Synthesis and biological evaluation of 4-aryl-5-cyano-2H-1,2,3-triazoles as inhibitor of HER2 tyrosine kinase

Article abstract of DOI:10.1016/j.bmc.2006.09.041

4-Aryl-5-cyano-2H-1,2,3-triazoles bearing a variety of substituting groups on 4-phenyl were synthesized. The chemicals, designed as HER2 tyrosine kinase inhibitors, were screened for bioactivity of inhibiting growth of breast cancer MDA-MB-453 cells. The

Full text of DOI:10.1016/j.bmc.2006.09.041

Bioorganic & Medicinal Chemistry 15 (2007) 1533–1538
Synthesis and biological evaluation of 4-aryl-5-cyano-2H-1,2,3-
triazoles as inhibitor of HER2 tyrosine kinase
Zhi-Yi Cheng,a,* Wen-Jie Li,a Feng He,a Jun-Min Zhoub and Xiao-Feng Zhub,*
aSchool of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510080, China
bState key laboratory of Oncology in South China, Cancer Center of the Sun Yat-sen University, Guangzhou 510060, China
Received 17 August 2006; revised 19 September 2006; accepted 21 September 2006
Available online 15 December 2006
Abstract—4-Aryl-5-cyano-2H-1,2,3-triazoles bearing a variety of substituting groups on 4-phenyl were synthesized. The chemicals,
designed as HER2 tyrosine kinase inhibitors, were screened for bioactivity of inhibiting growth of breast cancer MDA-MB-453 cells.
The lowest IC50 value of inhibiting HER2 tyrosine kinase phosphorylation in breast cancer cells is 6.6 lM and the IC50 value of cell
growth inhibition is correspondingly 30.9 lM. The lipophilicity of substituting groups on triazoles is the main factor to influence
their bioactivities.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
ing homodimer with itself or heterodimers with other rel-
atives. The dimerization activates the receptor tyrosine
kinases, which phosphorylate special tyrosine residues
on proteins.8 These phosphorylated tyrosine residues ini-
tiate downstream multiple signaling pathways associated
with cell growth (or differentiation) mainly including the
Ras/MAP kinase pathway and PKB/Akt pathway.9
Epidermal growth factor receptor (EGFR) family has
four members: HER2 (human epidermal growth factor
receptor-2; also known as erbB2) and its relatives
HER1 (epidermal growth factor receptor; EGFR),
HER3, and HER4. HER2, like other EGFR members,
is a transmembranous glycoprotein (p185neu) with
intrinsic tyrosine kinase activity encoded by the HER2
protooncogene located on the long arm of chromosome
17 (17q21).1 Being on the upstream of the signal path-
way, it acts as a mayor switch in different signal transduc-
tion processes which mediate and sustain the organ
physiologic activity.2 In normal cells, activation of this
receptor tyrosine kinase family triggers a rich network
of signaling pathways, which control normal cell growth,
differentiation, motility, and adhesion in several cell lin-
eages.3 HER2 overexpression is identified on many tu-
mor cells and the relationship between HER2 status
and clinicopathological characteristics in breast cancer
has been investigated.4 Statistically overexpression of
HER2 occurs in a number of human cancers, including
25–30% of breast cancer,5 28% in pulmonary adenocarci-
noma, and 17% of colorectal adenocarcinoma.6 Its
upregulated expression is also related to rapid disease
progression, chemoresistance, accelerated relapse as well
as poor prognosis and mortality.7 Signal-transduction
pathways depending on HER2 initiate with HER2 form-
The overexpression of HER2 promotes receptor dimer-
ization8 and results in cell transformation. Clearly,
HER2 triggers a diverse signaling network rather than
a single pathway. Therefore, HER2 is proving to be an
excellent target for antitumor therapy,10 specific to
HER2-overexpressing cancers such as breast cancer.6
We previously reported that 1,2,3-triazole was discov-
ered as an inhibitor of HER2 tyrosine kinase through
computer-aided drug design approach and searched
from molecule libraries (see Fig. 1 illustrating the inter-
action of 3k with HER2 tyrosine kinase).11 Recently,
some triazoles were synthesized and showed that the
triazole derivatives can inhibit HER2 tyrosine kinase
phosphorylation in the breast cancer cell, and it has
also been testified that the growth of human breast
cancer cell MDA-MB-453 can be inhibited by the
chemicals.
