Synthesis and anticancer activities of novel 1,2,4-triazolo[3,4-a] phthalazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.07.031

Trying to develop potent and selective anticancer agents, two series of novel 1,2,4-triazolo[3,4-a]phthalazine derivatives were designed and synthesized. Their antitumor activities were evaluated by MTT method against four selected human cancer cell lines (MGC-803, EC-9706, HeLa and MCF-7). Our results showed that compound 11h exhibited good anticancer activities compared to 5-fluorouracil against the four tested cell lines, with IC₅₀ values ranging from 2.0 to 4.5 μM. Flow cytometry analysis indicated that compound 11h induced the cellular early apoptosis and cell cycle arrest at G2/M phase in EC-9706.

Full text of DOI:10.1016/j.ejmech.2014.07.031

European Journal of Medicinal Chemistry 85 (2014) 235e244
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activities of novel 1,2,4-triazolo[3,4-a]
phthalazine derivatives
Deng-Qi Xue, Xu-Yao Zhang, Chao-Jie Wang, Li-Ying Ma, Nan Zhu, Peng He,
Kun-Peng Shao, Peng-Ju Chen, Yi-Fei Gu, Xiao-Song Zhang, Cai-Feng Wang, Cong-Hui Ji,
Qiu-Rong Zhang*, Hong-Min Liu*
New Drug Research & Development Center, School of Pharmaceutical Sciences, Zhengzhou University, No. 100, KeXue DaDao, Zhengzhou 450001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Trying to develop potent and selective anticancer agents, two series of novel 1,2,4-triazolo[3,4-a]
phthalazine derivatives were designed and synthesized. Their antitumor activities were evaluated by
MTT method against four selected human cancer cell lines (MGC-803, EC-9706, HeLa and MCF-7). Our
results showed that compound 11h exhibited good anticancer activities compared to 5-fluorouracil
against the four tested cell lines, with IC50 values ranging from 2.0 to 4.5 mM. Flow cytometry analysis
indicated that compound 11h induced the cellular early apoptosis and cell cycle arrest at G2/M phase in
Received 8 January 2014
Received in revised form
8 July 2014
Accepted 9 July 2014
Available online 10 July 2014
EC-9706.
Keywords:
Triazole
© 2014 Published by Elsevier Masson SAS.
Phthalazine
Anticancer
Apoptosis
Cell cycle arrest
1. Introduction
Triazole is a versatile moiety that found in a large variety of
bioactive molecules, such as anti-fungal [11], anti-bacterial [12],
Cancer is now considered as one of the most serious health
problems and leading cause of death over the world [1]. During the
last decade, multiple synthetic phthalazine derivatives have been
reported as antitumor agents [2e4]. Among them, Vatalanib (PTK-
787) (Fig. 1), a VEGFR (vascular endo-thelial growth factor receptor)
inhibitor, is currently in phase III clinical trials for advanced colo-
rectal cancer [5] and additional phase II studies for leukemia [6],
metastatic gastrointestinal stromal tumor (GIST) [7], and metastatic
melanoma [8].
In order to improve the antitumor efficacy of vatalanib, many
new phthalazine derivatives combined vatalanib with the piper-
azinyl group in one structure were developed. Such as compounds
I, II and III (Fig. 1), which have been described with highly potent
against human liver cancer in vitro [9,10]. Based on the structures of
phthalazine derivatives I, II and III, we supposed that piperazine
function in the position 1 of phthalazine was beneficial for the
bioactivity.
anti-allergic [13], anti-HIV [14], anti-tubercular [15] and anti-
inflammatory agents [16]. So we have designed a series of 6-
nitrogen-containing heterocyclic compound substituted-1,2,4-tri-
azolo[3,4-a]phthalazine derivatives and accessed to it by an effi-
cient synthetic route (Scheme 1).
Furthermore, we combined 1,2,4-triazolo[3,4-a]phthalazine
derivatives with the piperazinyl group in one structure, and inser-
ted an acetyl-flexible linker into the structure to get the new
chemical entities. Various substituents R1 were introduced into the
C-3. R3 was linked to the aniline ring (Fig. 1). Finally, we obtained
twenty-three novel 1,2,4-triazolo[3,4-a]phthalazine derivatives
and their cytotoxic activities against human cancer cell lines in vitro
and effects on the cell cycle and apoptosis were determined in this
study.
2. Results and discussion
2.1. Chemistry
The synthetic route was shown in Scheme 1. The compound 1
was prepared by the reaction of phthalic anhydride with hydrazine
hydrate in acetic acid, which reacted further with the refluxing
* Corresponding authors.
E-mail addresses: zqr406@sina.com (Q.-R. Zhang), liuhm@zzu.edu.cn
(H.-M. Liu).
http://dx.doi.org/10.1016/j.ejmech.2014.07.031
0223-5234/© 2014 Published by Elsevier Masson SAS.

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D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
Fig. 1. The structures of PTK-787, I, II, III and the target compounds.
phosphorus oxychloride to yield the compound 2 [17]. The com-
pound 3 was obtained by the reaction of 2 with hydrazine hydrate
in ethanol [18]. Compounds 4a, 4b and 4c were prepared by the
reaction of 3 with trimethoxy methane, benzoyl chloride and acetyl
chloride in dioxane, respectively [19,20]. According to our previ-
ously reported method [21], compounds 4a, 4b and 4c reacted
further with appropriate nitrogen-containing heterocyclic com-
pound in N, N-dimethylformamide to yield the target compounds
of 5aed, 6aeb and 7aed. The compounds 10aee were synthesized
via a series of substituted anilines with 2-chloroacetyl chloride in
the presence of Et3N in acetone at room temperature [10]. The
compounds 11aem successfully were obtained via the reaction of
intermediate 5a, 6a and 7a with 10aee in the presence of K2CO3 in
refluxing acetone, respectively.
with IC50 values ranging from 2.0 to 4.5
m
M. Compound 11f
M) was about 5.9-fold potent than 5-Fluorouracil
M) against MGC-803 cell line. Compound 11b was
(IC50 ¼ 1.7
m
m
(IC50 ¼ 10.0
more potent than compound 11a against the four tested cell lines.
