Synthesis and bioevaluation of 1-phenyl-pyrazole-4-carboxylic acid derivatives as potent xanthine oxidoreductase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.08.047

A diverse library of 1-phenyl-pyrazole-4-carboxylic acid derivatives were synthesized and evaluated for their inhibitory potency against xanthine oxidoreductase (XOR) in vitro and vivo, and the structure-activity relationship (SAR) analyses were also pres

Full text of DOI:10.1016/j.ejmech.2017.08.047

Accepted Manuscript
Synthesis and bioevaluation of 1-phenyl-pyrazole-4-carboxylic acid derivatives as
potent xanthine oxidoreductase inhibitors
Jing Li, Fangping Wu, Xingguo Liu, Yake Zou, Huixiong Chen, Zheng Li, Lei Zhang
PII:
S0223-5234(17)30658-X
10.1016/j.ejmech.2017.08.047
EJMECH 9691
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2017
Revised Date: 12 May 2017
Accepted Date: 21 August 2017
Please cite this article as: J. Li, F. Wu, X. Liu, Y. Zou, H. Chen, Z. Li, L. Zhang, Synthesis and
bioevaluation of 1-phenyl-pyrazole-4-carboxylic acid derivatives as potent xanthine oxidoreductase
inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.047.
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ACCEPTED MANUSCRIPT
Synthesis and bioevaluation of 1-phenyl-pyrazole-4-carboxylic acid
derivatives as potent xanthine oxidoreductase inhibitors
Jing Lia, Fangping Wua, Xingguo Liub, Yake Zoua, Huixiong Chenc, Zheng Lid, Lei Zhanga, *
a
School of Bioscience and Bioengineering, South China University of Technology,
Guangzhou 510006, P. R. China
b School of Chemical Engineering and Light Industry, Guangdong University of Technology
c CNRS UMR8601, Université Paris Descartes, PRES Sorbonne Paris Cité, UFR Biomédicale,
45 rue des Saints-Pères, 75270 Paris Cedex 06, France
d Centre of Drug Discovery, China Pharmaceutical University, Nanjing 210009, P. R. China
*Corresponding author.
Tel./fax: +86 20 39380678
E-mail address: lzhangce@scut.edu.cn (L. Zhang).

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Graphical abstract
1-Phenyl-pyrazole-4-carboxylic acid derivatives were synthesized and evaluated as xanthine
oxidoreductase inhibitors. Compound 16c was a mixed-type xanthine oxidoreductase inhibitor
with an IC50 of 5.7 nM and a considerable hypouricemic effect similar to febuxostat in mice.

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Highlights
Some 1-phenyl-pyrazole-4-carboxylic acid derivatives showed considerable xanthine
oxidoreductase inhibitory effects in vitro compared to febuxostat in the nanomolar range.
The derivative 16c exhibited a similar hypouricemic potency compared to febuxostat in a
potassium oxonate-hypoxanthine-induced hyperuricemia mice model.

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ABSTRACT
A diverse library of 1-phenyl-pyrazole-4-carboxylic acid derivatives were synthesized and
evaluated for their inhibitory potency against xanthine oxidoreductase (XOR) in vitro and vivo,
and the structure-activity relationship (SAR) analyses were also presented. Approximately
half of the target compounds exhibited the inhibitory potency for XOR at the nanomolar level.
Compounds 16c, 16d, and 16f emerged as the most potent xanthine oxidoreductase inhibitors
with IC50 values of 5.7, 5.7 and 4.2 nM, respectively, in comparison to febuxostat (IC50 of 5.4
nM). Steady-state kinetics measurements indicated that 16c is a mixed-type inhibitor. A
computer molecular docking study of 16c bound to XOR was performed to gain an insight
into its bind mode and SAR for the series. A potassium oxonate-hypoxanthine-induced
hyperuricemia model in mice was chosen to further confirm the hypouricemic effects of 16c
and 16f, and the results demonstrated that 16c exhibits similar hypouricemic potency to
febuxostat.
Keywords: 1-phenyl-pyrazole-4-carboxylic acid, xanthine oxidoreductase inhibitors,
hypouricemic effect, potassium oxonate

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1. Introduction
Xanthine oxidoreductase (XOR) is the key enzyme in the purine metabolic pathway that
catalyses the oxidation of hypoxanthine to xanthine, and follows to the final product uric acid.
The overproduction of uric acid could lead to hyperuricemia, which is the underlying cause of
gout. Thus, XOR is considered as one of the most promising targets for treating
hyperuricemia and gout [1, 2].
Allopurinol, a classical XOR inhibitor, has been used to treat gout for several decades. As a
purine analogue, it can potentially affect the activities of purine and pyrimidine metabolism
enzymes inducing severe side effects such as fulminant hepatitis, renal failure, and
Stevens-Johnson syndrome [3, 4]. Therefore, it is extremely important to discover novel
non-purine XOR inhibitors with higher XOR inhibitory potency and fewer side effects. In fact,
non-purine XOR inhibitors have drawn extensive attention from pharmaceutical industries
worldwide in recent years. Febuxostat, a thiazole-based carboxylic acid derivative, has
already been approved by the Food and Drug Administration (FDA) in 2009 for the
management of hyperuricemia in adults with chronic gout [5, 6]. Based on the success of
febuxostat, its analogues, such as pyrazoles (e.g., Y-700) [7], selenazoles [8], isoxazoles [9],
imidazoles [10], 2-arylbenzo[b]furan [11], etc., have been reported in the recent literatures.
Moreover, topiroxostat, a triazole derivative without a carboxyl moiety, has also been
successfully introduced into the market in Japan in 2013 (Fig. 1) [12].

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Encouraged by the outstanding potency of these inhibitors, we attempted to search for more
potent compounds using an isosteric substitution strategy with the hope of extending SAR to
help us identify non-purine XOR inhibitors. In this context, two series of
1-phenyl-pyrazole-4-carboxylic acid derivatives have been designed and synthesized. The
XOR inhibitory potencies of these compounds have been tested in vitro, with the
hypouricemic effects of compounds 16c and 16f evaluated in vivo (Fig. 2).
2. Results and discussion
2.1 Chemistry
The syntheses of the target derivatives (Series I) 7c-d, 8a-e, and 9a-e were performed as
outlined in Scheme 1. Commercially available 5-bromo-2-hydroxy benzonitrile 1 was
alkylated with the appropriate C2-C8 alkyl bromides or alkyl iodide in the presence of K2CO3
to provide 2a-e [13]. Compounds 2a-e were treated with NaI, CuI and N,
N’-dimethyl-ethylenediamine in dried toluene via a halogen-exchange reaction to give 3a-e.
The key intermediates 4c-d/5a-e/6a-e were obtained through a C-N coupling reaction with
3a-e catalyzed by trans-N, N’-dimethyl-1, 2-cyclohexanediamine, CuI and K2CO3, which
provided a higher regioselectivity and yield [14]. By hydrolysing those key intermediates,
target compounds 7c-d/8a-e/9a-e were obtained in good yields. It should be noted that 7c-d
have already been reported and that 7d is Y-700 [7].
Similarly, the series II derivatives, 16a-f, 17f-g, and 18f-g, were synthesized by the strategy
as shown in Scheme 2, starting from 5-bromo-2-fluorobenzonitrile/4-bromo-1-fluoro-2-

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nitrobenzene 10 without halogen-exchange reaction. The total yields ranged from 12 % to
49 %.
2.2 Biological activity in vitro
The in vitro bovine XOR inhibitory activity of all the derivatives were evaluated by
examining uric acid levels at 295 nm [15] and comparing these data with the standard
inhibitor febuxostat.
As shown in Table 1, all of the series I derivatives showed poorer XOR inhibitory activities
than febuxostat and 7d (Y-700), except 7c. It is obvious that the molecular volume at the
pyrazole moiety 3-position (R1) is responsible for the XOR inhibitory activity. For example,
derivatives 8a, 8e, and 9a-e (IC50 value >100 nM for all those compounds) with methyl- or
trifluoromethyl- groups at R1 exhibited much lower XOR inhibitory activities than 7c (IC50 of
2.4 nM) and 7d (IC50 of 3.5 nM) with a hydrogen at R1. Moreover, for derivatives 7d, 8d and
9d, with the same neopentyl- group at the phenyl 4-position (R), the IC50 values increased
from 3.5 nM, to 23 nM and to >100 nM with the R1 changed from hydrogen to methyl- and
trifluoromethyl- group. These results indicated that the XOR inhibitory activity decreased
with an increased R1 volume. Besides, different alkyl-substitution at the phenyl 4-position (R)
could also regulate the XOR inhibitory activity. For example, among the derivatives 8a-e with
the R of ethyl-, isopropyl-, isobutyl-, neopentyl-, and octyl- group respectively, the XOR
inhibitory activity increased first and then decreased, 8d showed the best XOR inhibitory
activity with the IC50 value of 23 nm, that is about 2-fold more potent than 8b (IC50 of 45 nm),

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5-fold more potent than 8a and 8e (IC50 >100 nm). Briefly, for the series I derivatives, the
modification at R1 was in reference to the structure of febuxostat, and the inhibitory activities
suggested that the febuxostat SAR was not fully suitable for those pyrazole derivatives, as the
results were disappointing.
Based on the above results, modifications were focused on the phenyl moiety by
bioisosterism and ring analogues. As shown in Table 2, all of the series II derivatives showed
some XOR inhibitory activity with IC50 values lower than 100 nM, except for 17g and 18g.
Compounds with the methyl- group at the pyrazole 3-position (R1) still exhibited poorer XOR
inhibitory activities than compounds with the H-group at R1, similar to series I derivatives.
That is, 16a-f with the H-group at R1 showed IC50 values about 10 nM or much lower, while
17f-g and 18f-g with methyl- group at R1 showed all IC50 values above 10 nM. The cyano-
group at the phenyl 3-position (R2) (17f, IC50 of 13.3 nM) resulted in a significant
improvement in the inhibitory activity with respect to the nitro- group (18f, IC50 value of 59.6
nM); the same effect had also been reported for Y-700 series [7], though it had been
confirmed that an electrophilic substituent, such as cyano- or nitro-group, at the phenyl
3-position in febuxostat analogues tended to induce their inhibitory potencies [5, 7, 9]. The
different alkyl- amine groups at the phenyl 4-position (NR) were introduced to verify the
influence of the substitution pattern on the inhibitory potency of these molecules. Within the
series, compounds 16c, 16d and 16f (IC50 values of 5.7, 5.7 and 4.2 nM, respectively) showed
considerable XOR inhibitory activity compared to febuxostat (IC50 value of 5.4 nM), and
compound 16f represented the most potent molecule with a combination of a hydrogen at R1,

