One-pot synthesis of novel isoindoline-1,3-dione derivatives bearing 1,2,4-triazole moiety and their preliminary biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.06.041

A series of novel isoindoline-1,3-diones containing 1,2,4-triazole moiety were synthesized via a one-pot reaction. Bioassay indicated that compounds 33, 35, 37 and 39 exhibited much higher activities against Botryodiplodia theobromae than commercial fungicide triadimefon at the dosage of 150 mg/L. Most interestingly, compounds 36, 37 and 45 displayed much stronger antitumor activities against four human cell lines than positive control Fluorouracil. Particularly, compound 37 had four-fold improvement compared to Fluorouracil in inhibiting A549 and HepG2 cell proliferation with IC₅₀ values of 6.76 and 9.44 μM, respectively. Further flow-activated cell sorting analysis revealed that compound 37 displayed apoptosis-inducing effect on HepG2 cells in a dose-dependent manner. These encouraging results could be helpful for the development of new antitumor compounds.

Full text of DOI:10.1016/j.ejmech.2012.06.041

European Journal of Medicinal Chemistry 54 (2012) 813e822
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
One-pot synthesis of novel isoindoline-1,3-dione derivatives bearing 1,2,4-triazole
moiety and their preliminary biological evaluation
,
,
,
Pei-Liang Zhao a 1, Wei-Feng Ma b 1, An-Na Duan a, Min Zou a, Yi-Chen Yan a, Wen-Wei You a
*
,
Shu-Guang Wu a
,
*
a Department of Chemistry, School of Pharmaceutical Science, Southern Medical University, Guangzhou 510515, PR China
b Department of Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel isoindoline-1,3-diones containing 1,2,4-triazole moiety were synthesized via a one-pot
reaction. Bioassay indicated that compounds 33, 35, 37 and 39 exhibited much higher activities against
Botryodiplodia theobromae than commercial fungicide triadimefon at the dosage of 150 mg/L. Most
interestingly, compounds 36, 37 and 45 displayed much stronger antitumor activities against four human
cell lines than positive control Fluorouracil. Particularly, compound 37 had four-fold improvement
compared to Fluorouracil in inhibiting A549 and HepG2 cell proliferation with IC50 values of 6.76 and
Received 24 April 2012
Received in revised form
19 June 2012
Accepted 21 June 2012
Available online 3 July 2012
9.44 mM, respectively. Further flow-activated cell sorting analysis revealed that compound 37 displayed
apoptosis-inducing effect on HepG2 cells in a dose-dependent manner. These encouraging results could
be helpful for the development of new antitumor compounds.
Keywords:
Isoindoline-1,3-dione
1,2,4-Triazole
One-pot synthesis
Antifungal
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antitumor activity
1. Introduction
recently compound 4 has emerged as potential antitumor agent
inhibiting cyclin-dependent kinases (CDKs) [16].
Recently, increasing interests have been focused on the cyclic
imides because of their broad spectrum of biological activities
[1e6]. Among the existing various N-substituted cyclic imides,
isoindoline-1,3-dione derivatives have been identified as one of the
most promising scaffold, which contain the general structure
eCOeN(R)eCOe, so that they are hydrophobic and neutral, and can
therefore cross biological membranes in vivo [7]. N-substituted
isoindoline-1,3-dione has been a very interesting and hot research
area due to its broad structural diversity and broad-spectrum bio-
logical activities [8e12]. As shown in Fig. 1, by introduction of
nitrogen-containing heterocycle into the isoindoline-1,3-diones,
compounds 1 and 2 have been found to exhibit potent antibacterial
and antifungal activities [7,13]. While halogen-substituted
compound 3 has been proved to be effective in treating prostate
cancer [14], which is the most common malignancy in American
men and the second leading cause of cancer mortality [15]. More
It is well-known that the derivatives of 1,2,4-triazole always
possess a wide range of biological activities including insecticidal
[17], antifungal [18], antimicrobial [19], antiviral [20], antitumor
[21], and anti-inflammatory [22] properties. Some of 1,2,4-triazole
derivatives have also been widely used in agriculture and medicine.
For example, bromuconazole (5) and fluconazole (6) have been
used as commercial antifungicides for many years [23,24] (Fig. 2),
while anastrozole (7) and vorozole (8) have been developed as
anticancer drugs recently [25,26]. Therefore, as a part of our
research work on the development of novel bioactive nitrogen-
containing heterocycles [27e29], as shown in Fig. 3, we are very
interested in the design and efficient synthesis of isoindoline-1,3-
diones bearing 1,2,4-triazole substructure, which will be expected
to exhibit interesting features due to the co-existence of two kinds
of pharmacophore. In this paper, we firstly described the one-pot
synthesis and biological activity of a series of new isoindoline-
1,3-dione derivatives containing 1,2,4-triazol moiety 9e46 (Fig. 3),
which have, to our knowledge, not been reported so far.
2. Chemistry
* Corresponding authors. Tel./fax: þ86 (0)20 61648196.
E-mail addresses: youww@smu.edu.cn (W.-W. You), shuguang@smu.edu.cn
(S.-G. Wu).
Due to the different substituents on the 1,2,4-triazole core, two
methods were used to synthesize the 4-amino-5-substituted-4H-
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.041

814
P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
O
O
O
O
O
F
O
O
N
N
S
N
4
F
N
SO2NH
N
N
N
N
F
O
O
O
O
F
Cl
2
1
3
4
Fig. 1. Structures of some bioactive isoindoline-1,3-dione.
1,2,4-triazole-3-thiols 47 with the starting materials of their cor-
responding carboxylic acids according to our previously reported
method [29,30]. Meanwhile, a variety of phenacyl bromides were
readily afforded by the bromination reaction of commercially
available acetophenones with 1.02 equiv. N-bromosuccinimide
(NBS) and catalytic amount of 2,20-azo-bisisobutyronitrile (AIBN) in
anhydrous carbon tetrachloride, respectively. The expected target
isoindoline-1,3-dione derivatives (9e46) were prepared by equi-
molar amounts of 4-amino-5-substituted-4H-1,2,4-triazole-3-thiol
(47), isobenzofuran-1,3-dione (48) and excess amount of haloge-
nated compounds (1.2 equiv) with equimolar amount of zinc
bromide and 2.0 equimolar amounts of triethylamine (TEA) in
absolute toluene (Scheme 1). It should be noted that the substitu-
ents at the 5-position of 1,2,4-triazole ring such as CH3, CH3CH2,
CH3CH3CH2, and 3,4,5-trimethoxyphenyl, have practically no
obvious effect on the yields.
The structures of the prepared isoindoline-1,3-dione derivatives
9e46 were characterized by 1H NMR, 13C NMR, ESI-MS spectrum
and elemental analysis, and the results are presented in the
Experimental section. In addition, the representative compound 37
was further confirmed by single-crystal X-ray diffraction. As shown
in Fig. 4, the dihedral angle of the 1,2,4-triazole ring and the adja-
cent pyrrolidine plane shows a gauche conformation with an angle
of 88.32ꢀ that is affected by the bulky 4-methylphenacylthio group.
Penz. and P. oryzae Cav. at the concentration of 150 mg/L. In most
cases, aryl-triazole derivatives displayed higher fungicidal activity
against B. theobromae and F. oxysporum f. sp. Niveum than alkyl-
triazole derivatives, for example, compounds 33 and 18
(R ¼ CH2CH2 ¼ CH2), 35 and 17 (R ¼ CH2CH^CH), 37 and 12 (R ¼ 4-
MeC6H4COCH2), 39 and 10 (R ¼ EtCO2CH2). However, when aryl-
triazole derivatives contain benzyl group, the antifungal activity
against B. theobromae decreased dramatically, such as compounds
34 (R ¼ 2-ClC6H4CH2) and 40 (R ¼ C6H5CH2), which exhibited much
lower antifungal activity than the corresponding alkyl-triazole
derivatives 16 and19, respectively.
It is showed in Table 1 that, within the series of alkyl-triazole
derivatives, antifungal activities were influenced by the S-substit-
uents on the 1,2,4-triazole ring and phenacyl substitution always
resulted in higher activities against F. oxysporum f. sp. Cubense and
P. oryzae Cav., and only compound 10 (R ¼ 4-ClC6H4COCH2) dis-
played good antifungal activities (ꢁ80%) against C. gloeosporioides
Penz. at the concentration of 150 mg/L. While most compounds of
aryl-triazole derivatives 33, 35, 37 and 39 displayed excellent
antifungal activities (ꢁ90%) against B. theobromae, which are
superior to that of Triadimefon, a commercial fungicide. Interest-
ingly, compounds 33 and 37 also displayed similar potent anti-
fungal activities (ꢁ80%) against C. gloeosporioides Penz. in
comparison with reference drug. Meanwhile, compound 33 was
also found to display fungicidal activity against other fungi. These
results indicated that compound 33 showed a broad spectrum
antifungal activity and further investigations are necessary to
optimize the potentially leading compound as more efficacious
antifungal agents.
