Design and synthesis of novel triazolo-lapatinib hybrids as inhibitors of breast cancer cells

Article abstract of DOI:10.1007/s00044-018-2247-0

A series of triazolo-lapatinib hybrids were synthesized via copper (II)-oxide nanoparticle (Cu₂O-NP)-catalyzed azide-alkyne cycloaddition. The ability of these compounds to reduce the viability of breast cancer SKBR3 and SUM159 cells and stem cell-like KG-1a leukemia cells was subsequently evaluated. Compared with lapatinib, compounds 6c–f were more potent than lapatinib against the three cell lines. Next, the toxicity of compounds 6c–f was assessed in zebrafish. Compound 6d had comparable toxicity with lapatinib at 200 μM (mortality rate = 10 vs. 10%), and compound 6e had lower toxicity than lapatinib at 200 μM (mortality rate = 0 vs. 10%). Thus, compound 6e is a promising lead compound worthy of further investigation. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-018-2247-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2247-0
ORIGINAL RESEARCH
Design and synthesis of novel triazolo-lapatinib hybrids as inhibitors
of breast cancer cells
YeHui Shi1 Wei Zhang2 Lixin Li2 ZhongSheng Tong1 CuiGai Bai2
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Received: 11 April 2018 / Accepted: 15 September 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A series of triazolo-lapatinib hybrids were synthesized via copper (II)-oxide nanoparticle (Cu2O-NP)-catalyzed azide-alkyne
cycloaddition. The ability of these compounds to reduce the viability of breast cancer SKBR3 and SUM159 cells and stem
cell-like KG-1a leukemia cells was subsequently evaluated. Compared with lapatinib, compounds 6c–f were more potent
than lapatinib against the three cell lines. Next, the toxicity of compounds 6c–f was assessed in zebrafish. Compound 6d had
comparable toxicity with lapatinib at 200 μM (mortality rate = 10 vs. 10%), and compound 6e had lower toxicity than
lapatinib at 200 μM (mortality rate = 0 vs. 10%). Thus, compound 6e is a promising lead compound worthy of further
investigation.
Graphical Abstract
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Keywords Lapatinib Triazolo-lapatinib hybrids Click chemistry Cu2O-NP Cancer stem cell
Introduction
Lapatinib is a quinazoline compound that can inhibit the
* ZhongSheng Tong
receptor tyrosine kinases ErbB-1 and ErbB-2 (Wood et al.
2004). Correspondingly, lapatinib has been shown to
potently inhibit the growth of ErbB-1/2-dependent tumor
cell lines, such as the breast cancer cell line SKBR3, which
constitutively overexpresses ErbB-2 (Kim et al. 2008). The
efficacy of lapatinib against breast cancer cells has also been
shown to correlate with a down-regulation of the mitogen-
activated protein kinases (MAPK) and phosphoinositide 3-
kinase (PI3K)/Akt signaling pathways, and an up-regulation
of the downstream FOXO3A signaling pathway (Hegde
et al. 2007; Lacerda et al. 2010).
tongzhongsheng@tjmuch.com
* CuiGai Bai
bj20081120@163.com
1
Department of Breast Oncology, Tianjin Medical University
Cancer Institute and Hospital, National Clinical Research Center
for Cancer, Key Laboratory of Breast Cancer Prevention and
Therapy, Ministry of Education, Key Laboratory of Cancer
Prevention and Therapy, Tianjin Medical University, 300060
Tianjin, China
2
High-throughput Molecular Drug Discovery Center, Tianjin
International Joint Academy of BioMedicine, 300457
Tianjin, China

Medicinal Chemistry Research
Among breast cancers, triple-negative breast-cancer has a
high rate of tumor recurrence and metastasis, and is usually
modeled in vitro using the SUM159 and MDA-MB-231 cell
lines (So et al. 2014). Cancer stem cells (CSCs) represent a
subpopulation of cancer cells that exhibit an indefinite
potential for self-renewal, and are thought to drive tumor-
igenesis (Reya et al. 2001). CSCs have also been associated
with drug resistance, tumor invasion, tumor relapse and
disease progression (Rasheed et al. 2011). Previously,
lapatinib was reported to increase the percentage of CSCs.
The KG-1a cell line is a human promyeloblastic leukemia
cell line that exhibits characteristics similar to those of
leukemia stem cells. KG-1a cells are also resistant to che-
motherapeutic drugs; therefore, the KG-1a cell line provides
a model for studying leukemia stem cells (Li et al. 2008).
Click chemistry is a frequently used strategy in chemical
synthesis, drug development and biomedical materials, and
the typical chemical reaction is copper-catalyzed azide–
alkyne cycloaddition (Moses and Moorhouse 2007; Hein
et al. 2008; Bonnet et al. 2006).
ethyl acetate (3 × 50 mL). The crude material was purified
by column chromatography to afford compound 2.