2. Chemistry
Synthesis of the object chemicals is via a two-step proce-
dure as outlined in Scheme 1. First, arylacetylenes (1)
Keywords: 1,2,3-Triazole; Tyrosine kinase inhibitor; Breast cancer.
*
Corresponding authors. E-mail: chengzhy@mail.sysu.edu.cn
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.041

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Z.-Y. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 1533–1538
Figure 1. Proposed binding mode of 3k with the kinase domain of HER2 obtained after docking and molecular dynamics simulation.
Table 1. The reaction time and yield of synthesis of 3-arylpropyne-
nitriles
R2
R4
R1
R3
N
Scheme 1. Reagents and conditions: (a) CuCN, (CH3)3SiCl, NaI (cat.);
DMSO/CH3CN/H2O, 50 ꢁC, 24–72 h; (b) NaN3, 90–120 ꢁC, 1.5 h.
Compound R1
R2
R3
R4
Time Yielda
(h)
(%)
2a
2b
2c
2d
2e
2f
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
48
48
72
24
24
24
58
76
80
85
80
70
75
75
50
50
80
80
reacted with cuprous cyanide in the presence of chloro-
trimethylsilane and sodium iodide to generate 3-aryl-
propynenitriles 2a–2l after more than one day’s
stirring in the mixture solvent of sulfoxide and acetoni-
trile.12 Chemicals 2a–2l on further reaction with NaN3
afford corresponding 2H-1,2,3-triazoles 3a–3l in excel-
lent yields.13
(CH3)2CH
CH3O
H
CH3O CH3O
H
H
H
H
H
H
H
CH3O CH3O
CH3O CH3O
2g
2h
2i
CH3O 24
H
H
H
H
F
F
Cl
H
H
H
H
H
72
48
48
72
72
2j
Br
The structure of newly synthesized compounds was con-
firmed by recording the IR, 1H NMR, and mass spectra.
Characterization data of triazoles are given in the text.
IR spectrum of compounds showed absorption bands
at 3200–2500, 2240, and 2210 cmÀ1 due to N–H,
C„N, and C„C groups, respectively. The absence of
the absorption band corresponding to C„C stretching
frequency of the reactants clearly confirmed the forma-
tion of triazoles 3a–3l. The 1H NMR spectrum of target
compounds showed a broad singlet at d 12.5 ppm corre-
sponding to active proton on the triazole ring. Mass
spectral data of the newly synthesized compounds were
recorded and are also given in the text. All the com-
pounds showed negative molecular ion peaks as the ma-
jor picks revealing the presence of the active proton. The
major peaks appearing in the spectra were due to the
loss of the proton on the triazole ring.
2k
2l
PhO
PhO
a Purified yield.
iles which bear a methoxy group were easily formed
comparatively, because after 24 h the reaction was al-
most finished and the yield of the product was relatively
high and did not increase by prolonging the reaction
time (see Table 1). It is suggested that the reaction can
be accelerated by the electron-donating substituting
groups.
Cycloaddition reactions were performed at different
temperatures (90 or 120 ꢁC) for the individual substitut-
ing group on arylpropynenitrile. Corresponding higher
temperature could enhance the reaction but result in
more side products and lower yields. Electron-donating
groups had a negative effect on the reaction results. For
multi-methoxy group trazoles were obtained at lower
yields even though at higher temperature.