3. Pharmacology results and discussion
3.1. Antifungal activity
The in vitro antifungal activities of compounds 9e46 against six
fungi including Botryodiplodia theobromae, Fusarium oxysporum f.
sp. Niveum, Fusarium oxysporum f. sp. Cubense, Colletotrichum
musae (Berk & Curt) Arx, Phyricularia oryzae Cav. and Colletotrichum
gloeosporioides Penz. were tested. The results are listed in Table 1, in
which the inhibition percentage was expressed as the mean of
values obtained in three independent experiments. Triadimefon,
a commercial fungicide, was used as a positive control. For the
3.2. Antitumor activity
The in vitro antitumor activities of the synthesized compounds
9e46 against four human cancer cell lines, including HepG2, A549,
PC-3M and MKN45, were assayed by MTT method. Fluorouracil was
used as the reference drug and the results expressed as IC50 (mM)
were summarized in Table 2. Here, the IC50 value represents the
concentration of one compound resulting in a 50% inhibition in cell
growth after 48 h incubation, and is the average of three inde-
pendent experiments.
As indicated in Table 2, the results indicated that only
compounds 36, 37 and 41e46 were found to be active. Among
them, three representative compounds 36, 37 and 45 displayed
much higher antitumor activities against HepG2, A549, PC-3M and
convenience
of
structureeactivity
relationship
analysis,
compounds 9e32 and 33e46 were defined as alkyl-triazole
derivatives and aryl-triazole derivatives, respectively. Although it
seems impossible to extract an obvious structureeactivity rela-
tionship from the data shown in Table 1, we can conclude clearly
that nearly all compounds exhibit significant antifungal activities
against B. theobromae, F. oxysporum f. sp. Niveum, C. gloeosporioides
OH
CN
N
N
N
N
N
N
N
N
N
N
N
N
F
N
N
N
O
N
N
Cl
Cl
N
NC
CH3
Br
5 (Bromuconazole)
F
6 (Fluconazole)
7 (Anastrozole)
8 (Vorozole)
Fig. 2. Structures of some commercial 1,2,4-triazole derivatives.

P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
815
45). Replacement of phenacyl group with methyl, benzyl, allyl,
propargyl and ester group (33e35 and 38e40) led to a dramatic
decrease in antitumor activity. It can be concluded that 3,4,5-
trimethoxyphenyl group at the 5-position of 1,2,4-triazole ring
and substituted phenacyl group at the 3-position play a crucial role
in modulating the antitumor activity.
To study the effect of the synthesized compounds on cell cycle
progression, flow-activated cell sorting analysis was performed. The
most promising compound 37 was tested against HepG2 cell lines at
S
R2
N
N
N
H2N
R1
4H-1,2,4-Triazole
R2
O
S
O
N
N
N
N
R
N
O
R1
O
9~46
given concentrations (4, 8,16 mM). Fig. 5 and Table 3 show the results
Lead compound
after 24 h treatment with 37. As can be seen in Fig. 5 and Table 3,
compound 37 had no obvious effect on the cell cycle arrest. However,
cells accumulated dramatically in sub-G0 phase at 15.05e32.58%,
suggesting that compound 37 might possess apoptosis-inducing
effect on HepG2 cells in a dose-dependent manner.
Fig. 3. Design strategy of the title compounds.
MKN45 cell lines than positive control Fluorouracil. Especially,
compound 37 showed pretty high potency against A549, HepG2
cell lines with IC50 values of 6.76, 9.44 mM, respectively, which was
4. Conclusion
found to be four times more potent than Fluorouracil. These
encouraging results could be helpful for the development of new
antitumor compounds.
In summary, a series of novel isoindoline-1,3-diones bearing
1,2,4-triazole ring were designed and efficiently synthesized via
a one-pot, three-component reaction of isobenzofuran-1,3-dione,
Further analysis on the structureeactivity relationship investi-
gated the effects of several substituents on 1,2,4-triazole ring. In
most cases, compounds bearing 3,4,5-trimethoxyphenyl group at
the 5-position of 1,2,4-triazole ring (36, 37 and 41e46) displayed
obviously higher cytotoxic activity than those of the corresponding
compounds with methyl, ethyl or n-propyl group at the same
position (9e32). Nevertheless, within the series of compounds
33e46 bearing 3,4,5-trimethoxyphenyl group, anticancer activities
were influenced by the C-3 substituents on the 1,2,4-triazole ring
and phenacyl substitution always gave higher activities (36, 37 and
4-amino-5-substituted-4H-1,2,4-triazole-3-thiol,
and
various
halogenated compounds. Subsequent biological evaluation indi-
cated that four compounds 33, 35, 37 and 39 exhibited more potent
in vitro antifungal activities than the reference drug against
B. theobromae. Among them, compound 33 was identified as the
most promising antifungal candidate for further study, due to its
broad spectrum and the highest antifungal activity. Most interest-
ingly, three representative compounds 36, 37 and 45 displayed
much higher antitumor activities against HepG2, A549, PC-3M and
R1
O
O
R1
NH2
O
TEA, ZnBr2
N
SH
N
R1
+
O
R2
N
N
N
N
Br
N
N
N
toluene, rt
N
N
90
O
HS
O
S
O
R2
49
47
48
9-46
Scheme 1. One-pot synthesis of compounds 9e46.
Fig. 4. Molecular structure of compound 37.

816
P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
Table 1
Structures and antifungal activities of compounds 9e46.
R
S
O
R
R
N
N
R
N
N
O
S
O
S
O
S
N
N
N
N
N
N
O
O
N
N
N
N
N
N
O
O
O
O
O
9~16
17~24
25~32
33~46
Comp.
R
150 mg/L (in vitro)b
BTa
FNa
FCa
CAa
PCa
CGa
9
C6H5CH2
EtCO2CH2
e
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þþ
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þþ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
þ
e
e
e
e
e
þ
e
þþ
e
e
e
þ
þ
þþ
e
þ
e
e
e
e
þþ
e
e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Triadimefon
e
e
þþ
þþþ
þ
4-ClC6H4COCH2
4-MeC6H4COCH2
C6H5COCH2
HC^CCH2
Me
2-ClC6H5CH2
HC^CCH2
H2C]CHCH2
C6H5CH2
C6H5COCH2
4-ClC6H4COCH2
4-MeC6H4COCH2
Me
e
þþ
þ
þþ
þ
e
þ
þ
þþ
e
þ
e
þ
þþ
þþ
þ
þþ
þþ
e
þ
þþ
þþ
þ
þþ
þþ
þþ
þ
þþ
þ
þ
e
þ
e
e
þþ
þþ
e
e
þþ
e
e
e
e
EtCO2CH2
H2C]CHCH2
C6H5CH2
e
þ
þ
e
þþ
þþ
e
þþ
þ
e
þþ
þ
e
4-ClC6H4COCH2
4-MeC6H4COCH2
HC^CCH2
2-ClC6H5CH2
C6H5COCH2
Me
H2C]CHCH2
2-ClC6H5CH2
HC^CCH2
4-ClC6H4COCH2
4-MeC6H4COCH2
Me
EtCO2CH2
C6H5CH2
C6H5COCH2
4-MeOC6H4COCH2
2-MeC6H4COCH2
4-FC6H4COCH2
4-BrC6H4COCH2
3,4-F2C6H4COCH2
þþ
þþ
þþ
þþ
þþ
þþ
þþþ
þþ
þþ
þþ
þþþ
þþ
þþ
þþ
þ
e
þþ
þ
þ
þþþ
e
þ
þþ
e
þ
e
e
e
þþ
e
e
e
þþþþ
e
þþ
e
þ
e
þþþþ
e
e
þþ
e
e
þþþþ
e
e
e
þ
þþ
þþ
e
þþþþ
þ
e
e
þ
þþ
e
þþþ
þþ
e
e
þ
e
þþ
þþ
þþþ
þþ
þþþþ
þ
e
e
e
e
þþ
e
þþ
þ
e
þþ
e
e
e
þþþ
þþ
þþþþ
þþþ
þþþ
a
BT for Botryodiplodia theobromae, FN for Fusarium oxysporum f. sp. Niveum, FC for Fusarium oxysporum f. sp. Cubense, CA for Colletotrichum musae (Berk & Curt) Arx, PC for
Phyricularia oryzae Cav., CG for Colletotrichum gloeosporioides Penz.
b
Rating system for the inhibition percentage: þþþþ, ꢁ90%; þþþ, ꢁ80%; þþ, ꢁ60%; þ, ꢁ50%; e, <50%.
MKN45 cell lines than positive control Fluorouracil. Particularly,
compound 37 was four-fold improvement compared to Fluoro-
uracil in inhibiting A549 and HepG2 cell proliferation with IC50
5. Experimental protocols
5.1. Chemistry
values of 6.76 and 9.44 mM, respectively.
These results indicated that compound 37 could be used as
a lead for further developing new isoindoline-1,3-diones deriva-
tives possessing both antifungal and antitumor activities. Flow-
activated cell sorting analysis revealed that compound 37 dis-
1H NMR and 13C NMR spectra were recorded on a Mercury-Plus
400 spectrometer in CDCl3 or DMSO-d6 solution and chemical
shifts were recorded in parts per million (ppm) with TMS as the
internal reference. MS spectra were determined using a Micromass
ZQ 4000 mass spectrometry, and signals were given in m/z.