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cyano- group at R2, and bulky piperidine- group at NR, which were favourable for the XOR
inhibitory activity by the series II derivatives. Additionally, it is not surprising to find that 17f
with the piperidine- group at NR still retained some inhibitory potency though the substituent
at R1 is methyl- group, indicating that appropriate substituent at NR could make up the lose of
inhibitory potency by the introduction of big volume group at R1 to some extent.
Furthermore, Lineweaver-Burk plots for enzyme inhibition in the presence or absence of
compound 16c with varying xanthine concentrations (10-50 µM) indicate that 16c acted as a
mixed-type XOR inhibitor (Fig. 3) [8, 10, 15].
2.3 Molecular modelling
To better understand the good XOR inhibitory potency of compound 16c at the molecular
level, and to gain an insight into its binding mode with XOR, experimental docking into the
binding pocket of the XOR/Y-700 complex (PBD entry code 1VDV) was performed. The
docking experiment was carried out using MOE (Molecular Operating Environment).
According to our docking study (Fig. 4), febuxostat could bind XOR with the carboxylate
moiety interacting with Arg880 and Thr1010, and the nitrile group of phenyl unit forming
hydrogen bond with Asn768 [5]. Y-700 could bind XOR with the carboxylate moiety
interacting with Arg880, the pyrazole ring as a whole sandwiching between Phe914 and
Phe1009, and the benzonitrile portion inserting between leu873 and leu1014 [15].
Accordingly, 16c was found to bind with XOR in a similar way to that of Y-700. This
explained the fairly good XOR inhibitory potency of 16c at the molecular level. However, the

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potency of 16c to inhibit XOR was weaker than that of Y-700. The difference in substituent
groups at the C-4 of the phenyl unit may be the reason, the hydrophobic 2, 2-dimethylpropoxy
tail of Y-700 surrounded by several hydrophobic amino acid residues was positioned at the
entrance of the active site-directed channel, while that hydrophobic interaction of 16c was
scattered, that may also explained the XOR inhibitory activity increased first and then
decreased of derivatives 8a-e with the R of ethyl-, isopropyl-, isobutyl-, neopentyl-, and octyl-
group respectively. Moreover, the methyl- group on the thiazole ring of febuxostat can
interact with the surrounding hydrophobic cavity, and the sandwich interaction of that thiazole
ring with Phe914 and Phe1009 was different from that of the pyrazole ring of 16c, indicating
methyl- or other molecular volume group substituted on the pyrazole ring might weaken the
binding of XOR.
2.4 Hypouricemic effect in vivo
Compounds 16c and 16f were further tested in vivo to verify their hypouricemic effects in
the acute hyperuricemia mouse model compared to the standard inhibitors febuxostat and 7d
(Y-700) by examining uric acid levels in serum (Fig. 5). As expected, subcutaneous injection
of 300 mg/kg potassium oxonate with an intraperitoneal injection of 500 mg/kg hypoxanthine
to mice caused a marked increase in serum uric acids levels in the hyperuricemia vehicle
group compared to normal vehicle group (data not shown as they were below the low limit of
detection); this was maintained throughout the experiments. At the 5 mg/kg dose, the tested
compounds significantly reduced uric acid serum levels in hyperuricemic mice compared with
the hyperuricemia vehicle group (P<0.01 for 16c and P<0.05 for 16f) at 1 h interval after

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administration, which is nearly equal to that of febuxostat. The two tested compounds also
showed continuous hypouricemic effects throughout the whole experiments. Specifically,
compound 16c and febuxostat could lower the uric acid levels from approximately 1000 µM
to 200 µM in the hyperuricemic mice.
2.5 Pharmacokinetic study
The pharmacokinetic study was carried out for compound 16c in rat preliminarily, and the
pharmacokinetic parameters were shown in Table 3. After oral administration of 16c at 10
mg/kg dose, the concentrations of 16c in plasma reached Cmax at 1.0 h and the t1/2 was 5.0 h.
However, Cmax and AUC0-∞ of 16c were remarkably lower than those of Y-700 [15],
suggesting that further study on the stability and metabolism of 16c was necessary.
3. Conclusions
Two series of 1-phenylpyrazole derivatives were designed, synthesized and evaluated as
XOR inhibitors. Compounds 16c, 16d and 16f emerged as the most potent XOR inhibitors,
with IC50 values at nanomolecule level compared to febuxostat. The molecular docking
studies of those compounds against XOR provided that 16c could bind XOR in a similar way
to that of Y-700, and some explanations of the effect of different substitution on the XOR
inhibitory potency. Specifically, compound 16c was found to be an orally effective XOR
inhibitor with potent and long-lasting hypouricemic effects in the hyperuricemia mouse model.
The preliminary pharmacokinetic study on rat suggested that further strategy to improve the
plasma concentration of 16c is required for a plausible candidate for XOR inhibitor.

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4. Experimental protocols
4.1 Chemistry
4.1.1 Materials and instruments
Unless otherwise indicated, the reagents were purchased from commercial suppliers and
used without further purification. Organic solvents were dried by standard methods when
necessary. All of the reactions were monitored by thin layer chromatography (TLC) using
silica gel glass cards (Qingdao Ocean Chemical, Qingdao, Shandong, P. R. China) with
ultraviolet indicator at 254 nm. The column chromatography was performed using silica gel
(200-300 mesh) from Qingdao Ocean Chemical (Qingdao, Shandong, P. R. China) with ethyl
acetate (EtOAc)/petroleum ether (60-90 °C) mixture as the eluent. Melting points were
recorded on a Stuart SMP40 melting point apparatus (Bibby Scientific, Britain) and are
uncorrected. HRMS spectra were determined on an Agilent 1290 instrument in ESI mode
(Agilent Technologies, USA). 1H (400 MHz) and 13C (100 MHz) NMR spectra were obtained
on Bruker Avance III HD 400 MHz spectrometers (Bruker, Germany) using TMS as the
internal standard. Infrared spectra were recorded using KBr plates on a Bruker Vertex 33
instrument (Bruker, Germany).
4.1.2. General procedure for the preparation of compounds 2a-e
K2CO3 (1.4 g, 10.1 mmol) was added to compound 1 (5.1 mmol) in DMF (15 mL) and
stirred at room temperature for 0.5 h. Alkyl halide (10.1 mmol) was added to the mixture and
heated at 30-100 °C for 3-12 h. The reaction was diluted with H2O (150 mL) and then
extracted with EtOAc (150 mL×2). The combined organic phases were washed with H2O
(200 mL×2) and brine (200 mL), dried over MgSO4 and concentrated in a vacuum. The
residue was purified by flash column chromatography (0-11% EtOAc in petroleum ether).
4.1.2.1. 5-bromo-2-ethyoxyl-benzonitrile (2a) Reaction temperature: 30 °C. White solid (yield:
99%), m.p.: 83.6-85.1 °C; 1H NMR (CDCl3): δ 7.61 (d, 1H, J = 1.9 Hz, Ar-H), 7.58 (dd, 1H, J
= 8.9 and 2.4 Hz, Ar-H), 6.83 ( d, 1H, J = 8.9 Hz, Ar-H), 4.12 (q, 2H, J = 7.0 Hz, CH2CH3),
and 1.46 (t, 3H, J = 7.0 Hz, CH2CH3).

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4.1.2.2. 5-bromo-isopropoxy-benzonitrile (2b) Reaction temperature: 50 °C. Colourless oil
1
(yield: 86%); H NMR (CDCl3): δ 7.61 (s, 1H, J = 1.6 Hz, Ar-H), 7.57 (dd, 1H, J = 9.0 and
2.4 Hz, Ar-H), 6.84 (d, 1H, J = 9.0 Hz, Ar-H), 4.60 (m, 1H, CH(CH3)2), and 1.38 (d, 6H, J =
6.1 Hz, CH(CH3)2).
4.1.2.3. 5-bromo-isobutoxy-benzonitrile (2c) Reaction temperature: 85 °C. Colourless oil
(yield: 80%); 1H NMR (CDCl3): δ 7.67 (d, 1H, J = 2.4 Hz, Ar-H), 7.62 (dd, 1H, J = 9.0 and
2.5 Hz, Ar-H), 6.86 (d, 1H, J = 9.0 Hz, Ar-H), 3.83 (d, 2H, J = 6.5 Hz, CH2CH(CH3)2), 2.19
(m, 1H, CH2CH(CH3)2), and 1.09 (d, 6H, J = 6.7 Hz, CH2CH(CH3)2).
4.1.2.4. 5-bromo-neopentyl-benzonitrile (2d) Reaction temperature: 85 °C. Colourless oil
1
(yield: 40%); H NMR (CDCl3): δ 7.62 (d, 1H, J = 2.5 Hz, Ar-H), 7.58 (dd, 1H, J = 8.9 and
2.5 Hz, Ar-H), 6.83 (d, 1H, J = 8.9 Hz, Ar-H), 3.69 (s, 2H, CH2C(CH3)3), and 1.07 (s, 9H,
CH2C(CH3)3).
4.1.2.5. 5-bromo-octyloxy-benzonitrile (2e) Reaction temperature: 100 °C. Colourless oil
1
(yield: 99%); H NMR (CDCl3): δ 7.63 (d, 1H, J = 1.6 Hz, Ar-H), 7.59 (dd, 1H, J = 8.9 and
2.4 Hz, Ar-H), 6.84 (d, 1H, J = 9.0 Hz, Ar-H), 4.04 (t, 2H, J = 6.5 Hz, CH2(CH2)6CH3),
1.88-1.77 (m, 2H, CH2CH2(CH2)5CH3), 1.53-1.40 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.30 (d,
8H, J = 17.7 Hz, CH2CH2CH2(CH2)4CH3), and 0.88 (t, 3H, J = 6.7 Hz, (CH2)7CH3).
4.1.3. General procedure for the preparation of compounds 3a-e
Compound (2a-e) (4.8 mmol), NaI (1.2 g, 8.0 mmol), N, N’-dimethyl-ethylenediamine (82
mg, 0.96 mmol), CuI (92 mg, 0.48 mmol) and dried toluene (5 mL) were added to a tube
filled with argon. The mixture was stirred at 110 °C for 24 h, diluted with H2O (20 mL),
extracted with EtOAc (15 mL×2), dried over MgSO4 and concentrated in a vacuum. The
residue was purified by flash column chromatography (5-10% EtOAc in petroleum ether).
4.1.3.1. 5-iodo-2-ethyoxyl-benzonitrile (3a) White solid (yield: 71%), m.p.: 85.1-86.7 °C; 1H
NMR (CDCl3): δ 7.80 (d, 1H, J = 2.2 Hz, Ar-H), 7.77 (dd, 1H, J = 8.9 and 1.2 Hz, Ar-H), 6.73
(d, 1H, J = 8.9 Hz, Ar-H), 4.13 (q, 2H, J = 7.0 Hz, CH2CH3), and 1.47 (t, 3H, J = 7.0 Hz,
CH2CH3).
4.1.3.2. 5-iodo-2-isopropoxy-benzonitrile (3b) Colourless oil (yield: 84%); 1H NMR (CDCl3):
δ 7.76 (d, 1H, J = 2.1 Hz, Ar-H), 7.73 (dd, 1H, J = 8.9 and 2.2 Hz, Ar-H), 6.73 (d, 1H, J = 8.9
Hz, Ar-H), 4.72-4.38 (m, 1H, CH(CH3)2), and 1.37 (d, 6H, J = 6.1 Hz, CH(CH3)2).