Compounds 11j and 11i have the similar SAR. The results showed
that the 4-Cl atom substitution on aniline was more beneficial for
the bioactivity than 4-F atom. Compounds 11c, 11e and 11k were
less potent against the four tested cell lines. The results showed
that the 4-OCH3 substitution on aniline was not beneficial for the
bioactivity. Compound 11f was more potent than compounds 11b
and 11j against the four tested cell lines. Compound 11h showed
better inhibitory activity against all the assayed cell lines than that
of compounds 11e and 11m. The results showed that the C-3-Ph
substitution was more beneficial for the bioactivity than C-3-H
and C-3-CH3 substitution. Compounds 11aee were more potent
than compound 5a against the four tested cell lines. Compound 11h
was more potent than compound 6a against the four tested cell
lines. Compounds 11j and 11m were more potent than compound
7a against the four tested cell lines. Compounds 11i and 11l showed
better inhibitory activity than compound 7a against three of the
four tested cell lines. The results showed that the target compounds
which (substituted-phenyl)acetamide was linked to the piperazino
showed better cytotoxic activities.
2.2. Evaluation of biological activity
2.2.1. Anticancer activity
The cytotoxic effects of synthesized compounds were evaluated
against four human cancer cell lines (MGC-803, EC9706, Hela and
MCF-7) by using MTT assay method [22,23]. 5-Fluorouracil was
used as reference compound. To examine the effects of synthesized
compounds (5ae7d), on cell growth, we conducted a dose escala-
tion experiment (Table 1). The results indicated that the four hu-
man cancer cell lines were refractory to 6a-induced cytotoxic
2.2.2. Apoptosis assay
effects at doses as high as 12e18.6
other synthesized compounds up to 37e128
inhibition of cell growth. The results showed that the C-3-Ph sub-
stitution is more beneficial for the bioactivity than C-3-H and C-3-
CH3 substitution. Compounds 5b, 7b and 7d had a weak inhibitory
effect against all the assayed cell lines with an IC50 higher than
m
M, whereas treatment with
M resulted in a 50%
Apoptosis is considered a major way that most of the anticancer
drugs kill tumor cells [24]. Compound 11h was chosen to further
explore its mechanism of action. In order to better characterize the
mode of cell death induced by compound 11h, we performed a
biparametric cytofluorimetric analysis using propidium iodide (PI)
and Annexin-V-FITC in EC-9706 cells. After treatment with com-
pound 11h for 12 and 24 h at different concentrations (0, 0.8, 1.6,
m
40
mM.
As shown in Table 2, the results suggested that most of them
3.2 mM), EC-9706 cells were labeled with the two dyes, and the
showed moderate to excellent cytotoxic activities against four
tested human cancer cell lines with the IC50 values ranging from 1.7
resulting red (in the web version) (PI) and green (in the web
version) (FITC) fluorescence was monitored by flow cytometry. As
shown in Fig. 2 and Fig. 3, after treatment for 12 h (Fig. 2), com-
pound 11h caused the extent of early apoptosis to increase
to 124.5
mM. Among them, the most potent compound 11h was
more potent than 5-Fluorouracil against the four tested cell lines,

D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
237
Scheme 1. Reagents and conditions: Ⅰ: H2NeNH2$H2O, CH3COOH, 120 ꢀC, 90%; Ⅱ: POCl3, 110 ꢀC, 90%; Ⅲ: C2H5OH, H2NeNH2$H2O, 78 ꢀC, 77%; Ⅳ: 4a: CH(OC2H5)3, dioxane, N(C2H5)3,
110 ꢀC, 82%; 4b: CH3COCl, dioxane, N(C2H5)3, 110 ꢀC, 76%; 4c: C6H5COCl, dioxane, N(C2H5)3, 110 ꢀC, 78%; Ⅴ: appropriate nitrogen-containing heterocyclic compound, NaOH, DMF,
100 ꢀC. Ⅵ: Et3N/acetone, r.t. 4e8 h; Ⅶ: K2CO3/acetone, 10aee, reflux, 7e12 h.
significantly from 7.9% (DMSO control) to 32.9%. After treatment for
24 h (Fig. 3), compound 11h caused the extent of early apoptosis to
increase significantly from 8.2% (DMSO control) to 41.4%. The re-
sults showed that compound 11h markedly increased the cellular
apoptosis in a concentration-dependent manner.
2.2.3. Cell cycle analysis
Molecules that inhibit the growth of cancer cells invariably
cause alteration of cell cycle distribution, with preferential G2/M
blockade [25]. We therefore examined the effect of different
Table 2
Anticancer activity of target compounds 11aem.
Compd R1
R3
IC50
MGC-803
(m
M)a
Table 1
Anticancer activity of target compounds 5aed, 6aeb and 7aed.
EC9706
Hela
MCF-7
Compd R1 R2
IC50
MGC-803 EC9706
Piperazin-1-yl >128 >128
(m
M)a
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
5-Fu
H
H
H
H
4-F
4-Cl
53.9 0.8 46.2 0.7 85.6 0.1
14.7 0.7 22.4 0.4 23.8 0.9
118.3 1.7
84.3 1.2
Hela
MCF-7
4-OCH3 61.1 0.8 67.7 0.9 105.1 1.5 85.4 0.5
3-CF3
2-F
4-Cl
3-CF3
2-F
5a
5b
5c
5d
6a
H
H
H
H
>128
>128
23.2 0.6 43.8 0.3 24.4 0.7
45.2 1.4 81.7 1.3 124.5 2.5 46.5 0.8
49.2 1.1
Piperidin-1-yl 61.4 0.7 66.9 0.8 55.4 0.9 108.5 1.2
Morpholin-4-yl >128
Pyrrolidin-1-yl >128
H
>128
>128
>128
>128
>128
>128
Ph
Ph
Ph
1.7 0.4
19.2 0.7 >128
4.5 0.3 2.0 0.2
11.9 0.6 13.4 0.9
27.7 0.2
>128
3.4 0.5
>128
29.5 0.9
>128
>128
39.0 1.1
2.0 0.1
Ph Piperazin-1-yl 12.2 0.3 9.5 0.4 17.0 0.1 18.6 0.8
Ph Morpholin-4-yl 53.5 1.1 37.2 1.2 107.9 2.1 101.5 1.4
6b
7a
7b
7c
7d
5-Fu
CH3 4-F
CH3 4-Cl
CH3 4-OCH3 >128
CH3 3-CF3
CH3 2-F
31.7 0.5 43.3 0.7 47.6 1.4
15.8 0.7 42.5 1.1 38.3 0.8
91.1 1.2 >128
43.2 1.2 31.2 0.8 57.4 0.6
27.2 0.9 54.3 0.4 46.0 1.1
CH3 Piperazin-1-yl >128
CH3 Piperidin-1-yl 40.1 0.4 61.3 0.7 57.1 0.6 107.5 1.1
CH3 Morpholin-4-yl >128 >128 >128 >128
CH3 Pyrrolidin-1-yl 92.1 0.9 >128 76.7 1.2 >128
10.0 0.4 4.7 0.5 12.0 0.7 4.6 0.8
>128
>128
>128
24.9 0.8
4.6 0.8
10.0 0.4 4.7 0.5
12.0 0.7
a
a
Anticancer activity was assayed by exposure for 72
h
to substances and
Anticancer activity was assayed by exposure for 72 h to substances and
expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50).
