Synthesis and antitumor activity of triazole-containing sorafenib analogs

Article abstract of DOI:10.3390/molecules22101759

Using a highly effective binuclear Cu complex as the catalyst, the 1,3-dipolar cycloaddition reactions between 16 alkynes and two azides were successfully performed and resulted in the production of 25 new triazole-containing sorafenib analogs. Several compounds were evaluated as potent antitumor agents. Among them, 4-(4-(4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)- N-methylpicolinamide (8f) potently suppressed the proliferation of HT-29 cancer cells by inducing apoptosis and almost completely inhibited colony formation at a low micromolar concentration.

Full text of DOI:10.3390/molecules22101759

molecules
Article
Synthesis and Antitumor Activity of
Triazole-Containing Sorafenib Analogs
Wenjing Ye, Qi Yao, Simiao Yu, Ping Gong and Mingze Qin *
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University),
Ministry of Education, 103 Wenhua Road, Shenyang 110016, China; yewenjing@syphu.edu.cn (W.Y.);
13664141520@163.com (Q.Y.); 18841498044m@sina.cn (S.Y.); gongpinggp@126.com (P.G.)
* Correspondence: qinmingze001@126.com; Tel./Fax: +86-24-2398-6426
Received: 20 September 2017; Accepted: 15 October 2017; Published: 24 October 2017
Abstract: Using a highly effective binuclear Cu complex as the catalyst, the 1,3-dipolar cycloaddition
reactions between 16 alkynes and two azides were successfully performed and resulted in the
production of 25 new triazole-containing sorafenib analogs. Several compounds were evaluated
as potent antitumor agents. Among them, 4-(4-(4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-
N-methylpicolinamide (8f) potently suppressed the proliferation of HT-29 cancer cells by inducing
apoptosis and almost completely inhibited colony formation at a low micromolar concentration.
Keywords: binuclear Cu catalyst; sorafenib analogs; antitumor agents
1. Introduction
Although significant progress has been achieved in the treatment of tumors, it is still a major
health concern worldwide. Sorafenib is a well-known antitumor agent, which potently targets several
kinases such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor
receptor (PDGFR), stem cell factor receptor (c-Kit), and the downstream protein Raf [
excellent activity against a broad range of tumor types including renal, hepatocellular, and colorectal
carcinomas [ ]. However, the relative bioavailability of sorafenib is merely 38 49%, mainly due to
its poor water solubility [ ]. Thus, a feasible strategy for identifying antitumor agents could be the
1]. It demonstrates
2–4
−
5,6
modification of sorafenib, paying equal attention to the potency and hydrophilicity of the derivatives.
The results of structure–activity relationship (SAR) analyses have indicated that the pyridine
facilitates a suitable chemical interaction with Cys531 in a hinge region of B-Raf kinase, while the
terminal phenyl ring occupies a hydrophobic pocket. The urea moiety connects both functional groups
and forms important hydrogen-bond interactions with Glu500 and Asp593 [7–10]. Considerable
studies were conducted on modification of the urea moiety, and potent compounds were identified
by introducing an amide [11], rhodanine [12], or benzimidazole [13] group. From the viewpoint
of medicinal chemistry, we considered that the urea moiety of sorafenib could be replaced by a
1,2,3-triazole fragment, which could act as a hydrogen-bond acceptor as well as a chemical linker
between functional groups [14].
The constructing of 1,2,3-triazole cycles has been intensively studied in recent years.
The Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction between alkynes and azides (CuAAC)
is known as one of the most efficient methods [15
been developed for accelerating the catalytic process and decreasing the amount of metal catalyst
needed over the past decade [17 19]. Very recently, we explored Cu complexes bearing an
unsymmetrical bipyridine-pyrazole-amine ligand and demonstrated their applications in the CuAAC
reaction (Scheme 1) [20 21]. To the best of our knowledge, the binuclear complex is currently one of
,16]. Numerous efficient catalytic systems have
–
1–3
,
2
the most active catalyst systems used in the CuAAC reaction.
Molecules 2017, 22, 1759; doi:10.3390/molecules22101759
www.mdpi.com/journal/molecules

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Scheme 1. Cu complexes bearing an unsymmetrical bipyridine-pyrazole-amine ligand and application
in CuAAC reaction.
Herein, we reported the application of our highly active catalyst (
2
) in the synthesis of novel
sorafenib analogs containing a 1,2,3-triazole moiety (Figure 1). The 3-CF3-4-Cl phenyl moiety in the
chemical structure of sorafenib was also modified and evaluated to identify an optimal substitution
pattern. The antiproliferative activity of these compounds was evaluated in HT-29 and MCF-7 cancer
cell lines. The biological evaluation led to the discovery of compound 8f, which potently suppressed
the proliferation of HT-29 cells by inducing apoptosis. Furthermore, 8f effectively inhibited colony
formation of HT-29 cells.
Figure 1. Design of novel sorafenib analogs.
2. Results and Discussion
2.1. Chemistry
The preparation of compounds 8a–y is described in Scheme 2. Most alkynes were obtained from
commercial sources and were used without further purification. Alkynes 5k and 5l were prepared
using the Seyferth–Gilbert reaction from the corresponding aldehydes 4k and 4l, which were treated
with 1.5 equivalents of the Bestmann–Ohira reagent in the presence of 2.0 equivalents of potassium
carbonate in methanol (MeOH) at room temperature [22]. The azides 7a and 7b were synthesized
using a previously reported method [20]. Next, a library of triazolic derivatives was synthesized by
cycloaddition reactions between various alkynes and azides using the binuclear complex
2 as the
catalyst with a 0.1–0.3 mol % catalyst loading and sodium L-ascorbate as the reductant in MeOH
(Scheme 2). Twenty-five triazoles were obtained with excellent isolated yields between 91 and 99%.
The chemical structures of the triazoles were confirmed using nuclear magnetic resonance (NMR)

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and high-resolution mass spectrometry (HRMS). The object products were 1,4-disubtituted triazoles,
which were determined by comparing the NMR spectra of 8a–y with those of our previously reported
triazoles [20].
