Synthesis of new triarylpyrazole derivatives possessing terminal sulfonamide moiety and their inhibitory effects on PGE2 and nitric oxide productions in lipopolysaccharide-induced RAW 264.7 macrophages

Article abstract of DOI:10.3390/molecules23102556

This article describes the design, synthesis, and in vitro anti-inflammatory screening of new triarylpyrazole derivatives. A total of 34 new compounds were synthesized containing a terminal arylsulfonamide moiety and a different linker between the sulfonamide and pyridine ring at position 4 of the pyrazole ring. All the target compounds were tested for both cytotoxicity and nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Compounds 1b, 1d, 1g, 2a, and 2c showed the highest NO inhibition percentages and the lowest cytotoxic effect. The most potent derivatives were tested for their ability to inhibit prostaglandin E2 (PGE2) in LPS-induced RAW 264.7 macrophages. The IC50 for nitric oxide inhibition, PGE2 inhibition, and cell viability were determined. In addition, 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) and Cyclooxygenase 2 (COX-2) protein expression as well as iNOS enzymatic activity.

Full text of DOI:10.3390/molecules23102556

Article
Synthesis of New Triarylpyrazole Derivatives
Possessing Terminal Sulfonamide Moiety and Their
Inhibitory Effects on PGE2 and Nitric Oxide
Productions in Lipopolysaccharide-Induced RAW
264.7 Macrophages
Mohammed S. Abdel-Maksoud 1, Mohammed I. El-Gamal 2,3,4, Mahmoud M. Gamal El-Din 1,
Yunji Choi 5, Jungseung Choi 5, Ji-Sun Shin 6,7, Shin-Young Kang 6,7, Kyung Ho Yoo 8, Kyung-Tae
Lee 6,7, Daejin Baek 5,* and Chang-Hyun Oh 9,10,
*
1
Medicinal & Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre (NRC), Dokki, Giza 12622, Egypt; ph_ss@hotmail.com (M.S.A.-M.);
dr.m.g.eldin@hotmail.com (M.M.G.E.-D.)
2
Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah 27272, United
Arab Emirates; drmelgamal2002@gmail.com
3
4
5
Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
Department of Chemistry, Hanseo University, Seosan 31962, Korea; cossmoss@paran.com (Y.C.);
lovely1131@nate.com (J.C.)
6
7
8
Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul 02792
Korea; jsshin@khu.ac.kr (J.-S.S.); kang940818@naver.com (S.-Y.K.); ktlee@khu.ac.kr (K.-T.L.)
Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul
130-650, Korea
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul 130-650, Korea;
khyoo@kist.re.kr
9
Center for Biomaterials, Korea Institute of Science and Technology, Cheongryang, Seoul 130-650, Korea
Department of Biomolecular Science, University of Science and Technology, Daejeon, Yuseong-gu 34113,
Korea
10
* Correspondence: djbaek@hanseo.ac.kr (D.B.); choh@kist.re.kr (C.-H.O.); Tel.: +82-010-8922-5160 (C.-H.O.);
Fax: +82-41-660-1119 (C.-H.O.)
Received: 17 September 2018; Accepted: 2 October 2018; Published: 7 October 2018
Abstract: This article describes the design, synthesis, and in vitro anti-inflammatory screening of
new triarylpyrazole derivatives. A total of 34 new compounds were synthesized containing a
terminal arylsulfonamide moiety and a different linker between the sulfonamide and pyridine ring
at position 4 of the pyrazole ring. All the target compounds were tested for both cytotoxicity and
nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7
macrophages. Compounds 1b, 1d, 1g, 2a, and 2c showed the highest NO inhibition percentages
and the lowest cytotoxic effect. The most potent derivatives were tested for their ability to inhibit
prostaglandin E2 (PGE2) in LPS-induced RAW 264.7 macrophages. The IC50 for nitric oxide
inhibition, PGE2 inhibition, and cell viability were determined. In addition, 1b, 1d, 1g, 2a, and 2c
were tested for their inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) and
Cyclooxygenase 2 (COX-2) protein expression as well as iNOS enzymatic activity.
Keywords: anti-inflammatory; inducible nitric oxide synthase (iNOS); nitric oxide; prostaglandine
E2; triarylpyrazole
Molecules 2018, 23, 2556; doi:10.3390/molecules23102556
www.mdpi.com/journal/molecules

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1. Introduction
Inflammation is one of the most important and complicated defense mechanisms. Inflammation
participates in vital pathological and physiological processes like infection and wound healing [1].
As a result of tissue damage, many chemical intermediates are released in the damaged area. The
chemical intermediates (such as E, L, and P-selectin and chemokines) initiate activation and
migration of white blood cells to the damaged area. Eosinophils and neutrophils are the first
leucocytes that migrate to the affected area followed by macrophages that release a number of
cytokines and growth factors that affect the surrounding tissues [2–5]. Inflammation can be acute,
occurring as part of a healing process, or chronic inflammation, which arises from the over response
of the immune system and can lead to tissue damage. Chronic inflammation contributes to several
physiological disorders such as neurodegenerative diseases [6], cancer [7], inflammatory bowel
disease [8], and arteriosclerosis [9].
At the inflammation site, monocytes are converted to macrophages that release a large amount
of nitric oxide (NO). Nitric oxide is produced as a result of oxidation of L-arginine by one of the nitric
oxide synthase family members: endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS),
which is a calcium-dependent enzyme, and inducible NOS (iNOS), which is calcium-independent
enzyme) [10,11]. The presence of pro-inflammatory and chemical stimuli, such as lipopolysaccharide
(LPS), leads to over-expression of iNOS [12]. Successful NO production inhibitory agents act
through inhibition of iNOS protein expression and/or inhibition of iNOS enzymatic activity.
In addition to nitric oxide, prostaglandins are another important inflammation phospholipid
by-product [13]. Prostaglandin E2 (PGE2) plays an important role in most inflammation conditions
[14], such as glomerulonephritis, which may lead to renal failure [15]. The production of PGE2 is
initiated by membrane phospholipids that are converted to arachidonic acid under the effect of the
phospholipases enzyme. Arachidonic acid is transformed first to prostaglandin H2, which finally
produces PGE2 [16]. The increase in both prostaglandin E2 and nitric oxide in chronic inflammation
cases can lead to severe complicated physiological disorders [17,18]. So, the inhibition of both PGE2
and nitric oxide could result in the discovery of new anti-inflammatory drug candidates.
Several scaffolds have been investigated for their antiinflammatory activity, such as thiadiazole
[19–21], chromones [22,23], triazoles [24], imidazole [25], and pyrazole. Many compounds with a
pyrazole backbone have been proven to exhibit both anticancer [26–31] and antiinflammatory effects
[32–35]. Celecoxib is an anti-inflammatory drug that contains diarylpyrazole as a back bone and
works through inhibition of the COX-2 enzyme [36,37]. Previously, we reported the synthesis of a
series of triarylpyrazoles [38–41], from which compound I (Figure 1) showed the highest activity for
both nitric oxide and PGE2 production inhibition [40]. In the current work and based on our previous
work, we synthesized a new series of triarylpyrazole derivatives. The new series contains
2-substituted pyridine at position 4 of the pyrazole ring. The substitutions contain a terminal
sulfonamide moiety and a different linker between the sulfonamide and pyridine ring. The linker we
used to investigate the effect of linker length on the activity was either ethylene or propylene. The
new series was screened for its ability to inhibit nitric oxide; their cytotoxicity on RAW 264.7
macrophages was also investigated. The most potent compounds were tested for their inhibitory
effect on PGE2 and iNOS expression.

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R2
O
N
N
H
O
S
O
O
N
H2N
HN
O
N
O
N
N
S
CF3
N
n
N
H
R1
1a-i, 2a-i, 3a-h, and 4a-h
N
I
Celecoxib
n = 1, 2
R1 = H, p-Br, p-Cl, p-F, p-OCH3,
p-CH3, p-CF3, m-F, fused benzene
R2= H, CH3
.
Figure 1. General structures of the target compounds, Celecoxib, and previously-reported pyrazole
compound [40].
2. Results and Discussion
2.1. Chemistry
The synthesis of the final target compounds 1a–i, 2a–i, 3a–h, and 4a–h was achieved by
adopting the synthetic strategy illustrated in Scheme 1. We first synthesized the side chains 8a–i and
9a–i. The main intermediate 5 was synthesized according to previously reported procedures [42,43].
Eventually, the target compounds 1a–i and 2a–i were obtained by coupling compound 5 with 8a–i
and 9a–i using pyridine as a solvent and refluxing for 12 h. Another pathway to obtain 1a–i and 2a–i
was refluxing 5 with 1,2-ethylenediamine or 1,3-propylenediamine to produce 6 and 7, which, upon
reaction with the appropriate arylsulfonyl chloride in the presence of triethylamine, produced the
desired final compounds 1a–i and 2a–i. Demethylation of compounds 1 and 2 using boron
tribromide produced the hydroxyl final analogues 3a–h and 4a–h (Scheme 1). The structures of the
final target compounds and their yields are represented in Table 1.
Scheme 1. Synthesis of final target compounds 1a–i, 2a–i, 3a–h, and 4a–h. Reagents and conditions:
(i) 1,2-ethylenediamine or 1,3-propylenediamine, reflux 8 h; (ii) appropriate aryl sulfonyl chloride,