Elementary analyses were performed on a Vario EL III elemental
analysis instrument. Melting points (mp) were taken on a Buchi B-
545 melting point apparatus and uncorrected. Unless otherwise
played apoptosis-inducing effect on HepG2 cells in
a dose-
dependent manner. Further structural optimization and fungicidal
and antitumor activities about the isoindoline-1,3-dione analogs
are well under way.

P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
817
Table 2
reaction was complete according to the TLC detection, solvent was
removed under reduced pressure and the residue was purified by
column chromatography using a mixture of petroleum ether and
ethyl acetate (10:1) as an eluent to give the target compounds in
yields of 62e81%.
Cytotoxic activities of compounds 36, 37 and 41e46 against human tumor cells.
Comp.
R
In vitro cytotoxicity IC50
HepG2 A549
22.03 9.03
(
m
M)a
PC-3M
20.69
MKN45
36
37
41
42
43
44
45
46
4-ClC6H4COCH2
4-MeC6H4COCH2
C6H5COCH2
4-MeOC6H4COCH2
2-MeC6H4COCH2
4-FC6H4COCH2
4-BrC6H4COCH2
3,4-F2C6H4COCH2
26.64
9.62
9.44
170.71
28.73
6.76
>200
30.03
11.71
>200
32.80
>200
>200
22.58
5.1.1.1. 2-(3-(Benzylthio)-5-methyl-4H-1,2,4-triazol-4-yl)isoindoline-
>200
37.23
>200
126.40
22.32
>200
41.73
1,3-dione (9). Yield, 74%; mp 111e113 ꢀC; 1H NMR (400 MHz, CDCl3)
>200
>200
27.71
>200
46.83
143.87
d: 2.34 (s, 3H, triazoleeCH3), 4.36 (s, 2H, SCH2), 7.22e7.28 (m, 5H,
>200
16.05
>200
35.41
ArH), 7.89 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH), 7.99 (dd, J ¼ 3.0 Hz,
J ¼ 5.4 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3):
d
¼ 10.0, 38.2,
163.02
22.63
Fluorouracil
124.8, 127.8, 128.6, 129.0, 129.1, 135.4, 135.7, 150.7, 152.5, 162.1. ESI-
MS: m/z ¼ 373.25 [M þ Na]þ. Anal. Calcd. for C18H14N4O2S: C 61.70,
H 4.03, N 15.99, S 9.15. Found C 61.47, H 3.71, N 16.16, S 9.46.
a
The IC50 values of all the other compounds that were not listed, were estimated
M.
to be greater than 200
m
5.1.1.2. Ethyl
triazol-3-ylthio)acetate (10). Yield, 76%; mp 105e106 ꢀC; 1H NMR
(400 MHz, CDCl3)
2-(4-(1,3-dioxoisoindolin-2-yl)-5-methyl-4H-1,2,4-
noted, reagents were purchased from commercial suppliers and
used without further purification while all solvents were redistilled
before use.
d: 1.20 (t, J ¼ 7.0 Hz, 3H, OCH2CH3), 2.33 (s, 3H,
triazole-CH3), 3.94 (s, 2H, SCH2), 4.13 (dd, J ¼ 7.0 Hz, J ¼ 14.2 Hz, 2H,
OCH2CH3), 7.91 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH), 8.01 (dd,
J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3):
¼ 9.9,
d
5.1.1. General procedure for the one-pot reactions of 4-amino-5-
substituted-4H-1,2,4-triazole-3-thiol, isobenzofuran-1,3-dione, and
various halogenated compounds
A mixture of 4-amino-5-substituted-4H-1,2,4-triazole-3-thiol
(1.0 mmol), isobenzofuran-1,3-dione (1.0 mmol, 0.163 g), zinc
bromide (1.0 mmol, 0.225 g) and triethylamine (2.0 mmol, 0.202 g)
in 3.0 mL of dry toluene was stirred at room temperature for about
0.5 h. Then halogenated compound (1.2 mmol) was added. The
reaction mixture was heated at 90 ꢀC for another 3e8 h. After the
13.9, 35.8, 62.1, 124.9, 129.1, 135.7, 149.7, 152.7, 162.1, 167.1. ESI-MS:
m/z ¼ 370.22 [M þ Na]þ, 347.08 [M þ 1]þ. Anal. Calcd. for
C15H14N4O4S: C 52.02, H4.07, N16.18, S9.26; Found C 52.26, H 4.28, N
16.36, S 9.52.
5.1.1.3. 2-(3-(2-(4-Chlorophenyl)-2-oxoethylthio)-5-methyl-4H-
1,2,4-triazol-4-yl)isoindoline-1,3-dione (11). Yield, 74%; mp
115e117 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.34 (s, 3H, triazole-
Fig. 5. Effect of compound 37 on cell cycle and apoptosis in HepG2 cells. Flow cytometry analysis of HepG2 cells treated with 37 for 24 h. (A) Control; (B) 37, 4
37, 16 M.
mM; (C) 37, 8 mM; (D)
m

818
P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
Table 3
(400 MHz, CDCl3)
d
: 1.28 (t, J ¼ 7.6 Hz, 3H, CH2CH3), 2.20 (t,
Effect of compound 37 on cell cycle distribution in HepG2 cells.
J ¼ 2.6 Hz, 1H, ^CH), 2.61 (dd, J ¼ 7.4 Hz, J ¼ 15.0 Hz, 2H, CH2CH3),
3.79 (d, J ¼ 2.8 Hz, 2H, SCH2), 7.90e7.93 (m, 2H, ArH), 7.98e8.01 (m,
Concentration
Sub-G0
G0/G1
S
G2/M
2H, ArH). 13C NMR (100 MHz, CDCl3):
d
¼ 10.8, 18.0, 22.8, 73.3, 77.6,
0
4
8
m
m
m
M
M
M
2.48
15.05
29.22
32.58
54.65
40.73
37.45
36.70
29.84
25.41
17.50
21.07
16.38
14.40
11.38
6.31
124.9, 129.1, 135.8, 149.3, 157.2, 162.3. ESI-MS: m/z ¼ 335.19
[M þ Na]þ, 313.26 [M þ 1]þ. Anal. Calcd. for C15H12N4O2S: C 57.68,
H 3.87, N 17.94, S 10.27. Found C 57.95, H 4.16, N 17.89, S 10.08.
16 mM
CH3), 4.83 (s, 2H, SCH2), 7.42 (d, J ¼ 8.4 Hz, 2H, ArH), 7.88e7.92
(m, 4H, ArH), 8.00 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR
5.1.1.10. 2-(3-(Allylthio)-5-ethyl-4H-1,2,4-triazol-4-yl)isoindoline-
1,3-dione (18). Yield,81%;mp 88e90ꢀC;1HNMR(400 MHz, CDCl3)
d:
(100 MHz, CDCl3):
d
¼ 9.9, 42.7, 124.9, 129.1, 129.2, 129.8, 133.0,
1.26 (t, J ¼ 7.6 Hz, 3H, eCH2CH3), 2.59 (dd, J ¼ 7.2 Hz, J ¼ 14.8 Hz, 2H,
eCH2CH3), 3.68 (d, J ¼ 6.8 Hz, 2H, SCH2), 5.07 (d, J ¼ 10.0 Hz, 1H, ]
CHa), 5.17(d, J¼ 16.8 Hz, 1H, ]CHb), 5.78e5.84(m,1H, CH]), 7.90(dd,
J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH), 8.00 (dd, J ¼ 3.0 Hz, J ¼ 5.0 Hz, 2H, ArH).
135.8, 140.6, 150.2, 152.7, 162.1, 191.6. ESI-MS: 413.09 [M þ 1]þ.
Anal. Calcd. for C19H13ClN4O3S: C 55.28, H 3.17, N 13.57, S 7.77.
Found C 55.59,
13C NMR (100 MHz, CDCl3):
d
¼ 10.9, 18.0, 36.8, 119.5, 124.9, 129.2,
H 3.53, N 13.74, S 7.65.
131.9, 135.7, 150.3, 156.8, 162.4. ESI-MS: m/z ¼ 337.23 [M þ Na]þ,
315.21 [M þ 1]þ. Anal. Calcd. for C15H14N4O2S: C 57.31, H 4.49, N 17.82,
S 10.20. Found C 57.58, H 4.75, N 17.56, S 10.43.
5.1.1.4. 2-(3-Methyl-5-(2-oxo-2-p-tolylethylthio)-4H-1,2,4-triazol-4-
yl)isoindoline-1,3-dione (12). Yield, 65%; mp 114e116 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
CH3), 4.86 (s, 2H, SCH2), 7.36 (d, J ¼ 8.0 Hz, 2H, ArH), 7.62e7.71 (m,
3H, ArH), 7.91e7.96 (m, 3H, ArH).13C NMR (100 MHz, CDCl3):
d: 2.33 (s, 3H, triazole-CH3), 2.39 (s, 3H, Ar-
5.1.1.11. 2-(3-(Benzylthio)-5-ethyl-4H-1,2,4-triazol-4-yl)isoindoline-
1,3-dione (19). Yield, 66%; mp 87e89 ꢀC; 1H NMR (400 MHz, CDCl3)
d
¼ 9.9,
21.7, 43.2, 124.9, 128.5, 129.2, 129.5, 132.2, 135.7, 145.2, 150.6, 152.6,
162.1,192.3. ESI-MS:393.16 [M þ 1]þ. Anal. Calcd. for C20H16ClN4O3S:
C 61.21, H 4.11, N 14.28, S 8.17. Found C 61.53, H 4.49, N 14.58, S 8.39.
d
: 1.30 (t, J ¼ 7.6 Hz, 3H, CH2CH3), 2.63 (dd, J ¼ 7.6 Hz, J ¼ 15.2 Hz, 2H,
eCH2CH3), 4.37 (s, 2H, SCH2), 7.22e7.30 (m, 5H, ArH), 7.90 (dd,
J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH), 7.99 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH).