CHO
(a)
Cl
Cl
R1
R1
4k: R1 = Me
4l: R1 = CF3
5k: R1 = Me
5l: R1 = CF3
8a: X = NH, R = Ph
8b
N
: X = NH, R = 3-MePh
N
N
8c: X = NH, R = 4-MePh
8d: X = NH, R = 4-OMePh
8e: X = NH, R = 2-FPh
8f: X = NH, R = 3-FPh
8g: X = NH, R = 4-ClPh
8h: X = NH, R = 4-BrPh
8i: X = NH, R = 2-CF3Ph
R
N3
NH2
8q: X = O, R = Ph
8r: X = O, R = 3-MePh
8s: X = O, R = 3-FPh
O
N
O
N
O
(c)
(b)
8t: X = O, R = 4-ClPh
8u: X = O, R = 2-CF3Ph
8v: X = O, R = 3-CF3Ph
8w: X = O, R = 4-Cl-3-MePh
8x: X = O, R = 4-Cl-3-CF3Ph
8y: X = O, R = 2-thienyl
X
X
X
N
O
O
O
8j
: X = NH, R = 3-CF3Ph
8k: X = NH, R = 4-Cl-3-MePh
8l: X = NH, R = 4-Cl-3-CF3Ph
8m: X = NH, R = 2-pyridyl
8n: X = NH, R = 2-thienyl
8o: X = NH, R = CH2OH
8p: X = NH, R = CMe2OH
7a: X = NH
7b: X = O
6a: X = NH
6b: X = O
Scheme 2. Synthesis route of the 1,2,3-triazole derivatives. Reagents and conditions: (
eq., Bestman–Ohira reagent 1.5 eq., MeOH, rt, 48 h, 74% yield in 5k; 84% yield in 5l; ( ) HCl/NaNO2,
) appropriate alkyne, 0.1–0.3 mol % Cat.
a) K2CO3 2.0
b
1.2 eq. NaN3, EtOAc, rt, 3 h, 61% yield in 7a; 97% yield in 7b; (
2, sodium L-ascorbate, MeOH, 25–50 C, 16 h, 91–99% yields.
c
◦
2.2. Biological Evaluation
2.2.1. In Vitro Antiproliferative Activity and SARs
The newly synthesized sorafenib analogs were screened for antiproliferative activity in two cancer
cell lines, namely the HT-29 (human colorectal cancer) and MCF-7 (human breast cancer) using a
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Sorafenib was
used as the positive control. The biological data, expressed as half-maximal inhibitory concentration
(IC50) values, are listed in Table 1. Interestingly, we discovered that most compounds exhibited
significant antiproliferative activity, especially in the HT-29 cell line. This selectivity in cell lines was
quite consistent with that of sorafenib. Seven compounds, 8a, 8e, 8f, 8i, 8j, 8m, and 8n, exhibited
more potent activity than sorafenib did in inhibiting HT-29 cells. Notably, compounds 8e
,
8f, and 8n
potently inhibited the proliferation of both selected cells, with IC50 values less than 1
µM, which
highlighted their potential for further investigations. Compound 8f was identified as the most
promising compound, which exhibited a 26.5- and 80-fold increased potency compared with that
of sorafenib in inhibiting the HT-29 and MCF-7 cell lines, respectively. In addition, compound 8f
possessed a ClogP of 3.6, which is quite suitable for drug development.
In addition, relatively clear SAR results were obtained for these compounds. An aromatic ring
at the terminal might be indispensable for antiproliferative activity. For example, both compounds
8o and 8p, which have aliphatic substituents connecting the triazole moiety were radically inactive
against the tested cancer cells. In contrast, most compounds (8a, 8b, 8e, 8f, and 8i–8k) bearing a phenyl
group exhibited obvious activity. Compounds bearing a pyridine (8m) or thiophene (8n) fragment
exhibited promising antiproliferative activity, indicating that the heterocyclic ring was also critical for
this region.
Further investigations of the phenyl ring showed close SARs. Notably, a substituent at the
para-position was detrimental to the activity of amide series of compounds. None of the related
compounds showed antiproliferative activity against the HT-29 or MCF-7 cell line, even when
an electron-withdrawing (8g, R = 4-ClPh and 8h, R = 4-BrPh) or donating (8c, R = 4-MePh;
8d, R = 4-OMePh) group was incorporated. For compound 8c, shifting the methyl group to the

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meta-position produced compound 8b, which exhibited significantly improved activity against the
HT-29 cancer cells (IC50 = 8.53 M). The results suggest that substituents at the meta-position were
better tolerated. A similar observation was reported when the activity of 8e (R = 2-FPh, IC50 = 0.84 M)
was compared with that of 8f (R = 3-FPh, IC50 = 0.20 M). However, the introduction of substituents
µ
µ
µ
at both the meta- and para-positions failed to generate potent compounds (8k and 8l), which further
confirmed that the para-position could not be substituted in this chemical series.
The analysis of the biological results showed that replacing the amide fragment at the 2-position
of pyridine with an ester sharply decreased the activity. For example, compound 8a (X = NH, R = Ph;
IC50: 0.74
was reported for compound 8q (X = O, R = Ph; IC50: 15.22
when 8f (X = NH, R = 3-FPh; IC50: 0.20 M) was compared with 8s (X = O, R = 3-FPh; IC50: 9.12
µ
M) exhibited potent inhibitory activity in HT-29 cells, but a 20.6-fold decrease in potency
M). Reduced potency was also observed
M).
µ
µ
µ
These results indicate that a hydrogen-bond donor was necessary for activity, which was in line with
the observation for sorafenib. In view of potent antiproliferative acitivity exhibited by 8f, it was
evaluated for cytotoxicity against normal cells derived from human embryonic kidney (HEK293T).
Attractively, compound 8f was less cytotoxic for HEK293T cells, with an IC50 value of 5.32
results indicated that compound 8f was a good candidate for subsequent studies.
µM. These
Table 1. Antiproliferative activities and calculated logP-values of compounds 8a–8y.