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Triethylamine, Dichloromethane, 0 °C, overnight; (iii) pyridine, 8a–i or 9a–i, reflux 12 h; and (vii)
BBr3, DCM, −78 °C; 0 °C, overnight.
Table 1. Structures and yields of the final target compounds.
Compound
n
1
1
1
1
1
1
1
R1
R2
H
Yield
65%
61%
60%
67%
62%
69%
74%
Compound
n
1
1
1
1
1
1
1
R1
H
H
H
H
H
H
H
R2
H
Yield
36%
30%
41%
40%
38%
40%
32%
1a
1b
1c
1d
1e
1f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3a
3b
3c
3d
3e
3f
4-Br
4-Br
4-Cl
4-Cl
4-F
4-F
p-OCH3
4-CH3
4-CF3
4-CH3
4-CF3
3-F
1g
3g
Fused
benzene
1h
1i
1
1
CH3
CH3
3-F
66%
71%
3h
4a
1
2
H
H
33%
37%
Fused
benzene
H
H
2a
2b
2c
2d
2e
2f
2
2
2
2
2
2
CH3
CH3
CH3
CH3
CH3
CH3
60%
62%
62%
75%
71%
72%
4b
4c
4d
4e
4f
2
2
2
2
2
2
H
H
H
H
H
H
4-Br
4-Cl
42%
52%
43%
33%
39%
41%
4-Br
4-Cl
4-F
4-F
4-CH3
4-CF3
3-F
4-OCH3
4-CH3
4g
Fused
benzene
2g
2h
2i
2
2
2
CH3
CH3
CH3
4-CF3
71%
76%
66%
4h
2
H
40%
3-F
Fused
benzene
2.2. Biology
The ability of a certain molecule to inhibit the production of inflammatory mediator(s) is one of
successful approach to treatment both chronic and acute inflammation. The final target compounds
1a–i, 2a–i, 3a–h, and 4a–h were tested for their ability to inhibit NO release in LPS-induced RAW
264.7 macrophages at three different concentrations (Table 2).
Table 2. Nitric oxide production inhibition of the final target compounds at different dose levels.
Nitric Oxide % Inhibition
Compound
1 μM
5 μM
10 μM
1a
1b
1c
1d
1e
1f
17.35 ± 0.09 35.36 ± 1.14 52.93 ± 3.12
12.05 ± 0.11 24.23 ± 0.98 68.66 ± 2.47
13.27 ± 0.08 24.76 ± 0.17 49.89 ± 1.25
19.79 ± 0.14 29.36 ± 0.77 61.28 ± 1.33
13.52 ± 0.11 16.10 ± 0.09 41.47 ± 0.99
16.29 ± 0.10 26.54 ± 0.65 53.09 ± 2.10
10.98 ± 0.07 23.62 ± 0.54 60.80 ± 1.75
11.12 ± 0.02 26.24 ± 0.99 41.25 ± 2.15
13.40 ± 0.18 19.90 ± 0.24 51.51 ± 0.14
0.17 ± 0.03 30.20 ± 0.27 62.76 ± 3.25
0.01 ± 0.03 28.06 ± 0.66 52.44 ± 1.62
0.02 ± 0.01 24.69 ± 0.81 59.09 ± 0.93
7.81 ± 0.03 23.03 ± 0.89 55.00 ± 4.01
2.02 ± 0.01 23.90 ± 1.12 50.20 ± 1.88
1g
1h
1i
2a
2b
2c
2d
2e

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2f
4.36 ± 0.04 19.76 ± 0.96 46.32 ± 3.21
2.91 ± 0.02 26.26 ± 1.01 56.03 ± 0.89
4.87 ± 0.05 24.07 ± 0.91 41.54 ± 1.12
16.15 ± 0.12 25.75 ± 0.49 51.93 ± 1.93
4.78 ± 0.03 21.49 ± 0.37 48.97 ± 1.10
15.82 ± 0.16 24.36 ± 0.52 48.69 ± 1.74
6.34 ± 0.06 11.68 ± 0.71 33.81 ± 0.89
10.58 ± 0.09 18.31 ± 0.26 48.18 ± 0.74
7.15 ± 0.04 12.40 ± 0.72 31.43 ± 0.33
6.08 ± 0.07 15.21 ± 0.91 36.51 ± 0.59
16.19 ± 0.17 19.06 ± 0.89 37.68 ± 0.85
10.33 ± 0.08 14.72 ± 0.42 16.07 ± 0.48
11.11 ± 0.10 24.13 ± 0.56 59.25 ± 1.31
8.19 ± 0.06 22.18 ± 0.72 63.82 ± 2.14
0.86 ± 0.09 22.02 ± 0.42 46.95 ± 1.34
9.02 ± 0.03 20.84 ± 0.36 48.49 ± 1.79
0.69 ± 0.01 18.57 ± 0.98 59.69 ± 0.70
4.87 ± 0.14 18.87 ± 1.02 53.82 ± 1.87
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
4g
4h
ND
29.49 ± 0.86 51.15 ± 1.45
0.97± 0.01 21.04 ± 0.22 50.21 ± 2.01
77.89 ± 4.25
L-NIL (40 μM)
ND: Not determined.
The tested derivatives exhibited diverse activity for NO production inhibition. All compounds
inhibited NO release in a dose-dependent manner. For series 1a–i, most of the compounds inhibited
the production of NO by 50% or more at 10 μM. Compound 1b (p-bromo) showed the highest
inhibition at 68.66% followed by 1d (p-flouro) with inhibition of 61.28%, then 1g (p-CF3) with
inhibition 60.80%. Compound 1a, 1f, and 1i had moderate activity with 52.93%, 53.09%, and 51.51%
inhibition, respectively. Regarding compounds 2a–i, the highest inhibition was obtained from
compounds 2a (62.76%), 2c (59.09%), 2g (56.03%), 2d (55.00%), and 2b (52.44%). Generally for
methoxy series, derivatives with ethylene bridges were more active compared to compounds with
propylene bridges. In addition, compounds with electron-withdrawing groups were more potent
compared to compounds with electron-donating groups, and para substitutions were slightly more
active than meta substitutions. The electronic nature and the position of the substituents were other
important factors that confer optimum affinity to the receptor site.
Derivatives containing hydroxyl group, 3a–h and 4a–h, were less active compared to methoxy
derivatives. The highest percent inhibition for 3a–h was exhibited by 3a (48.97%). Series 4a–h
showed good inhibition with the highest demonstrated by compound 4b (63.85%) followed by 4e
(59.69%), 4a (59.25%), 4f (53.82%), and 4g (51.15%), as illustrated in Table 2. The methoxy group is
more hydrophobic and bulkier than hydroxyl, and this might affect the activity by enhancing the
molecule’s ability to cross the cell membrane and/or increasing the affinity with the target receptor
site. Furthermore, the most active compounds (e.g., 1b) were more active than the lead compound
possessing no tether on the pyridyl ring [33]. So, this side chain is an important contributor to the
inhibition activity, which could improve the molecular affinity to its receptor site.
In addition to NO inhibition, the cytotoxic activity of compounds 1a–i, 2a–i, 3a–h, and 4a–h in
RAW
264.7
macrophages
were
measured
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to check whether the
effects on the production of NO was caused by nonspecific cytotoxicity. The IC50 values for both
nitric oxide inhibition and cell viability are presented in Table 3.

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Table 3. IC50 (μM) for nitric oxide production and cell viability of final target compounds.
Compound NO( IC50) a Cytotoxicity (IC50) a
1a
1b
1c
9.17 ± 0.52
7.90 ± 0.41
10.10 ± 0.63
8.23 ± 0.32
13.40 ± 0.72
9.42 ± 0.12
8.55 ± 0.14
12.62 ± 0.29
9.76 ± 0.21
8.04 ± 0.09
9.50 ± 0.34
8.68 ± 0.22
12.42 ± 0.40
9.96 ± 0.18
9.16 ± 0.27
10.45 ± 0.44
9.19 ± 0.25
9.63 ± 0.48
10.29 ± 0.23
10.55 ± 0.51
15.21 ± 0.17
10.72 ± 0.52
16.14 ± 0.72
22.21 ± 0.31
21.42 ± 0.17
>30
245.78 ± 1.91
254.15 ± 2.54
219.39 ± 0.14
169.15 ± 1.64
285.41 ± 4.18
291.01 ± 1.12
261.57 ± 1.57
244.21 ± 2.23
>400
1d
1e
1f
1g
1h
1i
2a
2b
2c
346.2 ± 4.21
384.69 ± 1.29
289.92 ± 1.87
260.32 ± 2.24
>400
2d
2e
2f
322.54 ± 3.35
252.64 ± 1.87
245.78 ± 2.71
>400
2g
2h
2i
3a
3b
3c
29.75 ± 1.91
24.31 ± 0.41
22.69 ± 0.30
26.58 ± 0.47
32.52 ± 0.75
24.1 5 ± 1.61
28.74 ± 0.74
100.21 ± 1.21
16.58 ± 0.91
19.54 ± 0.49
18.79 ± 0.68
12.58 ± 0.22
22.96 ± 0.63
17.73 ± 0.61
9.35 ± 0.32
10.92 ± 0.22
ND
3d
3e
3f
3g
3h
4a
4b
4c
8.86 ± 0.36
8.34 ± 0.11
13.25 ± 0.52
11.32 ± 0.16
8.82 ± 0.32
9.45 ± 0.15
8.24 ± 0.41
9.96 ± 0.48
29.32 ± 0.15
4d
4e
4f
4g
4h
L-NIL
a Values represent means ± SD of three independent experiments; ND: Not determined.
Compounds 1a–i and 2a–i had high IC50 values in the cell viability test and all compounds had
an IC50 of more than 169 μM. The IC50 for nitric oxide production inhibition was less than 14 μM. The
most potent compound among the methoxy derivatives was 1b with an IC50 of 7.90 μM followed by
2a, 1d, and 2c with IC50 values of 8.04, 8.2, and 8.68 μM, respectively. These most potent molecules
showed extreme safety expressed by very high IC50 values as cytotoxic agents. This means that their
inhibitory effect against NO production is not due to the cytotoxic effect. Compounds 3a–h and 4a–h
showed cytotoxic effects at low doses and the IC50s for nitric oxide inhibition were close to the IC50s
of the cell viability test. From Table 3, it can be predicted that the inhibitory effect of
hydroxyl-containing compounds is due to the cytotoxic effect.
Compounds 1b, 1d, 1g, 2a, and 2c, which exhibited the highest activities regarding nitric oxide
inhibition and the highest IC50 values in the cell viability test, were investigated for their ability to
inhibit PGE2 production in LPS-induced RAW 264.7 macrophages at 1, 5, and 10 μM. The

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investigation results are shown in Table 4. Compounds 1b, 1g, 2a, and 2c were able to inhibit more
than 50% of the prostaglandin production at a dose 5 μM. The five compounds were able to reduce
PGE2 production by over 75% at a dose of 10 μM. Compound 1g was the most potent compound
with an IC50 of 4.55 μM followed by 2c, 1b, 2a, and 1d with IC50 values of 4.68, 4.72, 4.87, and 5.06
μM, respectively.
Table 4. Inhibitory effect and IC50 values of compounds 1b, 1d, 1g, 2a, and 2c on prostaglandin E2
(PGE2) production.
PGE2 Inhibition (%) a
Compound
1 μM
5 μM
10 μM
IC50 (μM)
4.75 ± 0.25
5.06 ± 0.21
4.55 ± 0.39
4.87 ± 0.44
4.68 ± 0.37
1b
1d
16.24 ± 0.79 52.56 ± 2.21 76.92 ± 1.34
10.26 ± 0.81 49.57 ± 1.25 85.75 ± 2.64
19.94 ± 0.89 53.85 ± 1.91 79.34 ± 2.19
31.09 ± 1.14 50.70 ± 0.99 76.89 ± 4.99
17.51 ± 1.51 52.80 ± 2.01 81.37 ± 2.94
1g
2a
2c
NS-398
90.53 ± 0.53 95.78 ± 0.67 98.25 ± 1.50 6.25 × 10−3 ± 0.41 × 10−3
a Values represent means ± SD of three independent experiments.
The cumulative activities of compounds 1b, 1d, 1g, 2a, and 2c are illustrated in Figure 2 using
N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) and N6-(1-Iminoethyl)-L-lysine
(L-NIL) as standard compounds for PGE2 production inhibition and NO production inhibition,
respectively. The tested compounds showed low cytotoxic activity in the viability test. A significant
reduction in both nitric oxide and PGE2 production was observed starting from 5 μM. At 20 μM, the
production of both inflammatory mediators was restored to normal levels.