Dates are presented as the means SDs of three independent experiments.
expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50).
Dates are presented as the means SDs of three independent experiments.

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D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
Fig. 2. Representative flow cytometric histograms of apoptotic EC-9706 cells after 12 h treatment with 11h at different concentrations.
concentrations of compound 11h on cell cycle progression with EC-
9706 cell line (Figs. 4 and 5). A cell-cycle cytotoxicity assay was
performed by treating EC-9706 cells at various concentrations of
chromatography (TLC) was carried out on glass plates coated with
silica gel (Qingdao Haiyang Chemical Co., G60F-254) and visualized
by UV light (254 nm). Melting points were determined on an X-5
micromelting apparatus and are uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker 400 MHz and 100 MHz spec-
trometer respectively. High resolution mass spectra (HRMS) were
recorded on a Waters Micromass Q-Tof Micro mass spectrometer by
electrospray ionization (ESI).
compound 11h (0, 0.8, 1.6, 3.2 mM) for 12 h (Fig. 4) and 24 h (Fig. 5).
Compound 11h caused an increase in the proportion of cells in G2/
M phase in a concentration-dependent manner with a concomitant
decrease of cells in other phases of the cell cycle. Specifically, the
percentage of cells in G2/M phase at different concentrations was
12.49%, 12.47%, 19.82% and 26.22% for 12 h. The percentage of cells
in G2/M phase at different concentrations was 16.03%, 24.19%,
27.10% and 44.42% for 24 h respectively.
4.2. Preparation of 2,3-dihydrophthalazine-1,4-dione (1)
Hydrazine hydrate (2.8 mL, 44 mmol) was added to a stirred
solution of phthalic anhydride (6.01 g, 40 mmol) in acetic acid
(22 mL). The mixture was heated to 120 ꢀC for 4 h. The reaction
mixture was cooled to room temperature and filtered through a
buchner funnel. Then the filter cake was washed with petroleum
ether (2 ꢁ 10 mL) and dried in vacuum. 1 (5.91 g, 90%) was obtained
3. Conclusions
In summary, twenty-three novel 1,2,4-triazolo[3,4-a]phthala-
zine derivatives were designed and synthesized. The cytotoxic ef-
fects observed in response to compound 11h were associated with
the induction of apoptotic cell death. It was worth mentioning that
Ph at C-3 was most active in all series and the target compounds
which (substituted-phenyl) acetamide was linked to the piperazino
showed better cytotoxic activities.
as a white solid. Mp: 181e182 ꢀC. 1H NMR (400 MHz, DMSO)
d 11.55
(s, 2H, NH), 8.08 (dd, J ¼ 5.7, 3.4 Hz, 2H, ArH), 7.96e7.76 (m, 2H,
ArH).
4.3. Preparation of 1,4-dichlorophthalazine (2)
4. Experimental section
The compound 1 (1.68 g, 10 mmol) was added to a stirred so-
lution of phosphorus oxychloride (15 mL). The mixture was heated
to 110 ꢀC for 1 h. After the reaction was complete (monitored by
TLC), the reaction mixture was cooled to room temperature. The
mixture was added dropwise to crushed ice with stirring for
4.1. General
Reagents and solvents were purchased from commercial sour-
ces and were used without further purification. Thin-layer

D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
239
Fig. 3. Representative flow cytometric histograms of apoptotic EC-9706 cells after 24 h treatment with 11h at different concentrations.
10 min. Then the mixture was filtered through a buchner funnel.
The filter cake was washed with H2O until neutral and dried in
vacuum. 2 (1.85 g, 90%) was obtained as a white solid. Mp:
yield a solid. The residue obtained was purified by chromatography
on silica gel using petroleum ether/ethyl acetate to give derivatives
4 as a yellow solids.
192e193 ꢀC; 1H NMR (400 MHz, DMSO)
ArH), 8.11e7.94 (m, 3H, ArH).
d
8.29 (d, J ¼ 7.6 Hz, 1H,
4.5.1. 6-Chloro-1,2,4-triazolo[3,4-a]phthalazine (4a)
Yield 82%. Yellow solid. Mp: 176e177 ꢀC; 1H NMR (400 MHz,
4.4. Preparation of 1-chloro-4-hydrazinylphthalazine (3)
DMSO)
d
9.64 (d, J ¼ 37.1 Hz, 1H, CH), 8.52 (d, J ¼ 7.8 Hz, 1H, ArH),
8.30e8.18 (m, 1H, ArH), 8.16e8.06 (m, 1H, ArH), 7.97 (dt, J ¼ 21.7,
The compound 2 (1.98 g, 10 mmol) was added to a boiling so-
lution of hydrazine hydrate (3.73 mL, 76.5 mmol) in ethanol
(50 mL). The mixture was heated at reflux for 0.5 h. The reaction
mixture was cooled to room temperature and filtered through a
buchner funnel. Then the filter cake was washed with ether
(2 ꢁ 20 mL) and dried in vacuum. 3 (1.49 g, 77%) was obtained as a
yellow flocks. Mp: 259e260 ꢀC; 1H NMR (400 MHz, DMSO)
7.2 Hz, 1H, ArH).
4.5.2. 6-Chloro-3-phenyl-1,2,4-triazolo[3,4-a]phthalazine (4b)
Yield 76%. Yellow solid. Mp: 173e174 ꢀC; 1H NMR (400 MHz,
DMSO)
8.30 (dd, J ¼ 8.2, 1.4 Hz, 2H, ArH), 8.23e8.15 (m, 1H, ArH), 8.12e8.01
(m, 1H, ArH), 7.71e7.59 (m, 3H, ArH).
d
8.65 (d, J ¼ 7.5 Hz, 1H, ArH), 8.36 (d, J ¼ 7.3 Hz, 1H, ArH),
d
8.32e8.27 (m, 1H, ArH), 8.21 (s, 1H, NH), 8.07e8.03 (m, 1H, ArH),
8.03e7.99 (m, 1H, ArH), 7.98e7.95 (m, 1H, ArH), 7.89 (s, 2H, NH2).