IC50 (µM) a
ClogP b
Products
HT-29
MCF-7
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
0.74 ± 0.12
8.53 ± 1.09
>100
89.83 ± 3.28
0.84 ± 0.17
0.20 ± 0.01
>100
7.53 ± 0.65
10.17 ± 0.77
>100
3.45
3.97
3.97
3.30
3.60
3.60
4.06
4.22
4.33
4.33
4.57
4.94
2.62
3.23
0.74
1.74
4.18
4.70
4.33
4.79
5.06
5.06
5.30
5.67
3.96
4.34
>100
0.33 ± 0.02
0.61 ± 0.12
>100
>100
>100
0.55 ± 0.08
0.76 ± 0.13
13.51 ± 1.28
49.20 ± 2.39
0.60 ± 0.05
0.58 ± 0.13
>100
10.99 ± 1.66
26.70 ± 3.37
50.96 ± 2.76
99.19 ± 3.58
7.21± 0.96
0.73 ± 0.32
>100
>100
>100
15.22 ± 2.68
5.33 ± 0.31
9.12 ± 0.60
11.81 ± 2.31
44.54 ± 2.28
>100
9.57 ± 1.05
54.98 ± 2.70
8.79 ± 0.97
5.29 ± 0.32
71.92 ± 3.66
48.33 ± 2.56
17.06 ± 2.17
36.23 ± 1.70
43.93 ± 2.67
>100
44.08 ± 2.69
40.84 ± 3.56
47.36 ± 1.38
43.30 ± 1.36
8s
8t
8u
8v
8w
8x
8y
sorafenib
a
b
Biological data are generated from at least three independent experiments; ClogP-values calculated using
MarvinSketch 6.1.0.
2.2.2. Inhibition of Colony Formation by 8f
Considering the potent antiproliferative activity exhibited by 8f, a colony formation assay was
conducted for a more visual result. HT-29 cells were treated with 8f at different concentrations (0, 0.125,
0.25, 0.5, and 1 µM) for 14 days. As shown in Figure 2, colony formation was significantly impaired

Molecules 2017, 22, 1759
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in the 8f-treated group at a concentration of 0.25
µM. Furthermore, treatment with 0.5
µM
8f almost
completely suppressed the colony formation.
Figure 2. Representative images of colony formation of HT-29 cells treated with 8f. Relative colony
formation rate is shown as mean SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus vehicle group;
±
p-values were calculated using an unpaired two-tailed Student’s t-test.
2.2.3. Induction of Cell Apoptosis by 8f
The results of the MTT and colony formation assays clearly demonstrated that 8f potently inhibited
the proliferation of HT-29 cells. To determine whether 8f acted by inducing apoptosis, a biparametric
cytofluorimetric analysis using propidium iodide and annexin V-fluorescein isothiocyanate staining
was performed. HT-29 cells were treated with 8f at various concentrations (0, 0.1, 0.3, and 1
72 h before staining and flow cytometry. As shown in Figure 3, the apoptotic rate of the HT-29 cells
obviously increased from 7.57% (control) to 61.7% following treatment with 1 8f. The results
suggest that 8f induced significant apoptosis of HT-29 cells in a dose-dependent manner.
µM) for
µM
Figure 3. Effect of compound 8f on cell apoptosis in HT-29 cells.

Molecules 2017, 22, 1759
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3. Experimental Section
3.1. General Information
The 1H- and 13C-NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker Co., Ltd.,
Zurich, Switzerland), with TMS as an internal standard. Melting points were determined on a Büchi
Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland). The HRMS analysis was
obtained on an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS (Version Q-TOF B.05.01,
Agilent Technologies Inc., Santa Clara, CA, USA). All the alkynes except 5k and 5l were purchased
from commercial sources. Intermediates 5k and 5l were prepared according to a literature procedure.
The characterization of compounds 8a, 8h, and 8q have been reported in [20]. All of the solvents were
purified according to the routine methods before being used.
3.2. General Procedure for the Synthesis of 5k and 5l
Dimethyl-1-diazo-2-oxopropylphosphonate (3.6 mmol) was added to a solution of aldehyde
4
(3.0 mmol) and K2CO3 (6.0 mmol) in MeOH (3 mL) and stirring was continued for 24 h. The reaction
mixture was diluted with Et2O (60 mL), washed with an aq solution (5%) of NaHCO3 (85 mL) and
water (25 mL) successively and dried over Na2SO4. After filtration and evaporation of the solvent in
vacuo, the resulting residue was purified by flash column chromatography to afford the corresponding
alkyne (petroleum ether (PE) as an elution solvent).
◦
1-Chloro-4-ethynyl-2-methylbenzene (5k). Yield: 74%, white solid. m.p.: 29.4–31.2 C. 1H-NMR (600 MHz,
DMSO-d6) 7.36 (d, 1H, J = 1.1 Hz, ArH), 7.29–7.24 (m, 2H, ArH), 3.07 (s, 1H, C CH), 2.25 (s, 3H, CH3).
δ
≡
1-Chloro-4-ethynyl-2-(trifluoromethyl)benzene (5l). Yield: 84%, white solid. m.p.: 29.6–30.1 ◦C. 1H-NMR
(600 MHz, DMSO-d6) 7.80 (d, 1H, J = 1.8 Hz, ArH), 7.58 (dd, 1H, J = 8.3, 1.9 Hz, ArH), 7.48 (d, 1H,
δ
J = 8.3 Hz, ArH), 3.19 (s, 1H, C≡CH).
3.3. General Procedure for 2-Catalyzed 1,3-Dipolar Cycloaddition Reactions
Under N2 atmosphere, the mixture of alkyne (0.3 mmol), azide (0.3 mmol), Na ascorbate
(0.006 mmol, 2 mol %), and 0.3
×
10−3 M solution of catalyst
2 in MeOH (0.1–0.3 mol %) was stirred
in a 10 mL Schlenk tube at 25 ◦C for 16 h. Evaporation of the solvents followed by purification by
short column chromatography on silica gel provided the desired 1,4-disubstituted triazole product.
The unreacted alkyne and azide were first eluted out with 3/1 petroleum ether (PE)/ethyl acetate,
and the pure 1,4-disubstituted triazole product was then obtained by elution with pure ethyl acetate.