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Figure 2. (A) In vitro cytotoxicity, (B) nitric oxide inhibition, and (C) prostaglandin E2 inhibition of
compounds 1b, 1d, 1g, 2a, and 2c. Data are presented as the means ± SD of three independent
experiments. # p < 0.05 versus the control cells; *** p < 0.001 versus lipopolysaccharide-stimulated
cells; ** p < 0.05 versus lipopolysaccharide-stimulated cells; * statistical significances were compared
using ANOVA and Dunnett’s post hoc test.
As a result of their activity against both NO and PGE2 production and low cellular toxicity,
compounds 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on the expression of both
iNOS and COX-2. The cellular lysates were prepared from the with- and without-pretreatment
tested compounds (5, 10, 20 μM) for one hour and then with LPS (1 μg/mL) for 24 h, using β-actin as
a reference. The results are shown in Figure 3. Compound 1g, possessing an ethylene spacer,
3-methoxyphenyl at position 3 of the pyrazole ring, and a p-(trifluoromethyl)phenyl terminal ring,
showed complete inhibition of iNOS expression at 20 μM. Compounds 1b and 1d exhibited a partial
inhibitory effect against iNOS at the same concentration (Figures 3 and 4). Compound 1g might
express its inhibitory effect on NO production mainly through inhibition of iNOS protein expression
and partially through inhibition of iNOS enzyme activity.

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Figure 3. Inhibitory activity of compounds 1b, 1d, 1g, 2a, and 2c on inducible nitric oxide synthase
(iNOS) and Cyclooxygenase 2 (COX-2) Cellular lysates were prepared from the with/without
pretreatment tested compound (5, 10, and 20 μM) for one hour and then with LPS (1 μg/mL) for 24 h.
Total cellular proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to Polyvinylidene fluoride (PVDF) membranes, and
detected with specific iNOS and COX-2 antibodies. β-actin was used as an internal control.
Figure 4. Effect of compounds 1b, 1d, 1g, 2a, and 2c on iNOS activity. Following pretreatment with
lipopolysaccharide (LPS, 1 μg/mL) for 12 h and wash with phosphate buffer solution (PBS), cells
were treated with 1g (5, 10, or 20 μM) for 12 h N6-(1-Iminoethyl)-L-lysine. (L-NIL) (40 μM) was used
as the positive control in the assay. Levels of NO in culture media were quantified using the Griess
reaction assay. Data are presented as the means ± SD of three independent experiments. # p < 0.05
versus the control cells; *** p < 0.001 versus LPS-stimulated cells; * statistical significances were
compared using ANOVA and Dunnett’s post hoc test.

Molecules 2018, 23, 2556
3. Materials and Methods
3.1. General
10 of 20
All chemicals were commercially available and used with no further purification. The final
compounds and intermediates were purified by column chromatography using silica gel
(0.040–0.063 mm, 230–400 mesh) and technical grade solvents. Analytical thin layer chromatography
(TLC) was adopted on silica gel 60 F254 plates from Merck (Merck, Massachusetts, MA, USA). Purity
percentages of the target compounds were confirmed to be more than 96% by liquid
chromatography-mass spectrometry (LC-MS). Proton nuclear magnetic resonance (1H-NMR) and
carbon NMR (13C-NMR) spectra were recorded on a Bruker Avance 400 or 300 spectrometer
(Massachusetts, MA, USA) using tetramethylsilane as an internal standard and signals are described
as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet), brs (broad singlet), or dd
(doublet of doublets). LC-MS analysis was carried out using the following system: Waters 2998
photodiode array detector, Waters 3100 mass detector, Waters SFO system fluidics organizer, Waters
2545 binary gradient module, Waters reagent manager, Waters 2767 sample manager, Waters 2998
photodiode and Sunfire™ C18 column (4.6 × 50 mm, 5 μm particle size) (Waters, Massachusetts,
MA, USA). The solvent gradient = 95% A at 0 min, 1% A at 5 min. Solvent A was 0.035%
trifluoroacetic acid (TFA) in water, solvent B was 0.035% TFA in CH3OH, and the flow rate was 3.0
mL/min. The area under the curve (AUC) was calculated using Waters MassLynx 4.1 Waters,
Massachusetts, MA, USA) software. Solvents and liquid reagents were transferred using
hypodermic syringes. Melting points were obtained on a Walden Precision Apparatus Electro
thermal 9300 apparatus (Stone, Staffordshire, England) and were uncorrected.
3.2. Synthesis of N-(2-aminoethyl)benzenesulfonamide(8a), N-(2-aminoethyl)substituted
benzenesulfonamides (8b–i), N-(3-aminopropyl)benzenesulfonamide(9a), and
N-(3-aminopropyl)benzenesulfonamides (9b–i).
These compounds were synthesized performing the six-step procedure reported in the
literature [42].
3.3. Synthesis of 2-bromo-4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridine (5)
This compound was synthesized performing the four-step procedure reported in the literature
[40].
3.4. Synthesis of N1-(4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl) ethane1,2-diamine (6)
and N1-(4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl) propane-1,3-diamine (7)
These compounds were synthesized utilizing the five-step procedure reported in the literature
[43]. The detailed procedures are mentioned in the supplementary file.
3.5. General Procedure for Synthesis of the Target Compounds
N-(2-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)arylsulfonamides (1a–i)
and N-(3-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl)arylsulfonamides
(2a–i).
3.5.1. Method A
To a solution of compound 6 or 7 (0.2 mmol) in anhydrous dichloromethane (5 mL),
triethylamine (50.5 mg, 0.5 mmol) was added at 0 °C. A solution appropriate arylsulfonyl chloride
(0.21 mmol) in anhydrous dichloromethane (1 mL) was added dropwise. The reaction mixture was
stirred at room temperature for 24 h. When the reaction was finished, the solvent was removed in
vacuo, and the residue was partitioned between ethyl acetate (5 mL) and water (5 mL). The organic
layer was separated and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The
combined organic layer was washed with saturated saline (2 × 5 mL) and the organic solvent was