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4.1.3.3. 5-iodo-2-isobutoxy-benzonitrile (3c) Colourless oil (yield: 80%); 1H NMR (CDCl3): δ
7.80 (d, 1H, J = 2.2 Hz, Ar-H), 7.76 (dd, 1H, J = 8.8 and 2.2 Hz, Ar-H), 6.72 (d, 1H, J = 8.9
Hz, Ar-H), 3.80 (d, 2H, J = 6.5 Hz, CH2CH(CH3)2), 2.15 (m, 1H, CH2CH(CH3)2), and 1.05 (d,
6H, J = 6.7 Hz, CH2CH(CH3)2).
1
4.1.3.4. 5-iodo-2-neopentyloxy-benzonitrile (3d) Colourless oil (yield: 99%); H NMR
(CDCl3): δ 7.79 (s, 1H, Ar-H), 7.75 (dd, 1H, J = 8.9 and 1.9 Hz, Ar-H), 6.72 (d, 1H, J = 8.8
Hz, Ar-H), 3.66 (s, 2H, CH2C(CH3)3), and 1.07 (s, 9H, CH2C(CH3)3).
1
4.1.3.5. 5-iodo-2-octyloxy-benzonitrile (3e) Colourless oil (yield: 80%); H NMR (CDCl3): δ
7.80 (d, 1H, J = 2.2 Hz, Ar-H), 7.76 (dd, 1H, J = 8.8 and 2.2 Hz, Ar-H), 6.73 (d, 1H, J = 8.9
Hz, Ar-H), 4.03 (t, 2H, J = 6.5 Hz, CH2(CH2)6CH3), 1.83 (m, 2H, CH2CH2(CH2)5CH3),
1.54-1.41 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.31 (m, 8H, CH2CH2CH2(CH2)4CH3), and 0.88
(t, 3H, J = 6.9 Hz, (CH2)7CH3).
4.1.4. General procedure for the preparation of compounds 4c-d, 5a-e, and 6a-e
Compound (3a-e) (1.2 mmol), CuI (19.1 mg, 0.1 mmol), trans-N, N’-dimethyl-1,
2-cyclohexane-diamine (29 mg, 0.2 mmol), K2CO3 (0.29 g, 2.1 mmol) and DMF (3 mL) were
added to a tube filled with argon. The mixture was stirred at 110 °C for 24 h, diluted with H2O
(20 mL), and extracted with EtOAc (15 mL×2). The combined organic phases were washed
with H2O (20 mL×2) and brine (30 mL), dried over MgSO4 and concentrated in a vacuum.
The residue was purified by flash column chromatography (12-25% EtOAc in petroleum
ether).
4.1.4.1. 1-(3’-cyano-4’-isbutoxy-phenyl)-pyrazol-4-carboxylate (4c) White solid (yield: 87%),
m.p.: 120.3-121.4 °C; 1H NMR (CDCl3): δ 8.32 (s, 1H, CH), 8.08 (s, 1H, CH), 7.90 (d, 1H, J
= 2.7 Hz, Ar-H), 7.85 (dd, 1H, J = 9.1 and 2.8 Hz, Ar-H), 7.05 (d, 1H, J = 9.1 Hz, Ar-H), 4.34
(q, 2H, J = 7.1 Hz, CH2CH3), 3.88 (d, 2H, J = 6.5 Hz, CH2CH(CH3)2), 2.29-2.12 (m, 1H,
CH2CH(CH3)2), 1.38 (t, 3H, J = 7.1 Hz, CH2CH3), and 1.09 (d, 6H, J = 6.7 Hz,
CH2CH(CH3)2).
4.1.4.2. ethyl 1-(3’-cyano-4’-neopentyloxy-phenyl)-pyrazol-4-carboxylate (4d) White solid
1
(yield: 98%), m.p.: 148.8-149.7 °C; H NMR (CDCl3): δ 8.32 (s, 1H, CH), 8.08 (s, 1H, CH),
7.90 (d, 1H, J = 2.7 Hz, Ar-H), 7.85 (dd, 1H, J = 9.0 and 2.7 Hz, Ar-H), 7.05 (d, 1H, J = 9.1
Hz, Ar-H), 4.34 (q, 2H, J = 7.1 Hz, CH2CH3), 3.75 (s, 2H, CH2C(CH3)3), 1.38 (t, 3H, J = 7.1

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Hz, CH2CH3), and 1.11 (s, 9H, CH2C(CH3)3).
4.1.4.3. methyl 1-(3’-cyano-4’-ethyoxyl-phenyl)-3-methyl-pyrazol-4-carboxylate (5a) White
solid (yield: 96%), m.p.: 176.8-178.0 °C; 1H NMR (CDCl3): δ 8.25 (s, 1H, CH), 7.87 (d, 1H, J
= 2.7 Hz, Ar-H), 7.81 (dd, 1H, J = 9.1 and 2.7 Hz, Ar-H), 7.03 (d, 1H, J = 9.1 Hz, Ar-H), 4.20
(q, 2H, J = 7.0 Hz, CH2CH3), 3.85 (s, 3H, CH3), 2.54 (s, 3H, CH3), and 1.51 (t, 3H, J = 7.0 Hz,
CH2CH3).
4.1.4.4. methyl 1-(3’-cyano-4’-isopropoxy-phenyl)-3-methyl-pyrazol-4-carboxylate (5b) White
solid (yield: 74%), m.p.: 95.7-96.8 °C; 1H NMR (CDCl3): δ 8.25 (s, 1H, CH), 7.86 (d, 1H, J =
2.7 Hz, Ar-H), 7.80 (dd, 1H, J = 9.1 and 2.8 Hz, Ar-H), 7.04 (d, 1H, J = 9.1 Hz, Ar-H),
4.76-4.60 (m, 1H, CH(CH3)2), 3.85 (s, 3H, CH3), 2.54 (s, 3H, CH3), and 1.43 (d, 6H, J = 6.1
Hz, CH(CH3)2).
4.1.4.5. methyl 1-(3’-cyano-4’-isobutoxy-phenyl)-3-methyl-pyrazol-4-carboxylate (5c) White
solid (yield: 64%), m.p.: 152.3-153.4 °C; 1H NMR (CDCl3): δ 8.25 (s, 1H, CH), 7.87 (d, 1H, J
= 2.7 Hz, Ar-H), 7.81 (dd, 1H, J = 9.1 and 2.8 Hz, Ar-H), 7.03 (d, 1H, J = 9.1 Hz, Ar-H), 3.87
(d, 2H, J = 6.6 Hz, CH2CH(CH3)2), 3.86 (s, 3H, CH3), 2.54 (s, 3H, CH3), 2.28-2.10 (m, 1H,
CH2CH(CH3)2), and 1.09 (d, 6H, J = 6.7 Hz, CH2CH(CH3)2).
4.1.4.6. methyl 1-(3’-cyano-4’-neopentyloxy-phenyl)-3-methyl-pyrazol-4-carboxylate (5d)
1
White solid (yield: 94%), m.p.: 141.3-142.4 °C; H NMR (CDCl3): δ 8.25 (s, 1H, CH), 7.86
(d, J = 2.4 Hz, 1H, Ar-H), 7.81 (dd, J = 9.1 and 2.3 Hz, 1H, Ar-H), 7.02 (d, J = 9.1 Hz, 1H,
Ar-H), 3.85 (s, 3H, CH3), 3.73 (s, 2H, CH2C(CH3)3), 2.53 (s, 3H, CH3), and 1.10 (s, 9H,
CH2C(CH3)3).
4.1.4.7. methyl 1-(3’-cyano-4’-octyloxy-phenyl)-3-methyl-pyrazol-4-carboxylate (5e) White
solid (yield: 96%), m.p.: 84.8-86.1 °C; 1H NMR (CDCl3): δ 8.25 (s, 1H, CH), 7.87 (d, 1H, J =
2.7 Hz, Ar-H), 7.81 (dd, 1H, J = 9.1 and 2.8 Hz, Ar-H), 7.03 (d, 1H, J = 9.1 Hz, Ar-H),
4.17-4.03 (m, 2H, CH2(CH2)6CH3), 3.85 (s, 3H, CH3), 2.54 (s, 3H, CH3), 1.86 (dd, 2H, J =
14.8 and 6.8 Hz, CH2CH2(CH2)5CH3), 1.54-1.44 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.43-1.21
(m, 8H, CH2CH2CH2(CH2)4CH3), and 0.89 (t, 3H, J = 6.8 Hz, (CH2)7CH3).
4.1.4.8. ethyl 1-(3’-cyano-4’-ethyoxyl-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylate (6a)
1
White solid (yield: 81%), m.p.: 168.3-169.4 °C; H NMR (CDCl3): δ 8.41 (s, 1H, CH), 7.91
(d, 1H, J = 2.7 Hz, Ar-H), 7.88 (dd, 1H, J = 9.0 and 2.8 Hz, Ar-H), 7.08 (d, 1H, J = 9.0 Hz,

