2.5 Hz, ArH), 7.58 (dd, 1H, J = 8.9 and 2.5 Hz, ArH), 6.83
3
(
d, 1H, J = 8.9 Hz, ArH), 3.69 (s, 2H, -OCH C(CH ) ), 1.07 (s, 9H, -OCH C(CH ) ).
2 3 3 2 3 3
1
4
.1.4.6. 5-bromo-2-(pentan-3-yloxy)-benzonitrile (5f). Colourless oil (yield: 96%); H NMR
(
400 MHz, CDCl ) δ 7.66-7.54 (m, 2H, ArH), 6.84 (d, J = 9.0 Hz, 1H, ArH), 4.21 (p, J = 5.8
3
Hz, 1H, -OCH(CH ) (CH ) ), 1.79-1.69 (m, 4H, -OCH(CH ) (CH ) ), 0.98 (t, J = 7.4 Hz, 6H,
2
2
3 2
2 2
3 2
-
OCH(CH ) (CH ) ).
2 2 3 2
1
4
.1.4.7. 5-bromo-2-(2-ethylbutoxy)-benzonitrile (5g). Colourless oil (yield: 96%); H NMR
(
600 MHz, CDCl ) δ 7.64 (d, J = 2.5 Hz, 1H, ArH), 7.60 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 6.86
3
(
d, J = 9.0 Hz, 1H, ArH), 3.94 (d, J = 5.6 Hz, 2H, -OCH CH(CH ) (CH ) ), 1.73 (m, 1H, -
2
2 2
3 2
OCH CH(CH ) (CH ) ), 1.55-1.46 (m, 4H, -OCH CH(CH ) (CH ) ), 0.94 (t, J = 7.5 Hz, 6H,
2
2 2
3 2
2
2 2
3 2
-
OCH CH(CH ) (CH ) ).
2 2 2 3 2
1
4
.1.4.8. 5-bromo-2-(cyclopentyloxy)-benzonitrile (5h). Colourless oil (yield: 96%); H NMR
(
400 MHz, CDCl ) δ 8.20 (d, J = 2.2 Hz, 1H, ArH), 8.18-8.14 (m, 1H, ArH), 7.07-7.04 (m,
3
1
H, ArH), 5.01-4.92 (m, 1H, -OCH(CH ) ), 4.35 (s, 2H, -OCH(CH ) ), 2.05-1.81 (m, 6H, -
2 4 2 4
OCH(CH ) ).
2
4
1
4
.1.4.9. 5-bromo-2-(cyclohexyloxy)-benzonitrile (5i). Colourless oil (yield: 92%); H NMR
(
400 MHz, CDCl ) δ 7.64 (d, J = 2.5 Hz, 1H, ArH), 7.57 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 6.86
3
(
d, J = 9.0 Hz, 1H, ArH), 4.42-4.32 (m, 1H, -OCH(CH ) ), 1.98-1.76 (m, 4H, -OCH(CH ) ),
2
5
2 5
1
.67 (m, 2H, -OCH(CH ) ), 1.56-1.33 (m, 4H, -OCH(CH ) ).
2 5 2 5
4
.1.5. General procedure for the preparation of compounds 6a–i
24

A mixture of compound 5a–i (2.4 mmol), CuI (0.2 mmol), ethyl 1H-imidazole-4-
carboxylate (2.0 mmol), trans-N,N-dimethyl-1,2-cyclohexanediamine (0.4 mmol), and
K CO (4.2 mmol) in DMF (5 mL) was stirred at 120 °C for 24 h under a nitrogen
2
3
atmosphere. Subsequently, the reaction was diluted with H O (30 mL) and extracted with
2
ethyl acetate (20 mL × 2). The organic phases were combined, washed with brine (30 mL ×
3
), dried over anhydrous Na SO , and concentrated under vacuum. The residue was purified
2 4
by flash column chromatography (12–30% ethyl acetate in petroleum ether).
4
.1.5.1. ethyl 1-(3-cyano-4-ethoxyphenyl)-1H-imidazole-4-carboxylate (6a). White solid
1
(
yield: 32%); H NMR (400 MHz, CDCl ) δ 7.90-7.54 (m, 4H, -NCH, ArH), 7.10 (d, J = 8.9
3
Hz, 1H, ArH), 4.46-4.18 (m, 4H, -COOCH CH , -OCH CH ), 1.58-1.34 (m, 6H, -
2
3
2
3
COOCH CH , -OCH CH ).
