Synthesis and anticancer activity of a novel class of flavonoids: 2,4-Diarylchromane[4,3-d]-Δ1,9b-1,2,3-thiadiazolines

Article abstract of DOI:10.1016/j.ejmech.2006.10.004

A series of 2,4-diarylchromane[4,3-d]-Δ^1,9b-1,2,3-thiadiazolines have been synthesized by cyclization of corresponding 2-arylchroman-4-one-arylhydrazones with SOCl₂ then treated with alcohol. All the compounds have been tested for th

Full text of DOI:10.1016/j.ejmech.2006.10.004

European Journal of Medicinal Chemistry 42 (2007) 226e234
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of a novel class of flavonoids:
2,4-Diarylchromane[4,3-d]-D1,9b-1,2,3-thiadiazolines
b
b
b
Huazhou Ying a, Yongzhou Hu a, , Qiaojun He , Runping Li , Bo Yang
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
Received 5 July 2006; received in revised form 1 October 2006; accepted 5 October 2006
Available online 28 November 2006
Abstract
A series of 2,4-diarylchromane[4,3-d]-D1,9b-1,2,3-thiadiazolines have been synthesized by cyclization of corresponding 2-arylchroman-4-
one-arylhydrazones with SOCl2 then treated with alcohol. All the compounds have been tested for their antiproliferative activity in vitro against
six human tumor cell lines, and the highly potent derivative 11a exhibited in vivo inhibitory effect on tumor growth. Mechanism research in-
dicated that it is due to 11a that induces DNA fragmentation.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: 2,4-Diarylchromane[4,3-d]-D1,9b-1,2,3-thiadiazolines; Synthesis; Antitumor activity
1. Introduction
this paper, we describe the synthesis of the 2,4-diarylchro-
mane[4,3-d]-D1,9b-1,2,3-thiadiazolines and their antiprolifera-
tive activity in vitro against various human cancer cell lines,
and we also reported the antitumor activity of 11a in ICR
mice bearing sarcoma 180 cells.
Flavonoids are very well known family possessing a broad
range of pharmacological properties, including antihepato-
toxic, antitumor, anti-inflammatory and antiviral properties
[1]. In antitumor area, flavonoids can inhibit the metabolism
of the benze[a]pyrene by hamster embryo cells in tissue cul-
ture [2] and markedly augment the cytotoxicity of TNF (tumor
necrosis factor-a) [3]. Flavonoids are also found to have the
ability for inhibiting tyrosinase and aromatase [4,5], estra-
diol-induced DNA synthesis [6] and inducing apoptosis [7].
Furthermore, most of the flavonoids displayed enhanced bio-
activity after structure modification [8,9]. On the other hand,
many compounds containing 1,2,3-thiadiazole and 1,2,4-thia-
diazoline moieties were reported for their antitumor activity
in recent years [10]. But there were few literatures reported
that the bioactivity of 1,2,3-thiadiazoline derivatives. This
prompted us to design and synthesis a series of new flavonoids
with 1,2,3-thiadiazoline ring and studied their bioactivity. In
2. Results and discussions
2.1. Chemistry
2.1.1. Synthesis of derivatives
The method utilized for the synthesis of 2,4-diarylchro-
mane[4,3-d]-D1,9b-1,2,3-thiadiazolines 10e11 is outlined in
Scheme 1. The arylketones 1 and arylaldehydes 2 were commer-
cially available and the chalones 3 and flavonones 4 were easily
prepared by following the previously reported methods in high
yield [11]. The obtained flavonones 4 were reacted with arylhy-
drazines 5 in ethanol at reflux conditions to give flavanone-4-
arylhydrazones 6 or 7 in 83e96% yields. We tried to prepare
target compounds by following Mohamed’s method [12]. Com-
pounds 6 or 7 were treated with thionyl chloride at 20 ꢀC for
10 h, however, no products were obtained. Maybe the materials
* Corresponding author. Tel./fax: þ86 571 8820 8460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.10.004

H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
227
R2'
R2'
O
O
OH
OH
O
NaOAc/EtOH
KOH/EtOH
r.t. 24h
+
OHC
reflux, 24 h
R2'
R1'
R1'
R1'
O
1
2
3
4
R4O
O
R2
B
R2'
4
O
EtOH, HOAc
reflux, 6 h
1)SOCl2,reflux,0.5 h
A
C
3a
4
+
R3
NHNH2
OR4
2)R4OH,reflux,0.5 h
S
R1
E
N
R1'
N
NNH
R3
Cl
5
D
6 R3 = NO2
7 R3 = SO2Me
10 R3 = NO2
11 R3 = SO2Me
R3
R2'
O
Cl
S
N
R1'
N
8 R3 = NO2
9 R3 = SO2Me
R3
Scheme 1.