ACCEPTED MANUSCRIPT
Ar-H), 4.37 (q, 2H, J = 7.1 Hz, CH2CH3), 4.22 (q, 2H, J = 7.0 Hz, CH2CH3), 1.53 (t, 3H, J =
7.0 Hz, CH2CH3), and 1.38 (t, 3H, J = 7.1 Hz, CH2CH3).
4.1.4.9. ethyl 1-(3’-cyano-4’-isopropoxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylate (6b)
1
White solid (yield: 65%), m.p.: 137.4-138.8 °C; H NMR (CDCl3): δ 8.41 (s, 1H, CH), 7.90
(d, 1H, J = 2.7 Hz, Ar-H), 7.86 (dd, 1H, J = 9.0 and 2.8 Hz, Ar-H), 7.09 (d, 1H, J = 9.1 Hz,
Ar-H), 4.71 (m, 1H, CH(CH3)2), 4.35 (q, 2H, J = 7.1 Hz, CH2CH3), 1.44 (d, 6H, J = 6.1 Hz,
CH(CH3)2), and 1.38 (t, 3H, J = 7.1 Hz, CH2CH3).
4.1.4.10. ethyl 1-(3’-cyano-4’-isobutoxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylate (6c)
1
White solid (yield: 58%), m.p.: 138.5-139.6 °C; H NMR (CDCl3): δ 8.40 (s, 1H, CH), 7.91
(d, 1H, J = 2.7 Hz, Ar-H), 7.87 (dd, 1H, J = 9.0 and 2.8 Hz, Ar-H), 7.07 (d, 1H, J = 9.1 Hz,
Ar-H), 4.37 (q, 2H, J = 7.1 Hz, CH2CH3), 3.90 (d, 2H, J = 6.5 Hz, CH2CH(CH3)2), 2.22 (m,
1H, CH2CH(CH3)2), 1.38 (t, 3H, J = 7.1 Hz, CH2CH3), and 1.10 (d, 6H, J = 6.7 Hz,
CH2CH(CH3)2).
4.1.4.11. ethyl 1-(3’-cyano-4’-neopentyloxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylate
1
(6d) White solid (yield: 69%), m.p.: 138.1-139.0 °C; H NMR (CDCl3): δ 8.41 (s, 1H, CH),
7.91 (d, 1H, J = 2.5 Hz, Ar-H), 7.87 (dd, 1H, J = 9.0 and 2.7 Hz, Ar-H), 7.07 (d, 1H, J = 9.0
Hz, Ar-H), 4.37 (q, 2H, J = 7.1 Hz, CH2CH3), 3.76 (s, 2H, CH2C(CH3)3), 1.38 (t, 3H, J = 7.1
Hz, CH2CH3), and 1.11 (s, 9H, CH2C(CH3)3).
4.1.4.12. ethyl 1-(3’-cyano-4’-octyloxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylate (6e)
White solid (yield: 69%), m.p.: 83.3-84.1 °C; 1H NMR (CDCl3): δ 8.40 (s, 1H, CH), 7.90 (d,
1H, J = 2.7 Hz, Ar-H), 7.87 (dd, 1H, J = 9.0 and 2.8 Hz, Ar-H), 7.08 (d, 1H, J = 9.1 Hz, Ar-H),
4.37 (q, 2H, J = 7.1 Hz, CH2CH3), 4.13 (t, 2H, J = 6.5 Hz, CH2(CH2)6CH3), 2.00-1.77 (m, 2H,
CH2CH2(CH2)5CH3), 1.50 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.38 (t, 3H, J = 7.1 Hz, CH2CH3),
1.31 (m, 8H, CH2CH2CH2(CH2)4CH3), and 0.89 (t, 3H, J = 6.9 Hz, (CH2)7CH3).
4.1.5 General procedure for the preparation of compounds 7c-d, 8a-e, and 9a-e
A mixture of 4c-d, 5a-e, or 6a-e (1.0 mmol), 1 M NaOH solution (2.0 mL), and
THF/ethanol (1:1) (10 mL) was heated at 60 °C for 2 h. After the reaction was completed, the
mixture was diluted with H2O (80 mL) and acidified with 1 M HCl solution to pH = 2. The
suspension was stirred at room temperature for 20 min, and the solid was collected by
filtration, washed with water, and recrystallized in methanol to yield the desired product.

ACCEPTED MANUSCRIPT
4.1.5.1. 1-(3’-cyano-4’-isobutoxy-phenyl)-pyrazol-4-carboxylic acid (7c) White solid (yield:
70%), m.p.: 198.5-199.7 °C; 1H NMR (DMSO-d6): δ 12.65 (s, 1H, COOH), 9.04 (s, 1H, CH),
8.30 (s, 1H, Ar-H), 8.19 (d, 1H, J = 9.1 Hz, Ar-H), 8.09 (s, 1H, CH), 7.41 (d, 1H, J = 8.9 Hz,
Ar-H), 3.99 (d, 2H, J = 5.7 Hz, CH2CH(CH3)2), 2.09 (m, 1H, CH2CH(CH3)2), and 1.03 (d, 6H,
J = 6.4 Hz, CH2CH(CH3)2), which is consistent with ref. [7].
4.1.5.2. 1-(3’-cyano-4’-neopentyloxy-phenyl)-pyrazol-4-carboxylic acid (7d) White solid
(yield: 55%), m.p.: 200.9-201.9 °C; 1H NMR (DMSO-d6): δ 12.66 (s, 1H, COOH), 9.04 (s, 1H,
CH), 8.30 (s, 1H, Ar-H), 8.24-8.14 (m, 1H, Ar-H), 8.09 (s, 1H, CH), 7.40 (d, 1H, J = 9.2 Hz,
Ar-H), 3.87 (s, 2H, CH2C(CH3)3), and 1.05 (s, 9H, CH2C(CH3)3), which is consistent with ref.
[7].
4.1.5.3. 1-(3’-cyano-4’-ethyoxyl-phenyl)-3-methyl-pyrazol-4-carboxylic acid (8a) White solid
1
(yield: 36%), m.p.: 234.1-235.2 °C; H NMR (DMSO-d6): δ 12.49 (s, 1H, COOH), 8.91 (s,
1H, CH), 8.24 (d, 1H, J = 2.8 Hz, Ar-H), 8.14 (dd, 1H, J = 9.2 and 2.8 Hz, Ar-H), 7.36 (d, 1H,
J = 9.3 Hz, Ar-H), 4.24 (q, 2H, J = 7.0 Hz, CH2CH3), 2.42 (s, 3H, CH3), and 1.38 (t, 3H, J =
13
7.0 Hz, CH2CH3); C NMR (DMSO-d6): δ 164.8, 159.2, 151.9, 132.8, 126.0, 123.9, 116.1,
115.0, 114.6, 101.6, 65.6, 14.8, and 13.7; HRMS: Calcd for C14H13N3O3 [M+H]+ 272.1030,
Found 272.1013; IR vmax/cm-1: 3296 (υO-H), 3133 and 2985 (υC-H), 2231 (υC≡N), 1705 (υC=O),
1557 and 1511 (υC=C), 1202 and 1111 (υC-O), 816 (δC-H).
4.1.5.4. 1-(3’-cyano-4’-isopropoxy-phenyl)-3-methyl-pyrazol-4-carboxylic acid (8b) White
solid (yield: 56%), m.p.: 233.5-234.7 °C; 1H NMR (DMSO-d6): δ 12.50 (s, 1H, COOH), 8.92
(s, 1H, CH), 8.23 (d, 1H, J = 2.8 Hz, Ar-H), 8.13 (dd, 1H, J = 9.2 and 2.8 Hz, Ar-H), 7.41 (d,
1H, J = 9.4 Hz, Ar-H), 5.03-4.69 (m, 1H, CH(CH3)2), 2.43 (s, 3H, CH3), and 1.34 (d, 6H, J =
6.0 Hz, CH(CH3)2); 13C NMR (DMSO-d6): δ 164.7, 158.3, 151.9, 132.8, 132.7, 125.8, 124.0,
116.2, 115.8, 115.0, 102.5, 72.5, 22.1, and 13.7; HRMS: Calcd for C15H15N3O3 [M+H]+
286.1186, Found 286.1169; IR vmax/cm-1: 3455 (υO-H), 3132 and 2989 (υC-H), 2232 (υC≡N),
1677 (υC=O), 1551 and 1506 (υC=C), 1270 and 1120 (υC-O), 815 (δC-H).
4.1.5.5. 1-(3’-cyano-4’-isobutoxy-phenyl)-3-methyl-pyrazol-4-carboxylic acid (8c) White
solid (yield: 56%), m.p.: 254.6-255.8 °C; 1H NMR (DMSO-d6): δ 12.49 (s, 1H, COOH), 8.92
(s, 1H, CH), 8.25 (d, 1H, J = 2.6 Hz, Ar-H), 8.15 (dd, 1H, J = 9.2 and 2.6 Hz, Ar-H), 7.38 (d,
1H, J = 9.2 Hz, Ar-H), 3.98 (d, 2H, J = 6.4 Hz, CH2CH(CH3)2), 2.44 (s, 3H, CH3), 2.09 (m,

ACCEPTED MANUSCRIPT
13
1H, CH2CH(CH3)2), and 1.03 (d, 6H, J = 6.7 Hz, CH2CH(CH3)2); C NMR (DMSO-d6): δ
164.8, 159.1, 151.9, 132.8, 126.0, 123.8, 115.9, 115.0, 114.5, 101.8, 75.9, 28.0, 18.7, and 13.4;
HRMS: Calcd for C16H17N3O3 [M+H]+ 300.1343, Found 300.1325; IR vmax/cm-1: 3480 (υO-H),
3140 and 2964 (υC-H), 2226 (υC≡N), 1692 (υC=O), 1556 and 1517 (υC=C), 1274 and 1115 (υC-O),
817 (δC-H).
4.1.5.6. 1-(3’-cyano-4’-neopentyloxy-phenyl)-3-methyl-pyrazol-4-carboxylic acid (8d) White
solid (yield: 54%), m.p.: 225.7-226.7 °C; 1H NMR (DMSO-d6): δ 12.52 (s, 1H, COOH), 8.92
(s, 1H, CH), 8.24 (d, 1H, J = 2.5 Hz, Ar-H), 8.1 (d, 1H, J = 9.2 Hz, Ar-H), 7.36 (d, 1H, J = 9.2
13
Hz, Ar-H), 3.85 (s, 2H, CH2C(CH3)3), 2.42 (s, 3H, CH3), and 1.04 (s, 9H, CH2C(CH3)3). C
NMR (DMSO-d6): δ 164.7, 159.7, 151.9, 132.9, 126.0, 123.6, 116.0, 115.0, 114.1, 101.0, 79.2,
31.8, 26.5, and 13.9; HRMS: Calcd for C17H19N3O3 [M+H]+ 314.1499, Found 314.1482; IR
vmax/cm-1: 3403 (υO-H), 3080 and 2964 (υC-H), 2230 (υC≡N), 1681 (υC=O), 1551 and 1513 (υC=C),
1290 and 1116 (υC-O), 823 (δC-H).
4.1.5.7. 1-(3’-cyano-4’-octyloxy-phenyl)-3-methyl-pyrazol-4-carboxylic acid (8e) White solid
1
(yield: 67%), m.p.: 130.0-131.1 °C; H NMR (DMSO-d6): δ 12.50 (s, 1H, COOH), 8.88 (s,
1H, CH), 8.24 (s, 1H, Ar-H), 8.14 (d, 1H, J = 9.2 Hz, Ar-H), 7.37 (d, 1H, J = 9.0 Hz, Ar-H),
4.17 (t, 2H, J = 5.9 Hz, CH2(CH2)6CH3), 2.43 (s, 3H, CH3), 1.83-1.67 (m, 2H,
CH2CH2(CH2)5CH3), 1.49-1.38 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.30 (m, 8H,
13
CH2CH2CH2(CH2)4CH3), and 0.86 (t, 3H, J = 6.5 Hz, (CH2)7CH3); C NMR (DMSO-d6): δ
164.6, 159.3, 151.9, 132.6, 125.9, 123.9, 116.0, 115.0, 101.6, 69.7, 31.6, 29.1, 28.8, 25.7, 22.5,
14.4, and 13.7; HRMS: Calcd for C20H25N3O3 [M+Na]+ 378.1778, Found 378.1793; IR
vmax/cm-1: 3438 (υO-H), 2964, 2921 and 2852 (υC-H), 2229 (υC≡N), 1697 (υC=O), 1555, 1514 and
1469 (υC=C), 1294 and 1116 (υC-O), 813 (δC-H).
4.1.5.8. 1-(3’-cyano-4’-ethyoxyl-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylic acid (9a)
1
White solid (yield: 47%), m.p.: 227.1-227.6 °C; H NMR (DMSO-d6): δ 13.24 (s, 1H,
COOH), 9.24 (s, 1H, CH), 8.36 (d, 1H, J = 2.7 Hz, Ar-H), 8.20 (dd, 1H, J = 9.2 and 2.7 Hz,
Ar-H), 7.44 (d, 1H, J = 9.3 Hz, Ar-H), 4.29 (q, 2H, J = 6.9 Hz, CH2CH3), and 1.41 (t, 3H, J =
13
6.9 Hz, CH2CH3); C NMR (DMSO-d6): δ 161.8, 160.2, 135.7, 132.0, 127.2, 125.5, 115.9,
115.4, 114.6, 101.6, 65.8, and 14.4; HRMS: Calcd for C14H10F3N3O3 [M+Na]+ 348.0566,
Found 348.0567; IR vmax/cm-1: 3489 (υO-H), 3141 and 2996 (υC-H), 2234 (υC≡N), 1710 (υC=O),