3. Results and discussion
3.1. Chemistry
1,3-Dipolar cycloaddition between unsymmetrical acety-
lenes and sodium azide results in tautomers. The crystal
Arylpropynenitriles (2) were prepared by the method
reported previously.11 We found that arylpropynenitr-

Z.-Y. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 1533–1538
Table 2. In vitro bioactivity of compounds 3a–3l
1535
N
N
R1
NH
R2
R3
N
R4
Compound
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
a
IC50 (lM)
153.3
53.5
211.7
36.5
130.7
13.5
169.8
>50
241.9
>50
223.7
>50
329.1
>50
35.0
31.6
56.7
11.0
55.0
7.9
30.9
6.6
51.7
16.6
b
IC50 (lM)
a Inhibitory growth of MDA-MB-453 cells with MTT assay.
b Inhibitory on HER2 tyrosine kinase phosphorylation in MDA-MB-453 cells.
HER1-overexpressing breast cancer cell MDA-MB-468
treated with 3k was not observed to be reduced through
Western blot assay (see Fig. 2). It is suggested that
the cell growth inhibition was ascribed mainly to the
inhibition of HER2 tyrosine kinase, for decreases of
HER phosphorylation could inhibit proliferation of
the cell through HER signal-transduction pathway
(Fig. 3).
Figure 2. Inhibition of HER2 tyrosine kinase phosphorylation and
HER2 receptor protein expression by chemical 3k. Overexpressing
HER2 receptor MDA-MB-453 cells were treated with different
concentrations of 3k for 30 min, then collected and lysed. Western
blot analysts was conducted and probed with anti-phospho-HER2 or
anti-HER2 antibody.
As potential inhibitors, the structure–activity relation-
ships covering the chemicals and their HER2 phosphor-
ylation inhibition capability can be summarily deduced
from the data presented in Table 2. Lipophilic substitute
on 4-phenyl of the triazole is favorable for the inhibitory
activity and an electron-drawing group does not appear
to enhance the inhibitory activity.
structures of 4-phenyl-1,2,3-triazole have been deter-
mined.14 2H-Isomer is predominant in solid and gas
phases, however 1,2-diH equilibrium exists in neutral
solution and 1,3-diH+ equilibrium exists in cation solu-
tion, respectively, within these kinds of compounds.
3.2. Bioactivity
4. Conclusion
In vitro bioactivity of compounds 3a–3l was evaluated,
growth inhibition of the human breast cancer MDA-
MB-453 cells tested by MTT assay, and phosphorylation
inhibition of HER2 tyrosine kinase measured by Wes-
tern blot assay. The results are summarized in Table 2.
In summary, we have described the synthesis of 4-aryl-5-
cyano-2H-1,2,3-triazole derivatives and investigated
their bioactivity as HER2 tyrosine kinase inhibitor.
The simple method for synthesis of this series of triazole
opens wide possibility for easy modification of their
chemical structures and properties in the desired
direction.
The most potent chemical is compound 3k. Its IC50 val-
ue of inhibiting the breast cancer cell growth was
30.9 lM and its IC50 value of phosphorylation inhibi-
tion was 6.6 lM. However, the compounds with their
IC50 value of phosphorylation inhibition more than
50 lM showed relatively lower activity of cell growth
inhibition. Western blot assay indicated that the phos-
pho-HER2 protein in breast cancer cell MDA-MB-453
was markedly reduced by compound 3k, while the total
number of HER2 protein was not altered (see Fig. 1). In
the parallel analysis, the phospho-HER1 protein in the
5-Cyano-2H-1,2,3-triazole derivatives were testified to
inhibit the growth of the human breast cancer cell
MDA-MB-453 through inhibiting its phosphorylation
of HER2 in the cell. The lipophilicity of substituting
groups on derivatives is the main factor to influence
their bioactivities, which is rational by the value of IC50.
5. Experimental protocols
5.1. General procedure for chemistry
NMR spectra were recorded on a Varian INOVA500NB
500 MHz spectrometer with Me4Si as the internal stan-
dard. IR spectra were carried out on a FT-IR Tensor
37 spectrophotometer in KBr or as films on KBr pellet.