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evaporated under reduced pressure. The residue was purified by column chromatography (silica
gel, hexane-ethyl acetate 4:1 v/v) to produce the required product.
3.5.2. Method B
A mixture of compound 5 (81 mg, 0.2 mmol) and compound 8 or 9 (0.2 mmol) in pyridine was
heated at 100 °C for 24 h. After complete reaction, monitored by thin-layer chromatograph (TLC),
pyridine was removed under reduced pressure. The residue was partitioned between water (50 mL)
and ethylacetate (50 mL). The organic layer was separated and washed three times with distilled
water (3 × 50 mL) and dried over Na2SO4. The organic layer was evaporated and the residue was
purified using column chromatography.
N-(2-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide
1
(1a); White solid (65%); mp 104–106 °C; H-NMR (300 MHz, CDCl3) δ 7.93 (s, 1H, Ar-H), 7.86–7.79
(m, 4H, Ar-H), 7.51–7.40 (m, 3H, Ar-H), 7.25–7.21 (m, 5H, Ar-H), 6.92–6.88 (m, 1H, Ar-H), 6.77 (d, J =
9.0 Hz, 1H, Ar-H) , 6.70 (s, 1H, Ar-H), 6.42 (d, J = 6.0 Hz, 1H, Ar-H), 6.24 (s, 1H, Ar-H), 4.98 (brs, 1H,
NH), 3.65 (s, 3H, OCH3), 3.32 (d, J = 3.0 Hz, 2H, CH2), 3.09 (d, J = 6.0 Hz, 2H, CH2); 13C-NMR (75
MHz, CDCl3) δ 159.6, 158.6, 147.4, 141.9, 140.1, 140.1, 139.5, 139.4, 132.4, 131.0, 130.0, 129.9, 128.9,
128.8, 127.6, 126.9, 125.1, 122.7, 120.0, 115.8, 114.7, 112.4, 105.9 (Ar-C), 55.3 (OCH3), 43.9 (CH2), 41.6
(CH2); LC-MS (m/z) calculated for C29H27N5O3S: 525.18, found: 526.0 (M + 1)+.
4-Bromo-N-(2-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (1b); White solid (61%); mp 136–138 °C; 1H-NMR (300 MHz, CDCl3) δ 7.96 (s, 1H,
Ar-H), 7.89 (d, J = 6.0 Hz, 1H, Ar-H), 7.64 (d, J = 9 Hz, 2H, Ar-H), 7.58–7.55 (m, 2H, Ar-H), 7.31–7.28
(m, 6H, Ar-H), 6.93 (d, J = 6.0 Hz, 1H, Ar-H), 6.81 (d, J = 9.0 Hz, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 6.48 (d,
J = 6.0 Hz, 1H, Ar-H), 6.24 (s, 1H, Ar-H), 4.81 (brs, 1H, NH), 3.69 (s, 3H, OCH3), 3.36 (brs, 2H, CH2),
3.12 (brs, 2H, CH2); 13C-NMR (75 MHz, CDCl3) δ 159.7, 158.5, 147.2, 142.2, 140.2, 139.5, 139.3, 139.2,
132.6, 132.6, 132.1, 131.0, 130.0, 128.8, 128.5, 127.6, 127.1, 125.1, 122.7, 119.8, 115.8, 114.8, 112.6, 106.1
(Ar-C), 55.3 (OCH3), 44.4 (NH-CH2), 41.6 (CH2-NH); LC-MS (m/z) calculated for C29H26BrN5O3S:
603.09, found: 605.0 (M + 2)+.
4-Chloro-N-(2-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (1c); White solid (60%); mp 132–134 °C; 1H-NMR (300 MHz, CDCl3) δ 7.94 (s, 1H,
Ar-H), 7.85 (d, J = 6.0 Hz, 1H, Ar-H), 7.72–7.69 (m, 2H, Ar-H), 7.54-7.51 (m, 2H, Ar-H), 7.39–7.22 (m,
6H, Ar-H), 6.91 (d, J = 9.0 Hz, 1H, Ar-H), 6.78 (d, J = 9.0 Hz, 1H, Ar-H), 6.70 (s, 1H, Ar-H), 6.43 (d, J =
3.0 Hz, 1H, Ar-H), 6.24 (s, 1H, Ar-H), 4.95 (brs, 1H, NH), 3.66 (s, 3H, OCH3), 3.33 (d, J = 3.0 Hz, 2H,
NH-CH2), 3.09 (d, J = 3.0 Hz, 2H, -CH2-NH); 13C-NMR (75 MHz, CDCl3) δ 159.6, 158.5, 147.3, 142.0,
140.1, 139.5, 139.3, 138.6, 131.0, 129.9, 129.1, 128.8, 127.6, 125.1, 122.7, 119.9, 115.8, 114.7, 112.5, 106.9
(Ar-C), 55.2 (OCH3), 44.1 (NH-CH2), 41.5 (-CH2-NH).; LC-MS (m/z) calculated for C29H26ClN5O3S:
559.14, found: 560.0 (M + 1)+.
4-Fluoro-N-(2-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (1d); White solid (67%); mp 155–156 °C; 1H-NMR (400 MHz, CDCl3) δ 7.83 (s, 1H,
Ar-H), 7.71–7.67 (m, 3H, Ar-H), 7.19–7.15 (m, 5H, Ar-H), 6.99 (t, J = 8.4 Hz, 2H, Ar-H), 6.80 (d, J = 8.4
Hz, 1H, Ar-H) , 6.67 (d, J = 8.8 Hz, 1H, ar-H), 6.59 (s, 1H, Ar-H), 6.34 (d, J = 5.2 Hz, 1H, Ar-H), 6.13 (s,
1H, Ar-H), 4.85 (brs, NH), 3.56 (s, 3H, OCH3), 3.23 (s, 2H, NH-CH2), 2.98 (d, J = 4.4 Hz, 2H, -CH2-NH);
13C-NMR (100 MHz, CDCl3) δ 159.7, 158.6, 147.1, 142.2, 140.1, 139.5, 139.3, 136.1, 130.9, 130.0, 129.7,
129.6, 129.5, 128.9, 128.7, 125.2, 125.0, 122.7, 119.8, 116.0, 115.0, 112.5, 105.9 (Ar-C), 55.3 (OCH3), 44.2
(CH2), 41.6 (CH2); LC-MS (m/z) calculated for C29H26FN5O3S: 543.17, found: 544.0 (M + 1)+.
4-Methoxy-N-(2-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (1e); White solid (62%); mp 144–146 °C; 1H-NMR (400 MHz, CDCl3) δ 7.94 (s, 1H,
Ar-H), 7.88 (d, J = 4.0 Hz, 1H, Ar-H), 7.74 (d, J = 8.0 Hz, 2H, Ar-H), 7.33–7.23 (m, 6H, Ar-H), 6.91 (d, J
= 8.0 Hz, 3H, Ar-H), 6.79–6.77 (m, 1H, Ar-H), 6.70–6.69 (m, 1H, Ar-H), 6.43 (dd, J = 8.0, J =4.0 Hz, 1H,

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Ar-H), 6.23 (s, 1H, Ar-H), 4.85 (s, NH), 3.83 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.34 (t, J = 8.0 Hz, 2H,
NH-CH2-), 3.08 (t, J = 8.0 Hz , 2H, -CH2-NH); 13C-NMR (100 MHz, CDCl3) δ 162.6, 159.6, 158.6, 147.5,
141.9, 140.1, 139.5, 139.4, 131.6, 131.0, 129.9, 129.1, 128.8, 127.6, 125.1, 122.7, 120.0, 115.7, 114.7, 114.1,
112.4, 105.9 (Ar-C), 55.5 (OCH3), 55.3 (OCH3), 43.8 (CH2), 41.5 (CH2); LC-MS (m/z) calculated for
C30H29N5O4S: 555.19, found: 556.0 (M + 1)+.
N-(2-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)-4-methyl
benzenesulfonamide (1f); White solid (69%); mp 150–152 °C; 1H-NMR (300 MHz, CDCl3) δ 7.97 (s, 1H,
Ar-H), 7.91 (d, J = 6.0 Hz, 1H), 7.71 (d, J = 9.0 Hz, 2H, Ar-H), 7.32–7.26 (m, 7H), 6.94 (d, J = 9.0 Hz, 1H,
Ar-H), 6.81 (d, J = 6.0 Hz, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 6.49 (d, J = 3.0 Hz, 1H, Ar-H), 6.26 (s, 1H,
Ar-H), 4.97 (brs, 1H, NH), 3.70 (s, 3H, OCH3), 3.36 (brs, 2H, NH-CH2-), 3.12 (t, J = 6.0 Hz, 2H,
-CH2-NH), 2.43 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) δ 159.7, 158.5, 147.4, 143.1, 142.1, 140.1, 139.6,
139.3, 137.1, 131.1, 129.9, 129.5, 128.8, 127.6, 127.0, 125.1, 122.7, 120.0, 115.7, 114.7, 112., 105.5 (Ar-C),
55.3 (OCH3), 44.0 (CH2), 41.7 (CH2), 21.4 (CH3); LC-MS (m/z) calculated for C30H29N5O3S: 539.20,
found: 540.0 (M + 1)+.
N-(2-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)-4-(trifluoromethyl)
benzenesulfonamide (1g); White solid (74%); mp 132–134 °C; 1H-NMR (300 MHz, CDCl3) δ 7.91 (d, J =
9.0 Hz, 4H, Ar-H), 7.71 (s, 1H, Ar-H), 7.30 (brs, 6H, Ar-H), 6.93 (s, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 6.71
(s, 2H, Ar-H), 6.48 (s, 1H, Ar-H), 6.25 (s, 1H, Ar-H), 4.87 (brs, 1H, NH), 3.67 (s, 3H, OCH3), 3.37 (s, 2H,
NH-CH2-), 3.14 (s, 2H, -CH2-NHSO2); 13C-NMR (75 MHz, CDCl3) δ 159.7, 158.5, 147.1, 147.0, 143.8,
142.4, 139.5, 139.4, 139.2, 134.1, 133.7, 130.9, 130.1, 129.9, 128.9, 127.4, 126.0, 125.2, 125.0, 122.7, 119.7,
115.8, 114.7, 112.8, 106.2 (Ar-C), 55.3 (OCH3), 44.7 (CH2), 41.7 (CH2); LC-MS (m/z) calculated for
C30H26F3N5O3S: 593.17, found: 594.0 (M + 1)+.
3-Fluoro-N-(2-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (1h); White solid (66%); mp 118–120 °C; 1H-NMR (300 MHz, CDCl3) δ 7.94 (s, 1H,
Ar-H), 7.85 (d, J = 6.0 Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.46–7.37 (m, 2H, Ar-H),
7.29–7.21 (m, 6 H), 6.90 (d, J = 9.0 Hz, 1H, Ar-H), 6.78 (d, J = 9.0 Hz, 1H, Ar-H), 6.71 (s, 1H), 6.43 (d, J =
6.0 Hz, 1H, Ar-H), 6.25 (s, 1H, Ar-H), 4.99 (brs, 1H, NH), 3.65 (s, 3H, OCH3), 3.33 (brs, 2H, NH-CH2-),
3.10 (t, J = 6.0 Hz, 2H, -CH2-NH-SO2); 13C-NMR (75 MHz, CDCl3) δ 159.6, 158.6, 147.3, 142.2, 142.0,
140.2, 139.5, 139.4, 130.9, 130.8, 130.7, 129.9, 128.8, 127.6, 125.1, 122.7, 119.9, 119.6, 119.3, 115.7, 114.7,
114.4, 114.1, 112.5, 106.0 (Ar-C), 55.2 (OCH3), 44.2 (CH2), 41.5( CH2); LC-MS (m/z) calculated for
C29H26FN5O3S: 543.17, found: 544.0 (M + 1)+.
N-(2-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
1
naphthalene-1-sulfonamide (1i); White solid (71%); mp 178–180 °C; H-NMR (400 MHz, CDCl3) δ 8.38
(s, 1H, Ar-H), 7.91–7.85 (m, 4H, Ar-H), 7.75 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H, Ar-H), 7.64–7.55 (m, 2H,
Ar-H), 7.34–7.21 (m, 6H, Ar-H), 7.02 (brs, 1H, NH), 6.89 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, Ar-H), 6.76 (d,
J = 7.6 Hz, 1H, Ar-H), 6.68 (s, 1H, Ar-H), 6.42 (d, J = 4.8 Hz, 1H, Ar-H), 6.17 (s, 1H, Ar-H), 4.87 (brs,
1H, NH), 3.65 (s, 3H, OCH3), 3.34 (d, J = 4.4 Hz, 2H, NH-CH2-), 3.14 (t, J = 5.2 Hz, 2H, -CH2-NH-SO2 );
13C-NMR (100 MHz, CDCl3) δ 159.6, 158.4, 147.1, 142.1, 140.1, 139.5, 139.4, 136.8, 134.6, 132.1, 130.9,
129.8, 129.3, 129.1, 128.8, 128.6, 128.2, 127.8, 127.6, 127.4, 125.1, 122.7, 122.3, 119.9, 115.7, 114.7, 112.4,
105.9 (Ar-C), 55.2 (OCH3), 44.3 (CH2), 41.5 (CH2); LC-MS (m/z) calculated for C33H29N5O3S: 575.20,
found: 576.0 (M + 1)+.
N-(3-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl) benzenesulfonamide
(2a); White solid (60%); mp 119–120 °C; 1H-NMR (300 MHz, CDCl3) δ 7.98 (d, J = 12.0 Hz, 2H, Ar-H),
7.83 (d, J = 9.0 Hz, 2H, Ar-H), 7.52–7.45 (m, 3H, Ar-H), 7.31–7.24 (m, 6H, Ar-H), 6.93 (d, J = 6.0 Hz, 1H,
Ar-H), 6.79 (d, J = 9.0 Hz, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 6.45 (d, J = 3.0 Hz, 1H, Ar-H), 6.22 (s, 1H,
Ar-H), 4.73 (brs, 1H, NH), 3.68 (s, 3H, OCH3), 3.31 (brs, 2H, NH-CH2-), 2.97 (brs, 2H, -CH2-NH-SO2),
1.67–1.58 (m, 2H, -CH2-); 13C-NMR (75 MHz, CDCl3) δ 159.6, 158.7, 147.2, 142.1, 140.4, 140.1, 139.5,
139.3, 132.2, 131.0, 129.9, 128.9, 128.8, 127.6, 126.9, 125.1, 122.7, 120.0, 115.7, 114.8, 112.1, 105.7 (Ar-C),