Compound 2 Yield 21%, yellow powder; MS: [M+H]+
1
421.16; H-NMR (400 MHz, DMSO-D6) δ 9.82 (s, 1H),
8.56 (s, 1H), 8.26 (s, 1H), 8.00 (d, J = 2.8 Hz, 1H), 7.82 (d,
J = 8.8 Hz 1H), 7.72 (dd, J1 = 2.4 Hz, J2 = 8.8, 1H), 7.61
(dd, J1 = 2.4 Hz, J 2 = 8.8, 1H), 7.48 (m, 1H), 7.30 (m, 3H),
7.19 (m, 1H), 5.27 (s, 2H).
General procedure for the synthesis of compounds 5a–f
To a solution of compound 4a–f (12.53 mmol) and
NaHCO3 (2.63 g, 31.31 mmol) in ethanol (10 ml) was added
compound 3 (0.98 g, 13.15 mmol) in ethanol (6 ml) solution
at 78 °C for about 3 h. The reaction mixture was filtered
through a celite pad to remove NaHCO3. The solvent was
evaporated under vacuum. The crude material was purified
by column chromatography to obtain product.
1
In this study, a series of triazole derivatives of lapatinib
were synthesized, and then assayed for their capacity to
reduce the viability of SKBR3 breast cancer cells, which
constitutively express ErbB-2, SUM159 cells, which reflect
triple negative breast cancer and KG-1a cells, which exhibit
stem cell-like qualities. Next, the toxicity of triazole deri-
vatives was assessed in zebrafish.
Compound 5a Yield 33%, oil; MS: [M+H]+ 161.91; H-
NMR (400 MHz, DMSO-D6) δ 3.35 (d, J = 2.4 Hz, 2H),
3.22 (t, J = 6.8 Hz, 2H), 3.08 (t, J = 2.4 Hz, 1H), 2.99 (s,
3H), 2.96 (t, J = 6.6 Hz, 2H), 2.27 (bs, 1H). 13C-NMR (100
MHz, CDCl3-D) δ 81.3, 72.3, 54.7, 42.5, 42.40, 38.0.
1
Compound 5b Yield 54%, oil; MS: [M+H]+ 205.15; H-
NMR (400 MHz, CDCl3-D) δ 7.35 (s, 1H), 6.28 (s, 1H),
6.19 (d, J = 2.8 Hz, 1H), 3.54 (s, 2H), 3.26 (s, 2H); 2.61 (s,
4H), 2.53 (s, 4H), 2.21 (s, 1H); 13C-NMR (100 MHz,
CDCl3-D) δ 151.5, 142.1, 110.0, 108.8, 78.9, 73.1, 54.7,
52.5, 51.7, 46.7.
Materials and methods
Chemistry
1
All reagents were of analytical grade and were dried and
Compound 5d Yield 18%, oil; MS: [M+H]+ 181.65; H-
1
purified if necessary. H-NMR and 13C-NMR spectra were
NMR (400 MHz, CDCl3-D) δ 3.44 (d, J = 2.0 Hz, 2H), 2.86
(d, J = 12.0 Hz, 2H), 2.68 (m, 1H), 2.26 (m, 2H), 2.18 (t, J
= 2.4 Hz, 1H), 2.01 (t, J = 11.2 Hz, 2H), 1.82 (d, J = 12.4
Hz, 2H), 1.45 (m, 4H), 0.86 (t, J = 7.4 Hz, 3H). 13C-NMR
(100 MHz, CDCl3-D) δ 82.4, 71.3, 60.7, 52.1, 35.2, 31.9,
29.8, 20.1, 12.1.
recorded on a Bruker AV 400 or 600 spectrometer using
CDCl3-D, DMSO-D6 or MeOD-D4 Mas the solvent. Che-
mical shifts are reported in parts per million (ppm) relative
to either a tetramethylsilane internal standard or solvent
signals. Data are reported as follows: chemical shift, mul-
tiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br.
= broad, m = multiplet), coupling constants and integration.
High-resolution mass spectra were conducted using Agilent
6520 Q-TOF LC/MS by ESI-FTICR technique.
Compound 5e Yield 32%, oil; 1H-NMR (400 MHz,
CDCl3-D) δ 8.52 (s, 1H), 8.49 (d, J = 4.7 Hz, 1H), 7.66 (d,
J = 7.8 Hz, 2H), 7.24 (dd, J = 7.7, 4.8 Hz, 1H), 3.50 (s,
2H), 3.45 (d, J = 2.4 Hz, 3H), 2.90–2.77 (m, 2H), 2.75–
2.66 (m, 1H), 2.20 (t, J = 2.4 Hz, 1H), 2.13–2.03 (m, 4H),
1.87–1.78 (m, 2H), 1.46–1.33 (m, 3H); 13C-NMR (100
MHz, CDCl3-D) δ 150.5, 148.6, 136.7, 134.0, 123.4, 82.4,
71.3, 60.3, 53.1, 52.1, 35.2, 32.2.