2
3
2
3
4
.1.5.2. ethyl 1-(3-cyano-4-isopropoxyphenyl)-1H-imidazole-4-carboxylate (6b). White solid
1
(
yield: 35%); H NMR (400 MHz, CDCl ) δ 7.87 (d, J = 1.4 Hz, 1H, -NCH), 7.77 (d, J = 1.4
3
Hz, 1H, -NCH), 7.63-7.53 (m, 2H, ArH), 7.10 (d, J = 9.1 Hz, 1H, ArH), 4.77-4.66 (m, 1H, -
OCH(CH ) ), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 1.45 (d, J = 6.1 Hz, 6H, -OCH(CH ) ),
3
2
2
3
3 2
1
.41 (t, J = 7.1 Hz, 3H, -COOCH CH ).
2 3
4
.1.5.3. ethyl 1-(3-cyano-4-isobutoxyphenyl)-1H-imidazole-4-carboxylate (6c). White solid
1
(
yield: 45%); H NMR (400 MHz, CDCl ) δ 7.87 (d, J = 1.3 Hz, 1H, -NCH), 7.77 (d, J = 1.3
3
Hz, 1H, -NCH), 7.62 (d, J = 2.7 Hz, 1H, ArH), 7.56 (dd, J = 9.0, 2.8 Hz, 1H, ArH), 7.09 (d, J
9.0 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.90 (d, J = 6.5 Hz, 2H, -
=
2
3
OCH CH(CH ) ), 2.28-2.14 (m, 1H, -OCH CH(CH ) ), 1.41 (t, J = 7.1 Hz, 3H, -
2
3 2
2
3 2
COOCH CH ), 1.10 (d, J = 6.7 Hz, 6H, -OCH CH(CH ) ).
2
3
2
3 2
4
.1.5.4. ethyl 1-(4-(sec-butoxy)-3-cyanophenyl)-1H-imidazole-4-carboxylate (6d). White
1
solid (yield: 36%); H NMR (400 MHz, CDCl ) δ 7.87 (d, J = 1.4 Hz, 1H, -NCH), 7.77 (d, J
3
25

=
1.4 Hz, 1H, -NCH), 7.61 (d, J = 2.7 Hz, 1H, ArH), 7.55 (dd, J = 9.0, 2.8 Hz, 1H, ArH),
7
2
.09 (d, J = 9.1 Hz, 1H, ArH), 4.53-4.44 (m, 1H, -OCH(CH )CH CH ), 4.41 (q, J = 7.1 Hz,
3
2
3
H, -COOCH CH ), 1.92-1.71 (m, 2H, -OCH(CH )CH CH ), 1.41 (dd, J = 10.0, 4.2 Hz, 6H,
2
3
3
2
3
-
OCH(CH )CH CH , -COOCH CH ), 1.04 (t, J = 7.4 Hz, 3H, -OCH(CH )CH CH ).
3 2 3 2 3 3 2 3
4
.1.5.5. ethyl 1-(3-cyano-4-(neopentyloxy)phenyl)-1H-imidazole-4-carboxylate (6e). White
1
solid (yield: 34%); H NMR (600 MHz, CDCl ) δ 7.87 (d, J = 1.4 Hz, 1H, -NCH), 7.77 (d, J
3
=
1.4 Hz, 1H, -NCH), 7.62 (d, J = 2.7 Hz, 1H, ArH), 7.56 (dd, J = 9.0, 2.8 Hz, 1H, ArH),
7
.09 (d, J = 9.0 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 3.76 (s, 2H, -
2
3
OCH C(CH ) ), 1.41 (t, J = 7.1 Hz, 3H, -COOCH CH ), 1.12 (s, 9H, -OCH C(CH ) ).
2
3 3
2
3
2
3 3
4
.1.5.6. ethyl 1-(3-cyano-4-(pentan-3-yloxy)phenyl)-1H-imidazole-4-carboxylate (6f). White
1
solid (yield: 38%); H NMR (600 MHz, CDCl ) δ 7.86 (d, J = 1.3 Hz, 1H, -NCH), 7.77 (d, J
3
=
1.2 Hz, 1H, -NCH), 7.61 (d, J = 2.7 Hz, 1H, ArH), 7.56 (dd, J = 9.0, 2.8 Hz, 1H, ArH),
7
2
7
.11 (d, J = 9.0 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 4.04 (d, J = 5.6 Hz,
2
3
H), 1.78 (m, 1H, -OCH(CH ) (CH ) ), 1.58-1.48 (m, 4H, -OCH(CH ) (CH ) ), 1.41 (t, J =
2
2
3 2
2 2
3 2
.1 Hz, 3H, -COOCH CH ), 0.97 (t, J = 7.5 Hz, 6H, -OCH(CH ) (CH ) ).