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55.3 (OCH3), 40.1 (CH2), 38.3 (CH2), 29.9 (CH2); LC-MS (m/z) calculated for C30H29N5O3S: 539.20,
found: 540.0 (M + 1)+.
4-Bromo-N-(3-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
benzenesulfonamide (2b); White solid (62%); mp 117–119 °C; 1H-NMR (400 MHz, CDCl3) δ 7.98 (s, 1H,
Ar-H), 7.90 (d, J = 5.6 Hz, 1H, Ar-H), 7.69 (d, J = 8.8 Hz, 2H, Ar-H), 7.60 (d, J = 8.8 Hz, 2H, Ar-H),
7.35–7.20 (m, 6H, Ar-H), 6.95 (dd, J =8.0 , J = 2.0 Hz, 1H, Ar-H), 6.72 (t, J = 2.0 Hz, 1H, Ar-H), 6.49 (dd,
J = 6.0 Hz, J = 1.6 Hz, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.21 (brs, 1H, NH), 3.70 (s, 3H, OCH3), 3.36 (q, J =
6.4 Hz, 2H, NH-CH2-), 2.99 (d, J = 5.6 Hz, 2H, -CH2NHSO2), 1.68 (p, J = 6.4 Hz, 2H, CH2-CH2-CH2);
13C-NMR (75 MHz, CDCl3) δ 159.7, 158.9, 147.3, 142.1, 140.2, 139.6, 139.4, 132.2, 131.0, 130.0, 128.8,
128.5, 127.6, 127.1, 125.1, 122.8, 120.0, 115.8, 114.8, 112.1, 105.9 (Ar-C), 55.3 (OCH3), 40.2 (CH2),
38.3(CH2), 29.9(CH2); LC-MS (m/z) calculated for C30H28BrN5O3S: 617.11, found: 618.00 (M + 1)+.
4-Chloro-N-(3-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
1
benzenesulfonamide (2c); White solid (62%); mp 98–100 °C; H-NMR (400 MHz, CDCl3) δ 7.95 (s, 1H,
Ar-H), 7.93 (d, J = 5.2 Hz, 1H, Ar-H), 7.75 (d, J = 8.4 Hz, 2H, Ar-H), 7.42 (d, J = 8.4 Hz, 2H, Ar-H),
7.32–7.25 (m, 6H, Ar-H), 6.93 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H, Ar-H), 6.79 (d, J = 7.6 Hz, 1H, Ar-H), 6.71
(s, 1H, Ar-H), 6.45 (d, J = 5.6 Hz, 1H, Ar-H), 6.25 (s, 1H, Ar-H), 4.84 (brs, 1H, NH), 3.68 (s, 3H, OCH3),
3.34 (d, J = 5.6 Hz, 2H, NH-CH2-), 2.90 (brs, 2H, -CH2NHSO2), 1.66 (t, J = 6.0 Hz, 2H, -CH2-); 13C-NMR
(100 MHz, CDCl3) δ 159.4, 158.4, 146.8, 142.8, 140.2, 139.3, 139.3, 138.9, 138.6, 130.9, 130.0, 129.5, 129.2,
128.8, 128.4, 127.6, 125.1, 122.7, 119.8, 115.7, 114.8, 112.0, 105.9 (Ar-C), 55.2 (OCH3), 40.0 (CH2), 38.2
(CH2), 29.9 (CH2); LC-MS (m/z) calculated for C30H28ClN5O3S: 573.16, found: 574.0 (M + 1)+.
4-Fluoro-N-(3-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
1
benzenesulfonamide (2d); White solid (75%); mp 92–94 °C; H-NMR (300 MHz, CDCl3) δ 7.96 (s, 2H,
Ar-H), 7.82 (d, J = 3.0 Hz, 2H, Ar-H), 7.32–7.28 (m, 6H, Ar-H), 7.14 (t, J = 9.0 Hz, 2H, Ar-H), 6.94 (d, J =
6.0 Hz, 1H, Ar-H), 6.80 (d, J = 6.0 Hz, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 6.46 (d, J = 6.0 Hz, 1H, Ar-H), 6.24
(s, 1H, Ar-H), 4.60 (brs, 1H, NH), 3.69 (s, 3H, OCH3), 3.36 (s, 2H, NH-CH2-), 2.98 (brs, 2H,
-CH2NHSO2), 1.66 (d, J = 6.0 Hz, 2H, -CH2-); 13C-NMR (75 MHz, CDCl3) δ 159.6, 158.7, 147.2, 140.1,
139.3, 131.0, 129.9, 129.6, 129.5, 128.8, 127.6, 125.1, 122.7, 119.9, 116.2, 115.9, 115.7, 114.8, 112.2, 106.0
(Ar-C), 55.2 (OCH3), 40.0 (CH2), 38.2 (CH2), 30.0 (CH2); LC-MS (m/z) calculated for C30H28FN5O3S:
557.19, found: 558.0 (M + 1)+.
4-Methoxy-N-(3-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
benzenesulfonamide (2e); White solid (71%); mp 124–126 °C; 1H-NMR (300 MHz, CDCl3) δ 7.96 (s, 1H,
Ar-H), 7.94 (d, J = 6.0 Hz, 1H, Ar-H), 7.76 (d, J = 9.0 Hz, 2H, Ar-H),7.33–7.24 (m, 6H, Ar-H), 6.93 (d, J =
9.0 Hz, 3H, Ar-H), 6.79 (d, J = 9.0 Hz, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 6.54 (brs, 1H, NH), 6.45 (d, J = 3.0
Hz, 1H, Ar-H), 6.22 (s, 1H, Ar-H), 4.61 (brs, 1H, NH), 3.86 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 3.32 (t, J
= 6.0 Hz, 2H, NH-CH2-), 2.96 (t, J = 6.0 Hz, 2H, -CH2NHSO2), 1.65 (p, J = 6.0 Hz, 2H, -CH2-); 13C-NMR
(75 MHz, CDCl3) δ 159.7, 158.8, 147.5, 142.0, 140.1, 139.6, 139.4, 132.0, 131.1, 130.0, 128.8, 127.6, 125.1,
122.8, 120.1, 115.7, 114.8, 114.1, 112.2, 105.7 (Ar-C), 55.6 (OCH3), 55.3 (OCH3), 40.2 (CH2), 38.4(CH2),
30.0 (CH2); LC-MS (m/z) calculated for C31H31N5O3S: 569.21, found: 570.0 (M + 1)+.
N-(3-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl)-4-methylbenzenesulfo
namide (2f); Buff solid (72%); mp 94–96 °C; 1H-NMR (300 MHz, CDCl3) δ 7.95 (s, 1H, Ar-H), 7.91 (d, J
= 6.0 Hz, 1H, Ar-H), 7.71 (d, J = 9.0 Hz, 2H, Ar-H),7.30–7.24 (m, 7H, Ar-H), 6.92 (d, J = 6.0 Hz, 1H,
Ar-H), 6.79 (d, J = 6.0 Hz, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 6.44 (d, J = 6.0 Hz, 1H, Ar-H), 6.23 (s, 1H,
Ar-H), 4.78 (brs, 1H, NH), 3.67 (s, 3H, OCH3), 3.26 (brs, 2H, NH-CH2-), 2.94 (d, J = 3.0 Hz, 2H,
-CH2NHSO2), 2.60 (s, 3H, CH3), 1.62 (d, J = 6.0 Hz, 2H, -CH2-); 13C-NMR (75 MHz, CDCl3) δ 160.2,
159.3, 147.9, 143.5, 142.5, 140.6, 140.1, 139.9, 137.8, 131.5, 130.4, 130.1, 129.3, 128.1, 127.5, 125.6, 123.2,
120.6, 116.2, 115.3, 112.5, 106.1 (Ar-C), 55.8 (OCH3), 40.7 (CH2), 38.9 (CH2), 30.2 (CH2), 22.0 (CH3);
LC-MS (m/z) calculated for C31H31N5O4S: 553.21, found: 554.0 (M + 1)+.