Compounds 5c, 5f were prepared as described for
compound 5a, but the reaction temperature was 25 °C,
and they were low boiling point. So get the crude products
to synthesize the target products, directly.
General procedure for the synthesis of compound 2
A solution of compound 1 (0.1 g, 0.2 mmol) NaN3 (15 mg,
0.24 mmol), CuI (8 mg, 0.02 mmol), L-Pro (4.6 mg, 0.04
mmol), NaOH (1.6 mg, 0.04 mmol) in DMSO (10 ml) was
stirred at 50 °C for about 3 h. The reaction mixture was
poured into cold water, and the product was extracted with

Medicinal Chemistry Research
General procedure for the synthesis of compounds 6a–f
44.6; HRMS-ESI(+) calculated for C29H29ClFN8O,
559.2137; found 559.2131 [M+H]+.
A solution of compound 5a–f (0.26 mmol), compound 2
(110.80 mg, 0.26 mmol) and Cu2O-NP catalyst in acetoni-
trile (5 mL) and DMF (2 ml) was stirred for about 5 h. The
reaction mixture was filtered through a celite pad to remove
the Cu2O-NP catalyst. And the acetonitrile was evaporated
under reduced pressure. The residue in DMF was poured
into cold water, and the product was extracted with ethyl
acetate (3 × 20 ml). The organic layer was separated and the
solvent was removed under reduced pressure. The crude
material was purified by column chromatography to afford
product.
Compound 6d Yield 38%, yellow solid powder; MS: [M
1
−H]- 599; H-NMR (400 MHz, CDCl3-D) δ 9.19 (s, 1H),
8.74 (d, J = 5.2 Hz, 2H), 8.02 (s, 1H), 7.94 (s, 2H), 7.79 (s,
1H), 7.51 (d, J = 8.0 Hz, 1H), 7.34 (m, 1H), 7.20 (t, J = 4.4
Hz, 2H), 7.01 (t, J = 8.2 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H),
5.08 (s, 2H), 3.84 (s, 2H), 2.82 (d, J = 11.2 Hz, 2H), 2.43 (t,
J = 9.6 Hz, 1H), 2.22 (t, J = 7.6 Hz, 2H), 1.89 (t, J = 11.0
Hz, 2H), 1.78 (d, J = 11.6 Hz, 2H), 1.45 (m, 2H), 1.35 (m,
2H), 0.86 (t, J = 7.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3-
D) δ 164.2, 161.8, 158.3, 155.8, 151.2, 149.7, 148.6, 138.9,
134.2, 132.1, 130.2, 125.2, 124.8, 123.3, 122.4, 120.4,
115.6, 115.0, 114.8, 114.0, 113.9, 113.6, 70.3, 60.7, 54.8,
52.4, 41.7, 32.5, 20.2, 11.9; HRMS-ESI(+) calculated for
C32H34ClFN8O, 601.2606; found 601.2589 [M+H]+; MP:
265.3–266.0 °C.
Compound 6a Yield 32%, yellow solid powder; MS: [M
1
−H]- 580.36; H-NMR (400 MHz, DMSO-D6) δ 10.02 (s,
1H), 9.07 (s, 1H), 8.73 (s, 1H), 8.66 (s, 1H), 8.37 (d, J =
7.6 Hz, 1H), 8.04 (d, J = 2.4 Hz, 1H), 8.00 (d, J = 9.2 Hz,
1H), 7.76 (dd, J1 = 2.4 Hz, J2 = 8.8, 1H), 7.48 (m, 1H),
7.30 (q, J = 8.7 Hz, 3H), 7.20 (m, 1H), 5.27 (s, 2H), 3.94 (s,
2H), 3.31 (m, 2H, obscured by water peak), 3.05 (s, 5H);
13C-NMR (100 MHz, DMSO-D6) δ 163.9, 161.5, 158.2,
155.6, 150.4, 140.1, 134.6, 133.3, 131.1, 130.3, 125.7,
124.6, 123.8, 122.8, 121.8, 121.5, 115.3, 115.1, 114.8,
114.6, 114.4, 114.2, 69.9, 54.1, 43.9, 42.7, 41.9; HRMS-
ESI(+) calculated for C27H26ClFN7O3S, 582.1490; found
582.1484 [M+H]+.