N-Methy◦l-4-(4-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinamide (8b). Yield: 99%, white solid. m.p.:
185–187 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.33 (s, 1H, triazole-H), 8.82 (q, 1H, J = 4.8 Hz, NH), 8.57
(d, 1H, J = 5.6 Hz, ArH), 8.10 (dt, 2H, J = 8.9, 2.3 Hz, ArH), 7.79 (s, 1H, ArH), 7.75 (d, 1H, J = 7.7 Hz,
ArH), 7.53 (dt, 2H, J = 8.9, 2.2 Hz, ArH), 7.49 (d, 1H, J = 2.6 Hz, ArH), 7.40 (t, 1H, J = 7.7 Hz, ArH),
7.27 (dd, 1H, J = 5.6, 2.6 Hz, ArH), 7.22 (d, 1H, J = 7.5 Hz, ArH), 2.80 (d, 3H, J = 4.9 Hz, NCH3), 2.39
(s, 3H, CCH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.18, 163.72, 153.28, 152.66, 150.67, 147.49, 138.21,
134.15, 130.13, 128.96, 125.91, 122.55, 122.37, 122.27, 119.80, 114.59, 109.34, 26.06, 21.12. HRMS Calcd for
C22H19N5O2 [M]: 385.1539; found for C22H19N5O2 [M]: 385.1532.
N-Methyl-4-(4-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinamide (8c). Yield: 99%, white solid. m.p.:
◦
195–197 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.30 (s, 1H, triazole-H), 8.84 (q, 1H, J = 4.4 Hz, NH), 8.58
(d, 1H, J = 5.6 Hz, ArH), 8.10 (dt, 2H, J = 10.0, 3.2 Hz, ArH), 7.85 (d, 2H, J = 8.0 Hz, ArH), 7.54–7.49
(m, 3H, ArH), 7.33 (d, 2H, J = 7.9 Hz, ArH), 7.27 (dd, 1H, J = 5.6, 2.6 Hz, ArH), 2.80 (d, 3H, J = 4.9 Hz,
NHCH3), 2.36 (s, 3H, CCH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.18, 163.70, 153.25, 152.65, 150.66,
147.45, 137.69, 134.17, 129.59, 127.44, 125.30, 122.36, 122.30, 119.46, 114.58, 109.32, 26.05, 20.90. HRMS
Calcd for C22H19N5O2 [M]: 385.1539; found for C22H19N5O2 [M]: 385.1558.

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4-(4-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8d). Yield: 97%, white
◦
solid. m.p.: 215–217 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.24 (s, 1H, triazole-H), 8.83 (d, 1H, J = 4.8 Hz,
NH), 8.58 (d, 1H, J = 5.6 Hz, ArH), 8.10 (dt, 2H, J = 8.9, 3.2 Hz, ArH), 7.90 (dt, 2H, J = 8.8, 2.8 Hz, ArH),
7.54–7.49 (m, 3H, ArH), 7.27 (dd, 1H, J = 8.0, 2.6 Hz, ArH), 7.10 (dt, 2H, J = 8.8, 2.8 Hz, ArH), 3.82 (s,
3H, OCH3), 2.81(d, 3H, J = 4.9 Hz, NCH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.20, 163.72, 159.35,
153.21, 152.65, 150.68, 147.36, 134.21, 126.75, 122.75, 122.37, 122.26, 118.89, 114.58, 114.48, 109.33, 55.24,
26.07. HRMS Calcd for C22H19N5O3 [M]: 401.1488; found for C22H19N5O3 [M]: 401.1471.
4-(4-(4-(2-Fluorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8e). Yield: 98%, white solid.
m.p.: 182–183 ◦C. IR (KBr, cm−1): 3395.0, 1684.6, 1631.3, 1604.1, 1566.3, 1533.9, 1512.4, 901.8, 859.3,
1
837.4. H-NMR (600 MHz, DMSO-d6)
δ 9.14 (d, 1H, J = 2.9 Hz, triazole-H), 8.83 (q, 1H, J = 4.4 Hz, NH),
8.57(d, 1H, J = 5.6 Hz, ArH), 8.21–8.14 (m, 3H, ArH), 7.52–7.46 (m, 4H, ArH), 7.42–7.37 (m, 2H, ArH),
7.27 (dd, 1H, J = 5.5, 2.6 Hz, ArH), 2.80 (d, 3H, J = 4.9 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ
165.17, 163.72, 159.43, 157.79, 153.43, 152.66, 150.68, 141.01, 140.99, 134.01, 130.23, 130.18, 127.76, 127.74,
125.09, 125.07, 122.78, 122.29, 122.09, 122.02, 118.00, 117.91, 116.27, 116.13, 114.61, 109.34, 26.06. HRMS
Calcd for C21H17N5O2 [M]: 389.1288; found for C21H17N5O2 [M]: 389.1304.
4-(4-(4-(3-Fluorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8f). Yield: 99%, white solid.
m.p.: 209–210 ◦C. IR (KBr, cm−1): 3403.3, 1676.7, 1630.7, 1610.4, 1589.8, 1568.9, 1538.8, 1514.7, 897.2,
1
862.4, 836.4. H-NMR (600 MHz, DMSO-d6)
δ 9.42 (s, 1H, triazole-H), 8.83 (q, 1H, NH), 8.58 (d, 1H,
J = 5.6 Hz, ArH), 8.09 (d, 2H, J = 8.9 Hz, ArH), 7.82 (d, 1H, J = 7.8 Hz, ArH), 7.76 (d, 1H, J = 10.1 Hz
ArH), 7.59–7.52 (m, 3H, ArH), 7.49 (d, 1H, J = 2.6 Hz, ArH), 7.27–7.22 (m, 2H, ArH), 2.80 (d, 3H,
J = 4.9 Hz, CH3). 13C-NMR (150 MHz, DMSO-d6)
165.15, 163.72, 163.45, 161.83, 153.44, 152.67, 150.69,
,
δ
146.29 (d, J = 2.7 Hz), 134.03, 132.61 (d, J = 8.6 Hz), 132.55 (d, J = 8.5 Hz), 131.30, 131.25, 122.44 (d,
J = 2.8 Hz), 121.41 (d, J = 2.5 Hz), 120.71 (d, J = 2.6 Hz), 115.13, 114.99, 114.63, 112.03, 111.88, 109.36,
26.07. HRMS Calcd for C21H16FN5O2 [M]+: 389.1288; found for C21H16FN5O2 [M + H]+: 390.1355.
4-(4-(4-(4-Chlorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8g). Yield: 94%, white solid.