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N-(3-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl)-4-(trifluoromethyl)
benzenesulfonamide (2g); White solid (71%); mp 150–152°C; 1H-NMR (300 MHz, CDCl3) δ 7.84–7.81 (m,
4H, Ar-H), 7.61 (d, J = 6.0 Hz, 2H, Ar-H), 7.49 (brs, 1H, NH), 7.21–7.16 (m, 6H, Ar-H), 6.82 (d, J = 6.0
Hz, 1H, Ar-H), 6.68 (d, J = 5.4, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 6.33 (d, J = 3.9 Hz, Ar-H), 6.14 (s, 1H,
Ar-H), 4.52 (brs, 1H, NH), 3.56 (s, 3H, OCH3), 3.26 (brs, 2H, NH-CH2-), 2.89 (brs, 2H, CH2NHSO2),
1.95 (brs, 2H, -CH2-); 13C-NMR (75 MHz, CDCl3) δ 159.7, 158.8, 147.1, 142.2, 140.1, 139.5, 139.3, 131.0,
129.9, 128.8, 127.6, 127.4, 126.1, 126.0, 125.1, 122.7, 119.9, 115.7, 114.8, 112.2, 106.1 (Ar-C), 55.2 (OCH3),
40.1 (CH2), 38.3 (CH2), 30.1 (CH2); LC-MS (m/z) calculated for C31H28 F3N5O3S: 607.19, found: 608.0 (M
+ 1)+.
3-Fluoro-N-(3-((4-(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
benzenesulfonamide (2h); White solid (76%); mp 120–122 °C; 1H-NMR (300 MHz, CDCl3) δ 7.94 (s, 1H,
Ar-H), 7.91 (d, J = 6.0 Hz, 1H, Ar-H), 7.30-7.22 (m, 10H, Ar-H), 6.93 (dd, J = 2.0 Hz, J = 6.0 Hz, 1H,
Ar-H), 6.89 (d, J = 6.0 Hz, 1H, Ar-H), 6.70 (s, 1H, Ar-H), 6.44 (d, J = 6.0 Hz, 1H, Ar-H), 6.24 (s, 1H,
Ar-H), 4.74 (s, 1H, NH), 3.67 (s, 3H, OCH3), 3.31 (brs, 2H, NH-CH2-), 2.98 (t, J = 6.0 Hz, 2H,
CH2NHSO2), 1.65 (brs, 2H, -CH2-); 13C-NMR (75 MHz, CDCl3) δ 159.6, 158.8, 147.2, 142.5, 140.1, 139.5,
139.3, 131.0, 130.8, 130.7, 129.9, 128.8, 127.6, 125.1, 122.7, 122.7, 120.0, 119.5, 119.2, 115.7, 114.8, 114.4,
114.1, 112.1, 105.9 (Ar-C), 55.2 (OCH3), 40.1 (CH2), 38.2(CH2), 29.9 (CH2); LC-MS (m/z) calculated for
C30H28 FN5O3S: 557.19, found: 558.0 (M + 1)+.
N-(3-((4-(3-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl)
naphthalene-1-sulfonamide (2i); White solid (66%); mp 134–136 °C; 1H-NMR (300 MHz, CDCl3) δ 8.40
(s, 1H, Ar-H), 7.97-7.78 (m, 5H, Ar-H), 7.60 (d, J = 6.0 Hz, 2H, Ar-H), 730-7.22 (m, 6H, Ar-H), 6.91 (d, J
= 9.0 Hz, 1H, Ar-H), 6.77 (d, J = 6.0 Hz, 1H, Ar-H), 6.70 (s, 1H, Ar-H), 6.45 (d, J = 6.0 Hz, 1H, Ar-H),
6.20 (s, 1H, Ar-H) , 4.61 (s, 1H, NH), 3.65 (s, 3H, OCH3), 3.31 (brs, 2H, NH-CH2-), 3.00 (s, 2H,
CH2NHSO2), 1.64 (brs, 2H, -CH2-); 13C-NMR (75 MHz, CDCl3) δ 159.6, 158.8, 147.5, 142.0, 140.1, 139.6,
139.4, 137.2, 134.6, 132.1, 131.0, 129.9, 129.3, 129.1, 128.8, 128.5, 128.1, 127.8, 127.6, 127.4, 125.1, 122.7,
122.4, 120.0, 115.7, 114.8, 112.1, 105.8 (Ar-C), 55.2 (OCH3), 40.1 (CH2), 38.2 (CH2), 29.9 (CH2); LC-MS
(m/z) calculated for C30H28 FN5O3S: 589.21, found: 590.0 (M + 1)+.
3.6. General Procedure for Synthesis of
N-(2-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)benzenesulfonamide (3a),
N-(2-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl) benzenesulfonamide
(3b–h), N-(3-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)
amino)propyl)benzenesulfonamide (4a) and N-(3-((4-(3-(3-hydroxyphenyl)
-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl) (substituted)benzenesulfonamide (4b–h)
To a mixture of compound (1a–i) or (2a–i) (0.1 mmol) in methylene chloride (5 mL), BBr3 (0.13 g,
1.0 mmol) was added dropwise at −78 °C under nitrogen, and the reaction mixture was stirred at 0
°C for 24 h. The mixture was quenched with saturated aqueous NaHCO3. Ethyl acetate (10 mL) was
added and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (3 ×
10 mL). The combined organic layer extracts were washed with brine and dried over anhydrous
Na2SO4. The organic solvent was evaporated under reduced pressure and the residue was purified
by column chromatography.
N-(2-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide
(3a); light brown solid (36%); mp 100–102 °C; 1H-NMR (400 MHz,CD3OD) δ 8.05 (s, 1H, Ar-H), 7.83
(d, J = 7.2 Hz, 2H, Ar-H), 7.76 (d, J = 5.6 Hz, 1H, Ar-H), 7.54–7.50 (m, 3H, Ar-H), 7.37–7.34 (m, 3H,
Ar-H), 7.29–7.27 (m, 2H, Ar-H), 7.18 (t, J = 8.0 Hz, 1H, Ar-H), 6.84-6.81 (m, 1H, Ar-H), 6.67–6.64 (m,
2H, Ar-H), 6.46 (dd, J = 5.2 Hz, J = 1.2 Hz, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 3.26 (t, J = 6.0 Hz, 2H,
NH-CH2-), 2.99 (t, J = 6.0 Hz, 2H, -CH2NHSO2), 13C-NMR (100 MHz, CD3OD) δ 158.6, 157.6, 149.5,
141.9, 140.3, 139.3, 138.9, 132.1, 130.7, 129.7, 128.7, 128.5, 127.7, 126.5, 121.2, 119.7, 116.8, 115.9, 112.3,

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105.7 (Ar-C), 42.2 (CH2), 40.9 (CH2); LC-MS (m/z) calculated for C28H25N5O3S: 511.17, found: 512.0 (M
+ 1)+.
4-Bromo-N-(2-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
1
benzenesulfonamide (3b); Buff solid (30%); mp 178–180 °C; H-NMR (400 MHz,CDCl3) δ 7.88 (s, 1H,
Ar-H), 7.56 (d, J = 6.0 Hz, 3H, Ar-H), 7.42 (d, J = 8.1 Hz, 2H, Ar-H), 7.17–7.08 (m, 6H, Ar-H), 6.80 (d, J
= 7.6 Hz, 1H, Ar-H), 6.64 (s, 1H, Ar-H), 6.58 (d, J = 7.1 Hz, 1H, Ar-H), 6.37 (d, J = 4.7 Hz, 1H, Ar-H),
6.17 (s, 1H, Ar-H), 5.08 (brs, 1H, NH), 3.13 (brs, 2H, NH-CH2-), 2.93 (brs, 2H, -CH2NHSO2); 13C-NMR
(100 MHz, CD3OD) δ 158.0, 157.3, 146.6, 146.4, 142.4, 140.6, 139.1, 138.7, 132.3, 132.1, 130.7, 129.0,
128.5, 128.3, 127.4, 125.2, 124.9, 119.6, 117.4, 117.2, 116.2, 112.2, 105.4 (Ar-C), 42.9 (CH2), 41.5 (CH2);
LC-MS (m/z) calculated for C28H24BrN5O3S: 589.17, found: 590.0 (M + 1)+.
4-Chloro-N-(2-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (3c); Light brown solid (41%); mp 100–102°C; 1H-NMR (400 MHz, CD3OD) δ 8.05
(s, 1H, Ar-H), 7.78–7.74 (m, 3H, Ar-H), 7.47 (d, J = 8.8 Hz, 2H, Ar-H), 7.35–7.32 (m 3H, Ar-H),
7.28–7.27 (m, 2H, Ar-H), 7.18 (t, J = 8.0 Hz, 1H, Ar-H), 6.84–6.81 (m, 1H, Ar-H), 6.67 (s, 1H, Ar-H),
6.66 (t, J = 2.0 Hz, 1H, Ar-H), 6.47 (dd, J = 5.2 Hz, J = 1.2 Hz, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 3.25 (t, J =
6.0 Hz, 2H, NH-CH2-), 3.01 (t, J = 6.0 Hz, 2H, -CH2NHSO2), 13C-NMR (100 MHz, CD3OD) δ 158.5,
157.6, 146.5, 1141.9, 14.0.9, 139.3, 139.1, 138.9, 138.2, 130.7, 129.8, 129.2, 129.0, 128.9, 128.5, 128.2, 127.7,
126.2, 121.2, 119.7, 116.9, 115.9, 111.3, 105.6 (Ar-C), 42.2 (CH2), 40.8 (CH2); LC-MS (m/z) calculated for
C28H24ClN5O3S: 545.13, found: 546.0 (M + 1)+.
4-Fluoro-N-(2-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
benzenesulfonamide (3d); Light yellow solid (40%); mp 106–108 °C; 1H-NMR (400 MHz, CDCl3) δ 7.90
(s, 1H, Ar-H), 7.78–7.75 (m, 2H, Ar-H), 7.60 (d, J = 8.0 Hz, 1H, Ar-H), 7.20 (s, 5H, Ar-H), 7.12 (t, J = 8.0
Hz, 1H, Ar-H), 7.03 (t, J = 8.0 Hz, 2H, Ar-H), 6.83 (d, J = 8.0 Hz, 1H, Ar-H), 6.67 (s, 1H, Ar-H), 6.61 (d,
J = 8.0 Hz, 1H, Ar-H), 6.40 (d, J = 8.0 Hz, 1H, Ar-H), 6.22 (s, 1H, Ar-H) 3.18 (s, 2H, NH-CH2-), 2.97 (s,
2H, -CH2NHSO2), 13C-NMR (100 MHz, CDCl3) δ 166.1, 163.6, 158.1, 157.3, 146.6, 142.4, 140.6, 139.2,
135.7, 130.7, 130.2, 129.7, 129.6, 128.8, 127.8, 125.1, 121.7, 119.6, 116.7, 116.3, 116.1, 112.3, 105.5 (Ar-C),
42.9 (CH2), 41.6 (CH2); LC-MS (m/z) calculated for C28H24FN5O3S: 529.59, found: 530.0 (M + 1)+.
N-(2-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)-4-methylbenzenesulfon
amide (3e); Yellow solid (38%); mp 114–116 °C; 1H-NMR (400 MHz, CDCl3) δ 7.91 (s, 1H, Ar-H), 7.69
(d, J = 8.0 Hz, 3H, Ar-H), 7.29–7.21 (m, 6H, Ar-H), 7.15 (t, J = 8.0 Hz, 1H, Ar-H), 6.87 (d, J = 8.0 Hz, 1H,
Ar-H), 6.72 (s, 1H, Ar-H), 6.61 (d, J = 8.0 Hz, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 6.22 (s, 1H, Ar-H), 5.32
(brs, 1H, NH), 3.20 (brs, 2H, NH-CH2-), 3.00–2.98 (m, 2H, -CH2NHSO2), 2.78 (s, 3H, CH3); 13C-NMR
(100 MHz, CDCl3) δ 158.2, 157.1, 146.7, 143.4, 142.4, 140.4, 139.3, 139.2, 136.7, 130.8, 130.2, 129.7, 128.8,
127.6, 127.0, 125.0, 121.8, 119.7, 116.8, 112.3, 105.6 (Ar-C), 42.9 (CH2), 41.6 (CH2), 21.0(CH3); LC-MS
(m/z) calculated for C29H29N5O3S: 525.59, found: 526.0 (M + 1)+.
N-(2-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)-4-(trifluoromethyl)
1
benzenesulfonamide (3f); White solid (40%); mp 138–140 °C; H-NMR (400 MHz, CDCl3) δ 7.86 (d, J =
9.0 Hz, 3H, Ar-H), 7.62–7.57 (m, 3H, Ar-H), 7.17 (s, 5H, Ar-H), 7.10 (t, J = 7.6 Hz, 1H, Ar-H), 6.80 (d, J
= 7.1 Hz, 1H, Ar-H), 6.64 (s, 1H, Ar-H), 6.58 (d, J = 7.0 Hz, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 5.32 (brs, 1H,
NH), 3.20 (brs, 2H, NH-CH2-), 3.00–2.98 (m, 2H, -CH2NHSO2), 2.78 (s, 3H, CH3); LC-MS (m/z)
calculated for C29H24F3N5O3S: 579.59, found: 580.0 (M + 1)+.
3-Fluoro-N-(2-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
ethyl)
benzenesulfonamide (3g); White solid (32%); mp 146–148 °C; 1H-NMR (400 MHz, CD3OD) δ 8.05 (s, 1H,
Ar-H), 7.87 (d, J = 5.2 Hz, 1H, Ar-H), 7.64 (d, J = 7.6 Hz, 1H, Ar-H), 7.56–7.51 (m, 2H, Ar-H), 7.37–7.28
(m, 6H, Ar-H), 7.19 (t, J = 8.0 Hz, 1H, Ar-H), 6.82 (d, J = 8.0 Hz, 1H, Ar-H), 6.68–6.65 (m, 2H, Ar-H),
6.47 (d, J = 5.6 Hz, 1H, Ar-H), 6.37 (s, 1H, Ar-H), 3.27 (t, J = 5.2 Hz, 2H, NH-CH2-), 3.02 (t, J = 6.0 Hz,
2H, -CH2NHSO2); 13C-NMR (75 MHz, CD3OD) δ 163.6, 161.1, 158.6, 157.6, 146.6, 142.6, 141.9, 139.3,