2
3
2 2
3 2
4
.1.5.7. ethyl 1-(3-cyano-4-(2-ethylbutoxy)phenyl)-1H-imidazole-4-carboxylate (6g). White
1
solid (yield: 42%); H NMR (600 MHz, CDCl ) δ 7.86 (d, J = 1.3 Hz, 1H, -NCH), 7.77 (d, J
3
=
1.2 Hz, 1H, -NCH), 7.61 (d, J = 2.7 Hz, 1H, ArH), 7.56 (dd, J = 9.0, 2.8 Hz, 1H, ArH),
7
2
.11 (d, J = 9.0 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, -COOCH CH ), 4.04 (d, J = 5.6 Hz,
2
3
H, -OCH CH(CH ) (CH ) ), 1.78 (m, 1H, -OCH CH(CH ) (CH ) ), 1.57-1.49 (m, 4H, -
2
2 2
3 2
2
2 2
3 2
OCH CH(CH ) (CH ) ), 1.41 (t, J = 7.1 Hz, 3H, -COOCH CH ), 0.97 (t, J = 7.5 Hz, 6H, -
2
2 2
3 2
2
3
OCH CH(CH ) (CH ) ).
2
2 2
3 2
4
.1.5.8. ethyl 1-(3-cyano-4-(cyclopentyloxy)phenyl)-1H-imidazole-4-carboxylate (6h). White
1
solid (yield: 42%); H NMR (400 MHz, CDCl ) δ 7.87 (s, 1H, -NCH), 7.78 (s, 1H, -NCH),
3
26

7
.62-7.51 (m, 2H, ArH), 7.11 (d, J = 9.0 Hz, 1H, ArH), 4.97-4.86 (m, 1H, -OCH(CH ) ), 4.42
2 4
(
q, J = 7.1 Hz, 2H, -COOCH CH ), 2.02-1.84 (m, 6H, -OCH(CH ) ), 1.75-1.69 (m, 2H, -
2 3 2 4
OCH(CH ) ), 1.42 (t, J = 7.1 Hz, 3H, -COOCH CH ).
2
4
2
3
4
.1.5.9. ethyl 1-(3-cyano-4-(cyclohexyloxy)phenyl)-1H-imidazole-4-carboxylate (6i). White
1
solid (yield: 45%); H NMR (400 MHz, CDCl ) δ 7.86 (d, J = 1.3 Hz, 1H, -NCH), 7.77 (d, J
3
=
1.2 Hz, 1H, -NCH), 7.61 (d, J = 2.7 Hz, 2H, ArH), 7.54 (dd, J = 9.0, 2.8 Hz, 1H, ArH),
7
.11 (d, J = 8.8 Hz, 1H, ArH), 4.48 (m, 1H, -OCH(CH ) ), 4.41 (q, J = 7.1 Hz, 2H, -
2
5
COOCH CH ), 2.02-1.70 (m, 2H, -OCH(CH ) ), 1.59-1.37 (m, 7H, -OCH(CH ) , -
2
3
2 5
2 5
COOCH CH ).
2
3
4
.1.6. General procedure for the preparation of compounds IIa–i
THF/ethanol (1:1, 10 mL) was added to 6a–i (1.0 mmol), and the resulting mixture was
maintained at 70 °C for 2 h. After completion of the reaction, the mixture was diluted with
H O (100 mL) and acidified to pH 2 with 1 M HCl solution, and the resulting suspension was
2
stirred in an ice bath for 30 min. The resulting solid was filtered, washed with water, and
purified by recrystallization from methanol to yield the desired product.
4
9
2
.1.6.1. 1-(3-cyano-4-ethoxyphenyl)-1H-imidazole-4-carboxylic acid (IIa). White solid (yield:
1
5%); H NMR (400 MHz, DMSO-d ) δ 8.63 (s, 1H, -NCH), 8.52 (s, 1H, -NCH), 8.25 (d, J =
6
.7 Hz, 1H, ArH), 8.06 (dd, J = 9.2, 2.8 Hz, 1H, ArH), δ 7.42 (d, J = 9.2 Hz, 1H, ArH), 4.27
13
(
q, J = 6.9 Hz, 2H, -OCH CH ), 1.40 (t, J = 6.9 Hz, 3H, -OCH CH ). C NMR (101 MHz,
2 3 2 3
DMSO-d ) δ 167.10, 163.92, 156.65, 137.35, 134.79, 129.32, 125.79, 124.79, 123.67, 123.40,
6
+
1
15.17, 65.04, 14.98. HRMS(ESI): Calcd for C H N O [M+Na] 280.0698, Found
13
11
3
3
2
80.0691.