MS spectra were performed on a ZAB-HS instrument or
a TSQ QUANTUM. Melting points (mp) were obtained
on a WRR Melting point apparatus and the values are
Figure 3. Inhibition of HER1 tyrosine kinase phosphorylation and
HER1 receptor protein expression by chemical 3k. Overexpressing
HER1 receptor MDA-MB-468 cells were treated with different
concentrations of 3k for 1 h, then collected and lysed. Western blot
analysts was conducted and probed with anti-phospho-HER1 or anti-
HER1 antibody.

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Z.-Y. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 1533–1538
uncorrected. TLC was carried out on glass plates coated
with silica gel GF254. Compounds were purified by
chromatography on silica gel.
5.2.9. 3-(4-Chlorophenyl)propynenitrile (2i). IR(Nujol):
2260 cmÀ1 (C„N), 2142 cmÀ1 (C„C); 1H NMR
(CDCl3, d): 7.3 (d, 2H), 7.7 (d, 2H) ppm; MS-FAB
(m/z): 161 (M).
5.2. General procedure for the synthesis of 3-aryl-
propynenitriles (2a–2l)
5.2.10. 3-(4-Bromophenyl)propynenitrile (2j). IR(Nujol):
2276 cmÀ1 (C„N), 2146 cmÀ1 (C„C); 1H NMR
(CDCl3, d): 7.5 (d, 2H), 7.6 (d, 2H) ppm; MS-FAB
(m/z): 205 (M).
A 100 ml flask is charged with dimethylsulfoxide (30 ml)
and acetonitrile (10 ml). To this rapidly stirring solution
cuprous cyanide(10.7 g, 120 mmol), sodium iodide
(0.6 g, 6.0 mmol), and arylacetylene (40 mmol) were
added successively. Chlorotrimethylsilane (15.2 ml,
120 mmol) was dropped to the reaction mixture over a
5 min period, via the addition funnel, and the reaction
mixture then was heated at 50 ꢁC for 24–72 h. The reac-
tion was terminated by cooling the mixture to room
temperature, then 50 ml water was added to the reaction
mixture and was extracted with ether (5· 20 ml). The
combined organic layer was washed with saturated sodi-
um bicarbonate (50 ml), brine (50 ml) in turn, and then
dried with magnesium sulfate overnight. The residue ob-
tained after filtering and evaporation was chromato-
graphed on silica gel using hexane–ethyl acetate (100:5,
v/v) as eluent to yield 3-arylpropynenitriles.
5.2.11. 3-(3-Phenoxyphenyl)propynenitrile (2k). IR(Nu-
1
jol): 2265 cmÀ1 (C„N), 2146 cmÀ1 (C„C); H NMR
(CDCl3, d): 7.3 (m) ppm; MS-FAB (m/z): 219 (M).
5.2.12. 3-(4-Fluoro-3-phenoxylphenyl)propynenitrile (2l).
1
IR(Nujol): 2260 cmÀ1 (C„N), 2147 cmÀ1 (C„C); H
NMR (CDCl3, d): 7.2 (m) ppm; MS (FAB) m/z: 237 (M).
5.3. General procedure for the synthesis of 4-aryl-5-
cyano-2H-1,2,3-triazoles (3a–3l)
NaN3 (0.97 g, 15 mmol) suspended in DMF (10 ml) was
strongly stirred at 90 or 120 ꢁC (change according to the
variety of arylpropynenitriles). To this flask a solution
of arylpropynenitrile (10 mmol) in DMF (4 ml) was
added dropwise through an addition funnel and kept
the addition lasting 30 min. After the solution was add-
ed, the mixture was maintained stirring for another hour
at the reaction temperature. The residue obtained after
removal of the solvent was treated with water (10 ml).