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138.9, 130.8, 130.0, 130.7, 129.7, 128.5, 127.7, 125.2, 122.5, 121.1, 119.7, 119.1, 118.8, 116.8, 113.7, 113.5,
111.3, 105.7 (Ar-C), 42.2 (CH2), 40.8 (CH2); LC-MS (m/z) calculated for C28H24 FN5O3S: 529.16, found:
530.0 (M + 1)+.
N-(2-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)ethyl)
naphthalene-1-sulfonamide (3h); White solid (33%); mp 146–148 °C; 1H-NMR (400 MHz, CDCl3) δ 8.37
(s, 1H, Ar-H), 7.87–7.73 (m, 5H, Ar-H), 7.61–7.51 (m, 3H, Ar-H), 7.22 (d, J = 4.0 Hz, 5H, Ar-H), 7.11 (t,
J = 8.0 Hz, 1H, Ar-H), 6.84 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H, Ar-H), 6.69 (t, J = 4.0 Hz, 1H, Ar-H), 6.58 (d, J
= 8.0 Hz, 1H, Ar-H), 6.38 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H, Ar-H), 6.15 (s, 1H, Ar-H), 5.03 (brs, 1H, NH),
3.19 (brs, 2H, NH-CH2-), 3.02 (t, J = 8.0 Hz, 2H, -CH2NHSO2); 13C-NMR (75 MHz, CDCl3) δ 158.0,
157.2, 146.5, 142.4, 140.5, 139.2, 136.4, 134.7, 132.0, 130.7, 130.2, 129.4, 129.2, 128.8, 128.3, 127.8, 127.7,
127.4, 125.0, 122.1, 121.7, 119.6, 117.3, 116.8, 112.3, 105.5 (Ar-C), 43.0 (CH2), 41.6 (CH2); LC-MS (m/z)
calculated for C32H27N5O3S: 561.66, found: 562.0 (M + 1)+.
N-(3-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl) benzenesulfonamide
1
(4a); White solid (37%); mp 154–156 °C; H-NMR (400 MHz, CDCl3) δ 7.92 (s, 1H, Ar-H), 7.81–7.74
(m, 3H, Ar-H), 7.51 (d, J = 7.2 Hz, 1H, Ar-H), 7.44 (t, J = 7.6 Hz, 2H, Ar-H), 7.26 (s, 5H, Ar-H), 7.14 (t, J
= 7.9 Hz, 1H, Ar-H), 6.87 (d, J = 7.88 Hz, 1H, Ar-H), 6.74 (s, 1H, Ar-H), 6.60 (d, J = 7.4 Hz, 1H, Ar-H),
6.51 (d, J = 5.3 Hz, 1H, Ar-H), 6.11(s, 1H, Ar-H), 4.83 (brs, 1H, NH), 3.06 (brs, 2H, NH-CH2-), 2.88 (d, J
= 5.8 Hz, 2H, -CH2NHSO2), 1.53 (p, J = 5.8 Hz, 2H, CH2CH2CH2); LC-MS (m/z) calculated for
C29H27N5O3S: 525.63, found: 526.0 (M + 1)+.
4-Bromo-N-(3-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
benzenesulfonamide (4b); Light yellow solid (42%); mp 184–186 °C; 1H-NMR (400 MHz, CD3OD) δ 8.06
(s, 1H, Ar-H), 7.79 (d, J = 5.2 Hz, 1H, Ar-H), 7.80–7.67 (m, 4H, Ar-H), 7.37–7.34 (m, 3H, Ar-H),
7.29–7.27 (m, 2H, Ar-H), 7.19 (t, J = 8.4 Hz, 1H, Ar-H), 6.84–6.81 (m, 1H, Ar-H), 6.68 (s, 1H, Ar-H),
6.66 (s, 1H, Ar-H), 6.51 (dd, J = 5.6 Hz, J = 1.2 Hz, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 3.14 (t, J = 8.0 Hz, 2H,
NH-CH2-), 2.93 (t, J = 8.0 Hz, 2H, -CH2NHSO2); 1.61 (d, J = 6.0 Hz, 2H, CH2CH2CH2); 13C-NMR (100
MHz, CD3OD) δ 158.9, 157.6, 146.6, 141.9, 140.9, 139.7, 139.3, 138.9, 132.0, 130.8, 129.8, 128.5, 128.3,
127.7, 126.6, 125.2, 121.2, 119.8, 116.8, 115.9, 110.9, 105.4 (Ar-C), 40.2 (CH2), 38.1 (CH2), 28.8 (CH2);
LC-MS (m/z) calculated for C29H26BrN5O3S: 604.10, found: 605.0 (M + 1)+.
4-Chloro-N-(3-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
benzenesulfonamide (4c); White solid (52%); mp 166–168 °C; 1H-NMR (400 MHz, CD3OD) δ 8.06 (s, 1H,
Ar-H), 7.87–7.75 (m, 3H, Ar-H), 7.60–7.46 (m, 2H, Ar-H), 7.36 (t, J = 5.8 Hz, 3H, Ar-H), 7.32–7.26 (m,
2H, Ar-H), 7.19 (t, J = 7.9 Hz, 1H, Ar-H), 6.83 (ddd, J = 8.3 Hz, 2.3 Hz, 0.8 Hz, 1H, Ar-H), 6.67 (t, J = 5.0
Hz, 2H, Ar-H), 6.51 (dd, J = 5.5 Hz, 1.4 Hz, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 3.14 (t, J = 6.6 Hz, 2H,
NH-CH2-), 2.93 (t, J = 6.8 Hz, 2H, -CH2NHSO2), 1.61 (p, J = 6.7 Hz, 2H, CH2CH2CH2); 13C-NMR (100
MHz, CD3OD) δ 158.9, 157.6, 146.7, 141.9, 140.9, 139.3, 139.2, 138.9, 138.3, 130.8, 129.8, 129.0, 128.5,
128.2, 127.7, 125.2, 121.2, 119.8, 116.8, 115.9, 110.9, 105.4 (Ar-C), 40.2 (CH2), 38.1 (CH2), 28.8 (CH2);
LC-MS (m/z) calculated for C29H26ClN5O3S: 560.7, found: 561.0 (M + 1)+.
4-Fluoro-N-(3-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)benzenesulfonamide (4d); White solid (45%); mp 144–146 °C; 1H-NMR (400 MHz, CD3OD) δ 8.06
(s, 1H, Ar-H), 7.98–7.86 (m, 2H, Ar-H), 7.80 (d, J = 5.5 Hz, 1H, Ar-H), 7.37 (d, J = 6.6 Hz, 3H, Ar-H),
7.33–7.24 (m, 4H, Ar-H), 7.20 (t, J = 7.8 Hz, 1H, Ar-H), 6.83 (d, J = 8.2 Hz, 1H, Ar-H), 6.67 (d, J = 8.4 Hz,
2H, Ar-H), 6.51 (d, J = 5.5 Hz, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 3.15 (t, J = 6.6 Hz, 2H, NH-CH2-), 2.92 (t, J
= 6.7 Hz, 2H,-CH2NHSO2), 1.74–1.53 (m, 2H, CH2CH2CH2); 13C-NMR (100 MHz, CD3OD) δ 158.9,
157.6, 146.7, 141.9, 139.3, 138.8, 136.7, 130.8, 129.7, 129.5, 129.4, 128.5, 127.7, 125.2, 121.2, 119.8, 116.8,
115.9, 115.8, 115.6, 110.9, 105.4 (Ar-C), 40.2 (CH2), 38.2 (CH2), 28.8 (CH2); LC-MS (m/z) calculated for
C29H26FN5O3S: 543.17, found: 544.0 (M + 1)+.