Compound 6e Solid 23%, yellow solid powder; MS: [M
−H]- 648.4; 1H-NMR (600 MHz, MeOD-D4) δ 8.63 (s,
1H), 8.45 (s, 1H), 8.42 (s, 1H), 8.10 (d, J = 10.8 Hz, 1H),
7.87 (s, 1H), 7.69 (d, J = 15.0 Hz, 1H), 7.54 (d, J = 10.8
Hz, 1H), 7.31 (m, 3H), 7.19 (d, J = 7.8 Hz, 1H), 7.14 (d, J
= 9.6 Hz, 1H), 6.97 (t, J = 9.6 Hz, 1H), 6.88 (d, J = 9.0 Hz,
1H), 4.98 (s, 2H), 3.86 (s, 2H), 3.46 (s, 2H), 2.79 (d, J =
11.4 Hz, 2H), 2.52 (m, 1H), 2.02 (t, J = 11.1 Hz, 2H), 1.89
(d, J = 11.4 Hz, 2H), 1.44 (m, 2H). 13C-NMR (150 MHz,
MeOD-D4) δ 163.7, 162.1, 157.8, 154.8, 150.6, 148.9,
148.6, 147.1, 139.6, 134.4, 132.5, 129.9, 128.7, 124.8,
124.3, 122.3, 121.7, 120.7, 115.2, 114.2, 114.0, 113.5,
113.3, 112.8, 69.6, 61.1, 52.1, 40.6, 31.5. HRMS-ESI( + )
calculated for C35H33ClFN9O, 650.2559; found 650.2585
[M+H]+; MP: 148.1–148.8 °C.
Compound 6b Yield 22%, yellow solid powder; MS: [M
−H]- 623.3; 1H-NMR (400 MHz, CDCl3-D) δ 8.82 (s, 1H),
8.78 (s, 1H), 8.68 (s, 1H), 8.04 (s, 1H), 7.99 (s, 1H), 7.85 (s,
1H), 7.58 (d, J = 8.0 Hz, 1H), 7.35 (s, 2H), 7.22 (t, J = 4.4
Hz, 2H), 7.03 (t, J = 7.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H),
6.30 (s, 1H), 6.18 (s, 1H), 5.13 (s, 2H), 3.67 (s, 2H), 3.50 (s,
2H), 2.48 (d, J = 29.2 Hz, 8H); 13C-NMR (100 MHz,
CDCl3-D) δ 164.2, 161.8, 158.1, 155.9, 151.1, 149.7,
145.4, 142.3, 139.1, 134.2, 132.1, 130.2, 125.1, 123.4,
122.4, 122.2, 121.3, 115.6, 115.1, 114.8, 114.2, 114.0,
113.8, 113.5, 110.1, 109.0, 70.4, 54.6, 52.9, 52.5, 52.4;
HRMS-ESI(+) calculated for C33H30ClFN8O2, 625.2242;
found 625.2253 [M+H]+; MP: 84.5–85.1 °C.
Compound 6f Yield 31%, yellow solid powder; MS: [M
−H]- 528; 1H-NMR (400 MHz, DMSO-D6) δ 10.15 (s, 1H),
9.20 (s, 1H), 8.99 (s, 1H), 8.67 (s, 1H), 8.40 (d, J = 8.8 Hz,
1H), 8.07 (d, J = 2.4 Hz, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.80
(dd, J1 = 2.0 Hz, J2 = 8.8, 1H), 7.48 (q, J = 7.3 Hz, 1H),
7.32 (q, J = 8.3 Hz 3H), 7.20 (m, 1H), 5.27 (s, 2H), 4.21 (s,
2H), 2.96 (s, 4H), 1.84 (s, 4H). 13C-NMR (100 MHz,
DMSO-D6) δ 163.9, 161.5, 158.3, 150.3, 140.1, 133.3,
131.1, 131.0, 124.6, 123.8, 122.8, 121.5, 115.3, 115.1,
114.8, 114.6, 114.4, 69.9, 31.7, 29.5, 22.5, 14.4. HRMS-
ESI(+) calculated for C28H26ClFN7O, 530.1871; found
530.1866 [M+H]+.