13C NMR (100 MHz, CDCl3):
d
¼ 10.9, 17.9, 38.3, 124.8, 127.8, 128.6,
5.1.1.5. 2-(3-Methyl-5-(2-oxo-2-phenylethylthio)-4H-1,2,4-triazol-4-
129.0, 129.1, 135.5, 135.7, 150.7, 156.7, 162.3. ESI-MS: m/z ¼ 387.27
[M þ Na]þ, 365.26 [M þ 1]þ. Anal. Calcd. for C19H16N4O2S: C 62.62,
H 4.43, N 15.37, S 8.80. Found C 62.41, H 4.13, N 15.65, S 8.53.
yl)isoindoline-1,3-dione (13). Yield, 63%; mp 183e185 ꢀC; 1H NMR
(400 MHz, CDCl3)
d
: 2.34 (s, 3H, triazole-CH3), 4.89 (d, J ¼ 4.8 Hz,
2H, SCH2), 7.45 (t, J ¼ 8.0 Hz, 2H, ArH), 7.56 (t, J ¼ 4.2 Hz, 1H, ArH),
7.89e7.95 (m, 4H, ArH), 7.98e8.01 (m, 2H, ArH). 13C NMR (100 MHz,
5.1.1.12. 2-(3-Ethyl-5-(2-oxo-2-phenylethylthio)-4H-1,2,4-triazol-4-
yl)isoindoline-1,3-dione (20). Yield, 67%; mp 158e159 ꢀC; 1H NMR
CDCl3):
d
¼ 9.9, 43.2, 125.0, 128.5, 128.8, 129.2, 134.1, 134.7, 135.8,
150.5, 152.7, 162.2, 192.7. ESI-MS: m/z ¼ 401.34 [M þ Na]þ, 379.42
[M þ 1]þ. Anal. Calcd. for C20H16ClN4O3S: C 60.31, H 3.73, N 14.81,
S 8.47. Found C 60.69, H 3.99, N 14.51, S 8.30.
(400 MHz, CDCl3)
d
: 1.29 (t, J ¼ 7.6 Hz, 3H, CH2CH3), 2.61 (dd, 2H,
J ¼ 7.2 Hz, J ¼ 15.2 Hz, eCH2CH3), 4.88 (s, 2H, SCH2), 7.42e7.47 (t,
J ¼ 7.6 Hz, 2H, ArH), 7.56 (d, J ¼ 7.6 Hz, 1H, ArH), 7.89e7.94 (m, 4H,
ArH), 7.99 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (100 MHz,
5.1.1.6. 2-(3-Methyl-5-(prop-2-ynylthio)-4H-1,2,4-triazol-4-yl)iso-
CDCl3):
d
¼ 10.8, 17.8, 43.1, 124.8, 128.3, 128.7, 129.0, 134.0, 134.6,
indoline-1,3-dione (14). Yield, 75%;mp 96e98ꢀC;1H NMR (400 MHz,
135.7, 150.3, 156.9, 162.2, 192.6. ESI-MS: m/z ¼ 415.31 [M þ Na]þ,
393.47 [M]þ. Anal. Calcd. for C20H16N4O3S: C 61.21, H 4.11, N 14.28,
S 8.17. Found C 61.35, H 4.31, N 14.15, S 8.39.
CDCl3)
d
: 2.21 (t, J ¼ 2.6 Hz,1H, ^CH), 2.35 (s, 3H, triazoleeCH3), 3.81
(d, J ¼ 2.4 Hz, 2H, SCH2), 7.92 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH), 8.01
(dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3):
d
¼ 10.0, 22.7, 73.3, 77.5,124.9,129.1,135.8,149.3,152.9,162.2. ESI-MS:
5.1.1.13. 2-(3-(2-(4-Chlorophenyl)-2-oxoethylthio)-5-ethyl-4H-1,2,4-
m/z ¼ 299.09[Mþ 1]þ. Anal. Calcd. forC14H10N4O2S:C 56.37, H3.38, N
triazol-4-yl)isoindoline-1,3-dione (21). Yield, 72%; mp 170e171 ꢀC; 1H
18.78, S 10.75. Found C 56.70, H 3.62, N 18.58, S 10.48.
NMR (400 MHz, CDCl3)
d
: 1.28 (t, J ¼ 7.6 Hz, 3H, CH2CH3), 2.61 (dd,
J¼ 7.6 Hz, J¼ 15.2 Hz, 2H, CH2CH3), 4.82 (s, 2H, SCH2), 7.42(dd, J¼ 2.8Hz,
J ¼ 8.8 Hz, 2H, ArH), 7.86e7.92 (m, 4H, ArH), 7.98e8.00 (m, 2H, ArH).13C
5.1.1.7. 2-(3-Methyl-5-(methylthio)-4H-1,2,4-triazol-4-yl)isoindo-
line-1,3-dione (15). Yield, 78%; mp 113e115 ꢀC; 1H NMR (400 MHz,
NMR (100 MHz, CDCl3):
d
¼ 10.8, 17.8, 42.6, 124.8, 129.0, 129.1, 129.8,
CDCl3)
d: 2.32 (s, 3H, triazoleeCH3), 2.61 (s, 3H, SCH3), 7.91 (dd,
133.0, 135.7, 140.5, 150.0, 156.9, 162.2, 191.5. ESI-MS: m/z ¼ 449.26
[M þ Na]þ, 427.23 [M þ 1]þ. Anal. Calcd. for C20H15ClN4O3S: C 56.27, H
3.54, N 13.12, S 7.51. Found C 56.40, H 3.80, N 13.41, S 7.79.
J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH), 8.00 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H,
ArH). 13C NMR (150 MHz, CDCl3):
d
¼ 9.6, 15.4, 124.6, 128.8, 135.6,
151.6, 152.2, 162.0. ESI-MS: m/z ¼ 297.20 [M þ Na]þ, 275.15
[M þ 1]þ. Anal. Calcd. for C12H10N4O2S: C 52.54, H 3.67, N 20.43,
S 11.69. Found C 52.37, H 3.76, N 20.15, S 11.38.
5.1.1.14. 2-(3-Ethyl-5-(2-oxo-2-p-tolylethylthio)-4H-1,2,4-triazol-4-
yl)isoindoline-1,3-dione (22). Yield, 69%; mp 130e131 ꢀC; 1H NMR
(400 MHz, CDCl3)
d
: 1.29 (t, J ¼ 3.6 Hz, 3H, CH2CH3), 2.38 (s, 3H,
5.1.1.8. 2-(3-(2-Chlorobenzylthio)-5-methyl-4H-1,2,4-triazol-4-yl)
AreCH3), 2.62 (dd, J ¼ 7.6 Hz, J ¼ 14.8 Hz, 2H, CH2CH3), 4.86 (s, 2H,
SCH2), 7.24 (t, J ¼ 4.0 Hz, 2H, ArH), 7.83 (d, J ¼ 8.0 Hz, 2H, ArH), 7.90 (dd,
J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH), 7.99 (dd, J ¼ 3.0 Hz, J ¼ 5.6 Hz, 2H, ArH).
isoindoline-1,3-dione (16). Yield, 73%; mp 134e136 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 2.35 (s, 3H, triazoleeCH3), 4.42 (s, 2H, SCH2),
13C NMR (100 MHz, CDCl3):
d
¼ 10.9, 17.9, 21.7, 43.3, 124.9, 128.5, 129.2,
7.15e7.19 (m, 2H, ArH), 7.27 (t, J ¼ 2.8 Hz,1H, ArH), 7.39 (d, J ¼ 2.4 Hz,
1H, ArH), 7.90 (dd, J ¼ 3.2 Hz, J ¼ 5.2 Hz, 2H, ArH), 7.98 (d, J ¼ 3.0 Hz,
129.5,132.3,135.7,145.2,150.5,156.9,162.3,192.3. ESI-MS:m/z¼ 430.09
[M þ Na]þ, 407.13 [M þ 1]þ. Anal. Calcd. for C21H18N4O3S: C 62.05, H
4.46, N 13.78, S 7.89. Found C 62.16, H 4.56, N 13.56, S 7.98.
J ¼ 5.4 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3):
¼ 10.0, 36.1, 124.8,
d
127.0, 129.1, 129.4, 129.5, 131.4, 133.5, 134.1, 135.7, 150.3, 152.6, 162.1.