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N-(3-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl)
1
-4-methylbenzenesulfonamide (4e); White solid (33%); mp 158–160 °C; H-NMR (400 MHz, CD3OD) δ
8.06 (s, 1H, Ar-H), 7.79 (d, J = 5.5 Hz, 1H, Ar-H), 7.72 (d, J = 8.3 Hz, 2H, Ar-H), 7.41–7.25 (m, 7H,
Ar-H), 7.19 (t, J = 7.9 Hz, 1H, Ar-H), 6.83 (ddd, J = 8.3 Hz, 2.3 Hz, 1.0 Hz, 1H, Ar-H), 6.71–6.61 (m, 2H,
Ar-H), 6.51 (dd, J = 5.5, 1.5 Hz, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 3.13 (t, J = 6.7 Hz, 2H, NH-CH2-), 2.89 (t,
J = 6.8 Hz, 2H,-CH2NHSO2), 2.41 (s, 3H, CH3), 1.60 (p, J = 6.7 Hz, 2H, CH2CH2CH2); 13C-NMR (100
MHz, CD3OD) δ 158.9, 157.6, 146.7, 143.1, 141.9, 140.9, 139.3, 138.9, 137.4, 130.8, 129.8, 129.3, 128.5,
127.7, 126.6, 125.2, 121.2, 119.8, 116.8, 115.9, 110.9, 105.4 (Ar-C), 40.2 (CH2), 38.2 (CH2), 28.9 (CH2), 20.1
(CH3); LC-MS (m/z) calculated for C30H29N5O3S: 539.20, found: 540.0 (M + 1)+.
N-(3-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
1
propyl)-4-(trifluoromethyl)benzenesulfonamide (4f); White solid (39%); mp 150–152 °C; H-NMR (400
MHz, CD3OD) δ 8.09–7.99 (m, 3H, Ar-H), 7.87 (d, J = 8.3 Hz, 2H, Ar-H), 7.80 (d, J = 5.5 Hz, 1H, Ar-H),
7.41–7.34 (m, 3H, Ar-H), 7.34–7.25 (m, 2H, Ar-H), 7.20 (t, J = 7.9 Hz, 1H, Ar-H), 6.83 (ddd, J = 8.3 Hz,
2.4 Hz, 0.9 Hz, 1H, Ar-H), 6.71–6.63 (m, 2H, Ar-H), 6.52 (dd, J = 5.6 Hz, 1.5 Hz, 1H, Ar-H), 6.36 (d, J =
6.0 Hz, 1H, Ar-H), 3.16 (t, J = 6.7 Hz, 2H, NH-CH2-), 2.96 (t, J = 6.8 Hz, 2H, ,-CH2NHSO2), 1.64 (p, J =
6.7 Hz, 2H, CH2CH2CH2); 13C-NMR (100 MHz, CD3OD) δ 158.8, 157.6, 146.5, 142.0, 140.9, 139.3, 138.8,
130.7, 129.7, 128.5, 127.7, 127.3, 125.9, 125.2, 121.2, 119.7, 116.8, 115.9, 110.9, 105.5 (Ar-H), 40.2 (CH2),
38.1 (CH2), 28.9 (CH2); LC-MS (m/z) calculated for C30H26F3N5O3S: 593.20, found: 594.0 (M + 1)+.
3-Fluoro-N-(3-((4-(3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)
propyl)
benzenesulfonamide (4g); White solid (41%); mp 120–122 °C; 1H-NMR (400 MHz, CD3OD) δ 8.05 (s, 1H,
Ar-H), 7.79 (d, J = 5.5 Hz, 1H, Ar-H), 7.67 (d, J = 7.9 Hz, 1H, Ar-H), 7.63–7.51 (m, 2H, Ar-H), 7.41–7.25
(m, 6H, Ar-H), 7.19 (t, J = 7.9 Hz, 1H, Ar-H), 6.87–6.79 (m, 1H, Ar-H), 6.67 (t, J = 4.2 Hz, 2H, Ar-H),
6.50 (dd, J = 5.5 Hz, 1.3 Hz, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 3.15 (t, J = 6.7 Hz, 2H, NH-CH2-), 2.93 (t, J =
6.8 Hz, 2H, -CH2NHSO2), 1.63 (p, J = 6.7 Hz, 2H, CH2CH2CH2), 13C-NMR (100 MHz, CD3OD) δ 163.7,
161.2, 158.8, 157.6, 146.5, 142.7, 141.9, 140.9, 139.3, 138.9, 130.9, 130.8, 130.8, 129.8, 128.5, 127.7, 125.2,
122.5, 122.5, 121.2, 119.8, 119.1, 118.9, 116.8, 115.9, 113.7, 113.5, 110.9, 105.5 (Ar-C), 40.2 (CH2), 38.2
(CH2), 28.9 (CH2); LC-MS (m/z) calculated for C29H26FN5O3S: 543.17, found: 544.0 (M + 1)+.
N-(3-((4-(3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)propyl)
naphthalene-1-sulfonamide (4h); White solid (40%); mp 168–70 °C; 1H-NMR (400 MHz, CD3OD) δ 8.40
(s, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 7.97 (d, J = 8.4 Hz, 2H, Ar-H), 7.92 (d, J = 8.0 Hz, 1H, Ar-H), 7.81 (dd,
J = 8.8 Hz, 1.6 Hz, 1H, Ar-H), 7.73 (d, J = 5.2 Hz, 1H, Ar-H), 7.65-7.57 (m, 2H, Ar-H), 7.36-7.25 (m, 5H,
Ar-H), 7.13 (t, J = 8.0 Hz, 1H, Ar-H), 6.79 (dd, J = 8.4 Hz, 2.0 Hz, 1H, Ar-H), 6.62 (s, 1H, Ar-H), 6.59 (d,
J = 6.4 Hz, 1H, Ar-H), 6.46 (d, J = 4.8 Hz, 1H, Ar-H), 6.27 (s, 1H, Ar-H), 3.11 (t, J = 6.8 Hz, 2H), 2.94 (t, J
= 6.8 Hz, 2H, Ar-H), 1.60 (t, J = 6.8Hz, 2H, Ar-H); 13C-NMR (100 MHz, CD3OD) δ 158.7, 157.6, 146.4,
141.9, 140.9, 139.3, 138.9, 137.2, 134.7, 132.1, 130.7, 129.7, 129.1, 128.8, 128.5, 128.3, 127.7, 127.6, 127.5,
127.2, 125.2, 122.0, 121.2, 119.7, 116.8, 115.9, 110.9, 105.3 (Ar-C), 40.2 (CH2), 38.2 (CH2), 28.8 (CH2);
LC-MS (m/z) calculated for C33H29N5O3S: 575.20, found: 576.0 (M + 1)+.
3.7. Biological Evaluation
Cell culture and sample treatment were performed as reported in the literature [38–41]. The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability followed
the procedure previously described in the literature [38–41]. Nitrite determination was carried as
described in the literature [38–41], and the PGE2 assay was carried as previously described [38–41].
4. Conclusion
In this article, a new series of 1,3,4-triaylpyrazole derivatives were synthesized. The new
analogues were divided into four groups 1a–i, 2a–i, 3a–h, and 4a–h. All compounds were tested for
their ability to inhibit nitric oxide production in LPS-induced RAW 264.7 macrophages and cell
viability to measure their cytotoxic effects. Compounds 1a–i exhibited the highest NO production

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inhibitor activity with low toxicity profile, followed by 2a–I, then 4a–h, and finally 3a–h.
Compounds 3a–h and 4a–h showed high cellular toxicity. Compounds 1b, 1d, 1g, 2a, and 2c had the
highest activity and lowest toxicity. Compounds 1b, 1d, 1g, 2a, and 2c were tested for their PGE2
inhibition ability and showed IC50 values of 4.72, 5.06, 4.55, 4.87, and 4.68 μM, respectively.
Compounds 1b, 1d, 1g, 2a, and 2c were assayed for their ability to inhibit iNOS and COX-2
expressions. Compounds 1b, 1d, and 1g exhibited a potential iNOS inhibitory effect at 20 μM and
slightly inhibited iNOS enzyme activity in a dose-dependent manner. We concluded that these
compounds inhibit NO production by inhibiting iNOS protein expression and by inhibiting iNOS
enzyme activity to a lesser extent. These compounds with good activity and relatively low toxicity
profiles can be used as promising compounds for future optimization and development of potential
anti-inflammatory agents.
Supplementary Materials: Spectral data of the new synthesized compounds and intermediates are
available online.
Author Contributions: M.S.A.-M.: Performed the synthetic and spectroscopic parts, participating in design of
the synthetic compounds, and participated in manuscript preparation. M.I.E-G.: Target compounds design,
synthesis, data interpretation, direct lab supervision, and participated in manuscript preparation. M.M.G.E.-D.:
Performed mass analysis. Y.C.: Participated in synthetic and analysis part. J.C.: Participated in synthetic and
analysis part. J.-S.S. and S.-Y.K.: Performed biological activity. K.-T.L.: Design and supervision of biological
work. K.H.Y., D.B. and C.-H.O.: Synthesis and analysis advisor.
Funding: This work was supported by 2017 research program of Hanseo University in Korea.
Acknowledgments: The authors would like to thank Hanseo University for funding this work.
Conflicts of Interest: The authors declare no conflict of interest.
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