were polymerized and the byproducts were polymers. Different
reaction conditions were tried; at last, we found that the com-
pounds 10 or 11 were obtained in 37e76% yield via condensa-
tion of the corresponding hydrazones 6 or 7 in excess SOCl2
under N2 for 0.5 h, followed by refluxing in alcohol for 0.5 h.
It is unexpected that all the products 10 and 11 were substituted
with alkoxyl at 3a- and 4-positions, and chlorine at 200-position.
Besides that, as is listed in Table 1, some of the products were
substituted with chlorine at 6-position and 20-position: (1)
when the materials with 5,7-dimethoxyl at A-ring (6cef and
7d), the products (10cef, 10k and 11d) have a chlorine at the
6-position; (2) when the materials with 30,40-dioxmethylene
(6h) or 30,40-dimethoxyl (6i) at B-ring, the products (10h and
10i) were substituted with chlorine at 20-position (Table 1).
The other desired compounds 12 were obtained by reduc-
tion of 10 with Raney-Ni and H2 followed by acetylation
with acetic anhydride in the presence of pyridine (Scheme
2). All the new compounds synthesized were characterized
vitro against several tumor cell lines including Bel-72, ECA-
109, PC-3, MCF-7, HL-60, and A-549 cells. 2-Phenyl-
chroman-4-one, a kind of flavonone, was tested together as
a positive control in the assays. The results are summarized
in Table 2.
As is demonstrated in Table 2, compounds 11a and 11e ex-
hibited the most potent antiproliferative activity against all the
tested cell lines. Some general conclusions that can be drawn
from the results, based more on the consistency of effect than
on statistical significance, would be as follows: (1) the com-
pounds without substituent at A-ring (10aeb, 11a, 11c and
12aeb) showed similar antiproliferative activity, and all these
compounds displayed more effective activity than 2-phenyl-
chroman-4-one. (2) The effect of compounds 10aej, 11b
and 11e, which have methyl at 8 position, is not uniform.
Compounds 10gej were almost inactive, however, 11b and
11e showed high activity. In this series, methylsulfonyl group
at D-ring was important. (3) The compounds 10cef and 10k
with tri-substituent at A-ring showed low activity. (4)
The compounds with para substituent at B-ring were more
effective than those with ortho substituent or tri-substituent.
p-Methyl group substituted at B-ring might be the most
beneficial (cf. 10e, 10f and 10c). The compounds with
tri-substituted at B-ring (10h and 10i) or ortho substituent at
B-ring (10d) almost lost their antiproliferative activity. (5)
1
by H NMR, ESI-MS and IR spectroscopy, and the structure
of 10c was confirmed by X-ray analysis [13].
2.2. Biological activities
All the synthesized 2,4-diarylchromane[4,3-d]-D1,9b-1,2,3-
thiadiazolines were tested for antiproliferative activities in

228
H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
Table 1
The structure of hydrazones and products
Compound
Substituent
R10
Compound
Substituent
R1
R20
R2
R4
6a
6b
6c
6d
6e
6f
H
H
10a
10b
10c
10d
10e
10f
H
40-Cl
40-MeO
40-Cl
20-Cl
40-Me
40-MeO
40-Cl
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
Et
Et
Et
Et
Et
Et
Et
H
40-MeO
H
6-Cl-7,9-diMeO
5,7-diMeO
5,7-diMeO
5,7-diMeO
5,7-diMeO
6-Me
H
20-Cl
6-Cl-7,9-diMeO
6-Cl-7,9-diMeO
40-Me
40-MeO
6-Cl-7,9-diMeO
8-Me
6g
6h
6i
H
10g
10h
10i
6-Me
6-Me
6-Me
30,40-OCH2Oe
8-Me
8-Me
20-Cl-40,50-OCH2O-
20-Cl-40,50-diMeO
40-MeO
40-Cl
30,40-diMeO
6j
40-MeO
10j
8-Me
6-Cl-7,9-diMeO
6c
7a
7b
7c
7d
7e
5,7-diMeO
H
6-Me
H
10k
11a
11b
11c
11d
11e
12a
12b
H
H
8-Me
40-Cl
40-MeO
40-MeO
H
40-MeO
40-MeO
40-Cl
H
H
6-Cl-7,9-diMeO
5,7-diMeO
6-Me
H
8-Me
H
8-Me
40-Cl
40-Cl
40-Cl
The compound with dimethoxyl at 3a- and 4-positions (10c)
might be more active than that with diethoxyl (10k).