ESI-MS: m/z ¼ 385.34 [M þ 1]þ. Anal. Calcd. for C18H13ClN4O2S: C
56.18, H 3.40, N 14.56, S 8.33. Found C 55.95, H 3.64, N 14.35, S 8.15.
5.1.1.15. 2-(3-Ethyl-5-(methylthio)-4H-1,2,4-triazol-4-yl)isoindoline-
1,3-dione (23). Yield, 73%; mp 82e83 ꢀC; 1H NMR (400 MHz, CDCl3)
5.1.1.9. 2-(3-Ethyl-5-(prop-2-ynylthio)-4H-1,2,4-triazol-4-yl)iso-
d
: 1.29 (t, J ¼ 3.6 Hz, 3H, eCH2CH3), 2.61 (dd, J ¼ 4.8 Hz, J ¼ 7.2 Hz,
indoline-1,3-dione (17). Yield, 71%; mp 128e130 ꢀC; 1H NMR
2H, eCH2CH3), 2.65 (s, 3H, SCH3), 7.91 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H,

P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
819
ArH), 8.01 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (150 MHz,
J ¼ 7.6 Hz, J ¼ 15.2 Hz, 2H, eCH2CH2CH3), 2.21 (t, J ¼ 2.6 Hz, 1H,
^CH), 2.58 (t, J ¼ 7.6 Hz, 2H, eCH2CH2CH3), 3.82 (d, J ¼ 2.8 Hz, 2H,
SCH2), 7.92 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH), 8.02 (dd, J ¼ 3.0 Hz,
DMSO-d6):
d
¼ 11.0, 15.7, 17.9, 124.9, 129.2, 135.7, 151.8, 156.7, 162.4.
ESI-MS: m/z ¼ 311.12 [M þ Na]þ, 289.20 [M þ 1]þ. Anal. Calcd. for
C13H12N4O2S: C 54.15, H 4.20, N 19.43, S 11.12. Found C 53.98,
H 4.48, N 19.20, S 11.43.
J ¼ 16.0 Hz, 2H, ArH). 13C NMR (100 MHz, DMSO-d6):
¼ 13.5, 19.8,
d
20.4, 25.2, 74.8, 79.3, 128.1, 130.0, 130.5, 130.8, 132.1, 134.8, 149.0,
156.3, 167.1, 167.5. ESI-MS: m/z ¼ 350.06 [M þ Na]þ, 327.13
[M þ 1]þ. Anal. Calcd. for C16H14N4O2S: C 58.88, H 4.32, N 17.17,
S 9.82. Found C 59.06, H 4.52, N 16.96, S 9.49.
5.1.1.16. Ethyl
triazol-3-ylthio)acetate (24). Yield, 69%; mp 110e111 ꢀC; 1H NMR
(400 MHz, CDCl3)
2-(4-(1,3-dioxoisoindolin-2-yl)-5-ethyl-4H-1,2,4-
d
: 1.20 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.28 (t,
J ¼ 7.6 Hz, 3H, eCH2CH3), 2.60 (dd, J ¼ 7.6 Hz, J ¼ 15.2 Hz, 2H,
eCH2CH3), 3.94 (s, 2H, SCH2), 4.13 (dd, J ¼ 7.2 Hz, J ¼ 14.2 Hz, 2H,
OCH2CH3), 7.90e7.93 (m, 2H, ArH), 8.00 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz,
5.1.1.22. 2-(3-(2-Chlorobenzylthio)-5-propyl-4H-1,2,4-triazol-4-yl)
isoindoline-1,3-dione (30). Yield, 66%; mp 91e93 ꢀC; 1H NMR
(400 MHz, CDCl3)
d
: 0.93 (t, J ¼ 7.6 Hz, 3H, eCH2CH2CH3), 1.71 (m,
2H, ArH). 13C NMR (100 MHz, CDCl3):
d
¼ 10.6, 13.7, 17.7, 35.5, 61.9,
2H, eCH2CH2CH3), 2.54 (t, J ¼ 7.6 Hz, 2H, eCH2CH2CH3), 4.39 (s, 2H,
SCH2), 7.13e7.17 (m, 2H, ArH), 7.25 (t, J ¼ 4.8 Hz, 1H, ArH), 7.38 (d,
J ¼ 2.0 Hz, 1H, ArH), 7.89 (dd, J ¼ 3.2 Hz, J ¼ 5.2 Hz, 2H, ArH), 7.97
(dd, J ¼ 2.8 Hz, J ¼ 5.2 Hz, 2H, ArH). 13C NMR (150 MHz, CDCl3):
124.7, 128.9, 135.7, 149.5, 156.9, 162.1, 167.5. ESI-MS: m/z ¼ 384.26
[M þ Na]þ, 361.21 [M þ 1]þ. Anal. Calcd. for C16H16N4O4S: C, 53.32;
H, 4.47; N, 15.55; S, 8.90. Found C 53.47, H 4.68, N 15.09, S 8.78.
d
¼ 13.6, 19.9, 26.2, 36.2, 124.9, 127.1, 129.2, 129.4, 129.6, 131.5, 133.6,
5.1.1.17. 2-(3-(Allylthio)-5-propyl-4H-1,2,4-triazol-4-yl)isoindoline-
134.2, 135.7, 150.3, 155.9, 162.3. ESI-MS: m/z ¼ 415.26 [M þ 1]þ.
Anal. Calcd. for C20H17ClN4O2S: C 58.18, H 4.15, N 13.57, S 7.77.
Found C 58.46, H 4.38, N 13.43, S 7.98.
1,3-dione (25). Yield, 64%; mp 125e127 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
: 0.93 (t, J ¼ 7.4 Hz, 3H, eCH2CH2CH3), 1.71 (dd, J ¼ 7.6 Hz,
J ¼ 15.2 Hz, 2H, eCH2CH2CH3), 2.55 (t, J ¼ 7.8 Hz, 2H, eCH2CH2CH3),
3.69 (d, J ¼ 7.2 Hz, 2H, SCH2), 5.076e5.20 (m, 2H, CH]CH2),
5.78e5.87 (m, 1H, CH]CH2), 7.90e7.93 (m, 2H, ArH), 7.98e8.02 (m,
5.1.1.23. 2-(3-(2-Oxo-2-phenylethylthio)-5-propyl-4H-1,2,4-triazol-
4-yl)isoindoline-1,3-dione (31). Yield, 64%; mp 96e97 ꢀC; 1H NMR
2H, ArH). 13C NMR (100 MHz, CDCl3):
d
¼ 13.6,19.9, 26.2, 36.8, 119.4,
(400 MHz, DMSO-d6)
d
: 0.93 (t, J ¼ 7.4 Hz, 3H, eCH2CH2CH3), 1.67
124.9, 129.1, 131.9, 135.7, 150.1, 155.7, 162.4. ESI-MS: m/z ¼ 351.31
[M þ Na]þ, 329.22 [Mþ1]þ. Anal. Calcd. for C16H16N4O2S: C 58.52,
H 4.91, N 17.06, S 9.76. Found C 58.60, H 5.15, N 17.10, S 9.93.
(m, 2H, eCH2CH2CH3), 2.69 (t, J ¼ 7.0 Hz, 2H, eCH2CH2CH3), 4.92 (d,
J ¼ 23.6 Hz, 2H, SCH2), 7.56 (t, J ¼ 7.8 Hz, 3H, ArH), 7.69 (t, J ¼ 3.8 Hz,
2H, ArH), 7.75 (dd, J ¼ 1.2 Hz, J ¼ 7.6 Hz, 1H, ArH), 7.94 (dd,
J ¼ 0.8 Hz, J ¼ 7.6 Hz, 1H, ArH), 8.02 (t, J ¼ 4.2 Hz, 2H, ArH). 13C NMR
5.1.1.18. 2-(3-(Benzylthio)-5-propyl-4H-1,2,4-triazol-4-yl)isoindo-
(150 MHz, CDCl3 þ DMSO-d6): ¼ 11.8, 18.1, 23.6, 38.3, 126.3, 126.6,
d
line-1,3-dione (26). Yield, 67%; mp 106e107 ꢀC; 1H NMR (400 MHz,
126.9, 128.2, 128.7, 128.8, 130.0, 131.8, 133.3, 133.6, 148.3, 154.4,
165.3, 165.8, 191.2. ESI-MS: m/z ¼ 430.06 [M þ Na]þ, 407.13
[M þ 1]þ. Anal. Calcd. for C21H18N4O3S: C 62.05, H 4.46, N 13.78,
S 7.89. Found C 62.23, H 4.64, N 13.62, S 7.73.