4.5.3. 6-Chloro-3-methyl-1,2,4-triazolo[3,4-a]phthalazine (4c)
Yield 78%. Yellow solid. Mp: 169e170 ꢀC; 1H NMR (400 MHz,
4.5. General procedure for synthesis of 4 (4a, 4b, 4c)
CDCl3)
d
8.64 (d, J ¼ 7.9 Hz, 1H, ArH), 8.24 (d, J ¼ 8.2 Hz, 1H, ArH),
7.99 (t, J ¼ 7.6 Hz, 1H, ArH), 7.85 (t, J ¼ 7.8 Hz, 1H, ArH), 2.81 (s, 3H,
The compound 3 (1.49 g, 7.73 mmol) was added to a stirred
solution of triethylamine (1.08 mL, 7.73 mmol) and triethoxy
methane or appropriate acyl chloride (9.28 mmol) in dioxane. The
mixture was heated to 110 ꢀC. After the reaction was complete
(monitored by TLC), the solvent was removed under reduced
pressure, and the solid obtained was partitioned between
dichloromethane (150 mL) and water (3 ꢁ 100 mL). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo to
CH3).
4.6. General procedure for the synthesis of compounds 5aed, 6aeb
and 7aed
NaOH (0.12 g, 3 mmol) and appropriate nitrogen-containing
heterocyclic compound (1.2 mmol) were added to a stirred solu-
tion of 4 (1 mmol) in DMF (1.5 mL). The reaction mixture was

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D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
Fig. 4. Effect of compound 11h on cell cycle in EC-9706 cells incubated for 12 h.
heated to 100 ꢀC. After the reaction was complete (monitored by
TLC), the reaction mixture was cooled to room temperature. The
solvent was poured into water (10 mL) and separated by filtration.
The residue obtained was recrystallized from DMF to give 5aed,
6aeb and 7aed. The yield, 1H NMR, 13C NMR, HR-MS (ESI) data of
each compound were given below.
J ¼ 7.7 Hz, 1H, ArH), 4.00 (d, J ¼ 3.9 Hz, 4H, CH2), 3.43 (d, J ¼ 4.0 Hz,
4H, CH2). 13C NMR (101 MHz, CDCl3)
158.14, 139.37, 133.18, 130.31,
d
127.02, 126.34, 124.70, 124.15, 120.45, 66.57, 51.78.HR-MS (ESI):
Calcd. C13H13N5O, [MþH]þ m/z: 256.1198, found: 256.1194.
4.6.4. 6-(Pyrrolidin-1-yl)-1,2,4-triazolo[3,4-a]phthalazine (5d)
Yield 78%. White solid. Mp: 312e313 ꢀC; 1H NMR (400 MHz,
4.6.1. 6-(Piperazin-1-yl)-1,2,4-triazolo[3,4-a]phthalazine (5a)
Yield 83%. White solid. Mp: 309e310 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.74 (s, 1H, CH), 8.59 (d, J ¼ 7.8 Hz, 1H, ArH), 8.16 (d,
J ¼ 8.3 Hz, 1H, ArH), 7.80 (t, J ¼ 7.3 Hz, 1H, ArH), 7.67 (m, 1H, ArH),
CDCl3)
d
8.87 (s, 1H, CH), 8.66 (d, J ¼ 7.9 Hz, 1H, ArH), 8.06 (d,
3.75 (t, J ¼ 6.5 Hz, 4H, CH2), 2.03 (t, J ¼ 6.5 Hz, 4H, CH2). 13C NMR
J ¼ 8.1 Hz, 1H, ArH), 7.90 (dd, J ¼ 19.0, 11.5 Hz, 1H, ArH), 7.77 (t,
(101 MHz, CDCl3) d 154.88, 142.16, 138.84, 132.17, 129.50, 126.78,
J ¼ 7.7 Hz,1H, ArH), 3.41 (dd, J ¼ 29.3, 25.1 Hz, 4H, CH2), 3.18 (m, 4H,
124.54, 123.67, 120.85, 51.55, 25.79. HR-MS (ESI):Calcd. C13H13N5,
CH2), 1.86 (s, 1H, NH). 13C NMR (101 MHz, CDCl3)
d
158.57, 142.37,
[MþNa]þ m/z: 262.1069, found: 262.1066.
139.39, 132.99, 130.20, 126.54, 124.67, 124.03, 120.73, 52.70, 45.85.
HR-MS (ESI): Calcd. C13H14N6, [MþH]þ m/z: 255.1358, found:
255.1350.
4.6.5. 3-Phenyl-6-(piperazin-1-yl)-1,2,4-triazolo[3,4-a]phthalazine
(6a)
Yield 75%. White solid. Mp: 251e252 ꢀC; 1H NMR (400 MHz,
4.6.2. 6-(Piperidin-1-yl)-1,2,4-triazolo[3,4-a]phthalazine (5b)
Yield 76%. White solid. Mp: 316e317 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.71 (d, J ¼ 7.6 Hz, 1H, ArH), 8.52 (dd, J ¼ 5.3, 3.3 Hz, 2H,
ArH), 8.08 (d, J ¼ 8.1 Hz, 1H, ArH), 7.92e7.85 (m, 1H, ArH), 7.81e7.74
(m, 1H, ArH), 7.60e7.48 (m, 3H, ArH), 3.49e3.40 (m, 4H, CH2),
3.25e3.17 (m, 4H, CH2), 1.96 (s, 1H, NH). 13C NMR (101 MHz, CDCl3)
CDCl3)
d
8.84 (s, 1H, CH), 8.61 (d, J ¼ 7.6 Hz, 1H, ArH), 8.02 (d,
J ¼ 8.1 Hz,1H, ArH), 7.84 (m,1H, ArH), 7.74 (m,1H, ArH), 3.33 (m, 4H,
CH2),1.85 (m, 4H, CH2),1.73 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3)
d 158.09, 148.62, 143.42, 132.93, 130.05, 129.78, 128.54, 127.70,
d
159.12, 142.37, 139.31, 132.76, 130.12, 126.68, 124.51, 123.80,
126.92, 126.31, 125.12, 124.05, 119.96, 52.77, 45.86. HR-MS (ESI):
121.09, 52.63, 25.76, 24.41. HR-MS (ESI): Calcd. C14H15N5, [MþNa]þ
m/z: 276.1225, found: 276.1221.
Calcd. C19H18N6, [MþH]þ m/z: 331.1671, found: 331.1669.
4.6.6. 4-(3-Phenyl-1,2,4-triazolo[3,4-a]phthalazin-6-yl)morpholine
(6b)
4.6.3. 4-(1,2,4-Triazolo[3,4-a]phthalazin-6-yl)morpholine (5c)
Yield 59%. Yellow solid. Mp: 174e175 ꢀC; 1H NMR (400 MHz,
Yield 58%. White solid. Mp: 249e250 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.88 (s, 1H, CH), 8.67 (d, J ¼ 8.2 Hz, 1H, ArH), 8.06 (d,
CDCl3)
d
8.72 (d, J ¼ 7.9 Hz, 1H, ArH), 8.49 (d, J ¼ 7.4 Hz, 2H, ArH),
J ¼ 8.1 Hz, 1H, ArH), 7.89 (dd, J ¼ 14.0, 6.4 Hz, 1H, ArH), 7.78 (t,
8.07 (d, J ¼ 8.1 Hz, 1H, ArH), 7.90 (t, J ¼ 7.6 Hz, 1H, ArH), 7.77 (t,

D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
241
Fig. 5. Effect of compound 11h on cell cycle in EC-9706 cells incubated for 24 h.