Because compound 11a have highly antiproliferative activity
in vitro, we tested its antiproliferative activities on S180 tumor
bearing mice, Cyclophosphamide (CTX), a well-known anti-
cancer agent, was tested together as positive control (Table 3).
Since apoptosis is an important response to most chemo-
therapeutic agents [14,15], we evaluated the role of these com-
pounds in inducing apoptosis. To confirm whether cell death
was caused by apoptotic processes, we assessed the DNA frag-
mentation in the cells exposed to 11a (0.5 mg/mL, 1.0 mg/mL,
2.0 mg/mL) for 24 h. DNA ladder on an agarose gel was ob-
served in 11a (Fig. 1). By flow cytometry, the percentages
of apoptotic cells were 3.9, 25.3 and 68.3 after treatment
with 11a (0.5, 1.0, 2.0 mg/mL, respectively) for 48 hours
(Fig. 2). The induction of DNA ladder and the increased
sub-G0/G1 contents indicate that 11a is able to induce apopto-
sis in HL-60 cells.
compounds had antiproliferative activity in vitro and com-
pound 11a showed in vitro as well as in vivo the inhibitory ef-
fect on tumor growth. Further mechanism research indicated
that compound 11a has the abilities to induce apoptosis in
HL-60 cells. It provided an available approach to develop
a novel class of flavonoids with potential as anticancer drugs.
4. Experimental protocols
Melting points were not corrected and were recorded on
a Buchi apparatus. IR spectra, KBr pellets, 400e4000 cmꢁ1
,
were recorded on a Bruker VECTOR 22 FTIR spectrophotom-
eter. 1H NMR and 13C NMR spectra were recorded on a Bruker
AM 400 instrument at 400 and 500 MHz (chemical shifts are
expressed as d values relative to TMS as internal standard).
Mass spectra (MS), ESI (positive) were recorded on an Es-
quire-LC-00075 spectrometer.
4.1. Synthesis
3. Conclusion
4.1.1. General procedure for the preparation of flavanone-
4-arylhydrazones 6 and 7
An equimolar of flavonones and arylhydrozine (5 mmol) in
ethanol (50 mL) and acetic acid (0.5 mL) was heated under
In conclusion, we synthesized a series of new flavonoids,
2,4-diarylchromane[4,3-d]-D1,9b-1,2,3-thiadiazolines, and stud-
ied their ability of antiproliferative activity. Most of these
R2
1)Raney-Ni, H2,12 h
OEt
OEt
R2
O
O
OEt
OEt
S
R1
S
R1
2)Ac2O, C5H5N, r.t., 12 h
N
N
Cl
N
N
Cl
NO2
NHAc
10
12
Scheme 2.