Then the water layer was extracted three times with
CH2Cl2 to remove the oil and give a transparent light
color solution. Finally, the aqueous solution was acidi-
fied with 10% HCl resulting in light yellow precipitate
which was washed with iced water to yield crude 4-ar-
yl-5-cyano-2H-1,2,3-triazoles. The products were puri-
fied by column chromatography on silica gel eluted
with petrol ether and ethyl acetate (100:10, v/v).
5.2.1. 3-Phenylpropynenitrile (2a). IR(Nujol): 2269 cmÀ1
1
(C„N), 2145 cmÀ1 (C„C); H NMR (CDCl3, d): 7.4
(3H), 7.6 (2H); MS-FAB (m/z): 127 (M).
5.2.2. 3-(4-Methylphenyl)propynenitrile (2b). IR(Nujol):
2262 cmÀ1 (C„N), 2137 cmÀ1 (C„C); 1H NMR
(CDCl3, d): 2.4 (s, 6H), 7.3 (d, 2H), 7.6 (d, 2H) ppm;
MS-FAB (m/z): 141 (M).
5.2.3. 3-(4-Isopropylphenyl)propynenitrile (2c). IR(Nu-
1
jol): 2266 cmÀ1 (C„N), 2244 cmÀ1 (C„C); H NMR
(CDCl3, d): 1.3 (d, 6H), 3.2 (m, 1H), 7.3 (d, 2H), 7.5
(d, 2H) ppm; MS-FAB (m/z): 169 (M).
5.2.4. 3-(4-Methoxyphenyl)propynenitrile (2d). IR(Nu-
jol): 2262 cmÀ1 (C„N), 2137 cmÀ1 (C„C); 1H
NMR(CDCl3, d): 3.8 (s, 3H), 7.0 (d, 2H), 7.5 (d,
2H) ppm; MS-FAB (m/z): 157 (M).
5.3.1. 4-Phenyl-5-cyano-1,2,3-triazoles (3a). Reaction
temperature: 90ꢁC; yield: 80%; mp: 165–166 ꢁC (lit.13
166–167 ꢁC); IR(KBr): 3250–2500 cmÀ1 (N–H),
2240 cmÀ1 (C„N) cmÀ1 1H NMR (CD3COCD3, d):
;
7.6 (m, 3H), 8.0 (m, 2H), 15.2 (br s, 1H) ppm; ESI-MS
(m/z): 169.145 (MÀH)À. Anal. Calcd for C9H6N4
(170.17): C, 63.52; H, 3.55; N, 32.92. Found: C, 63.75;
H, 4.13; N, 32.74.
5.2.5. 3-(2,3-Dimethoxyphenyl)propynenitrile (2e). IR-
(Nujol): 2262 cmÀ1 (C„N), 2145 cmÀ1 (C„C); 1H
NMR (CDCl3, d): 3.8 (s, 6H), 6.9 (m, 2H), 7.2 (d,
1H) ppm; MS-FAB (m/z): 187 (M).
5.3.2.
(3b). Reaction temperature: 90 ꢁC; yield: 80%; mp:
4-(4-Methylphenyl)-5-cyano-2H-1,2,3-triazoles
3250–2500 cmÀ1
5.2.6. 3-(3,4-Dimethoxyphenyl)propynenitrile (2f). IR-
(Nujol): 2255 cmÀ1 (C„N), 2140 cmÀ1 (C„C); 1H
NMR (CDCl3, d): 3.9 (s, 6H), 6.9 (d, 1H), 7.1 (m,
2H) ppm; MS-FAB (m/z): 187 (M).
173–174 ꢁC;
IR(KBr):
(N–H),
2240 cmÀ1 (C„N); 1H NMR (CD3COCD3, d): 2.4
(3H, s), 7.8 (d, 2H, J = 8.1 Hz), 7.3 (d, 2H, J = 7.9 Hz),
12.6 (br s, 1H) ppm; ESI-MS (m/z): 184.156 (MÀH)À.