ACCEPTED MANUSCRIPT
1553, 1505 and 1480 (υC=C), 1268 and 1152 (υC-O), 821 (δC-H).
4.1.5.9. 1-(3’-cyano-4’-isopropoxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylic acid (9b)
1
White solid (yield: 52%), m.p.: 238.3-239.3 °C; H NMR (DMSO-d6): δ 13.24 (s, 1H,
COOH), 9.22 (s, 1H, CH), 8.33 (d, 1H, J = 2.8 Hz, Ar-H), 8.17 (dd, 1H, J = 9.2 and 2.8 Hz,
Ar-H), 7.47 (d, 1H, J = 9.4 Hz, Ar-H), 5.09-4.66 (m, 1H, CH(CH3)2), and 1.35 (d, 6H, J = 6.0
Hz, CH(CH3)2); 13C NMR (DMSO-d6): δ 161.9, 159.4, 135.6, 131.8, 127.1, 125.6, 115.9,
115.4, 102.5, 72.7, and 22.0; HRMS: Calcd for C15H12F3N3O3 [M+Na]+ 362.0723, Found
362.0718; IR vmax/cm-1: 3479 (υO-H), 3137, 2989 and 2897 (υC-H), 2233 (υC≡N), 1722 (υC=O),
1555 and 1502 (υC=C), 1268 and 1151 (υC-O), 880, 820 (δC-H).
4.1.5.10. 1-(3’-cyano-4’-isobutoxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylic acid (9c)
1
White solid (yield: 52%), m.p.: 236.2-237.8 °C; H NMR (DMSO-d6): δ 13.29 (s, 1H,
COOH), 9.30 (s, 1H, CH), 8.42 (d, 1H, J = 2.8 Hz, Ar-H), 8.26 (dd, 1H, J = 9.2 and 2.8 Hz,
Ar-H), 7.52 (d, 1H, J = 9.3 Hz, Ar-H), 4.08 (d, 2H, J = 6.5 Hz, CH2CH(CH3)2), 2.25-2.04 (m,
13
1H, CH2CH(CH3)2), and 1.10 (d, 6H, J = 6.7 Hz, CH2CH(CH3)2); C NMR (DMSO-d6): δ
161.9, 160.4, 135.6, 131.9, 127.2, 127.1, 125.4, 115.4, 114.7, 102.0, 75.7, 28.1, and 19.4;
HRMS: Calcd for C16H14F3N3O3 [M+Na]+ 376.0879, Found 376.0894; IR vmax/cm-1: 3454
(υO-H), 3122 and 2977 (υC-H), 2236 (υC≡N), 1707 (υC=O), 1552 and 1505 (υC=C), 1245 and 1150
(υC-O), 821 (δC-H).
4.1.5.11. 1-(3’-cyano-4’-neopentyloxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylic acid
(9d) White solid (yield: 45%), m.p.: 231.7-233.5 °C; 1H NMR (DMSO-d6): δ 9.23 (s, 1H, CH),
8.35 (d, 1H, J = 2.8 Hz, Ar-H), 8.19 (dd, 1H, J = 9.2 and 2.8 Hz, Ar-H), 7.43 (d, 1H, J = 9.3
Hz, Ar-H), 3.88 (s, 2H, CH2C(CH3)3), and 1.05 (s, 9H, CH2C(CH3)3); 13C NMR (DMSO-d6):
δ 161.9, 161.1, 135.6, 131.9, 127.4, 125.3, 115.6, 115.4, 114.7, 101.7, 79.0, 32.3, and 26.6;
HRMS: Calcd for C17H16F3N3O3 [M+Na]+ 390.1036, Found 390.1042; IR vmax/cm-1: 3454
(υO-H), 3122 and 2977 (υC-H), 2236 (υC≡N), 1707 (υC=O), 1552, 1507 and 1471 (υC=C), 1245 and
1150 (υC-O), 821 (δC-H).
4.1.5.12. 1-(3’-cyano-4’-octyloxy-phenyl)-3-trifluoromethyl-pyrazol-4-carboxylic acid (9e)
1
White solid (yield: 64%), m.p.: 122.6-123.3 °C; H NMR (DMSO-d6): δ 13.24 (s, 1H,
COOH), 9.24 (s, 1H, CH), 8.35 (d, 1H, J = 2.6 Hz, Ar-H), 8.20 (dd, 1H, J = 9.2 and 2.6 Hz,
Ar-H), 7.45 (d, 1H, J = 9.3 Hz, Ar-H), 4.23 (t, 2H, J = 6.3 Hz, CH2(CH2)6CH3), 1.89-1.70 (m,

ACCEPTED MANUSCRIPT
2H, CH2CH2(CH2)5CH3), 1.54-1.42 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.42-1.21 (m, 8H,
13
CH2CH2CH2(CH2)4CH3), and 0.88 (t, 3H, J = 6.0 Hz, (CH2)7CH3); C NMR (DMSO-d6): δ
161.9, 160.4, 135.7, 131.9, 127.2, 125.5, 115.7, 115.3, 114.7, 101.8, 69.9, 31.6, 29.1, 28.7,
25.7, 22.5, and 14.4; HRMS: Calcd for C20H22F3N3O3 [M+Na]+ 390.1036, Found 390.1042;
IR vmax/cm-1: 3486 (υO-H), 3123 and 2919 (υCH), 2233 (υC≡N), 1712 (υC=O), 1552, 1507 and
1471 (υC=C), 1265 and 1148 (υC-O), 819 (δC-H).
4.1.6. General procedure for the preparation of compounds 11a-g and 12f-g
Alkylamine (30 mmol) and K2CO3 (4.1 g, 30 mmol) or CsCO3 (9.8 g, 30 mmol) was added
to compound 10 (10 mmol) in DMF or DMSO (20 mL). The mixture was heated at 25-120 °C
for 2-12 h. The reaction was diluted with H2O (200 mL) and then extracted with EtOAc (200
mL×2). The combined organic phases were washed with H2O (200 mL×2) and brine (200
mL), dried over MgSO4 and concentrated in a vacuum. The residue was purified by flash
column chromatography (0-10% EtOAc in petroleum ether).
4.1.6.1. 5-bromo-dimethylamino-benzonitrile (11a) Reaction temperature: 25 °C. Reagent:
CsCO3 and DMF. Yellow solid (yield: 65%), m.p.: 43.3-44.3 °C; 1H NMR (CDCl3): δ 7.57 (d,
1H, J = 2.5 Hz, Ar-H), 7.46 (dd, 1H, J = 9.1 and 2.5 Hz, Ar-H), 6.75 (d, 1H, J = 9.1 Hz), and
3.05 (s, 6H, N(CH3)2).
4.1.6.2. 5-bromo-isobutylamino-benzonitrile (11b) Reaction temperature: 70 °C. Reagent:
CsCO3 and DMSO. Orange solid (yield: 88%), m.p.: 74.4-75.0 °C; 1H NMR (CDCl3): δ 7.46
(d, 1H, J = 2.3 Hz, Ar-H), 7.43 (dd, 1H, J = 9.0 and 2.3 Hz, Ar-H), 6.54 (d, 1H, J = 9.0 Hz,
Ar-H), 4.65 (s, 1H, NHCH2CH(CH3)2), 3.07-2.91 (m, 2H, NHCH2CH(CH3)2), 1.91 (m, 1H,
NHCH2CH(CH3)2), and 1.00 (d, J = 6.7 Hz, 6H, NHCH2CH(CH3)2).
4.1.6.3. 5-bromo-(N-methyl)isobutylamino-benzonitrile (11c) Reaction temperature: 78 °C.
1
Reagent: CsCO3 and DMSO. Yellow oil (yield: 99%); H NMR (CDCl3): δ 7.54 (d, 1H, J =
1.3 Hz, Ar-H), 7.42 (dd, 1H, J = 9.1 and 3.3 Hz, Ar-H), 6.75 (d, 1H, J = 9.1 Hz, Ar-H), 3.19 (d,
2H, J = 7.5 Hz, NCH3CH2CH(CH3)2), 3.05 (s, 3H, NCH3CH2CH(CH3)2), 2.01 (m, 1H,
NCH3CH2CH(CH3)2), and 0.89 (d, 6H, J = 6.6 Hz, NCH3CH2CH(CH3)2).
4.1.6.4. 5-bromo-pyrrolidin-benzonitrile (11d) Reaction temperature: 86 °C. Reagent: K2CO3
and DMF. Orange solid (yield: 96%), m.p.: 67.2-67.4 °C; 1H NMR (CDCl3): δ 7.50 (d, 1H, J
= 2.5 Hz, Ar-H), 7.36 (dd, 1H, J = 9.2 and 2.5 Hz, Ar-H), 6.51 (d, 1H, J = 9.2 Hz, Ar-H),