CDCl3)
d
: 0.95 (t, J ¼ 7.4 Hz, 3H, eCH2CH2CH3), 1.70e1.76 (m, 2H,
eCH2CH2CH3), 2.57 (t, J ¼ 7.8 Hz, 2H, eCH2CH2CH3), 4.36 (s, 2H,
SCH2), 7.20e7.28 (m, 5H, ArH), 7.89 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H,
ArH), 7.99 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (100 MHz,
CDCl3):
d
¼ 13.6, 20.0, 26.1, 38.3, 124.9, 127.9, 128.6, 129.0, 129.1,
5.1.1.24. 2-(3-(Methylthio)-5-propyl-4H-1,2,4-triazol-4-yl)isoindo-
135.4, 135.7, 150.7, 155.7, 162.3. ESI-MS: m/z ¼ 412.27 [M þ Na]þ,
379.46 [M þ 1]þ. Anal. Calcd. for C20H18N4O2S: C 63.47, H 4.79, N
14.80, S 8.47. Found C 63.62, H 4.81, N 14.62, S 8.83.
line-1,3-dione (32). Yield, 67%; mp 77e79 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H, eCH2CH2CH3), 1.67e1.77 (m, 2H,
CH2CH2CH3), 2.55 (t, J ¼ 7.2 Hz, 2H, eCH2CH2CH3), 2.62 (s, 3H,
SCH3), 7.89e7.94 (m, 2H, ArH), 7.99e8.03 (m, 2H, ArH). 13C NMR
5.1.1.19. 2-(3-(2-(4-Chlorophenyl)-2-oxoethylthio)-5-propyl-4H-
(150 MHz, CDCl3):
d
¼ 13.5, 15.6, 19.9, 26.0, 124.8, 129.1, 135.7, 151.7,
1,2,4-triazol-4-yl)isoindoline-1,3-dione
(27). Yield,
: 0.94 (t, J ¼ 7.2 Hz, 3H,
65%;
mp
155.6, 162.4. ESI-MS: m/z ¼ 325.13 [M þ Na]þ, 303.32 [M þ 1]þ.
Anal. Calcd. for C14H14N4O2S: C 55.61, H 4.67, N 18.53, S 10.61. Found
C 55.57, H 4.94, N 18.26, S 10.83.
91e92 ꢀC; 1H NMR (400 MHz, CDCl3)
d
eCH2CH2CH3), 1.69e1.75 (m, 2H, eCH2CH2CH3), 2.56 (t, J ¼ 7.6 Hz, 2H,
eCH2CH2CH3), 4.83 (s, 2H, SCH2), 7.42 (d, J¼ 8.4 Hz, 2H, ArH), 7.87e7.93
(m, 4H, ArH), 8.00 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR
5.1.1.25. 2-(3-(Allylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-tria-
(150 MHz, CDCl3 þ DMSO-d6):
d
¼ 11.8, 18.1, 23.5, 38.1, 126.3, 127.0,
zol-4-yl)isoindoline-1,3-dione (33). Yield, 73%; mp 72e74 ꢀC; 1H
128.1, 128.4, 128.7, 130.0, 132.1, 133.2, 137.2, 148.1, 154.4, 165.3, 165.7,
190.3. ESI-MS: m/z ¼ 441.09 [M þ 1]þ. Anal. Calcd. for C21H17ClN4O3S: C
57.21, H 3.89, N 12.71, S 7.27. Found C 57.49, H 3.67, N 12.59, S 7.60.
NMR (400 MHz, CDCl3)
d: 3.71 (s, 6H, 2 ꢂ OCH3), 3.80 (s, 3H, OCH3),
3.83 (d, J ¼ 7.2 Hz, 2H, ]CH2), 5.16 (dd, J ¼ 0.6 Hz, J ¼ 9.8 Hz, 1H,
triazoleeSCHa), 5.28 (dd, J ¼ 1.2 Hz, J ¼ 16.8 Hz, 1H, triazoleeSCHb),
5.87e5.95 (m, 1H, CH]), 6.95 (s, 2H, ArH), 7.89 (dd, J ¼ 3.2 Hz,
J ¼ 5.6 Hz, 2H, ArH), 7.96 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C
5.1.1.20. 2-(3-(2-Oxo-2-p-tolylethylthio)-5-propyl-4H-1,2,4-triazol-
4-yl)isoindoline-1,3-dione (28). Yield, 70%; mp 98e99 ꢀC; 1H NMR
NMR (150 MHz, CDCl3):
d
¼ 36.5, 56.0, 60.8, 105.0, 119.7, 119.8,
(400 MHz, CDCl3)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.70e1.76
124.8, 129.1, 131.8, 135.8, 140.1, 152.0, 153.5, 155.2, 162.7. ESI-MS:
m/z (%): m/z ¼ 475.04 [M þ Na]þ, 453.08 [M þ 1]þ. Anal. Calcd.
for C22H20N4O5S: C 58.40, H 4.46, N 12.38, S, 7.09. Found C 58.15, H
4.74, N 12.50, S 6.83.
(m, 2H, CH2CH2CH3), 2.39 (s, 3H, AreCH3), 2.57 (t, J ¼ 7.2 Hz, 2H,
eCH2CH2CH3), 4.88 (s, 2H, SCH2), 7.25 (d, J ¼ 7.2 Hz, 2H, ArH), 7.84
(d, J ¼ 8.4 Hz, 2H, ArH), 7.91 (dd, J ¼ 2.8 Hz, J ¼ 5.2 Hz, 2H, ArH), 8.00
(dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (150 MHz, CDCl3):
d
¼ 13.6, 20.1, 21.7, 26.2, 43.3, 125.0, 128.6, 128.7, 129.3, 129.6, 132.3,
5.1.1.26. 2-(3-(2-Chlorobenzylthio)-5-(3,4,5-trimethoxyphenyl)-4H-
135.8, 145.2, 150.6, 155.9, 162.3, 192.4. ESI-MS: m/z ¼ 434.26
[M þ Na]þ, 421.13 [M þ 1]þ. Anal. Calcd. for C22H20N4O3S: C 62.84,
H 4.79, N 13.32, S 7.63. Found C 62.75, H 4.59, N 13.12, S 7.82.
1,2,4-triazol-4-yl)isoindoline-1,3-dione
(34). Yield,
62%;
mp
76e77 ꢀC; 1H NMR (400 MHz, CDCl3)
d
: 3.70 (s, 6H, 2 ꢂ OCH3), 3.79
(s, 3H, OCH3), 4.53 (s, 2H, SCH2), 6.79 (s, 2H, ArH), 7.18e7.31 (m, 3H,
ArH), 7.50 (t, J ¼ 4.6 Hz, 1H, ArH), 7.87 (dd, J ¼ 3.0 Hz, J ¼ 6.0 Hz, 2H,
ArH), 7.94 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH). 13C NMR (150 MHz,
5.1.1.21. 2-(3-(Prop-2-ynylthio)-5-propyl-4H-1,2,4-triazol-4-yl)iso-
indoline-1,3-dione (29). Yield, 77%; mp 98e99 ꢀC; 1H NMR
CDCl3):
d
¼ 35.7, 56.0, 60.7, 104.9, 119.7, 124.7, 127.0, 128.9, 129.4,
(400 MHz, CDCl3)
d
: 0.95 (t, J ¼ 7.4 Hz, 3H, eCH2CH2CH3), 1.74 (dd,
129.5, 131.5, 133.4, 134.1, 135.8, 140.0, 152.2, 153.5, 155.2, 162.5. ESI-

820
P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
MS: m/z (%): m/z ¼ 559.10 [M þ Na]þ, 537.12 [M þ 1]þ. Anal. Calcd.
for C26H21CN4O5S: C 58.15, H 3.94, N 10.43, S, 5.97. Found C 58.45,
H 4.23, N 10.67, S 6.16.
135.3, 135.8, 140.1, 152.5, 153.5, 155.1, 162.5. ESI-MS: m/z ¼ 525.13
[M þ Na]þ, 503.06 [M þ 1]þ. Anal. Calcd. for C26H22N4O5S: C 62.14, H
4.41, N 11.15, S 6.38. Found C 62.40, H 4.68, N 10.87, S 6.71.
5.1.1.27. 2-(3-(Prop-2-ynylthio)-5-(3,4,5-trimethoxyphenyl)-4H-
5.1.1.33. 2-(3-(2-Oxo-2-phenylethylthio)-5-(3,4,5-trimethoxyph-
1,2,4-triazol-4-yl)isoindoline-1,3-dione
(35). Yield,
: 2.25 (s, 2H, ^CH), 3.70 (s,
69%;
mp
enyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione (41). Yield, 73%;
80e81 ꢀC; 1H NMR (400 MHz, CDCl3)
d
mp 161e162 ꢀC; 1H NMR (400 MHz, CDCl3)
d
: 3.71 (s, 6H, 2 ꢂ OCH3),
6H, 2 ꢂ OCH3), 3.79 (s, 3H, OCH3), 3.93 (s, 2H, SCH2), 6.79 (s, 2H,
3.80 (s, 3H, OCH3), 5.01 (s, 2H, SCH2), 6.80 (s, 2H, ArH), 7.48 (t,
J ¼ 7.6 Hz, 2H, ArH), 7.61 (t, J ¼ 7.4 Hz, 1H, ArH), 7.88e7.91 (m, 2H,
ArH), 7.95e8.00 (m, 4H, ArH). ESI-MS: m/z ¼ 553.3 [M þ Na]þ, 531.4
ArH), 7.88e7.91 (m, 2H, ArH), 7.94e7.97 (m, 2H, ArH). 13C NMR
(100 MHz, CDCl3):
d
¼ 22.4, 55.9, 60.7, 104.9, 119.5, 124.8, 128.8,
135.9, 140.1, 151.0, 153.4, 155.4, 162.5. ESI-MS: m/z (%): m/z ¼ 473.01
[M þ Na]þ, 451.01 [M þ 1]þ. Anal. Calcd. for C22H18N4O5S: C 58.66, H
4.03, N 12.44, S, 7.12. Found C 58.88, H 3.87, N 12.31, S 7.41.