Anal. Calcd for C10H8N4 (184.20): C, 65.21; H, 4.38;
N, 30.42. Found: C, 65.45; H, 4.59; N, 30.17.
5.2.7. 3-(3,4,5-Trimethoxyphenyl)propynenitrile (2g).
1
IR(Nujol): 2262 cmÀ1 (C„N), 2146 cmÀ1 (C„C); H
NMR (CDCl3, d): 3.8 (9H), 6.7 (s, 2H) ppm; MS-FAB
(m/z): 217 (M).
5.3.3. 4-(4-Isopropylphenyl)-5-cyano-2H-1,2,3-triazoles
(3c). Reaction temperature: 90 ꢁC; yield: 80%; mp:
5.2.8. 3-(4-Fluorophenyl)propynenitrile (2h). IR(Nujol):
2268 cmÀ1 (C„N), 2145 cmÀ1 (C„C); 1H NMR
(CDCl3, d): 7.1 (d, 2H), 7.6 (d, 2H) ppm; MS-FAB
(m/z): 145 (M).
147–148 ꢁC;
IR(KBr):
3250–2500 cmÀ1
(N–H),
1
2240 cmÀ1 (C„N); H NMR (CD3COCD3, d): 1.3 (d,
6H, J = 7.0 Hz), 3.0 (m, 1H), 7.4 (d, 2H, J = 8.0 Hz),
7.9 (d, 2H, J = 8.5 Hz), 12.9 (br s, 1H) ppm; ESI-MS

Z.-Y. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 1533–1538
1537
(m/z): 211.226 (MÀH)À. Anal. Calcd for C12H12N4
(212.25): C, 67.90; H, 5.70; N, 26.40. Found: C, 68.18;
H, 6.15; N, 26.04.
2H, J = 8.9 Hz), 7.9 (d, 2H, J = 8.8 Hz), 15.3 (br s,
1H); ESI-MS (m/z): 245.925 (MÀH)À. Anal. Calcd for
C9H5BrN4 (249.07): C, 43.40; H, 2.02; N, 22.49. Found:
C, 43.14; H, 2.71; N, 22.12.
5.3.4. 4-(4-Methoxyphenyl)-5-cyano-2H-1,2,3-triazoles
(3d). Reaction temperature: 90 ꢁC; yield: 75%; mp:
5.3.11. 4-(3-Phenoxylphenyl)-5-cyano-2H-1,2,3-triazoles
(3k). Reaction temperature: 120 ꢁC; yield: 80%; mp:
202–203 ꢁC;
IR(KBr):
3252–2500 cmÀ1
(N–H),
1
2237 cmÀ1 (C„N); H NMR (CD3COCD3, d): 4.0 (s,
3H), 7.0 (d, 1H, J = 8.9 Hz), 7.9 (d, 1H, J = 8.8 Hz),
12.20 (br s, 1H) ppm; ESI-MS (m/z): 198.105 (MÀH)À.
Anal. Calcd for C10H8N4O (200.20): C, 59.99; H, 4.03;
N, 27.99. Found: C, 60.20; H, 4.61; N, 27.62.
159–160 ꢁC;
IR(KBr):
3250–2500 cmÀ1
(N–H),
2242 cmÀ1 (C„N); 1H NMR (CD3COCD3, d): 7.07
(dd, 2H, J = 1.0 Hz, J = 8.6 Hz), 7.1 (m, 2H), 7.4 (dd,
1H, J = 7.5, 8.5 Hz), 7.5 (t, 1H, J = 8.0 Hz), 7.6 (s,
1H), 7.7 (d, 1H, J = 7.8 Hz), 12.4 (br s, 1H) ppm; ESI-
MS (m/z): 260.125 (MÀH)À. Anal. Calcd for
C15H10N4O (262.27): C, 68.69; H, 3.84; N, 21.36.
Found: C, 68.41; H, 4.11; N 21.45.