J ¼ 7.7 Hz, 1H, ArH), 7.55 (dq, J ¼ 14.4, 7.2 Hz, 3H), 4.03 (m, 4H, CH2),
3.48 (m, 4H, CH2). 13C NMR (101 MHz, CDCl3)
157.65, 148.70,
143.38, 133.13, 130.14, 129.87, 128.59, 127.71, 126.84, 126.12, 125.20,
124.18, 119.68, 66.61, 51.85. HR-MS (ESI): Calcd. C19H17N5O, [MþH]þ
m/z: 332.1511, found: 332.1510.
4.6.9. 4-(3-Methyl-1,2,4-triazolo[3,4-a]phthalazin-6-yl)
morpholine (7c)
Yield 68%. White solid. Mp: 218e219 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
d
8.64 (d, J ¼ 8.1 Hz, 1H, ArH), 8.04 (d, J ¼ 8.1 Hz, 1H, ArH),
7.87 (m, 1H, ArH), 7.75 (dd, J ¼ 11.4, 4.1 Hz, 1H, ArH), 4.00 (m, 4H,
CH2), 3.43 (m, 4H, CH2), 2.76 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3)
d
157.47, 147.76, 142.47, 132.98, 129.96, 126.20, 125.11, 123.91, 119.86,
4.6.7. 3-Methyl-6-(piperazin-1-yl)-1,2,4-triazolo[3,4-a]phthalazine
(7a)
66.60, 51.76, 9.81. HR-MS (ESI): Calcd. C14H15N5O, [MþH]þ m/z:
270.1355, found: 270.1350.
Yield 63%. White solid. Mp: 221e222 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.63 (d, J ¼ 8.0 Hz, 1H, ArH), 8.05 (d, J ¼ 8.1 Hz, 1H, ArH),
4.6.10. 3-Methyl-6-(pyrrolidin-1-yl)-1,2,4-triazolo[3,4-a]
phthalazine (7d)
7.86 (m, 1H, ArH), 7.74 (m, 1H, ArH), 3.46 (s, 1H, NH), 3.41 (m, 4H,
CH2), 3.19 (m, 4H, CH2), 2.76 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3)
Yield 71%. White solid. Mp: 238e239 ꢀC; 1H NMR (400 MHz,
d
157.90, 147.74, 142.50, 132.85, 129.90, 126.38, 125.05, 123.86,
CDCl3)
d
8.61 (m, 1H, ArH), 8.18 (d, J ¼ 8.3 Hz, 1H, ArH), 7.80 (dd,
120.15, 52.55, 45.75, 9.78. HR-MS (ESI): Calcd. C14H16N6, [MþH]þ m/
z: 269.1515, found: 269.1515.
J ¼ 11.2, 4.0 Hz, 1H, ArH), 7.67 (m, 1H, ArH), 3.78 (t, J ¼ 6.6 Hz, 4H,
CH2), 2.70 (s, 3H, CH3), 2.05 (m, 4H, CH2). 13C NMR (101 MHz, CDCl3)
d
154.50, 147.07, 142.35, 132.06, 129.19, 126.69, 125.07, 123.58,
120.43, 51.48, 25.78, 9.76. HR-MS (ESI): Calcd. C14H15N5, [MþH]þ m/
z: 254.1406, found: 254.1403.
4.6.8. 3-Methyl-6-(piperidin-1-yl)-1,2,4-triazolo[3,4-a]phthalazine
(7b)
Yield 68%. White solid. Mp: 230e231 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.61 (d, J ¼ 7.8 Hz, 1H, ArH), 8.02 (d, J ¼ 8.1 Hz, 1H, ArH),
4.7. General procedure for the synthesis of compounds 10aee
7.84 (t, J ¼ 7.5 Hz, 1H, ArH), 7.72 (t, J ¼ 7.6 Hz, 1H, ArH), 3.27 (d,
J ¼ 63.8 Hz, 4H, CH2), 2.75 (s, 3H, CH3), 1.86 (d, J ¼ 4.5 Hz, 4H, CH2),
Triethylamine (3.67 mL, 0.03 mol) was added to a solution of
substituted aniline (0.02 mol) in acetone (25 mL), and then 2-
chloroacetyl chloride (2.14 mL, 0.026 mol) was added dropwise to
the reaction mixture at 0e10 ꢀC. The reaction mixture was stirred at
room temperature for 4e8 h. The mixture was concentrated under
1.75 (d, J ¼ 4.8 Hz, 2H, CH2). 13C NMR (101 MHz, CDCl3)
d 158.47,
147.67, 142.55, 132.58, 129.77, 126.55, 124.99, 123.69, 120.56, 52.61,
25.80, 24.50, 9.80. HR-MS (ESI): Calcd. C15H17N5, [MþH]þ m/z:
268.1562, found: 268.1560.

242
D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
reduced pressure. The residue was poured into ice water (20 mL),
stirred at room temperature for 1 h and separated by filtration to
obtain 10aee.
4.8.3. 2-(4-(1,2,4-Triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)-N-
(4-methoxy-phenyl) acetamide (11c)
Yield 67%. Yellow solid. Mp: 178e179 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.98 (s, 1H, NH), 8.87 (s, 1H, CH), 8.65 (d, J ¼ 7.6 Hz, 1H,
ArH), 8.04 (d, J ¼ 7.9 Hz,1H, ArH), 7.90 (t, J ¼ 7.3 Hz,1H, ArH), 7.78 (t,
J ¼ 7.3 Hz, 1H, ArH), 7.50 (d, J ¼ 8.3 Hz, 2H, ArH), 6.87 (t, J ¼ 11.6 Hz,
2H, ArH), 3.80 (s, 3H, OCH3), 3.50 (s, 4H, CH2), 3.30 (s, 2H, NCH2CO),
4.7.1. 2-Chloro-N-(4-fluorophenyl)acetamide
Yield 78%. White solid. Mp: 113e114 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.24 (s, 1H, NH), 7.53 (d, J ¼ 4.5 Hz, 2H, ArH), 7.08 (t,
2.94 (s, 4H, CH2). 13C NMR (101 MHz, CDCl3)
d 167.53, 158.17 156.47,
J ¼ 8.5 Hz, 2H, ArH), 4.22 (s, 2H, CH2).