ACCEPTED MANUSCRIPT
3.62-3.51 (m, 4H, N(CH2)2(CH2)2), and 2.03-1.95 (m, 4H, N(CH2)2(CH2)2).
4.1.6.5. 5-bromo-(2-methyl-pyrrolidin)-benzonitrile (11e) Reaction temperature: 85 °C.
1
Reagent: K2CO3 and DMSO. Yellow oil (yield: 76%); H NMR (CDCl3): δ 7.51 (d, 1H, J =
2.5 Hz, Ar-H), 7.36 (dd, 1H, J = 9.2 and 2.5 Hz, Ar-H), 6.56 (d, 1H, J = 9.3 Hz, Ar-H),
4.25-4.15 (m, 1H, NCHCH3CH2CH2CH2), 3.95-3.35 (m, 2H, NCHCH3CH2CH2CH2),
2.22-1.98 (m, 2H, NCHCH3CH2CH2CH2), 1.96-1.62 (m, 2H, NCHCH3CH2CH2CH2), and
1.19 (d, 3H, J = 6.1 Hz, NCHCH3CH2CH2CH2).
4.1.6.6. 5-bromo-piperidyl-benzonitrile (11f) Reaction temperature: 100 °C. Reagent: K2CO3
and DMSO. Yellow oil (yield: 96%); 1H NMR (CDCl3): δ 7.60 (d, 1H, J = 2.2 Hz, Ar-H), 7.51
(dd, 1H, J = 8.9 and 2.3 Hz, Ar-H), 6.85 (d, 1H, J = 8.9 Hz, Ar-H), 3.19-3.06 (m, 4H,
N(CH2)2(CH2)2CH2), 1.81-1.70 (m, 4H, N(CH2)2(CH2)2CH2), and 1.65- 1.53 (m, 2H,
N(CH2)2(CH2)2CH2).
4.1.6.7. 5-bromo-morpholinyl-benzonitrile (11g) Reaction temperature: 100 °C. Reagent:
1
K2CO3 and DMSO. Yellow oil (yield: 99%); H NMR (CDCl3): δ 7.62 (d, 1H, J = 1.6 Hz,
Ar-H), 7.55 (dd, 1H, J = 8.8 and 2.0 Hz, Ar-H), 6.86 (d, 1H, J = 8.8 Hz, Ar-H), 3.92-3.74 (m,
4H, N(CH2)2O(CH2)2), and 3.21-3.08 (m, 4H, N(CH2)2O(CH2)2).
4.1.6.8. 5-bromo-2-piperidyl-1-nitro-benzene (12f) Reaction temperature: 100 °C. Reagent:
1
K2CO3 and DMSO. Orange oil (yield: 99%); H NMR (CDCl3): δ 7.88 (d, 1H, J = 2.3 Hz,
Ar-H), 7.51 (dd, 1H, J = 8.9 and 2.3 Hz, Ar-H), 6.98 (d, 1H, J = 8.9 Hz, Ar-H), 3.06-2.93 (m,
4H, N(CH2)2(CH2)2CH2), and 1.75-1.65 (m, 4H, N(CH2)2(CH2)2CH2), 1.63-1.55 (m, 2H,
N(CH2)2(CH2)2CH2).
4.1.6.9. 5-bromo-2-morpholinyl-1-nitro-benzene (12g) Reaction temperature: 120 °C. Reagent:
1
K2CO3 and DMSO. Orange oil (yield: 98%); H NMR (CDCl3): δ 7.82 (d, 1H, J = 2.3 Hz,
Ar-H), 7.51 (dd, 1H, J = 8.8 and 2.3 Hz, Ar-H), 6.96 (d, 1H, J = 8.8 Hz, Ar-H), 3.80-3.67 (m,
4H, N(CH2)2O(CH2)2), and 3.06-2.89 (m, 4H, N(CH2)2O(CH2)2).
4.1.7. General procedure for the preparation of compounds 13a-f, 14f-g, and 15f-g
Compound (11a-g or 12f-g) (1.2 mmol), CuI (19.1 mg, 0.1 mmol), trans-N, N’-dimethyl-1,
2-cyclohexane-diamine (29 mg, 0.2 mmol), K2CO3 (0.29 g, 2.1 mmol) and DMF (3 mL) were
added to a tube filled with argon. The mixture was stirred at 110 °C for 24 h. The reaction was
diluted with H2O (20 mL) and extracted with EtOAc (15 mL×2). The combined organic

ACCEPTED MANUSCRIPT
phases were washed with H2O (20 mL×2) and brine (30 mL), dried over MgSO4 and
concentrated in a vacuum. The residue was purified by flash column chromatography
(12-25% EtOAc in petroleum ether).
4.1.7.1. ethyl 1-(3’-cyano-4’-dimethylamino-phenyl)-pyrazol-4-carboxylate (13a) White solid
1
(yield: 65%), m.p.: 136.0-137.3 °C; H NMR (CDCl3): δ 8.28 (s, 1H, CH), 8.07 (s, 1H, CH),
7.82 (d, 1H, J = 2.7 Hz, Ar-H), 7.72 (dd, 1H, J = 9.2 and 2.7 Hz, Ar-H), 6.95 (d, 1H, J = 9.2
Hz, Ar-H), 4.33 (q, 2H, J = 7.1 Hz, CH2CH3), 3.13 (s, 6H, N(CH3)2), and 1.37 (t, 3H, J = 7.1
Hz, CH2CH3).
4.1.7.2. ethyl 1-(3’-cyano-4’-isobutylamino-phenyl)-pyrazol-4-carboxylate (13b) White solid
1
(yield: 30%), m.p.: 129.8-131.1 °C; H NMR (CDCl3): δ 8.24 (s, 1H, CH), 8.05 (s, 1H, CH),
7.71 (d, 1H, J = 2.1 Hz, Ar-H), 7.71-7.59 (overlap, 1H, Ar-H), 6.74 (d, 1H, J = 8.8 Hz, Ar-H),
4.77 (s, 1H, NHCH2CH(CH3)2), 4.33 (q, 2H, J = 7.1 Hz, CH2CH3), 3.18-2.91 (m, 2H,
NHCH2CH(CH3)2), 1.95 (m, 1H, NHCH2CH(CH3)2), 1.37 (t, 3H, J = 7.1 Hz, CH2CH3), and
1.03 (d, 6H, J = 6.7 Hz, NHCH2CH(CH3)2).
4.1.7.3. ethyl 1-(3’-cyano-4’-(N-methyl)isobutylamino-phenyl)-pyrazol-4-carboxylate (13c)
1
White solid (yield: 28%), m.p.: 81.3-82.4 °C; H NMR (CDCl3): δ 8.27 (s, 1H, CH), 8.06 (s,
1H, CH), 7.79 (d, 1H, J = 2.7 Hz, Ar-H), 7.69 (dd, 1H, J = 9.2 and 2.8 Hz, Ar-H), 6.95 (d, 1H,
J = 9.2 Hz, Ar-H), 4.33 (q, 2H, J = 7.1 Hz, CH2CH3), 3.28 (d, 2H, J = 7.5 Hz,
NCH3CH2CH(CH3)2), 3.12 (d, 3H, J = 13.8 Hz, NCH3CH2CH(CH3)2), 2.15-1.98 (m, 1H,
NCH3CH2CH(CH3)2), 1.37 (t, 3H, J = 7.1 Hz, CH2CH3), and 0.93 (d, 6H, J = 6.6 Hz,
NCH3CH2CH(CH3)2).
4.1.7.4. ethyl 1-(3’-cyano-4’-pyrrolidin-phenyl)-pyrazol-4-carboxylate (13d) White solid
1
(yield: 37%), m.p.: 159.9-161.0 °C; H NMR (CDCl3): δ 8.25 (s, 1H, CH), 8.05 (s, 1H, CH),
7.73 (d, 1H, J = 2.7 Hz, Ar-H), 7.64 (dd, 1H, J = 9.3 and 2.7 Hz, Ar-H), 6.71 (d, 1H, J = 9.3
Hz, Ar-H), 4.33 (q, 2H, J = 7.1 Hz, CH2CH3), 3.74-3.55 (m, 4H, N(CH2)2(CH2)2), 2.13-1.94
(m, 4H, N(CH2)2(CH2)2), and 1.37 (t, 3H, J = 7.1 Hz, CH2CH3).
4.1.7.5. ethyl 1-(3’-cyano-4’-(2-methyl-pyrrolidin)-phenyl)-pyrazol-4-carboxylate (13e) White
1
solid (yield: 62%), m.p.: 108.7-109.1 °C; H NMR (CDCl3): δ 8.25 (s, 1H, CH), 8.05 (s, 1H,
CH), 7.74 (d, 1H, J = 2.7 Hz, Ar-H), 7.65 (dd, 1H, J = 9.3 and 2.8 Hz, Ar-H), 6.77 (d, 1H, J =
9.4 Hz, Ar-H), 4.37-4.30 (overlap, 2H, CH2CH3), 4.31- 4.25 (m, 1H, NCHCH3CH2CH2CH2),

ACCEPTED MANUSCRIPT
3.96-3.50 (m, 2H, NCHCH3CH2CH2CH2), 2.25 -2.01 (m, 2H, NCHCH3CH2CH2CH2),
2.01-1.67 (m, 2H, NCHCH3CH2CH2CH2), 1.37 (t, 3H, J = 7.1 Hz, CH2CH3), and 1.24 (d, 3H,
J = 6.1 Hz, NCHCH3CH2CH2CH2).
4.1.5.6. ethyl 1-(3’-cyano-4’-piperidyl-phenyl)-pyrazol-4-carboxylate (13f) White solid (yield:
55%), m.p.: 129.3-130.4 °C; 1H NMR (CDCl3): δ 8.31 (s, 1H, CH), 8.07 (s, 1H, CH), 7.87 (d,
1H, J = 2.7 Hz, Ar-H), 7.76 (dd, 1H, J = 9.0 and 2.7 Hz, Ar-H), 7.07 (d, 1H, J = 9.0 Hz, Ar-H),
4.33 (q, 2H, J = 7.1 Hz, CH2CH3), 3.28-3.10 (m, 4H, N(CH2)2(CH2)2CH2), 1.85-1.71 (m, 4H,
N(CH2)2(CH2)2CH2), 1.68-1.56 (m, 2H, N(CH2)2(CH2)2CH2), and 1.37 (t, 3H, J = 7.1 Hz,
CH2CH3).
4.1.7.7. methyl 1-(3’-cyano-4’-piperidyl-phenyl)-3-methyl-pyrazol-4-carboxylate (14f) White
solid (yield: 47%), m.p.: 152.5-153.9 °C; 1H NMR (CDCl3): δ 8.24 (s, 1H, CH), 7.85 (d, 1H, J
= 2.6 Hz, Ar-H), 7.73 (dd, 1H, J = 9.0 and 2.7 Hz, Ar-H), 7.05 (d, 1H, J = 9.0 Hz, Ar-H), 3.85
(s, 3H, CH3), 3.27-3.12 (m, 4H, N(CH2)2(CH2)2CH2), 1.88-1.73 (m, 4H, N(CH2)2(CH2)2CH2),
and 1.63 (m, 2H, N(CH2)2(CH2)2CH2).
4.1.7.8. methyl 1-(3’-cyano-4’-morpholinyl-phenyl)-3-methyl-pyrazol-4-carboxylate (14g)
1
yellow solid (yield: 46%), m.p.: 199.9-201.7 °C; H NMR (CDCl3): δ 8.27 (s, 1H, CH), 7.91
(d, 1H, J = 2.6 Hz, Ar-H), 7.79 (dd, 1H, J = 8.9 and 2.7 Hz, Ar-H), 7.08 (d, 1H, J = 9.0 Hz,
Ar-H), 3.95-3.88 (m, 4H, N(CH2)2O(CH2)2), 3.85 (s, 3H, CH3), 3.27-3.21 (m, 4H,
N(CH2)2O(CH2)2), and 2.54 (s, 3H, CH3).
4.1.7.9. methyl 1-(3’-nitro-4’-piperidyl-phenyl)-3-methyl-pyrazol-4-carboxylate (15f) Orange
solid (yield: 58%), m.p.: 131.5-132.3 °C; 1H NMR (CDCl3): δ 8.28 (s, 1H, CH), 8.09 (d, 1H, J
= 2.5 Hz, Ar-H), 7.75 (dd, 1H, J = 9.0 and 2.6 Hz, Ar-H), 7.19 (d, 1H, J = 9.0 Hz, Ar-H), 3.85
(s, 3H, CH3), 3.11-3.01 (m, 4H, N(CH2)2(CH2)2CH2), 2.53 (s, 3H, CH3), 1.72 (m, 4H,
N(CH2)2(CH2)2CH2), and 1.66-1.56 (m, 2H, N(CH2)2(CH2)2CH2).
4.1.7.10. methyl 1-(3’-nitro-4’-morpholinyl-phenyl)-3-methyl-pyrazol-4-carboxylate (15g)
Orange solid (yield: 47%), m.p.: 167.2-168.4 °C; 1H NMR (CDCl3): δ 8.31 (s, 1H, CH), 8.13
(d, 1H, J = 2.6 Hz, Ar-H), 7.82 (dd, 1H, J = 8.9 and 2.6 Hz, Ar-H), 7.23 (d, 1H, J = 8.9 Hz,
Ar-H), 3.93-3.76 (m, 7H, N(CH2)2O(CH2)2, CH3), 3.14-3.00 (m, 4H, N(CH2)2O(CH2)2), and
2.54 (s, 3H, CH3).
4.1.8 General procedure for the preparation of compounds 16a-f, 17f-g and 18f-g