5.3.5. 4-(2,3-Dimethoxyphenyl)-5-cyano-2H-1,2,3-tria-
zoles (3e). Reaction temperature: 120 ꢁC; yield: 60%;
mp: 179–180 ꢁC, IR(KBr): 3255–2968 cmÀ1 (N–H),
1
2242 cmÀ1 (C„N); H NMR (CD3COCD3, d): 3.9 (s,
5.3.12. 4-(4-Fluoro-3-phenoxylphenyl)-5-cyano-2H-1,2,3-
triazoles (3l). Reaction temperature: 120 ꢁC; yield: 80%;
mp: 127–129 ꢁC; IR(KBr): 3250–2500 cmÀ1 (N–H),
2245 cmÀ1 (C„N); 1H NMR (CD3COCD3, d): 7.05
(dd, 2H, J = 1.0 Hz, J = 8.7 Hz), 7.15 (t, 1H,
J = 7.4 Hz), 7.35 (m, 3H), 7.63 (dd, 1H, J = 1.9 Hz,
J = 7.5 Hz), 7.74 (dd, 1H, J = 2.2, 4.1, and 8.5 Hz),
12.34 (1H, br s) ppm; ESI-MS (m/z): 279.161 (MÀH)À.
Anal. Calcd for C15H9FN4O (280.26): C, 64.28; H,
3.24; N, 19.99. Found: C, 63.87; H, 3.53; N, 19.78.
3H), 4.0 (s, 3H), 7.3 (m, 3H), 15.1 (br s, 1H) ppm;
ESI-MS (m/z): 229.211 (MÀH)À. Anal. Calcd for
C11H10N4O2 (230.22): C, 57.39; H, 4.38; N, 24.34.
Found: C, 57.70; H, 4.81; N, 24.01.
5.3.6. 4-(3,4-Dimethoxyphenyl)-5-cyano-2H-1,2,3-tria-
zoles (3f). Reaction temperature: 120 ꢁC; yield: 60%;
mp: 247–248 ꢁC; IR(KBr): 3249–2939 cmÀ1 (N–H),
1
2237 cmÀ1 (C„N); H NMR (CD3COCD3, d): 3.912
(3H, s), 3.910 (3H, s), 7.56 (2H, m), 7.16 (dd, 1H,
J = 8.3 Hz, J = 17.6 Hz), 15.3 (1H, br s) ppm; ESI-MS
(m/z): 230.202 (MÀH)À. Anal. Calcd for C11H10N4O2
(230.22): C, 57.39; H, 4.38; N, 24.34. Found: C, 57.68;
H, 4.76; N, 24.13.
5.4. Biology
5.4.1. Cell and cell culture. Human breast cancer cell line
MDA-MB-453 was obtained from ATCC (USA).
MDA-MB-453 cells (overexpressing HER2 receptor)
were cultured in RPMI-1640 containing 10% of fetal bo-
vine serum (FBS). The cultures were maintained at
37 ꢁC in 5% CO2. All experiments were conducted on
cells in logarithmic growth phase.
5.3.7. 4-(3,4,5-Trimethoxyphenyl)-5-cyano-2H-1,2,3-tria-
zoles (3g). Reaction temperature: 120 ꢁC; yield 54%; mp:
222–223 ꢁC;
IR(KBr):
3197–2940 cmÀ1
(N–H),
1
2241 cmÀ1 (C„N); H NMR (CD3COCD3, d): 3.8 (s,
3H), 3.9 (s, 6H), 7.3 (s, 2H), 15.2 (br s, 1H) ppm; ESI-
MS (m/z): 259.191 (MÀH)À. Anal. Calcd for
C12H12N4O3 (260.25): C, 55.38; H, 4.65; N, 21.53.
Found: C, 55.29; H, 5.21; N, 21.01.