◦
m.p.: 244–246 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.40 (s, 1H, triazole-H), 8.83 (d, 1H, J = 4.8 Hz,
NH), 8.58 (d, 1H, J = 5.6 Hz, ArH), 8.09 (d, 2H, J = 8.9 Hz, ArH), 7.98 (d, 2H, J = 8.5 Hz, ArH), 7.60 (d,
2H, J = 8.5 Hz, ArH), 7.54 (d, 2H, J = 8.9 Hz, ArH), 7.49 (d, 1H, J = 2.5 Hz, ArH), 7.28 (dd, 1H, J = 5.6,
2.6 Hz, ArH), 2.81 (d, 3H, J = 4.9 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.16, 163.72, 153.39,
152.66, 150.69, 146.30, 134.06, 132.77, 129.17, 129.14, 127.05, 122.42, 122.40, 120.33, 120.31, 114.62, 109.35,
26.07. HRMS Calcd for C21H16ClN5O2 [M]: 405.0993; found for C21H16ClN5O2 [M]: 405.0991.
N-Methyl-4-(4-(4-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinamide (8i). Yield: 99%,
◦
1
white solid. m.p.: 51–54 C. H-NMR (600 MHz, DMSO-d6)
J = 4.8 Hz, NH), 8.58 (d, 1H, J = 5.6 Hz, ArH), 8.14 (dt, 2H, J = 8.9, 3.2 Hz, ArH), 7.94 (d, 1H, J = 8.0 Hz,
ArH), 7.84–7.83 (m, 2H, ArH), 7.73–7.71 (m, 1H, ArH), 7.53–7.50 (m, 3H, ArH), 7.27 (dd, 1H, J = 4.9
2.5 Hz, ArH), 2.81 (d, 1H, J = 4.9 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6)
165.17, 163.71, 153.45,
δ 9.07 (s, 1H, triazole-H), 8.83 (q, 1H,
,
δ
152.67, 150.68, 144.84, 133.90, 132.76, 132.22, 129.36, 128.98, 127.08 (q, J = 30.2 Hz), 126.51 (q, J = 5.5 Hz),
124.89, 123.08, 122.59, 122.41, 114.59, 109.37, 26.06. HRMS Calcd for C21H16F3N5O2 [M]: 439.1256;
found for C21H16F3N5O2 [M]: 439.1245.
N-Methyl-4-(4-(4-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinamide (8j). Yield: 99%,
white solid. m.p.: 216–218 C. IR (KBr, cm−1): 3391.6, 1678.7, 1594.3, 1573.2, 1542.9, 835.8, 800.3.
◦
1H-NMR (600 MHz, DMSO-d6)
δ 9.56 (s, 1H, triazole-H), 8.83 (d, 1H, J = 4.9 Hz, NH), 8.59 (d, 1H,
J = 5.6 Hz, ArH), 8.29 (d, 2H, J = 4.9 Hz, ArH), 8.11 (dt, 2H, J = 8.9, 2.0 Hz, ArH), 7.78–7.77 (m, 2H,
ArH), 7.56 (dt, 2H, J = 8.9, 2.0 Hz, ArH), 7.50 (d, 1H, J = 2.5 Hz, ArH), 7.28 (dd, 1H, J = 5.6, 2.6 Hz,
ArH), 2.81 (d, 3H, J = 4.9 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.14, 163.71, 153.47, 152.67,
150.69, 145.96, 134.00, 131.32, 130.36, 130.23 (q, JF = 31.8 Hz), 129.11, 126.87, 125.06, 124.84, 124.81,
123.26, 122.42, 122.38, 121.71, 121.68, 121.45, 120.96, 120.94, 114.64, 109.38, 26.06. HRMS Calcd for
C21H16F3N5O2 [M]: 439.1256; found for C21H16F3N5O2 [M]: 439.1245.

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4-(4-(4-(4-Chloro-3-methylphenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8k). Yield: 99%,
white solid. m.p.: 237–238 ◦C. 1H-NMR (600 MHz, DMSO-d6)
9.38 (s, 1H, triazole-H), 8.83 (d, 1H,
δ
J = 4.3 Hz, NH), 8.58 (d, 1H, J = 5.4 Hz, ArH), 8.09 (d, 1H, J = 8.5 Hz, ArH), 7.96 (s, 1H, ArH), 7.80
(d, 1H, J = 7.8 Hz, ArH), 7.57–7.49 (m, 4H, ArH), 7.28 (d, 1H, J = 2.9 Hz, ArH), 2.81 (d, 3H, J = 4.6 Hz,
NCH3), 2.43 (s, 3H, CCH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.16, 163.71, 153.37, 152.66, 150.69,
146.42, 136.19, 134.07, 133.00, 129.59, 129.16, 127.93, 124.51, 122.39, 122.34, 120.20, 114.62, 109.35, 26.06,
19.75. HRMS Calcd for C22H18ClN5O2 [M]: 419.1149; found for C22H18ClN5O2 [M]: 419.1137.
4-(4-(4-(4-Chloro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8l). Yield:
98%, white solid. m.p.: 226–228 ◦C. 1H-NMR (600 MHz, DMSO-d6)
δ
9.58 (s, 1H, triazole-H), 8.83 (d,
1H, J = 4.9 Hz, NH), 8.59 (d, 1H, J = 5.6 Hz, ArH), 8.38 (d, 1H, J = 1.9 Hz, ArH), 8.27 (dd, 1H, J = 8.3
,
1.8 Hz, ArH), 8.10 (dt, 2H, J = 8.9, 3.2 Hz, ArH), 7.91 (d, 1H, J = 8.4 Hz, ArH), 7.56–7.50 (m, 3H, ArH),
7.28 (dd, 1H, J = 5.6, 2.6 Hz, ArH), 2.81 (d, 3H, J = 4.9 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ
165.12, 163.71, 153.54, 152.67, 150.69, 145.06, 133.91, 132.62, 130.45, 130.09, 129.93, 127.52, 127.31,
124.27, 124.23, 123.70, 122.42, 122.41, 121.89, 121.28, 121.26, 114.65, 109.38, 26.06. HRMS Calcd for
C22H15ClF3N5O2 [M]: 473.0866; found for C22H15ClF3N5O2 [M]: 473.0883.