142.34, 139.33, 133.26, 130.61, 130.44, 126.37, 124.60, 124.09, 121.24,
120.51, 114.17, 61.99, 55.51, 53.18, 51.43. HR-MS (ESI): Calcd.
C
22H23N7O2, [MþH]þ m/z: 418.1191, found: 418.1189.
4.7.2. 2-Chloro-N-(4-chlorophenyl)acetamide
Yield 80%. Yellow solid. Mp: 141e142 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.27 (s, 1H, NH), 7.63e7.47 (m, 2H, ArH), 7.35 (dd, J ¼ 9.3,
4.8.4. 2-(4-(1,2,4-Triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)-N-
(3-(trifluoro-methyl)phenyl) acetamide (11d)
2.4 Hz, 2H, ArH), 4.21 (s, 2H, CH2).
Yield 69%. Yellow solid. Mp: 196e197 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
9.29 (s, 1H, NH), 8.87 (s, 1H, CH), 8.65 (d, J ¼ 7.8 Hz, 1H,
4.7.3. 2-Chloro-N-(4-methoxyphenyl)acetamide
Yield 74%. Yellow solid. Mp: 96e97 ꢀC; 1H NMR (400 MHz,
ArH), 8.04 (d, J ¼ 8.0 Hz, 1H, ArH), 7.90 (m, 2H, ArH), 7.80 (m, 2H,
ArH), 7.47 (t, J ¼ 7.8 Hz,1H, ArH), 7.38 (d, J ¼ 7.5 Hz,1H, ArH), 3.52 (s,
4H, CH2), 3.34 (s, 2H, NCH2CO), 2.97 (s, 4H, CH2). 13C NMR (101 MHz,
CDCl3)
d
8.23 (s, 1H, NH), 7.46 (d, J ¼ 8.9 Hz, 2H, ArH), 6.90 (d,
J ¼ 8.9 Hz, 2H, ArH), 4.19 (s, 2H, CH2), 3.82 (s, 3H, OCH3).
CDCl3) d 168.17, 158.13, 142.32, 139.32, 137.99, 133.29, 131.41, 130.47,
129.65, 126.34, 124.56, 124.09, 122.57, 120.91, 120.47, 116.18, 61.98,
4.7.4. 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide
53.17, 51.35. HR-MS (ESI): Calcd.
456.1760, found: 456.1760.
C
22H20F3N7O, [MþH]þ m/z:
Yield 83%. Yellow solid. Mp: 73e74 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
8.42 (s, 1H, NH), 7.87 (t, J ¼ 26.6 Hz, 2H, ArH), 7.69e7.42 (m,
2H, ArH), 4.31 (s, 2H, CH2).
4.8.5. 2-(4-(1,2,4-Triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)-N-
(2-fluoro-phenyl) acetamide (11e)
Yield 71%. Yellow solid. Mp: 192e194 ꢀC; 1H NMR (400 MHz,
4.7.5. 2-Chloro-N-(2-fluorophenyl)acetamide
Yield 70%. Yellow solid. Mp: 96e97 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
9.53 (s, 1H, NH), 8.89 (s, 1H, CH), 8.66 (d, J ¼ 7.9 Hz, 1H,
CDCl3)
d
8.55 (s, 1H, NH), 8.30 (t, J ¼ 7.6 Hz, 1H, ArH), 7.17 (dd,
ArH), 8.39 (t, J ¼ 7.5 Hz,1H, ArH), 8.05 (d, J ¼ 8.1 Hz,1H, ArH), 7.90 (t,
J ¼ 7.5 Hz, 1H, ArH), 7.78 (t, J ¼ 7.5 Hz, 1H, ArH), 7.12 (m, 3H, ArH),
3.53 (s, 4H, CH2), 3.35 (s, 2H, NCH2CO), 2.97 (s, 4H, CH2). 13C NMR
J ¼ 10.7, 5.6 Hz, 3H, ArH), 4.25 (s, 2H, CH2).
(101 MHz, CDCl3)
d 167.91, 158.07, 153.72, 151.30, 142.33, 139.38,
4.8. General procedure for the synthesis of compounds 11aem
133.21, 130.37, 126.35, 125.94, 124.66, 124.49, 124.11, 121.32, 120.50,
114.87, 61.99, 53.09, 51.43. HR-MS (ESI):Calcd. C21H20FN7O, [MþH]þ
m/z: 406.1792, found: 406.1789.
K2CO3 (0.345 g, 2.5 mmol) was added to a stirred solution of
compound (5a, 6a, 7a) (1 mmol) and 2-chloro-N-(substituted-
phenyl) acetamide (1.5 mmol) in acetone (5 mL). The reaction
mixture was heated to reflux for 7e12 h. The mixture was cooled,
separated by filtration. The filter liquor was concentrated under
reduced pressure, and purified by chromatography on silica gel
using MeOH/CH2Cl2 to obtain 11aem.
4.8.6. N-(4-Chlorophenyl)-2-(4-(3-phenyl-1,2,4-triazolo[3,4-a]
phthalazin-6-yl) piperazin-1-yl) acetamide (11f)
Yield 65%. Green solid. Mp: 238e239 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
9.12 (s, 1H, NH), 8.68 (d, J ¼ 7.9 Hz, 1H, ArH), 8.45 (d,
J ¼ 7.2 Hz, 2H, ArH), 8.04 (d, J ¼ 8.1 Hz, 1H, ArH), 7.87 (dd, J ¼ 14.1,
6.8 Hz, 1H, ArH), 7.77 (t, J ¼ 7.7 Hz, 1H, ArH), 7.54 (m, 5H, ArH), 7.31
(m, 2H, ArH), 3.55 (s, 4H, CH2), 3.33 (s, 2H, NCH2CO), 2.98 (m, 4H,
4.8.1. 2-(4-(1,2,4-Triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)-N-
(4-fluoro-phenyl) acetamide (11a)
CH2). 13C NMR (101 MHz, CDCl3)
d 167.85, 157.58, 152.67, 148.67,
Yield 69%. Yellow solid. Mp: 202e203 ꢀC; 1H NMR (400 MHz,
143.36, 136.03, 133.23, 130.30, 129.97, 129.37, 129.11, 128.63, 127.69,
126.71, 126.10, 125.07, 124.12, 120.72, 119.68, 62.06, 53.24, 51.43.
HR-MS (ESI): Calcd. C27H24ClN7O, [MþH]þ m/z: 498.1809, found:
498.1806.