Compound 6c Yield 29%, yellow solid powder; MS: [M
−H]- 557.3; 1H-NMR (600 MHz, MeOD-D4) δ 8.72 (s,
1H), 8.52 (s, 1H), 8.49 (s, 1H), 8.21 (dd, J1 = 3.6 Hz, J2 =
13.2, 1H), 7.89 (s, 1H), 7.81 (d, J = 9.6 Hz, 1H), 7.56 (d, J
= 11.4 Hz, 1H), 7.36 (m, 1H), 7.24 (d, J = 7.2 Hz, 1H),
7.19 (d, J = 9.6 Hz, 1H), 7.02 (m, 1H), 6.98 (d, J = 9.0 Hz,
1H), 5.07 (s, 2H), 3.75 (s, 2H), 2.58 (d, J = 66.0 Hz, 8H),
2.28 (s, 3H); 13C-NMR (150 MHz, MeOD-D4) δ 163.7,
162.1, 158.1, 154.9, 150.8, 148.6, 144.5, 139.6, 134.6,
132.4, 129.9, 128.8, 125.1, 124.3, 122.4, 121.8, 114.2,
114.0, 113.7, 113.5, 113.4, 113.1, 69.7, 54.3, 52.2, 51,9,
General procedure for the synthesis of compound 9
A solution of compound 7 (0.1 g, 0.41 mmol) in methanol
was stirred at room temperature for 10 min. Then compound
8 (95 mg, 0.49 mmol) and K2CO3 (113 mg, 0.82 mmol)

Medicinal Chemistry Research
were added and stirred at room temperature for 18 h. The
reaction mixture was filtered through a celite pad to remove
K2CO3. The solvent was evaporated under vacuum. Then
water was added and the product was extracted by ethyl
acetate (3 × 30 ml). The crude material was purified by
column chromatography to obtain product.
Compound 13 Yield 43%, yellow solid powder; MS:
1
[M-H]- 624.0; H-NMR (400 MHz, DMSO-D6) δ 10.09
(s, 1H), 9.12 (s, 1H), 8.76 (s, 1H), 8.64 (s, 1H), 8.35 (d,
J = 10.8 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.96 (d, J =
8.8 Hz, 1H), 7.80 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 7.47
(m, 2H), 7.30 (m, 3H), 7.19 (m, 1H), 7.00 (m, 2H), 5.36
(s, 2H), 3.81 (s, 2H), 3.00 (d, J = 11.2 Hz, 2H), 2.84 (m,
1H), 2.25 (t, J = 10.8 Hz, 1H), 2.05 (d, J = 11.2 Hz, 2H),
1.76 (m, 2H). 13C-NMR (100 MHz, DMSO-D6) δ 163.9,
161.5, 158.3, 155.5, 152.8, 150.3, 149.5, 140.1, 134.7,
133.4, 131.1, 130.2, 127.1, 126.2, 125.7, 124.6, 123.8,
122.8, 121.5, 120.1, 115.8, 115.3, 115.1, 114.7, 114.6,
114.4, 114.2, 69.9, 56.7, 52.9, 33.0, 31.7. HRMS-ESI(+)
calculated for C33H29ClFN7OS, 626.1905; found
626.1882 [M+H]+. MP: 207.8–208.3 °C.
Compound 9 Yield 81%, white solid powder; MS: [M+H]
+
1
210.09; H-NMR (400 MHz, CDCl3-D) δ 3.72 (m, 2H),
3.20 (m, 2H), 2.06 (s, 1H), 2.12 (d, J = 2.4 Hz, 1H), 1.81
(m, 2H), 1.60 (m, 2H), 1.47 (s, 9H). 13C-NMR (100 MHz,
CDCl3-D) δ 154.8, 86.4, 79.5, 69.5, 31.2, 28.4, 26.7.
General procedure for the synthesis of compound 12
A solution of compound 9 (0.38 g, 1.82 mmol) and HCl
(0.2 g, 5.46 mmol) in dichloromethane was stirred at room
temperature for 3 h. The solvent was evaporated under
reduced pressure. Water (80 ml) was added and the PH was
adjusted to 9 by adding 1 M NaOH solution. The product
was extracted into ethylacetate and separated the organic
layer. The solvent was removed under vacuum. Then we get
the rough product compound 10 (low boiling point). And
then the solution of compound 10, compound 11 (0.70 g,
6.24 mmol), acetic acid (0.05 g, 0.91 mmol) in methanol
was added sodium tris(acetoxy)borohydride (0.54 g, 2.60
mmol) in methanol at room temperature for 18 h. Then the
solvent was evaporated under reduced pressure. Then water
was added and the product was extracted by ethyl acetate
(3 × 50 ml). The crude material was purified by column
chromatography to obtain compound 12 (98 mg, 28%).
General procedure for the synthesis of compounds 15a–c
A solution of compound 14a–c (0.58 mmol), compound
2 (0.2 g, 0.48 mmol), Cu2O-NP catalyst in acetonitrile (6
ml) and DMF (2 ml), was stirred at room temperature for
about 5 h. The reaction mixture was filtered through a
celite pad to remove the Cu2O-NP catalyst. The acet-
onitrile was evaporated under reduced pressure. The
residue in DMF was poured into cold water, and the
product was extracted with ethyl acetate (3 × 20 ml). The
organic layer was separated and the solvent distilled off
completely under reduced pressure. The crude material
was purified by column chromatography to afford
product.