5.4.2. MTT assay. A total of 2000 cells in 100 ll culture
media with 10% fetal bovine serum were seeded to each
well of a 96-well plate. After the addition of each drug
in triplicate the cells were incubated at 37 ꢁC. Ten micro-
liters of 5 mg/ml MTT (Sigma) was add to each well. After
the addition of MTT, further incubation was carried out
for 4 h. At the end of the incubation the culture media
were removed and 0.2 ml DMSO was added. The optical
density (OD) was measured at 570 nm when the formazan
crystals were dissolved in DMSO. The concentrations re-
quired to inhibit growth by 50% (IC50 values) were calcu-
lated from the cytotoxicity curves (Bliss’s software).
5.3.8.
(3h). Reaction temperature: 120 ꢁC; yield: 80%; mp:
4-(4-Fluorophenyl)-5-cyano-2H-1,2,3-triazoles
3250–2500 cmÀ1
193–195 ꢁC;
IR(KBr):
(N–H),
1
2240 cmÀ1 (C„N); H NMR(CD3COCD3, d): 7.3 (d,
2H, J = 7.9 Hz), 7.8 (d, 2H, J = 8.1 Hz), 12.3 (br s,
1H) ppm; ESI-MS (m/z): 186.136 (MÀH)À. Anal. Calcd
for C9H5FN4(188.16): C, 57.45; H, 2.68; N, 29.78.
Found: C, 57.41; H, 2.94; N, 29.49.
5.3.9. 4-(4-Chlorophenyl)-5-cyano-2H-1,2,3-triazoles (3i).
Reaction temperature: 120 ꢁC; yield: 80%; mp: 189–
190 ꢁC; IR(KBr): 3250–2560 cmÀ1 (N–H), 2243 cmÀ1
(C„N); 1H NMR (CD3COCD3, d): 7.5 (d, 2H,
J = 8.6 Hz), 7.9 (d, 2H, J = 8.6 Hz), 15.1 (br s,
1H) ppm; ESI-MS (m/z): 202.143 (MÀH)À. Anal. Calcd
for C9H5ClN4 (204.62): C, 52.83; H, 2.46; N, 27.38.
Found: C, 52.46; H, 2.96; N, 27.17.
5.4.3. Determining phosphorylation inhibition of HER2
tyrosine kinase (Western blot method). One milliliter of
diluted cells 1· 106/ml was seeded into 6-well plate per
well. Cells treated with different concentration of drug
at the same time were washed twice with PBS, lysed in
100 ll lysis buffer, and then centrifuged at 14,000g for
10 min. Protein concentrations were measured by the
method of BCA (piece) and the IC50 values of phosphor-
ylation inhibition were calculated, respectively.
5.3.10.
(3j). Reaction temperature: 120 ꢁC; yield: 80%; mp:
182–183 ꢁC; IR(KBr): (N–H),
3224–2534 cmÀ1
2256 cmÀ1 (C„N); H NMR (CD3COCD3, d): 7.8 (d,
4-(4-Bromophenyl)-5-cyano-2H-1,2,3-triazoles
To explore the change of HER, 40 mg of the protein pre-
pared from aforesaid approach was used per lane, for
SDS–PAGE. Sample treatment buffer was added and
1

1538
Z.-Y. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 1533–1538
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incubated at 95 ꢁC for 10 min. Following the seperation
by 10% SDS–polyacrylamide gel electrophoresis proteins
were transferred onto an equilibrated polyvinylidene
difluoride membrane (Amersham) by electro-blotting.
Afterwards membranes were blocked by 5% non-fat milk
and consequently treated with primary antibodies and
secondary antibodies. When all these were done the mem-
brane was incubated at room temperature for 2 h. Before
illuminant chemical was added, the blot was washed with
TBST buffer (10 mmol/l Tris–HCl, pH 7.4, 150 mmol/l
NaCl, and 0.1% Tween 20) for 10 min once. And then
it was flaked, put into dark box to develop.
11. Zhu, X. F.; Zeng, Y. X.; Yang, D. J. Chin. J. Cancer 2001,
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