CDCl3)
d
9.09 (s, 1H, NH), 8.89 (s, 1H, CH), 8.68 (d, J ¼ 7.7 Hz, 1H,
ArH), 8.05 (d, J ¼ 8.0 Hz,1H, ArH), 7.92 (t, J ¼ 7.4 Hz,1H, ArH), 7.79 (t,
J ¼ 7.4 Hz, 1H, ArH), 7.58 (dd, J ¼ 8.0, 4.6 Hz, 2H, ArH), 7.06 (t,
J ¼ 8.4 Hz, 2H, ArH), 3.52 (s, 4H, CH2), 3.33 (s, 2H, NCH2CO), 2.97 (s,
4H, CH2). 13C NMR (101 MHz, CDCl3)
d 167.67, 160.61, 158.13, 139.36,
4.8.7. 2-(4-(3-Phenyl-1,2,4-triazolo[3,4-a]phthalazin-6-yl)
piperazin-1-yl)-N-(3-(trifluoromethyl)phenyl) acetamide (11g)
Yield 73%. White solid. Mp: 285e286 ꢀC; 1H NMR (400 MHz,
133.51, 133.29, 130.42, 126.31, 124.68, 124.16, 121.23, 121.16, 120.48,
115.87, 115.64, 61.95, 53.19, 51.42. HR-MS (ESI): Calcd. C21H20FN7O,
[MþH]þ m/z: 406.1792, found: 406.1788.
CDCl3)
d
9.28 (s, 1H, NH), 8.68 (d, J ¼ 7.4 Hz, 1H, ArH), 8.45 (dd,
J ¼ 8.2, 1.2 Hz, 2H, ArH), 8.04 (d, J ¼ 8.0 Hz, 1H, ArH), 7.86 (m, 3H,
ArH), 7.77 (m, 1H, ArH), 7.52 (m, 4H, ArH), 7.38 (m, 1H, ArH), 3.56 (s,
4H, CH2), 3.35 (s, 2H, NCH2CO), 2.99 (m, 4H, CH2). 13C NMR
4.8.2. 2-(4-(1,2,4-Triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)-N-
(4-chloro-phenyl) acetamide (11b)
Yield 71%. Yellow solid. Mp: 198e199 ꢀC; 1H NMR (400 MHz,
(101 MHz, CDCl3) d 168.22, 157.59, 148.62, 143.35, 138.01, 133.20,
CDCl3)
d
9.15 (s, 1H, NH), 8.86 (s, 1H, ArH), 8.63 (d, J ¼ 7.8 Hz, 1H,
131.45, 130.29, 129.94, 129.64, 128.61, 127.66, 126.71, 126.12, 125.03,
124.08, 122.62, 120.90, 119.67, 116.24, 62.05, 53.23, 51.36. HR-MS
(ESI): Calcd. C28H24F3N7O, [MþH]þ m/z: 532.2073, found: 532.2070.
ArH), 8.03 (d, J ¼ 8.1 Hz, 1H, ArH), 7.89 (m, 1H, ArH), 7.77 (dt,
J ¼ 22.4, 7.6 Hz, 1H, ArH), 7.55 (m, 2H, ArH), 7.30 (m, 2H, ArH), 3.50
(s, 4H, CH2), 3.31 (s, 2H, NCH2CO), 2.95 (m, 4H, CH2). 13C NMR
(101 MHz, CDCl3)
130.46, 129.25, 129.05, 126.35, 124.56, 124.04, 120.72, 120.46, 61.97,
53.15, 51.38. HR-MS (ESI): Calcd.
422.1496,found: 422.1494.
d
167.88, 158.12, 142.31, 139.31, 136.06, 133.27,
4.8.8. N-(2-Fluorophenyl)-2-(4-(3-phenyl-1,2,4-triazolo[3,4-a]
phthalazin-6-yl) piperazin-1-yl) acetamide (11h)
C
21H20ClN7O,[MþH]þ m/z:
Yield 68%. White solid. Mp: 178e179 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
9.51 (s, 1H, NH), 8.69 (d, J ¼ 7.7 Hz, 1H, ArH), 8.49 (m, 2H,

D.-Q. Xue et al. / European Journal of Medicinal Chemistry 85 (2014) 235e244
243
ArH), 8.38 (tt, J ¼ 12.8, 6.5 Hz, 1H, ArH), 8.03 (d, J ¼ 8.1 Hz, 1H, ArH),
7.87 (dd, J ¼ 11.2, 4.0 Hz, 1H, ArH), 7.76 (m, 1H, ArH), 7.53 (m, 3H,
ArH), 7.16 (m, 1H, ArH), 7.09 (m, 2H, ArH), 3.55 (d, J ¼ 19.4 Hz, 4H,
CH2), 3.34 (d, J ¼ 12.0 Hz, 2H, NCH2CO), 2.99 (m, 4H, CH2). 13C NMR
3.55 (s, 4H, CH2), 3.36 (s, 2H, NCH2CO), 2.99 (d, J ¼ 4.3 Hz, 4H, CH2),
2.79 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3)
168.00, 157.48, 147.64,
d
142.44, 133.04, 130.01, 126.19, 125.14, 124.71, 124.42, 123.97, 121.36,
119.93, 114.96, 62.07, 53.15, 51.41, 9.81. HR-MS (ESI): Calcd.
(101 MHz, CDCl3)
d
167.99, 157.54, 153.73, 151.31, 148.61, 143.34,
C
22H22FN7O, [MþH]þ m/z: 420.1948, found: 420.1943.
133.13, 130.21, 129.89, 128.61, 127.68, 126.78, 126.13, 125.08, 124.64,
124.50, 124.10, 121.38, 119.70, 114.88, 62.05, 53.11, 51.46. HR-MS
(ESI): Calcd. C27H24FN7O, [MþH]þ m/z: 482.2105, found: 482.2100.
4.9. Effect of compounds on cell viability
Exponentially growing cells were seeded at 5 ꢁ103 cells per well
into 96-well plates. After 24 h incubation at 37 ꢀC, the culture
medium was removed and replaced with fresh medium containing
the candidate compounds in different concentrations. The cells
were incubated for another 72 h. Then, 20 mL of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution
(5 mg/mL) was added to all wells and incubated for 4 h at 37 ꢀC. The
medium containing MTT was discarded, 150 mL of dimethyl sulf-
oxide (DMSO) was added to each well and the plates agitated until
the dark blue crystals (formazan) had completely dissolved; the
absorbance was measured using a microplate reader at a wave-
length of 570 nm. Each concentration was analyzed in triplicate and
the experiment was repeated three times. The average 50% inhib-
itory concentration (IC50) was determined from the concen-
trationeresponse curves according to the inhibition ratio for each
concentration.