Compound 15a Yield 36%, yellow solid powder; 1H-
NMR (400 MHz, DMSO-D6) δ 10.01 (s, 1H), 9.04 (s, 1H),
8.64 (s, 2H), 8.36 (d, J = 6.8 Hz, 1H), 8.04 (d, J = 2.4 Hz,
1H), 7.98 (s, 1H), 7.75 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H),
7.48 (q, J = 7.3 Hz, 1H), 7.31 (q, J = 8.7 Hz, 3H), 7.19 (m,
1H), 5.27 (s, 2H), 4.84 (t, J = 5.2 Hz, 1H), 3.76 (q, J = 6.3
Hz, 1H), 2.93 (t, J = 6.8 Hz, 2H). 13C-NMR (100 MHz,
DMSO-D6) δ 163.9, 161.5, 158.2, 155.5, 150.4, 146.6,
140.1, 134.7, 133.3, 131.1, 130.3, 125.7, 124.6, 123.8,
122.8, 121.5, 115.3, 115.1, 114.8, 114.6, 114.4, 114.0,
69.9, 60.6, 29.6; HRMS-ESI(+) calculated for
C25H20ClFN6O2, 491.1399; found 491.1379 [M+H]+; MP:
200.4–201.1 °C.
Compound 12 Yield 28%, white solid powder; MS: [M
1
+H]+ 206.03; H-NMR (400 MHz, CDCl3-D) δ 7.23 (d, J
= 4.8 Hz, 1H), 6.94 (m, 2H), 3.73 (s, 2H), 2.77 (m, 2H),
2.41 (s, 1H), 2.25 (s, 2H), 2.09 (d, J = 2.0 Hz, 1H), 1.89 (m,
2H), 1.72 (m, 2H); 13C-NMR (100 MHz, CDCl3-D) δ
141.5, 126.3, 125.1, 87.4, 69.1, 57.3, 51.7, 31.5, 29.7, 26.7.
General procedure for the synthesis of compound 13
A solution of compound 12 (0.07 g, 0.37 mmol), compound
2 (0.14 g, 0.33 mmol), Cu2O-NP catalyst in acetonitrile (5
ml) and DMF (2 ml) was stirred at room temperature for
about 5 h. The reaction mixture was filtered through a celite
pad to remove the Cu2O-NP catalyst. The acetonitrile was
evaporated under reduced pressure. The residue in DMF
was poured into cold water, and the product was extracted
with ethyl acetate (3 × 20 ml). The organic layer was sepa-
rated and the solvent distilled off completely under reduced
pressure. The crude material was purified by column chro-
matography to obtain product.
Compound 15b Yield 22%, yellow solid powder; 1H-
NMR (400 MHz, DMSO-D6) δ 9.99 (s, 1H), 9.04 (s, 1H),
8.62 (s, 2H), 8.35 (d, J = 14.0 Hz, 1H), 8.03 (d, J = 2.4 Hz,
1H), 7.98 (s, 1H), 7.76 (dd, J1 = 2.4 Hz, J2 = 9.2 Hz, 1H),
7.48 (m, 1H), 7.31 (q, J = 9.2 Hz, 3H), 7.19 (m, 1H), 5.26
(s, 2H), 4.43 (t, J = 5.0 Hz, 1H), 3.47 (q, J = 6.0 Hz, 2H),
2.77 (t, J = 7.6 Hz, 2H), 1.75 (m, 2H), 1.55 (m, 2H). 13C-

Medicinal Chemistry Research
Scheme 1 Synthesis of target
products with 1, 2, 3-triazol-1-yl
(6a–f). Reagents and conditions:
a NaN3, CuI, L-Pro, NaOH,
DMSO, 50–65 °C, 21%. b
NaHCO3, ethanol, 25–78 °C,
18–54%. c Cu2O-NP, r.t, 22–
38%
NMR (100 MHz, DMSO-D6) δ 163.9, 161.5, 158.2, 155.5,
150.4, 149.0, 140.1, 134.7, 133.3, 131.1, 130.3, 125.7,
124.6, 123.8, 122.8, 121.6, 120.8, 115.3, 115.1, 114.8,
114.6, 114.4, 114.1, 69.9, 60.9, 32.5, 25.9, 25.3; HRMS-
ESI(+) calculated for C27H25ClFN6O2, 519.1722; found
519.1707 [M+H]+; MP: 175.3–175.8 °C.
Inhibition of cell growth in vitro
SKBR3, SUM-159 and KG-1a cells were cultured in RPMI
1640 supplemented with 10% FBS at 37 °C in a 5% CO2
incubator. Cells were seeded in 96-well plates at a density
of 3000–20,000 cells per well. SKBr3, SUM-159, KG-1a
cells were treated for 72 h with increasing concentrations of
different compounds. Cell viability was measured using
MTT assay. The number of living cells was directly pro-
portional to the absorbance at 492 or 570 nm of a formazan
product reduced from MTT by living cells. The IC50 value
was obtained using SPSS 14.0 software. The results were
derived from at least three independent experiments
performed.