H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
229
Table 2
Antiproliferative activities of 2,4-diarylchromane[4,3-d]-D1,9b-1,2,3-thiadiazolines
Compound
Cell line (IC50, mg/ml)a
Bel-7402
ECA-109
PC-3
MCF-7
HL-60
A-549
10a
10b
11.8 (ꢂ0.7)
8.2 (ꢂ0.3)
>50
6.9 (ꢂ0.7)
9.6 (ꢂ0.6)
>50
4.1 (ꢂ0.4)
5.7 (ꢂ0.9)
15.8 (ꢂ3.3)
>50
15.0 (ꢂ0.6)
23.3 (ꢂ1.8)
>25
2.2 (ꢂ2.6)
3.3 (ꢂ3.4)
6.7 (ꢂ0.1)
>25
3.8 (ꢂ4.4)
5.1 (ꢂ6.4)
10c
10d
>50
>50
>50
>50
>50
7.3 (ꢂ1.0)
>50
>25
>25
10e
10f
7.3 (ꢂ1.0)
19.2 (ꢂ13.1)
>50
16.8 (ꢂ0.4)
23.2 (ꢂ0.04)
>25
>50
>50
>25
>25
17.7 (ꢂ2.0)
7.8 (ꢂ9.8)
>50
10g
10h
18.6 (ꢂ5.9)
>50
>50
14.8 (ꢂ0.9)
>50
>50
>50
>50
>25
>25
>25
>25
10i
10j
>50
>50
21.8 (ꢂ1.3)
>50
>50
>50
>25
>25
10k
11a
5.7 (ꢂ5.1)
5.9 (ꢂ4.0)
1.5 (ꢂ0.7)
5.9 (ꢂ0.7)
20.1 (ꢂ8.9)
11.5 (ꢂ9.1)
9.6 (ꢂ0.7)
4.1 (ꢂ1.0)
27.7 (ꢂ0.7)
2.9 (ꢂ3.5)
3.1 (ꢂ2.5)
0.6 (ꢂ0.3)
3.5 (ꢂ0.2)
>50
5.5 (ꢂ0.1)
4.1 (ꢂ0.2)
>25
3.2 (ꢂ0.4)
2.8 (ꢂ0.4)
0.6 (ꢂ0.4)
1.8 (ꢂ2.3)
5.6 (ꢂ0.3)
6.0 (ꢂ0.03)
2.2 (ꢂ2.9)
1.3 (ꢂ1.5)
3.4 (ꢂ10.0)
5.4 (ꢂ0.5)
4.9 (ꢂ1.4)
1.1 (ꢂ0.6)
3.3 (ꢂ3.6)
14.5 (ꢂ7.9)
10.3 (ꢂ1.7)
4.2 (ꢂ4.4)
2.5 (ꢂ2.2)
14.2 (ꢂ19.0)
10.3 (ꢂ0.3)
40.4 (ꢂ13.9)
23.5 (ꢂ17.5)
24.8 (ꢂ0.4)
7.3 (ꢂ0.9)
21.3 (ꢂ0.3)
10.5 (ꢂ0.6)
>50
11b
11c
>25
11d
11e
12.6 (ꢂ2.5)
2.5 (ꢂ1.2)
23.3 (ꢂ1.8)
19.8 (ꢂ1.2)
>25
6.0 (ꢂ6.0)
4.1 (ꢂ0.4)
2.4 (ꢂ0.2)
16.6 (ꢂ3.4)
12a
12b
2-Phenylchroman-4-one
a
Values are means of three experiments, standard deviation is given in parentheses.
reflux for 6 h. After cooling to r.t., the product 6 or 7 that pre-
cipitated was separated by suction and washed with ethanol.
J ¼ 7.6 Hz, 5-H), 8.11 (d, 2H, J ¼ 7.6 Hz, 300-H and 500-H),
10.31 (s, 1H, NH).
4.1.1.1. 2-Phenylchroman-4-one-(4-nitrophenyl)hydrozone 6a.
Yield: 87%, m.p. 249e250 ꢀC. ESI-MS: m/z ¼ 360
4.1.1.3. 5,7-Dimethoxyl-2-phenylchroman-4-one-(4-nitrophe-
nyl)hydrozone 6c. Yield: 89%, m.p. 214e216 ꢀC. ESI-MS:
[M þ 1]. IR (KBr): 3310, 1601 cmꢁ1
.
1H NMR (DMSO-
m/z ¼ 420 [M þ 1]. IR (KBr): 3321, 1589 cmꢁ1
.
1H NMR
d6) d: 2.77e2.84 (m, 1H, 3-H), 3.35e3.40 (m, 1H, 3-H),
5.25 (dd, 1H, J ¼ 12.0 Hz, 3.2 Hz, 2-H), 6.96 (d, 1H,
J ¼ 8.0 Hz, 8-H), 7.03 (t, 1H, J ¼ 8.0 Hz, 7-H), 7.27 (t,
1H, J ¼ 8.0 Hz, 6-H), 7.35 (m, 2H, 20-H and 60-H), 7.38
(d, 1H, J ¼ 7.2 Hz, 40-H), 7.44 (t, 2H, J ¼ 7.2 Hz, 30-H
and 50-H), 7.56 (d, 2H, J ¼ 8.0 Hz, 200-H and 600-H), 8.07
(d, 1H, J ¼ 8.0 Hz, 5-H), 8.11 (d, 2H, J ¼ 8.0 Hz, 300-H
and 500-H), 10.31 (s, 1H, NH).