27

4
.1.6.2. 1-(3-cyano-4-isopropoxyphenyl)-1H-imidazole-4-carboxylic acid (IIb). White solid
1
(
yield: 90%); H NMR (400 MHz, DMSO-d ) δ 8.40 (d, J = 1.4 Hz, 1H, -NCH), 8.33 (d, J =
6
1
.4 Hz, 1H, -NCH), 8.21 (d, J = 2.8 Hz, 1H, ArH), 8.01 (dd, J = 9.2, 2.9 Hz, 1H, ArH), 7.44
(
d, J = 9.3 Hz, 1H, ArH), 4.92-4.83 (m, 1H, -OCH(CH ) ), 1.34 (d, J = 6.0 Hz, 6H, -
3
2
13
OCH(CH ) ). C NMR (101 MHz, DMSO-d ) δ 163.88, 158.91, 137.27, 134.95, 129.81,
3
2
6
1
28.12, 126.66, 124.66, 116.10, 115.92, 102.70, 72.55, 22.04. HRMS(ESI): Calcd for
+
C H N O [M+H] 272.1035, Found 272.1025.
14
13
3
3
4
.1.6.3. 1-(3-cyano-4-isobutoxyphenyl)-1H-imidazole-4-carboxylic acid (IIc). White solid
1
(
yield: 100%); H NMR (400 MHz, DMSO-d ) δ 8.40 (s, 1H, -NCH), 8.33 (s, 1H, -NCH),
6
8
.22 (d, J = 2.6 Hz, 1H, ArH), 8.03 (dd, J = 9.1, 2.6 Hz, 1H, ArH), 7.40 (d, J = 9.2 Hz, 1H,
ArH), 3.97 (dd, J = 13.3, 6.5 Hz, 2H, -OCH CH(CH ) ), 2.08 (m, 1H, -OCH CH(CH ) ),
2
3 2
2
3 2
1
3
1
1
.02 (d, J = 6.7 Hz, 6H, -OCH CH(CH ) ). C NMR (101 MHz, DMSO-d ) δ 163.87, 159.91,
2 3 2 6
37.25, 134.95, 129.93, 128.14, 126.44, 124.65, 115.87, 114.82, 101.85, 75.67, 28.06, 19.20.
+
HRMS(ESI): Calcd for C H N O [M+Na] 308.1011, Found 308.1017.
1
5
15
3
3
4
.1.6.4. 1-(4-(sec-butoxy)-3-cyanophenyl)-1H-imidazole-4-carboxylic acid (IId). White solid
1
(
(
yield: 94%); H NMR (400 MHz, DMSO-d ) δ 8.40 (s, 1H, -NCH), 8.33 (s, 1H, -NCH), 8.20
6
d, J = 2.8 Hz, 1H, ArH), 8.01 (dd, J = 9.2, 2.8 Hz, 1H, ArH), 7.44 (d, J = 9.3 Hz, 1H, ArH),
4
.72-4.64 (m, 1H, -OCH(CH )CH CH ), 1.77-1.61 (m, 2H, -OCH(CH )CH CH ), 1.31 (d, J
3 2 3 3 2 3
13
=
6.1 Hz, 3H, -OCH(CH )CH CH ), 0.96 (t, J = 7.4 Hz, 3H, -OCH(CH )CH CH ). C NMR
3 2 3 3 2 3
(
101 MHz, DMSO-d ) δ 163.85, 159.24, 137.30, 134.89, 129.78, 128.18, 126.71, 124.69,
6
1
16.04, 115.86, 102.65, 77.18, 28.82, 19.27, 9.63. HRMS(ESI): Calcd for C H N O
15 15 3 3
+
[
M+Na] 308.1011, Found 308.1013.
4
.1.6.5. 1-(3-cyano-4-(neopentyloxy)phenyl)-1H-imidazole-4-carboxylic acid (IIe). White
1
solid (yield: 92%); H NMR (600 MHz, DMSO-d ) δ 8.40 (d, J = 1.3 Hz, 1H, -NCH), 8.33 (d,
6
28