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A mixture of 13a-f, 14f-g or 15f-g (1.0 mmol), 1 M NaOH solution (2.0 mL), and
THF/ethanol (1:1) (10 mL) was heated at 60 °C for 2 h. After the reaction was completed, the
mixture was diluted with H2O (80 mL) and acidified with 1 M HCl solution to pH = 2. The
suspension was stirred at room temperature for 20 min, and the solid was collected by
filtration, washed with water, and recrystallized in ethanol to yield the desired product.
4.1.8.1. 1-(3’-cyano-4’-dimethylamino-phenyl)-pyrazol-4-carboxylic acid (16a) White solid
1
(yield: 92%), m.p.: 210.5-211.2 °C; H NMR (DMSO-d6): δ 12.63 (s, 1H, COOH), 8.97 (s,
1H, CH), 8.13 (d, 1H, J = 2.7 Hz, Ar-H), 8.04 (s, 1H, CH), 8.01 (dd, 1H, J = 9.2 and 2.7 Hz,
13
Ar-H), 7.14 (d, 1H, J = 9.3 Hz, Ar-H), and 3.02 (s, 6H, N(CH3)2); C NMR (DMSO-d6): δ
164.0, 153.9, 142.3, 131.3, 131.2, 125.4, 125.2, 119.1, 118.4, 117.4, 99.9, and 43.1; HRMS:
Calcd for C13H12N4O2 [M+H]+ 257.1033, Found 257.1021; IR vmax/cm-1: 3473 (υO-H), 3137
and 2950 (υC-H), 2221 (υC≡N), 1692 (υC=O), 1557, 1523 and 1460 (υC=C), 1275 and 1184 (υC-N),
884 and 818 (δC-H).
4.1.8.2. 1-(3’-cyano-4’-isobutylamino-phenyl)-pyrazol-4-carboxylic acid (16b) White solid
(yield: 88%), m.p.: 189.2-190.6 °C; 1H NMR (DMSO-d6): δ 8.79 (s, 1H, COOH), 7.94 (s, 1H,
H), 7.91 (d, 1H, J = 2.7 Hz, Ar-H), 7.83 (dd, 1H, J = 9.2 and 2.7 Hz, Ar-H), 6.84 (d, 1H, J =
9.3 Hz, Ar-H), 6.35 (t, 1H, J = 5.9 Hz, NHCH2CH(CH3)2), 2.97 (t, 2H, J = 6.5 Hz,
NHCH2CH(CH3)2), 1.83 (m, 1H, NHCH2CH(CH3)2), and 0.84 (d, 6H, J = 6.6 Hz,
NHCH2CH(CH3)2); 13C NMR (DMSO-d6): δ 164.0, 150.3, 142.0, 131.1, 128.7, 126.5, 124.2,
117.8, 117.1, 112.8, 94.3, 50.5, 27.6, and 20.5; HRMS: Calcd for C15H16N4O2 [M+H]+
285.1346, Found 285.1329; IR vmax/cm-1: 3372 (υO-H), 2956 (υC-H), 2218 (υC≡N), 1688 (υC=O),
1584 and 1537 (υC=C), 1269 and 1172 (υC-N), 875 and 813 (δC-H).
4.1.8.3. 1-(3’-cyano-4’-(N-methyl)isobutylamino-phenyl)-pyrazol-4-carboxylic acid (16c)
1
White solid (yield: 90%), m.p.: 139.1-140.7 °C; H NMR (DMSO-d6): δ 12.59 (s, 1H,
COOH), 8.95 (s, 1H, CH), 8.08 (d, 1H, J = 2.8 Hz, Ar-H), 8.02 (s, 1H, CH), 7.97 (dd, 1H, J =
9.3 and 2.8 Hz, Ar-H), 7.15 (d, 1H, J = 9.3 Hz, Ar-H), 3.24 (d, 2H, J = 7.5 Hz,
NCH3CH2CH(CH3)2), 3.02 (s, 3H, NCH3CH2CH(CH3)2), 2.06-1.89 (m, 1H,
NCH3CH2CH(CH3)2), and 0.84 (d, 6H, J = 6.6 Hz, NCH3CH2CH(CH3)2); 13C NMR
(DMSO-d6): δ 164.0, 53.1, 142.3, 131.3, 130.9, 125.5, 125.4, 119.3, 119.2, 117.5, 99.4, 61.6,
41.8, 27.0, and 20.2; HRMS: Calcd for C16H18N4O2 [M+H]+ 299.1503, Found 299.1486; IR

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vmax/cm-1: 3398 (υO-H), 3113 and 2957 (υC-H), 2867 (υC-H), 2208 (υC≡N), 1694 (υC=O), 1516 and
1457 (υC=C), 1264 and 1188 (υC-N), 802 (δC-H).
4.1.8.4. 1-(3’-cyano-4’-pyrrolidin-phenyl)-pyrazol-4-carboxylic acid (16d) White solid (yield:
74%), m.p.: 236.0-236.8 °C; 1H NMR (DMSO-d6): δ 12.62 (s, 1H, COOH), 8.91 (s, 1H, CH),
8.00 (d, 1H, J = 0.5 Hz, Ar-H), 7.99 (s, 1H, CH), 7.91 (dd, 1H, J = 9.3 and 2.8 Hz, Ar-H),
6.87 (d, 1H, J = 9.4 Hz, Ar-H), 3.54 (m, 4H, N(CH2)2(CH2)2), and 2.08-1.78 (m, 4H,
N(CH2)2(CH2)2); 13C NMR (DMSO-d6): δ 164.0, 149.1, 142.0, 131.0, 128.9, 125.7, 125.6,
120.5, 117.2, 116.2, 93.4, 50.2, and 25.7; HRMS: Calcd for C15H14N4O2 [M+H]+ 283.1190,
Found 283.1172; IR vmax/cm-1: 3448(υO-H), 3142, 2972 and 2865 (υC-H), 2215 (υC≡N), 1691
(υC=O), 1570, 1527 and 1464 (υC=C), 1289 and 1180 (υC-N), 888 and 809 (δC-H).
4.1.8.5. 1-(3’-cyano-4’-(2-methyl-pyrrolidin)-phenyl)-pyrazol-4-carboxylic acid (16e) White
solid (yield: 62%), m.p.: 199.7-200.1 °C; 1H NMR (DMSO-d6): δ 12.54 (s, 1H, COOH), 8.89
(s, 1H, CH), 8.01 (d, 1H, J = 1.9 Hz, Ar-H), 8.01 (s, 1H, CH), 7.92 (dd, 1H, J = 9.4 and 2.8
Hz, Ar-H), 6.94 (d, 1H, J = 9.5 Hz, Ar-H), 4.26 (m, 1H, NCHCH3CH2CH2CH2), 3.86-3.39 (m,
2H, NCHCH3CH2CH2CH2), 2.19-1.93 (m, 2H, NCHCH3CH2CH2CH2), 1.93-1.57 (m, 2H,
NCHCH3CH2CH2CH2), and 1.14 (d, 3H, J = 6.1 Hz, NCHCH3CH2CH2CH2); 13C NMR
(DMSO-d6): δ 164.0, 148.6, 142.1, 131.1, 129.1, 125.8, 120.3, 117.3, 117.1, 94.8, 54.7, 51.3,
33.5, 23.9, and 19.6; HRMS: Calcd for C16H16N4O2 [M+H]+ 297.1346, Found 297.1329; IR
vmax/cm-1:3449 (υO-H), 3124, 2968 and 2872 (υC-H), 2206 (υC≡N), 1676 (υC=O), 1565, 1518 and
1454 (υC=C),1254 and 1179 (υC-N), 810 (δC-H).
4.1.8.6. 1-(3’-cyano-4’-piperidyl-phenyl)-pyrazol-4-carboxylic acid (16f) White solid (yield:
1
72%), m.p.: 215 °C (dec.); H NMR (DMSO-d6): δ 12.66 (s, 1H, COOH), 9.00 (s, 1H, CH),
8.22 (d, 1H, J = 2.7 Hz, Ar-H), 8.08 (dd, 1H, J = 9.1 and 2.8 Hz, Ar-H), 8.06 (s, 1H, CH),
7.25 (d, 1H, J = 9.1 Hz, Ar-H), 3.14 (m, 4H, N(CH2)2(CH2)2CH2), 1.67(m, 4H,
N(CH2)2(CH2)2CH2), and 1.60-1.51 (m, 2H, N(CH2)2(CH2)2CH2); 13C NMR (DMSO-d6): δ
164.0, 155.4, 142.5, 133.0, 131.6, 125.4, 124.7, 120.6, 118.0, 117.7, 105.3, 52.9, 26.1, and
23.8; HRMS: Calcd for C16H16N4O2 [M+H]+ 297.1346, Found 297.1330; IR vmax/cm-1: 3462
(υO-H), 3139, 2934 and 2848 (υC-H), 2225 (υC≡N), 1671 (υC=O), 1557, 1518 and 1449 (υC=C),
1274 and 1238 (υC-N), 896 and 826 (δC-H).
4.1.8.7. 1-(3’-cynao-4’-piperidyl-phenyl)-3-methyl-pyrazol-4-carboxylic acid (17f) White