[M þ 1]þ. 13C NMR (150 MHz, CDCl3):
d
¼ 42.6, 55.8, 60.6, 104.7,
119.4, 124.6, 128.2, 128.6, 128.7, 133.9, 134.5, 135.7, 140.0, 152.0,
153.3, 155.0, 162.4, 192.3. Anal. Calcd. for C27H22N4O6S: C 61.12,
H 4.18, N 10.56, S 6.04. Found C 60.90, H 3.91, N 10.72, S 6.31.
5.1.1.28. 2-(3-(2-(4-Chlorophenyl)-2-oxoethylthio)-5-(3,4,5-trime-
thoxyphenyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione (36). Yield,
5.1.1.34. 2-(3-(2-(4-Methoxyphenyl)-2-oxoethylthio)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione
78%; mp 79e80 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.71 (s, 6H,
2 ꢂ OCH3), 3.80 (s, 3H, OCH3), 4.94 (s, 2H, SCH2), 6.79 (s, 2H, ArH),
(42). Yield, 86%; mp 187e188 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.71
7.45 (d, J ¼ 8.8 Hz, 2H, ArH), 7.88e7.97 (m, 6H, ArH). 13C NMR
(s, 6H, 2 ꢂ OCH3), 3.80 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.98 (s, 2H,
SCH2), 6.79 (s, 2H, Ar), 6.93 (d, J ¼ 8.8 Hz, 2H, ArH), 7.89 (dd, J ¼ 3.0 Hz,
J ¼ 5.4 Hz, 2H, ArH), 7.94e7.98 (m, 4H, ArH). 13C NMR (150 MHz,
(150 MHz, CDCl3):
d
¼ 42.2, 55.8, 60.6, 104.7, 119.4, 124.7, 128.7,
128.9, 129.7,132.9,135.7,139.9,140.4,151.8,153.3,155.1,162.4, 191.2.
ESI-MS: m/z ¼ 587.09 [M þ Na]þ, 565.13 [M þ 1]þ. Anal. Calcd. for
C27H21ClN4O6S: C 57.40, H 3.75, N 9.92, S, 5.68. Found C 57.12,
H 3.98, N 9.71, S 5.97.
CDCl3):
d
¼ 12.7, 55.4, 55.9, 60.7,104.9,114.0,119.6,124.8,127.7,128.9,
130.8, 135.8, 140.0, 152.3, 153.5, 155.1, 162.5, 164.2, 190.9. ESI-MS:
m/z ¼ 583.2 [M þ Na]þ, 561.3 [Mþ1]þ. Anal. Calcd. for C28H24N4O7S:
C 59.99, H 4.32, N 9.99, S 5.72. Found C 59.78, H 4.59, N 10.02, S 5.92.
5.1.1.29. 2-(3-(2-Oxo-2-p-tolylethylthio)-5-(3,4,5-trimethoxyph-
enyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione (37). Yield, 69%;
5.1.1.35. 2-(3-(2-Oxo-2-o-tolylethylthio)-5-(3,4,5-trimethoxyph-
mp 197e198 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.40 (s, 3H, AreCH3),
enyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione (43). Yield, 69%;
3.71 (s, 6H, 2 ꢂ OCH3), 3.80 (s, 3H, OCH3), 4.97 (s, 2H, CH2), 6.79 (s,
mp 170e172 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.49 (s, 3H, AreCH3),
2H, ArH), 7.26 (t, J ¼ 8.0 Hz, 2H, ArH), 7.87e7.89 (m, 4H, ArH), 7.96 (dd,
3.71 (s, 6H, 2 ꢂ OCH3), 3.80 (s, 3H, OCH3), 4.95 (s, 2H, SCH2), 6.80
(s, 2H, ArH), 7.26e7.32 (m, 2H, ArH), 7.42 (t, J ¼ 7.2 Hz,1H, ArH), 7.83
(d, J ¼ 7.6 Hz, 1H, ArH), 7.89 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH),
J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (150 MHz, CDCl3):
¼ 21.7,
d
43.0, 56.0, 60.7, 104.9, 119.6, 124.8, 128.5, 129.0, 129.5, 132.2, 135.8,
140.0, 145.2, 152.3, 153.5, 155.3, 162.5, 192.1. ESI-MS: m/z ¼ 567.20
[M þ Na]þ, 545.21 [M þ 1]þ. Anal. Calcd. for C28H24N4O6S: C 61.75, H
4.44, N 10.29, S 5.89. Found C 61.79, H 4.72, N 9.99, S 6.27.
7.95e7.98 (m, 2H, ArH). 13C NMR (150 MHz, CDCl3):
d
¼ 21.6, 44.9,
56.0, 60.8, 105.0, 119.6, 124.8, 126.0, 129.0, 129.6, 132.3, 132.7, 134.5,
135.8, 139.6, 140.1, 152.3, 153.5, 155.3, 162.5, 195.2. ESI-MS:
m/z ¼ 567.4 [M þ Na]þ, 545.5 [M þ 1]þ. Anal. Calcd. for C28H24N4O6S:
C 61.75, H 4.44, N 10.29, S 5.89. Found C 61.50, H 4.27, N 10.05, S 6.20.
5.1.1.30. 2-(3-(Methylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-
triazol-4-yl)isoindoline-1,3-dione (38). Yield, 72%; mp 77e79 ꢀC; 1H
NMR (400 MHz, CDCl3)
d
: 2.70 (s, 3H, SCH3), 3.71 (s, 6H, 2 ꢂ OCH3),
5.1.1.36. 2-(3-(2-(4-Fluorophenyl)-2-oxoethylthio)-5-(3,4,5-trime-
3.79 (s, 3H, OCH3), 6.79 (s, 2H, ArH), 7.89 (dd, J ¼ 2.8 Hz, J ¼ 5.2 Hz,
thoxyphenyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione (44). Yield,
2H, ArH), 7.96 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR
79%; mp 168e169 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.71 (s, 6H,
(150 MHz, CDCl3):
d
¼ 15.4, 55.9, 60.7, 104.9, 119.7, 124.8, 128.9,
2 ꢂ OCH3), 3.80 (s, 3H, OCH3), 4.96 (s, 2H, SCH2), 6.79 (s, 2H, ArH),
7.15 (t, J ¼ 8.6 Hz, 2H, ArH), 7.89 (dd, J ¼ 3.2 Hz, J ¼ 5.2 Hz, 2H, ArH),
7.97 (dd, J ¼ 3.0 Hz, J ¼ 5.4 Hz, 2H, ArH), 8.03 (dd, J ¼ 5.4 Hz,
135.8,140.0,153.4,153.5,155.1,162.6. ESI-MS: m/z (%): m/z ¼ 449.02
[M þ Na]þ, 427.06 [M þ 1]þ. Anal. Calcd. for C20H18N4O5S: C 56.33,
H 4.25, N 13.14, S 7.52. Found C 56.68, H 4.16, N 13.21, S 7.90.
J ¼ 8.6 Hz, 2H, ArH). 13C NMR (150 MHz, CDCl3):
¼ 42.4, 55.9, 60.6,
d
104.8, 115.8, 116.0, 119.4, 124.7, 128.8, 131.1, 131.2, 135.8, 140.0, 152.0,
153.4,155.1,162.4,165.2,166.9,190.9. ESI-MS: m/z ¼ 587.4 [M þ K]þ,
549.5 [M þ 1]þ. Anal. Calcd. for C27H21FN4O6S: C 59.12, H 3.86,
N 10.21, S 5.85; Found C 59.06, H 3.99, N 10.03, S 6.04.
5.1.1.31. Ethyl 2-(4-(1,3-dioxoisoindolin-2-yl)-5-(3,4,5-trimethoxyph
enyl)-4H-1,2,4-triazol-3-ylthio)acetate
(39). Yield,
66%;
mp
59e61 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 1.24 (t, J ¼ 7.2 Hz, 2H,
OCH2CH3), 3.69 (d, J ¼ 3.2 Hz, 6H, 2 ꢂ OCH3), 3.78 (s, 3H, OCH3),
4.05 (s, 2H, SCH2), 4.17 (dd, J ¼ 7.2 Hz, J ¼ 14.2 Hz, 2H, OCH2CH3),
6.77 (d, J ¼ 2.0 Hz, 2H, ArH), 7.88 (dd, J ¼ 2.8 Hz, J ¼ 5.2 Hz, 2H, ArH),
7.96 (dd, J ¼ 3.2 Hz, J ¼ 5.6 Hz, 2H, ArH). 13C NMR (150 MHz, CDCl3):
5.1.1.37. 2-(3-(2-(4-Bromophenyl)-2-oxoethylthio)-5-(3,4,5-trime-
thoxyphenyl)-4H-1,2,4-triazol-4-yl )isoindoline-1,3-dione (45). Yield,
76%; mp 189e190 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.71 (s, 6H,
d
¼ 13.9, 35.3, 55.9, 60.7, 62.1, 104.9, 119.5, 124.8, 128.9, 135.8, 140.1,
2 ꢂ OCH3), 3.80 (s, 3H, OCH3), 4.96 (s, 2H, SCH2), 6.79 (s, 2H, ArH), 7.63
151.4, 153.4, 155.2, 162.4, 167.5. ESI-MS: m/z ¼ 521.12 [M þ Na]þ,
499.09 [M þ 1]þ. Anal. Calcd. for C23H22N4O7S: C 55.41, H 4.45,
N 11.24, S 6.43. Found C 55.58, H 4.70, N 11.17, S 6.28.