N-Methyl-4-(4-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinamide (8m). Yield: 94%, white solid.
◦
m.p.: 200–201 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.40 (s, 1H, triazole-H), 8.84 (q, 1H, J = 4.5 Hz, NH),
8.68 (d, 1H, J = 4.7 Hz, ArH), 8.58 (d, 1H, J = 5.6 Hz, ArH), 8.19–8.14 (m, 3H, ArH), 7.98 (dt, 1H, J = 7.7,
1.6 Hz, ArH), 7.52 (m, 3H, ArH), 7.43 (dd, 1H, J = 7.4, 4.9 Hz, ArH), 7.28 (dd, 1H, J = 5.5, 2.6 Hz, ArH),
2.81, 2.80 (d, 3H, J = 4.9 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.16, 163.72, 153.41, 152.65,
150.67, 149.73, 149.48, 148.29, 137.41, 134.05, 123.43, 122.54, 122.29, 121.52, 121.51, 119.86, 114.63, 109.36,
26.07. HRMS Calcd for C20H16N6O2 [M]: 372.1335; found for C20H16N6O2 [M]: 372.1340.
N-methyl-4-(4-(4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinamide (8n). Yield: 92%, white solid.
m.p.: 185–188 ◦C. IR (KBr, cm−1): 3381.7, 1677.0, 1631.4, 1587.3, 1571.9, 1530.8, 1511.8, 849.6, 838.6,
1
816.1. H-NMR (600 MHz, DMSO-d6)
δ
9.26 (s, 1H, triazole-H), 8.83 (d, 1H, J = 4.8 Hz, NH), 8.58 (d,
1H, J = 5.6 Hz, ArH), 8.09 (d, 2H, J = 8.9 Hz, ArH), 7.63 (dd, 1H, J = 5.0, 0.8 Hz, ArH), 7.54–7.49 (m, 4H,
ArH), 7.27 (dd, 1H, J = 5.5, 2.6 Hz, ArH), 7.21 (dd, 1H, J = 4.9, 3.6 Hz, ArH), 2.81 (d, 3H, J = 4.9 Hz,
CH3). 13C-NMR (151 MHz, DMSO-d6)
δ 165.14, 163.69, 153.39, 152.65, 150.66, 142.79, 133.95, 132.30,
128.06, 126.01, 124.68, 122.43, 122.34, 119.14, 114.60, 109.35, 26.05. HRMS Calcd for C19H15N5O2S [M]:
377.0946; found for C19H15N5O2S [M]: 377.0938.
4-(4-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8o). Yield: 92%, white solid.
◦
m.p.: 191–192 C. 1H-NMR (600 MHz, DMSO-d6)
δ
8.82 (d, 1H, J = 4.9 Hz, NH), 8.74 (s, 1H, triazole-H),
8.57 (d, 2H, J = 5.6 Hz, ArH), 8.06 (dt, 2H, J = 8.9, 3.2 Hz, ArH), 7.49–7.46 (m, 3H, ArH), 7.26 (dd, 1H,
J = 5.6, 2.6 Hz, ArH), 5.39 (t, 1H, J = 5.6 Hz, OH), 4.64 (d, 2H, J = 5.5 Hz, CH2), 2.80 (d, 3H, J = 4.9 Hz
CH3). 13C-NMR (151 MHz, DMSO-d6)
165.18, 163.72, 153.11, 152.64, 150.66, 149.25, 134.27, 122.29,
,
δ
122.26, 121.23, 114.58, 109.32, 54.98, 26.06. HRMS Calcd for C16H15N5O3 [M]: 325.1175; found for
C16H15N5O3 [M]: 325.1191.
4-(4-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)phenoxy)-N-methylpicolinamide (8p). Yield: 99%,
white solid. m.p.: 150–152 ◦C. 1H-NMR (600 MHz, DMSO-d6)
δ 8.83 (q, 1H, J = 4.5 Hz, NH), 8.65 (s,
1H, triazole-H), 8.57 (d, 1H, J = 5.6 Hz, ArH), 8.06 (dt, 2H, J = 8.9, 3.2 Hz, ArH), 7.48–7.45 (m, 3H,
ArH), 7.25 (dd, 1H, J = 5.6, 2.6 Hz, ArH), 5.28 (s, 1H, OH), 2.81 (d, 3H, J = 4.9 Hz, NCH3), 1.55 (s, 6H,
C(CH3)2). 13C-NMR (151 MHz, DMSO-d6)
δ 165.19, 163.72, 157.03, 153.01, 152.64, 150.66, 134.34, 122.25,
122.17, 119.10, 114.57, 109.33, 67.02, 30.60, 26.06. HRMS Calcd for C18H19N5O3 [M]: 353.1488; found for
C18H19N5O3 [M]: 353.1504.
Methyl 4-(4-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8r). Yield: 98%, white solid. m.p.:
147–149 ◦C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.34 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.5 Hz, ArH),
8.11 (d, 2H, J = 8.6 Hz, ArH), 7.79–7.74 (m, 2H, ArH), 7.56–7.52 (m, 3H, ArH), 7.41 (t, 1H, J = 7.6 Hz,
ArH), 7.32 (dd, 1H, J = 5.2, 2.0 Hz, ArH), 7.22 (d, 1H, J = 7.44 Hz, ArH), 3.87 (s, 3H, OCH3), 2.40 (s, 3H,

Molecules 2017, 22, 1759
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CCH3). 13C-NMR (151 MHz, DMSO-d6)
δ164.82, 164.72, 153.22, 151.92, 149.74, 147.53, 138.24, 134.19,
130.13, 128.99, 125.93, 122.57, 122.28, 119.78, 115.51, 112.89, 52.67, 21.14. HRMS Calcd for C22H18N4O3
[M]: 386.1379; found for C22H18N4O3 [M]: 386.1370.