(DMSO-d6) d: 2.73e2.80 (m, 1H, 3-H), 3.30e3.35 (m, 1H,
3-H), 3.77 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 5.18 (dd, 1H,
J ¼ 12.0 Hz, 3.2 Hz, 2-H), 6.23 (d, 1H, J ¼ 2.4 Hz, 8-H),
6.30 (d, 1H, J ¼ 2.4 Hz, 6-H), 7.29 (d, 2H, J ¼ 2.4 Hz, 20-H
and 60-H), 7.39 (t, 1H, J ¼ 7.2 Hz, 40-H), 7.46 (t, 2H,
J ¼ 7.2 Hz, 30-H and 50-H), 7.56 (d, 2H, J ¼ 8.0 Hz, 200-H
and 600-H), 8.13 (d, 2H, J ¼ 8.0 Hz, 300-H and 500-H), 10.12
(s, 1H, NH).
4.1.1.2.
(40-Methoxylphenyl)chroman-4-one-(4-nitrophenyl)
4.1.1.4.
5,7-Dimethoxyl-2-(4-chlorophenyl)chroman-4-one-
hydrozone 6b. Yield: 93%, m.p. 237e239 ꢀC. ESI-MS: m/z ¼
(4-nitrophenyl)hydrozone 6d. Yield: 85%, m.p. 185e187 ꢀC.
390 [M þ 1]. IR: 3299, 1597 cmꢁ1
.
1H NMR (DMSO-
ESI-MS: m/z ¼ 454 [M þ 1]. IR (KBr): 3235, 1593 cmꢁ1. H
1
d6) d: 2.80e2.87 (m, 1H, 3-H), 3.29e3.34 (m, 1H, 3-H),
3.75 (s, 3H, OCH3), 5.18 (dd, 1H, J ¼ 12.0 Hz, 2.8 Hz, 2-H),
6.93 (d, 1H, J ¼ 8.0 Hz, 8-H), 6.98 (d, 2H, J ¼ 8.8 Hz, 30-H
and 50-H), 7.27 (t, 1H, J ¼ 8.0 Hz, 7-H), 7.32 (t, 1H,
J ¼ 8.0 Hz, 6-H), 7.47 (d, 2H, J ¼ 8.8 Hz, 20-H and 60-H),
7.56 (d, 2H, J ¼ 7.6 Hz, 200-H and 600-H), 8.06 (d, 1H,
NMR (DMSO-d6) d: 2.67e2.74 (m, 1H, 3-H), 3.31e3.36
Table 3
Effects of 11a on S180 tumor bearing mice
Groups
No. of animals Tumor weight (g) Inhibition rate (%)
Control
11a, 80 mg/kg
20
10
1.21 (ꢂ0.33)
0.63 (ꢂ0.23)
0.66 (ꢂ0.20)
e
48.2*
45.4*
CTX, 50 mg/kg 10
Fig. 1. DNA fragmentation induced by 11a in HL-60 cells.
*P < 0.01 versus control.

230
H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
60-H), 7.45 (d, 2H, J ¼ 8.8 Hz, 200-H and 600-H), 8.10 (d, 2H,
J ¼ 8.8 Hz, 300-H and 500-H), 10.12 (s, 1H, NH).
4.1.1.7. 6-Methyl-4-2-phenylchroman-4-one-(4-nitrophenyl)-
hydrozone 6g. Yield: 85%, m.p. > 250 ꢀC. ESI-MS: m/
z ¼ 374 [M þ 1]. IR (KBr): 3307, 1591 cmꢁ1
.
1H NMR
(DMSO-d6) d: 2.32 (s, 3H, CH3), 2.76e2.83 (m, 1H, 3-H),
3.35e3.40 (m, 1H, 3-H), 5.22 (dd, J ¼ 12.0 Hz, 2.4 Hz, 1H,
2-H), 6.88 (d, 1H, J ¼ 8.0 Hz, 8-H), 7.11 (dd, 1H, J ¼ 8.0,
1.2 Hz, 7-H), 7.35 (d, 2H, J ¼ 7.2 Hz, 20-H and 60-H), 7.40
(t, 1H, J ¼ 7.2 Hz, 40-H), 7.45 (t, 2H, J ¼ 7.2 Hz, 30-H and
50-H), 7.57 (d, 2H, J ¼ 7.6 Hz, 200-H and 600-H), 7.89 (d, 1H,
J ¼ 1.2 Hz, 5-H), 8.14 (d, 2H, J ¼ 7.6 Hz, 300-H and 500-H),
10.29 (s, 1H, NH).