Compound 15c Yield 32%, yellow solid powder; 1H-
NMR (400 MHz, DMSO-D6) δ 9.99 (s, 1H), 9.09 (s, 1H),
8.70 (s, 1H), 8.42 (s, 1H), 8.03 (s, 1H), 7.75 (d, J = 10.0 Hz,
1H), 7.48 (q, J = 7.3 Hz, 1H), 7.32 (q, J = 8.5 Hz, 3H), 7.19
(m, 1H), 5.56 (s, 1H), 5.27 (s, 2H), 5.00 (m, 1H), 1.52 (d, J
= 6.4 Hz, 3H). 13C-NMR (100 MHz, DMSO-D6) δ 163.9,
161.5, 158.2, 154.8, 150.4, 140.1, 134.7, 133.3, 131.1,
125.6, 124.7, 123.8, 122.8, 121.6, 120.3, 115.3, 115.1,
114.8, 114.6, 114.4, 114.0, 69.9, 62.2, 24.3; HRMS-ESI(+)
calculated for C25H21ClFN6O2, 491.1399; found 491.1395
[M+H]+; MP: 186.4–187.0 °C.
Acute toxicity test of Zebrafish
Zebrafish were fed regularly for at least 3 days later, two
males and two females were placed in a shallow water

Medicinal Chemistry Research
Scheme 2 Synthesis of target
products with 1, 2, 3-triazol-1yl
(13). Reagents and conditions: a
K2CO3, methanol, r.t., 81%. b
HCl, dichloromethane, r.t.; c
NaHCO3, KI,ethanol., 40.16%.
d Cu2O-NP, r.t., 43%
environment, and the experiment was conducted in three
groups. The next day zebrafish eggs were collected into the
culture medium. The zebrafish eggs were cultured at 27 °C
in an incubator for 48 h, and then they were inhaled in 24-
well plates at a density of 10 per well. The zebrafish were
treated with increasing concentrations of different com-
pounds. The number of dead fish was recorded after 24 h.
Scheme 3 Synthesis of target products with 1, 2, 3-triazol-1yl (15a–c).
Reagents and conditions: a Cu2O-NP, r.t., 22–36%
Results and discussion
Compounds 15a–c were prepared with a similar strategy
Chemistry
and click reactions (Scheme 3).
Compound 1 (Ramanadham et al. 2010) was treated with
NaN3 to obtain compound 2, and the addition of L-proline
and NaOH improved the reaction yield and rate (Zhu and
Ma 2004). Treatment of the alkylating compounds 4a–f
with propargyl chloride 3 in K2CO3 and ethanol provided
compounds 5a–f. Compounds 6a–f were synthesized
though an azide-alkyne cycloaddition of compounds 5a–f
and compound 2 with copper (II)-oxide nanoparticles
(Cu2O-NP) as a catalyst (Scheme 1) (Zhang et al. 2010).
Inhibition of cell growth in vitro
For viability assays, the synthesized compounds were
converted to a methyl sulfonic acid salt form to improve
their solubility prior to testing (Table 1).
All compounds were evaluated against the SKBR3,
SUM159, and KG-1a cells (Table 1). Lapatinib was used as
a positive control in these assays. Compounds with tertiary
amino group 6b–f are the most active compounds in the
series, which could be protonated and form quaternary
ammonium ion in the solvation state at physiological PH.
Similarly, compounds with hydroxyl groups 15a–c are least
active or inactive. It suggests requirement of a particularly
favorable electrostatic environment for the activity.
Compound 6a showed poorer activity than lapatinib, its
IC50 values were >30 µM against SKBR3 and SUM159
cells. Against the KG-1a cell line, it showed an approximate
2-fold reduction in biological activity compared with lapa-
tinib (IC50 = 21.54 µM vs. 9.76 µM).
A
Seyferth-Gilbert homologation reaction between
compounds 7 and 8 in dry methanol produced compound 9
(Quesada and Taylor 2005). Then, deprotection of the Boc
group with HCl provided amine 10. Due to the instability of
compound 10, the crude product of compound 10 was
coupled with compound 11 to form compound 12 (Bamford
and Dean 2004). Finally, compound 13 was synthesized
through an azide-alkyne cycloaddition with compounds 12
and 2 (Scheme 2).