(d, J ¼ 8.4 Hz, 2H, ArH), 7.86e7.91 (m, 4H, ArH), 7.97 (dd, J ¼ 3.0 Hz,
J ¼ 5.4 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3):
¼ 42.4, 56.0, 60.7,
d
104.9, 119.5, 124.8, 128.9, 129.4, 129.9, 132.4, 133.4, 135.8, 140.1, 151.9,
153.5, 155.3, 162.5, 191.6. ESI-MS: m/z ¼ 611.3 [M þ Na]þ, 609.2
[Mþ 1]þ. Anal. Calcd.forC27H21BrN4O6S:C 53.21, H3.47, N9.19, S5.26.
Found C 53.10, H 3.66, N 9.32, S 5.48.
5.1.1.32. 2-(3-(Benzylthio)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-
triazol-4-yl)isoindoline-1,3-dione (40). Yield, 79%; mp 77e78 ꢀC; 1H
NMR (400 MHz, CDCl3)
d
: 3.70 (s, 6H, 2 ꢂ OCH3), 3.79 (s, 3H, OCH3),
4.45 (s, 2H, SCH2), 6.79 (s, 2H, ArH), 7.25 (t, J ¼ 3.8 Hz, 3H, ArH), 7.31
(dd, J ¼ 1.2 Hz, J ¼ 7.6 Hz, 2H, ArH), 7.86 (dd, J ¼ 3.0 Hz, J ¼ 4.4 Hz, 2H,
ArH), 7.93 (dd, J ¼ 3.0 Hz, J ¼ 4.4 Hz, 2H, ArH). 13C NMR (150 MHz,
5.1.1.38. 2-(3-(2-(3,4-Difluorophenyl)-2-oxoethylthio)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione
(46). Yield, 64%; mp 166e168 ꢀC; 1H NMR (400 MHz, CDCl3)
d:
CDCl3):
d
¼ 37.9, 56.0, 60.8,104.9,119.7,124.7,127.9,128.6,128.9,129.0,
3.70 (s, 6H, 2 ꢂ OCH3), 3.79 (s, 3H, OCH3), 4.89 (s, 2H, SCH2), 6.77

P.-L. Zhao et al. / European Journal of Medicinal Chemistry 54 (2012) 813e822
821
(s, 2H, ArH), 7.26 (t, J ¼ 8.0 Hz, 1H, ArH), 7.79e7.84 (m, 2H, ArH),
7.88e7.91 (m, 2H, ArH), 7.94e7.97 (m, 2H, ArH). 13C NMR
10% fetal bovine serum (FBS). Cells were passaged at 70e80%
confluence, about twice a week by trypsinization.
(100 MHz, CDCl3):
d
¼ 41.9, 55.9, 60.6, 104.8, 117.6, 117.7, 117.8,
Exponentially growing cells were plated in 96-well plates (1 ꢂ 104
cells/well for HepG2 cells, 1.6 ꢂ 104 cells/well for A549, PC-3M and
MKN45 cells) and incubated at 37 ꢀC for 24 h for attachment. Test
compounds were prepared by dissolving in dimethyl sulfoxide (DMSO)
at 10 mM and diluted with the medium. Then, culture medium was
changed, and cells grew in medium with the test compounds. DMSO
(0.1%) was used as negative control. Cells were incubated at 37 ꢀC for
48 h. Then the medium was replaced with MTT solution (5 mg/ml,
119.3, 124.7, 125.6, 125.7, 128.8, 131.7, 131.8, 135.8, 140.0, 149.4,
149.5, 151.1, 151.2, 151.7, 153.1, 153.2, 153.4, 154.8, 154.9, 155.2,
162.4, 190.1. ESI-MS: m/z ¼ 589.3 [M þ Na]þ, 567.4 [M þ 1]þ. Anal.
Calcd. for C27H20F2N4O6S: C 57.24, H 3.56, N 9.89, S 5.66. Found C
57.51, H 3.79, N 10.06, S 5.91.
5.2. Crystallographic analysis
100
mL) followed by incubation for another 3 h. The medium was then
Colorless blocks of 37 were mounted on a quartz fiber. Cell
dimensions and intensities were measured at 293 K on a Bruker
SMART CCD area detector diffractometer with graphite mono-
aspirated and formazan crystals were dissolved in DMSO (100
m
L) for
about 10 min. The absorbance at 570 nm (Abs) of the suspension was
measured by an enzyme-linked immunosorbent assay (ELISA) reader.
The inhibitionpercentage was calculated using the following formula: %
inhibition ¼ (Abscontrol ꢃ Abscompound)/Abscontrol ꢂ 100%. The IC50 values
of the test compounds and Fluorouracil were measured by treating cells
with drugs of varying concentrations, and analyzed by use of the prism
statistical package (GraphPad Software, San Diego, CA, U.S.A.).
chromated Mo K
a
radiation (
l
¼ 0.71073 Å); qmax ¼ 27.48; 13,548
measured reflections; 6130 independent reflections (Rint ¼ 0.0514) of
which 3868 had jFoj > 2jFoj. Data were corrected for Lorentz and
polarization effects and for absorption (Tmin ¼ 0.4415; Tmax ¼ 0.6353).
The structure was solved by direct methods using SHELXS-97 [31]; all
other calculations were performed with Bruker SAINT System and
Bruker SMART programs [32]. Full-matrix least-squares refinement
5.3.3. Flow-activating cell sorting analysis (FACS)
based on F2 using the weight of 1/[
s
2(Fo2) þ (0.1230P)2 þ 8.0218P] gave
The effect of compound 37 on cell cycle phase distribution of
human hepatoma HepG2 was assessed using flow cytometry. When
the cells were grown to about 70% confluence in 6-well microplates
final values of R ¼ 0.067,
uR ¼ 0.206, and GOF(F) ¼ 0.760 for 356
variables and 5896 contributing reflections. Maximum shift/
error ¼ 0.000(3), max/min residual electron density ¼ 0.21/
and treated with compound 37 at given concentrations (4, 8, 16 mM).
ꢀꢃ3
ꢃ0.16 e A . Hydrogen atoms were observed and refined with a fixed
After 24 h, cells were harvested by trypsinization, washed in PBS, and
value of their isotropic displacement parameter.
fixed in 70% ice cold (4 ꢀC) ethanol overnight. Then, they werewashed
Crystallographic data for the structure 37 has been deposited
with the Cambridge Crystallographic Data Center (CCDC) under the
number 866294. Copies of the data can be obtained, free of charge,
on application to CCDC 12 Union Road, Cambridge CB2 1EZ, UK [fax:
þ44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk or at www.ccdc.
cam.ac.uk].
with PBS, incubated with RNase (50
m
g/mL final concentration) at
37 ꢀC for 30 min, stained with propidium iodide (50
mg/mL final
concentration), and analyzed by flow cytometry (Beckman Coulter).
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (21102069 and 81101732), Natural Science Foundation
of Guangdong Province (10451051501004725), and the project of
science and technology new star in Zhujiang Guangzhou city
(No. 2012J2200051).
5.3. Pharmacology evaluation
5.3.1. Antifungal activity
The fungicidal activities of the compounds 9e46 were tested
in vitro against C. musae, F. oxysporum f. sp. Niveum,
C. gloeosporioides Penz., B. theobromae, F. oxysporum f. sp. Cubense
and P. oryzae Cav. and their relative inhibitory ratio (%) had been
determined by using the mycelium growth rate method [33,34].
Briefly, each tested sample was dissolved in DMF, added to the
thawed potato glucose agar culture medium (PDA) under 50 ꢀC,
mixed to homogeneity and transferred to sterile Petri dishes to
solidify. For primary screenings, compounds were used at
a concentration of 150 mg/L. At the same time, Triadimefon (a
commercial fungicide) and 1 equiv of DMF were used as drug and
blank controls, respectively. After the dishes were cooled, the
solidified plates were incubated with 5 mm mycelium disk, inver-
ted, and incubated at 26 ꢄ 2ꢀC for 48 h. Three replicates of each test
were carried out. The mycelial elongation radius (mm) of fungi
settlements was measured after 48 h of culture. The growth inhi-
bition rates were calculated with the following equation:
I ¼ [(C ꢃ T)/C] ꢂ 100% where I is the growth inhibition rate (%), C
is the average diameter of mycelia in the blank test, and T is the
average diameter of mycelia in the presence of those compounds.
The inhibition ratio of those compounds at the dose of 150 mg/L
was summarized in Table 2.
Appendix A. Supplementary material
Supplementary material associated with this article can be
found in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.06.041.
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