4.8.9. N-(4-Fluorophenyl)-2-(4-(3-methyl-1,2,4-triazolo[3,4-a]
phthalazin-6-yl) piperazin-1-yl) acetamide (11i)
Yield 74%. Yellow solid. Mp: 201e202 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
9.12 (s, 1H, NH), 8.60 (d, J ¼ 7.9 Hz, 1H, ArH), 8.02 (d,
J ¼ 8.1 Hz, 1H, ArH), 7.86 (t, J ¼ 7.3 Hz, 1H, ArH), 7.74 (m, 1H, ArH),
7.56 (m, 2H, ArH), 7.03 (m, 2H, ArH), 3.51 (s, 4H, CH2), 3.31 (s, 2H,
NCH2CO), 2.95 (m, 4H, CH2), 2.74 (s, 3H, CH3). 13C NMR (101 MHz,
CDCl3) d 167.82, 160.59, 158.15, 157.47, 147.69, 142.45, 133.53, 133.07,
130.09, 126.21, 124.99, 123.87, 121.24, 119.91, 115.70, 61.97, 53.22,
51.38, 9.78. HR-MS (ESI): Calcd.
420.1948, found: 420.1946.
C
22H22FN7O, [MþH]þ m/z:
4.8.10. N-(4-Chlorophenyl)-2-(4-(3-methyl-1,2,4-triazolo[3,4-a]
phthalazin-6-yl) piperazin-1-yl) acetamide (11j)
Yield 69%. Yellow solid. Mp: 205e206 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
9.15 (s, 1H, NH), 8.63 (d, J ¼ 7.9 Hz, 1H, ArH), 8.03 (d,
J ¼ 8.1 Hz, 1H, ArH), 7.88 (t, J ¼ 7.4 Hz, 1H, ArH), 7.76 (dd, J ¼ 11.3,
4.0 Hz,1H, ArH), 7.56 (dd, J ¼ 9.5, 6.7 Hz, 2H, ArH), 7.33 (t, J ¼ 5.8 Hz,
2H, ArH), 3.52 (s, 4H, CH2), 3.32 (s, 2H, NCH2CO), 2.96 (dd, J ¼ 11.5,
7.0 Hz, 4H, CH2), 2.77 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3)
4.10. Analysis of cellular apoptosis
EC-9706 cells were plated in 6-well plates (1.0 ꢁ 106 cells/well)
and incubated at 37 ꢀC for 24 h. Exponentially growing cells were
then incubated for 12 or 24 h with complete medium (blank) or
with the compound 11h. Cells were then harvested and the
Annexin-V-FITC/PI apoptosis kit (Biovision) was used according to
the manufacturer's instructions to detect apoptotic cells. Ten
thousand events were collected for each sample and analyzed by
Accuri C6 flow cytometer.
d
167.85, 157.42, 147.69, 142.46, 136.05, 133.07, 130.05, 129.33,
129.09, 126.17, 125.08, 123.90, 120.71, 119.89, 62.02, 53.22, 51.37,
9.80. HR-MS (ESI): Calcd. C22H22ClN7O, [MþH]þ m/z: 436.1653,
found:436.1650.
4.8.11. N-(4-Methoxyphenyl)-2-(4-(3-methyl-1,2,4-triazolo[3,4-a]
phthalazin-6-yl) piperazin-1-yl) acetamide (11k)
Yield 79%. Yellow solid. Mp: 169e170 ꢀC; 1H NMR (400 MHz,
4.11. Flow cytometric analysis of cell cycle distribution
CDCl3)
d
8.98 (s, 1H, NH), 8.64 (d, J ¼ 7.9 Hz, 1H, ArH), 8.04 (d,
For flow cytometric analysis of DNA content, 5 ꢁ 105 EC-9706
cells in exponential growth were treated with different concen-
trations of the test compounds for 12 or 24 h. After an incubation
period, the cells were collected, centrifuged and fixed with ice-cold
ethanol (70%). The cells were then treated with buffer containing
RNAse A and 0.1% Triton X-100 and then stained with PI. Samples
were analyzed on Accuri C6 flow cytometer (Becton, Dickinson).
Data obtained from the flow cytometer was analyzed using the
FlowJo software (Tree Star, Inc., Ashland, OR, USA).
J ¼ 8.1 Hz,1H, ArH), 7.88 (t, J ¼ 7.6 Hz,1H, ArH), 7.76 (t, J ¼ 7.7 Hz,1H,
ArH), 7.52 (d, J ¼ 8.9 Hz, 2H, ArH), 6.91 (d, J ¼ 8.9 Hz, 2H, ArH), 3.82
(s, 3H, OCH3), 3.52 (s, 4H, CH2), 3.31 (s, 2H, NCH2CO), 2.96 (m, 4H,
CH2), 2.77 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3)
d 167.58, 157.51,
156.50, 147.72, 142.48, 133.09, 130.60, 130.09, 126.21, 125.04, 123.94,
121.27, 119.95, 114.25, 62.01, 55.52, 53.23, 51.42, 9.79. HR-MS (ESI):
Calcd.C23H25N7O2, [MþH]þ m/z: 432.2148, found: 432.2143.
4.8.12. 2-(4-(3-Methyl-1,2,4-triazolo[3,4-a]phthalazin-6-yl)
piperazin-1-yl)-N-(3-(trifluoromethyl) phenyl) acetamide (11l)
Yield 65%. Yellow solid. Mp: 199e200 ꢀC; 1H NMR (400 MHz,
Acknowledgments
CDCl3)
d
9.26 (s, 1H, NH), 8.65 (d, J ¼ 7.8 Hz, 1H, ArH), 8.04 (d,
J ¼ 8.1 Hz, 1H, ArH), 7.89 (dd, J ¼ 13.6, 6.2 Hz, 3H, ArH), 7.77 (t,
J ¼ 7.7 Hz, 1H, ArH), 7.50 (t, J ¼ 8.2 Hz, 1H, ArH), 7.41 (d, J ¼ 7.7 Hz,
1H, ArH), 3.55 (s, 4H, CH2), 3.35 (s, 2H, NCH2CO), 2.98 (m, 4H, CH2),
Financial supports by National Nature Science Foundation of
China, No. 81172937.
2.78 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3)
d 168.16, 157.45, 147.74,
Appendix A. Supplementary data
142.48, 137.95, 133.14, 131.68, 131.33, 130.11, 129.70, 126.16, 125.07,
123.98, 122.57, 121.00, 119.91, 116.14, 62.05, 53.27, 51.36, 9.80. HR-
MS (ESI): Calcd. C23H22F3N7O, [MþH]þ m/z: 470.1916, found:
470.1915.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.031.
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