ACCEPTED MANUSCRIPT
solid (yield: 82%), m.p.: 216 °C (dec.); 1H NMR (DMSO-d6): δ 12.50 (s, 1H, COOH), 8.89 (s,
1H, CH), 8.17 (d, 1H, J = 2.7 Hz, Ar-H), 8.04 (dd, 1H, J = 9.0 and 2.7 Hz, Ar-H), 7.22 (d, 1H,
J = 9.1 Hz, Ar-H), 3.17-3.05 (m, 4H, N(CH2)2(CH2)2CH2), 2.40 (s, 3H, CH3), 1.66 (m, 4H,
13
N(CH2)2(CH2)2CH2), and 1.55 (m, 2H, N(CH2)2(CH2)2CH2); C NMR: 164.7, 155.1, 151.8,
133.0, 132.6, 125.0, 124.2, 120.6, 118.0, 114.9, 105.5, 53.0, 26.1, 23.9, and 13.7; HRMS:
Calcd for C17H18N4O2 [M+H]+ 311.1503, Found 311.1484; IR vmax/cm-1: 3453 (υO-H), 3136,
2947 and 2845 (υC-H), 2215 (υC≡N), 1686 (υC=O), 1551, 1497 and 1449 (υC=C), 1296 and 1117
(υC-N), 865 and 822 (δC-H).
4.1.8.8. 1-(3’-cyano-4’-morpholinyl-phenyl)-3-methyl-pyrazol-4-carboxylic acid (17g) White
solid (yield: 81%), m.p.: 255.1-255.8 °C; 1H NMR (DMSO-d6): δ 12.52 (s, 1H, COOH), 8.93
(s, 1H, CH), 8.24 (d, 1H, J = 2.6 Hz, Ar-H), 8.11 (dd, 1H, J = 9.0 and 2.6 Hz, Ar-H), 7.28 (d,
1H, J = 9.1 Hz, Ar-H), 3.77 (m, 4H, N(CH2)2O(CH2)2), 3.17 (m, 4H, N(CH2)2O(CH2)2), and
13
2.41 (s, 3H, CH3); C NMR (DMSO-d6): δ 164.7, 153.9, 151.9, 133.7, 132.1, 125.1, 124.3,
120.6, 117.9, 115.0, 105.6, 66.6, 51.9, and 13.7; HRMS: Calcd for C16H16N4O3 [M+H]+
313.1295, Found 313.1277; IR vmax/cm-1: 3454 (υO-H), 3139 and 2912 (υC-H), 2224 (υC≡N),
1683 (υC=O), 1555 and 1502 (υC=C), 1301 (υC-O), 1236 and 1121 (υC-N).
4.1.8.9. 1-(3’-nitro-4’-piperidyl-phenyl)-3-methyl-pyrazol-4-carboxylic acid (18f) Orange
solid (yield: 50%), m.p.: 218.2-219.0 °C; 1H NMR (DMSO-d6): δ 12.52 (s, 1H, COOH), 8.94
(s, 1H, CH), 8.28 (d, 1H, J = 2.6 Hz, Ar-H), 8.06 (dd, 1H, J = 9.0 and 2.6 Hz, Ar-H), 7.40 (d,
1H, J = 9.1 Hz, Ar-H), 2.98 (m, 4H, N(CH2)2(CH2)2CH2), 2.42 (s, 3H, CH3), 1.60 (m, 4H,
13
N(CH2)2(CH2)2CH2), and 1.54 (m, 2H, N(CH2)2(CH2)2CH2); C NMR (DMSO-d6): δ 164.6,
152.0, 144.9, 132.8, 132.6, 124.0, 123.0, 116.1, 115.0, 52.8, 26.0, 23.6, 19.0, and 13.7; HRMS:
Calcd for C16H18N4O4 [M+H]+ 331.1401, Found 331.1382; IR vmax/cm-1: 3453 (υO-H), 3131,
2937 and 2829 (υC-H), 1690 (υC=O), 1549 and 1486 (υC=C), 1331 (υNO2), 1289 and 1119 (υC-N),
831 (δC-H).
4.1.8.10. 1-(3’-nitro-4’-morpholinyl-phenyl)-3-methyl-pyrazol-4-carboxylic acid (18g)
1
Orange solid (yield: 75%), m.p.: 223.9-224.8 °C; H NMR (DMSO-d6): δ 12.54 (s, 1H,
COOH), 8.95 (s, 1H, CH), 8.31 (s, 1H, Ar-H), 8.10 (d, 1H, J = 9.0 Hz, Ar-H), 7.44 (d, 1H, J =
9.0 Hz, Ar-H), 3.68 (m, 4H, N(CH2)2O(CH2)2), 2.99 (m, 4H, N(CH2)2O(CH2)2), and 2.41 (s,
13
3H, CH3); C NMR (DMSO-d6): δ 164.8, 152.1, 143.8, 143.3, 133.5, 132.9, 124.0, 123.2,

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116.0, 115.2, 66.6, 52.0, and 13.8; HRMS: Calcd for C15H16N4O5[M+H]+ 333.1193, Found
333.1174; IR vmax/cm-1: 3454 (υO-H), 3139 and 2873 (υC-H), 1680 (υC=O), 1529 and 1445 (υC=C),
1355 (υNO2) , 1297 (υC-O), 1233 and 1120 (υC-N), 871 (δC-H).
4.2 Assay of the in vitro XOR inhibitory activity.
The XOR activity with xanthine as the substrate was measured spectrophotometrically
according to the procedure reported by Fukunari et al. [14]. 10× PBS (pH 7.4) was diluted to
1× PBS (unless special version, PBS referred in the assay system is 1× PBS). Xanthine (15.2
mg) was dissolved and diluted with PBS to obtain the substrate solution (0.5 mM). XOR (10.2
µL, Sigma, from bovine milk) was diluted with PBS (20 mL) to make the enzyme solution
(0.5 µL/100 µL). Tested compounds were diluted with PBS to the required concentrations.
The PBS, sample or blank solution (PBS), and enzyme solution (100 µL) were added to
96-well plates and incubated at 25 °C for 3 min. Then, the substrate solution was added to the
plates to a total volume of 200 µL in the plate. The plate was scanned at 295 nm on an
Ensipre-2300 microplate reader (Perkin Elmer, USA) at 1 min intervals for 10 min. Three
replicates were completed for each concentration. The IC50 values were calculated using
Excel 2007 and GraphPad 6.0 statistical system (GraphPad Software, Inc., San Diego, CA) .
The steady-state kinetics assay was also completed similar to above.
4.3 Molecular modelling
Molecular docking was carried out using MOE. 16c, febuxostat, and the X-ray crystal
structure of Y-700/XOR complex (PBD entry code 1VDV) were imported. Conformations of
compounds generated with the bond rotation method are placed in the active site with the
Triangle Matcher method, which ranked with the London dG function. The MMFF94x
forcefield is chosen to refine receptor-ligand complexes, and docked poses were analyzed by
MOE software package.
4.4 In vivo hypouricemic effect assay.
All of the animal protocols adhered to the Guide for the Care and Use of Laboratory
Animals published by the National Institutes of Health (NIH Publication 85-23, revised 1986).
Our experiments have been approved by the institutional committee of China Pharmaceutical

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University.
ICR mice (18-22 g) were purchased from the animal centre of Qing-Longshan (Nanjing,
Jiangsu, P. R. China). They were housed in plastic cages with free access to food and water at
room temperature (22 ± 2 °C) with relative humidity (55 ± 5 %) under a normal 12 h
light/dark cycle (light period 6:30 AM to 6:30 PM). They were allowed at least 1 week to
adapt to the environment before the experiments began.
Acute hyperuricemia was induced in the mice via the subcutaneous injection of 300 mg/kg
potassium oxonate with an intraperitoneal injection of 500 mg/kg hypoxanthine according to a
modified procedures reported by Ishibuchi et al. [7] and Xu et al. [15]. Febuxostat and Y-700
were used as positive drugs. Potassium oxonate and hypoxanthine were dissolved in saline
and febuxostat, Y-700, 16c and 16f were suspended in 0.5 % CMC-Na solution for
intragastric administration. Eight mice were allocated to each group. The animals were treated
with the potassium oxonate and hypoxanthine 1 h before drug administration (5 mg/kg).
Under halothane anaesthesia, one drop of blood was collected from the tail into a
heparin-coated tube after potassium oxonate (which was marked as 1 h) treatment and at 1, 2,
3 and 4 h after drug administration. The serum was obtained by centrifugation (3000 × g, 10
min) at 4 °C. Serum urate levels were determined with a uric acid assay kit (SanNuo,
Changsha, P. R. China).
All of the data were expressed as the mean ± S.D., and statistical analysis was performed
using Student’s t-test. The figures were obtained with the GraphPad 6.0 statistical system
(GraphPad Software, Inc., San Diego, CA).
4.5 Pharmacokinetics assessment
Various concentrations of 16c (2000, 1000, 500, 250, 125, 50, 25, 10 ng·mL−1) were
prepared to establish a standard curve, with febuxostat (5.0 µg·mL−1) as the internal standard
(IS). The standard curve was drawn using various concentrations of 16c along the ratio of 16c
area under the peak (AUC) to that of IS. The drugs were dissolved in 0.5 % CMC-Na solution
and treated to the rats by oral administration (10 mg/kg). After administration, blood was
collected from the retro-orbital plexus into heparin-coated tubes at 5, 15, 30, and 45 min, and

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1, 2, 4, 6, 8, 12, and 24 h. The blood samples were centrifuged at 9000 rpm for 10 min to
separate the plasma, which was stored at -78 °C until analysis. 50 µL IS solution and 150 µL
methanol were respectively added into 50 µL aliquot of plasma sample, well mixed, and
centrifuged at 9000 rpm for 10 min. Finally, 5.0 µL of the supernatant was analyzed on a
Waters liquid chromatography-mass spectrometry system (LC-MS/MS). Pharmacokinetic
profiles were performed using DAS 2.1.1 statistical software program.
Acknowledgments
This work was supported by the Science and Technology Project of Guangdong Province
(2015A020211005 and 2015B090901029) and the Fundamental Research Funds for The
Central Universities (2015ZM176).
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