Methyl 4-(4-(4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8s). Yield: 96%, white solid. m.p.:
◦
154–157 C. 1H-NMR (600 MHz, CDCl3)
δ 9.42 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.5 Hz, ArH), 8.08 (d,
2H, J = 8.6 Hz, ArH), 7.81 (d, 1H, J = 7.6 Hz, ArH), 7.75 (d, 1H, J = 9.9 Hz, ArH), 7.58–7.53 (m, 4H, ArH),
7.32 (dd, 1H, J = 5.2, 2.0 Hz, ArH), 7.25 (t, 1H, J = 7.1 Hz, ArH), 3.86 (s, 3H, CH3). 13C-NMR (150 MHz,
DMSO-d6) δ 164.84, 164.70, 163.47, 161.86, 153.39, 151.96, 149.76, 146.34, 134.07, 132.62, 132.56, 131.35,
131.29, 122.46, 122.34, 121.46, 120.70, 115.56, 115.17, 115.03, 112.93, 112.06, 111.91, 52.70. HRMS Calcd
for C21H15FN4O3 [M]: 390.1128; found for C21H15FN4O3 [M]: 390.1142.
Methyl 4-(4-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8t). Yield: 99%, white solid. m.p.:
1
173–175 ◦C. IR (KCl, cm−1): 3415.6, 1717.4, 1631.5, 1608.1, 1586.3, 884.0, 866.4, 855.6, 835.6. H-NMR
(600 MHz, DMSO-d6)
ArH), 7.98 (d, 2H, J = 8.3 Hz, ArH), 7.60 (d, 2H, J = 8.2 Hz, ArH), 7.56–7.53 (m, 3H, ArH), 7.32 (dd, 1H,
J = 5.3, 1.7 Hz, ArH), 3.86 (s, 3H, CH3). 13C-NMR (151 MHz, DMSO-d6)
164.82, 164.69, 153.34, 151.93,
δ 9.40 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.6 Hz, ArH), 8.09 (d, 2H, J = 8.7 Hz,
δ
149.75, 146.35, 134.09, 132.80, 129.18, 129.14, 127.07, 122.42, 122.30, 120.28, 115.53, 112.90, 52.67. HRMS
Calcd for C21H15ClN4O3 [M]: 406.0833; found for C21H15ClN4O3 [M]: 406.0827.
Methyl 4-(4-(4-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8u). Yield: 99%, white
solid. m.p.: 87–88 ◦C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.06 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.5 Hz,
ArH), 8.14 (d, 2H, J = 8.8 Hz, ArH), 7.93 (d, 1H, J = 7.9 Hz, ArH), 7.83–7.82 (m, 2H, ArH), 7.72 (t,
1H, J = 6.8 Hz, ArH), 7.55–7.52 (m, 3H, ArH), 7.32 (dd, 1H, J = 5.5, 2.4 Hz, ArH), 3.86 (s, 3H, CH3).
13C-NMR (151 MHz, DMSO-d6)
δ 164.81, 164.71, 153.39, 151.92, 149.75, 144.88, 133.93, 132.78, 132.25,
129.39, 128.98, 127.11 (q, J = 30.0 Hz), 126.53 (q, J = 5.4 Hz), 124.91, 123.10, 122.58, 122.33, 115.51, 112.89,
52.67. HRMS Calcd for C22H15F3N4O3 [M]: 440.1096; found for C22H15F3N4O3 [M]: 440.1062.
Methyl 4-(4-(4-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8v). Yield: 93%, white
solid. m.p.: 172–174 ◦C. 1H-NMR (600 MHz, CDCl3)
δ
9.55 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.6 Hz,
ArH), 8.28 (d, 2H, J = 5.0 Hz, ArH), 8.11 (dt, 2H, J = 8.9, 3.3 Hz, ArH), 7.77–7.76 (m, 2H, ArH), 7.55–7.53
(m, 3H, ArH), 7.32 (dd, 1H, J = 5.6, 2.6 Hz, ArH), 3.86 (s, 3H, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ
164.78, 164.64, 153.39, 151.90, 149.73, 145.97, 134.00, 131.29, 130.33, 130.21 (q, J = 31.9 Hz), 129.08, 126.85,
125.05, 124.82, 124.80, 123.24, 122.34, 122.28, 121.69, 121.67, 121.44, 120.90, 120.88, 115.51, 112.90, 52.63.
HRMS Calcd for C22H15F3N4O3 [M]: 440.1096; found for C22H15F3N4O3 [M]: 440.1102.
Methyl 4-(4-(4-(4-chloro-3-methylphenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8w). Yield: 99%, white
◦
solid. m.p.: 177–178 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.38 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.5 Hz,
ArH), 8.10 (dt, 2H, J = 6.8, 3.3 Hz, ArH), 7.95 (d, 1H, J = 1.5 Hz, ArH), 7.80 (dd, 1H, J = 8.2, 1.8 Hz,
ArH), 7.57–7.53 (m, 4H, ArH), 7.32 (dd, 1H, J = 5.6, 2.6 Hz, ArH), 3.86 (s, 3H, OCH3), 2.42 (s, 3H,
CCH3). 13C-NMR (151 MHz, DMSO-d6)
δ 164.84, 164.71, 153.33, 151.95, 149.76, 146.48, 136.23, 134.11,
133.06, 129.62, 129.16, 127.97, 124.55, 122.37, 122.32, 120.17, 115.55, 112.93, 52.70, 19.78. HRMS Calcd for
C22H17ClN4O3 [M]: 420.0989; found for C22H17ClN4O3 [M]: 420.1007.
Methyl 4-(4-(4-(4-chloro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8x). Yield: 99%,
white solid. m.p.: 175–177 ◦C. 1H-NMR (600 MHz, DMSO-d6)
δ
9.59 (s, 1H, triazole-H), 8.66 (d, 1H,
J = 5.6 Hz, ArH), 8.38 (d, 1H, J = 1.8 Hz, ArH), 8.27 (dd, 1H, J = 8.3, 1.9 Hz, ArH), 8.09 (dt, 2H, J = 8.9
1.9 Hz, ArH), 7.92 (d, 1H, J = 8.4 Hz, ArH), 7.56–7.54 (m, 3H, ArH), 7.33 (dd, 1H, J = 5.5, 2.5 Hz,
ArH), 3.87 (s, 3H, CH3). 13C-NMR (151 MHz, DMSO-d6)
164.81, 164.64, 153.47, 151.93, 149.75, 145.09,
,
δ
133.93, 132.62, 130.46, 130.12, 129.92, 127.52 (q, J = 30.8 Hz), 124.27, 123.71, 122.39, 122.31, 121.90, 121.20,
115.55, 112.94, 52.67. HRMS Calcd for C22H14ClF3N4O3 [M]: 474.0707; found for C22H14ClF3N4O3 [M]:
474.0730.