Medicinal Chemistry Research
Table 1 Inhibitory activity of triazole lapatinib derivatives against SKBR3, UM159 and KG-1a cells in vitro for 72 h
SKBR3
SUM159
IC50(μΜ)
KG-1a
Compound
lapatinib
R
IC50(μΜ)
IC50(μΜ)
_
20.11 1.37
12.14 0.88
9.76 0.98
21.54 4.30
12.19 2.96
5.48 0.25
6.42 1.07
5.87 0.69
2.06 0.44
3.28 0.24
>30
6a
6b
6c
>30
>30
14.69 1.82
7.47 0.49
4.79 0.57
5.80 0.20
6.24 2.05
>30
3.35 0.30
5.16 0.27
5.19 1.40
2.54 0.39
5.39 0.12
2.21 0.33
>30
6d
6e
6f
13
15a
15b
15c
>30
10.66 1.36
11.18 1.41
27.05 3.77
>30
>30
>30

Medicinal Chemistry Research
Among compounds 6c–f, compound 6c had the highest
toxicity, at a concentration of 50 μΜ for 24 h the fatality
rate of zebrafish was as high as 50%. In contrast, compound
6e had the lowest toxicity; at a concentration of 200 μΜ for
24 h the fatality rate of zebrafish was 0%. Compound 6d
had a lower toxicity than compound 6f, at a concentration of
200 μΜ for 24 h the fatality rates of zebrafish were 10 and
60%, respectively. Therefore, compound 6e is a promising
lead compound.
Conclusion
Fig. 1 Toxicity of lapatinib and triazol derivatives 6c–f in zebrafish for
24 h
A series of triazole derivatives of lapatinib were efficiently
synthesized via an azide-alkyne cycloaddition catalyzed by
Cu2O-NP. The resulting derivatives were subsequently
evaluated for their anti-cancer activities against the breast
cancer cell lines SKBR3 and SUM159, and the stem cell-
like leukemia cell line KG-1a. Based on cell viability
assays, replacement of the furan ring with a triazole 6a
resulted in a lower level of anti-cancer activity, while the
further introduction of amino moieties, compounds 6b–f
and 13, maintained or enhanced the anti-cancer activities.
However, the introduction of hydroxyl groups in com-
pounds 15a–c made them essentially inactive against
SKBR3, SUM159 and KG-1a cell lines. Compound 15b
exhibited a significant reduction in activity against SKBR3
cells compared with KG-1a cells. Finally, the toxicities of
compounds 6c–f were evaluated in zebrafish; compound 6e
had the lowest toxicity and was less toxic than lapatinib to
zebrafish. Thus, compound 6e is a promising lead com-
pound worthy of further investigation.
Compounds 6b–f contained amino moieties in their side
chains and had the same activities against SKBR3,
SUM159, and KG-1a cells. For SKBR3 and KG-1a cells,
the activity of compound 6b was almost the same as laptinib
(IC50 = 14.69 µM vs. 20.11 µM, and 12.19 µM vs. 9.76 µM,
respectively). However, compound 6b was specific for the
SUM159 cell line; the IC50 value was approximately 4
times less than lapatinib (IC50 = 3.35 µM vs. 12.14 µM).
The IC50 for compounds 6c-f against the SKBR3 cell line
were 7.47 µM, 4.79 µM, 5.80 µM, and 6.24 µM, respec-
tively, which were 3–5 times more potent than lapatinib
(IC50 = 20.11 µM). Against the SUM159 cell line, com-
pounds 6c–f had IC50 values of 5.16 µM, 5.19 µM, 2.55 µM,
and 5.39 µM, respectively, which were 2–4 times more
potent than lapatinib (IC50 = 12.14 µM). Against the KG-1a
cell line, compounds 6c–f had IC50 values of 2.06–6.42 µM,
which represented slightly stronger activity than lapatinib
(IC50 = 9.76 µM).
Compound 13 likewise contained amino moieties in its
side chains and had stronger activities than lapatinib against
SUM159 and KG-1a cells (IC50 = 2.21 µM vs. 12.14 µM
and 3.28 µM vs. 9.76 µM, respectively), but against SKBR3
cells, it exhibited no activity (IC50 > 30 μΜ).
Compounds 15a–c contained hydroxyl groups. Against
SKBR3, SUM159 and KG-1a cells, compounds 15a and
15c were essentially inactive (IC50 > 30 µM). Compound
Availability of data and materials
All data generated or analysed during this study are inclu-
ded in this published article.
Funding This work was supported by the Natural Science Foundation
of Tianjin (No. 16JCQNJC13800 to CGB), National Science &
Technology Pillar Program (No. 2015BAI12B15 to ZST), and Higher
Learning Basic Scientific Research Business Funded Projects of
Tianjin Medical University of Tianjin (No. 2016YD03 to YHS).
15b was two times more potent than lapatinib (IC50
=
10.66 µM vs. 20.11 µM) against SKBR3 cells, while against
KG-1a cells, compound 15b was three times less potent
than lapatinib (IC50 = 27.05 µM vs. 9.76 µM). The activity
of 15b was comparable to lapatinib against SUM159 cells.
Thus, compounds 6c–f are worth further study.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Acute toxicity test of Zebrafish
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