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Methyl 4◦-(4-(4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)phenoxy)picolinate (8y). Yield: 99%, white solid. m.p.:
152–153 C. 1H-NMR (600 MHz, DMSO-d6)
δ 9.26 (s, 1H, triazole-H), 8.65 (d, 1H, J = 5.5 Hz, ArH), 8.09
(d, 2H, J = 8.9 Hz, ArH), 7.63 (d, 1H, J = 5.0 Hz, ArH), 7.56–7.52 (m, 4H, ArH), 7.32 (dd, 1H, J = 5.5,
2.5 Hz, ArH), 7.21 (dt, 1H, J = 3.7, 1.2 Hz, ArH), 3.87 (s, 3H, CH3). 13C-NMR (151 MHz, DMSO-d6)
δ
164.82, 164.69, 153.36, 151.93, 149.75, 142.85, 133.99, 132.29, 128.11, 126.06, 124.74, 122.46, 122.27,
119.12, 115.53, 112.91, 52.67. HRMS Calcd for C19H14N4O3S [M]: 378.0787; found for C19H14N4O3S
[M]: 378.0797.
3.4. Cell Viability Assay
The viability of HT-29, MCF-7, and HEK293T cells was determined using an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. These cancer cell lines were
cultured in minimum essential medium supplemented with 10% foetal bovine serum.
Approximately 4
incubated at 37 ◦C in 5% CO2 for 24 h. The test compounds were added to the culture medium and
incubated for a further 72 h. Fresh MTT was then added to each well at a final concentration of 5 g/mL,
and incubated with the cells at 37 ◦C for 4 h. The formazan crystals in each well were dissolved in
100 L DMSO, and the absorbance at 492 nm (MTT formazan wavelength) and 630 nm (reference
×
103 cells were suspended in cell culture medium, plated in a 96-well plate and
µ
µ
wavelength) was measured by a microplate reader. The reported IC50 results represented averages
of at least three determinations and were calculated using the Bacus Laboratories Incorporated Slide
Scanner (Bliss) software.
3.5. Colony Formation Assay
Logarithmic phase HT-29 cells were seeded into 6-well plates at a concentration of 500 cells per
well. The next day, cells were treated with 8f at 0.125, 0.25, 0.5, and 1 µM. Colonies were fixed with
methanol at room temperature for 15 min and then stained with a solution of 0.1% crystal violet diluted
by phosphate buffered saline (PBS) from 0.5% crystal violet (dissolved in methanol) for 15 min and
then washed by water. The stained colonies were then photographed and dissolved by 33% acetic acid.
Optical density of absorbance was read at 570 nm.
3.6. Apoptosis Assay
Apoptosis of HT-29 cells was detected using a flow cytometric assay. Briefly, cells were seeded
in 6-well plates and incubated overnight. The following day, cells were treated with different
concentrations of compound 8f for 72 h. The cells and supernatants were harvested and washed
twice with cold PBS and then resuspended in 100
µ
L 1
×
Binding Buffer. 5 µL of FITC Annexin V and
5
µL◦ PI were added in each tube and the cells were then gently vortexed incubated for 15 min at RT
(25 C) in the dark. In addition, 400
µL of 1
×
Binding Buffer was then added to each tube. The stained
cells were analyzed by a flow cytometer (Thermo Fisher Scientific, San Jose, CA, USA).
4. Conclusions
In summary, we demonstrated the synthetic application of our highly active CuAAC reaction
catalyst in preparing a novel series of sorafenib analogs. The binuclear Cu catalyst exhibited a
satisfactory performance in the synthesis of the target compounds. At 0.1–0.3 mol % catalyst
loading, all triazole products were obtained at 91–99% isolated yields. Using this catalytic system,
25 new compounds were prepared effectively and were evaluated biologically. The MTT and colony
formation assays demonstrated that compound 8f potently inhibited the proliferation of HT-29
cells. The flow cytometric assay suggested that 8f caused significant apoptosis of HT-29 cells in
a dose-dependent manner.
According to the chemical structural characteristics of 8f, we considered it to be a kinase inhibitor.
Actually, we attempted to identify its biological target by screening it against a panel of kinases
including B-Raf, VEGFR-3, PDGFRα, fibroblast growth factor receptor 1 (FGFR1), c-Kit, TRAF2 and

Molecules 2017, 22, 1759
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NCK interacting kinase (TNIK), ret proto-oncogene (RET), cyclin-dependent 2 (CDK2), checkpoint
kinase 1 (CHK1), and cyclin-dependent kinase 4 (CDK4). However, none of these kinases were
significantly inhibited (Supplementary materials Table S1). These results indicated that 8f acted
quite differently from sorafenib. More detailed investigations of the mechanisms of action of these
compounds are in progress in our laboratory. Studies of the SARs indicated that a fluorine atom at
the meta-position of the terminal phenyl ring was well tolerated, rather than the 3-CF3-4-Cl group in
sorafenib. The hydrogen-bond donor at the 2-position of pyridine was indispensable for the activity
of this chemical series. These findings all provide potentially valuable guidance for designing more
powerful compounds.
Supplementary Materials: Supplementary materials are available online. Table S1: Enzymatic screening of
selected compounds.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (81502924
and 21502120) and the Innovation Training Project of Liaoning Province (201610163013).
Author Contributions: Mingze Qin and Wenjing Ye designed and wrote the paper; Qi Yao, Simiao Yu,
and Ping Gong carried out the experiments. All authors have read and approved the final manuscript.
Conflicts of Interest: The authors have declared no conflict of interest.
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Sample Availability: Samples of the compounds 8a–8y are available from the authors.
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