4.1.1.8. 2-(Benzo[d][1,3]dioxol-5-yl)-6-methylchroman-4-one-
(4-nitrophenyl)hydrozone 6h. Yield: 93%, m.p. > 250 ꢀC.
ESI-MS: m/z ¼ 418 [M þ 1]. IR (KBr): 3219, 1602 cmꢁ1
.
1H NMR (DMSO-d6) d: 2.29 (s, 3H, CH3), 2.74e2.81 (m,
1H, 3-H), 3.27e3.32 (m, 1H, 3-H), 5.10 (dd, 1H,
J ¼ 12.0 Hz, 2.8 Hz, 2-H), 6.02 (s, 2H, CH2), 6.83 (d, 1H,
J ¼ 8.0 Hz, 8-H), 6.94 (d, 1H, J ¼ 8.0 Hz, 7-H), 7.01 (d,
1H, J ¼ 8.0 Hz, 50-H), 7.08 (d, 1H, J ¼ 8.0 Hz, 60-H), 7.14
(s, 1H, 20-H), 7.33 (d, 2H, J ¼ 8.4 Hz, 200-H and 600-H),
7.84 (s, 1H, 5-H), 8.12 (d, 2H, J ¼ 8.4 Hz, 300-H and 500-
H), 10.26 (s, 1H, NH).
4.1.1.9. 2-(3,4-Dimethoxyphenyl)-6-methylchroman-4-one-(4-
nitrophenyl)hydrozone 6i. Yield: 90%, m.p. > 250 ꢀC. ESI-
Fig. 2. Induction of apoptosis by 11a in HL-60 cells.
MS: m/z ¼ 434 [M þ 1]. IR (KBr): 3317, 1595 cmꢁ1
.
1H
NMR (DMSO-d6) d: 2.29 (s, 3H, CH3), 2.82e2.89 (m,
1H, 3-H), 3.27e3.33 (m, 1H, 3-H), 3.74 (s, 3H, OCH3),
3.76 (s, 3H, OCH3), 5.10 (dd, 1H, J ¼ 10.0 Hz, 2.4 Hz, 2-
H), 6.84 (d, 1H, J ¼ 8.4 Hz, 8-H), 6.97 (d, 1H, J ¼ 8.4 Hz,
7-H), 7.04e7.09 (m, 2H, 50-H and 60-H), 7.16 (s, 1H, 20-
H), 7.33 (d, 2H, J ¼ 8.8 Hz, 200-H and 600-H), 7.85 (s, 1H,
5-H), 8.11 (d, 2H, J ¼ 8.8 Hz, 300-H and 500-H), 10.27 (s,
1H, NH).
(m, 1H, 3-H), 3.75 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 5.37
(dd, 1H, J ¼ 12.0 Hz, 2.4 Hz, 2-H), 6.22 (d, 1H, J ¼ 2.4 Hz,
8-H), 6.31 (d, 1H, J ¼ 2.4 Hz, 6-H), 7.27 (brs, 2H, 40-H and
60-H), 7.40e7.54 (m, 3H, 50-H, 200-H and 600-H), 7.74 (d,
1H, J ¼ 8.0 Hz, 30-H), 8.10 (d, 2H, J ¼ 8.8 Hz, 300-H and 500-
H), 10.10 (s, 1H, NH).
4.1.1.5. 5,7-Dimethoxyl-2-(4-methylphenyl)chroman-4-one-(4-
nitrophenyl)hydrozone 6e. Yield: 90%, m.p. 208e210 ꢀC.
4.1.1.10. 2-(4-Methoxyphenyl)-6-methylchroman-4-one-(4-ni-
1
ESI-MS: m/z ¼ 434 [M þ 1]. IR (KBr): 3322, 1602 cmꢁ1. H
trophenyl)hydrozone 6j. Yield: 89%, m.p. > 250 ꢀC. ESI-
NMR (DMSO-d6) d: 2.31 (s, 3H, CH3), 2.69e2.76 (m, 1H,
3-H), 3.23e3.28 (m, 1H, 3-H), 3.74 (s, 3H, OCH3), 3.88 (s,
3H, OCH3), 5.10 (dd, 1H, J ¼ 12.0 Hz, 3.2 Hz, 2-H), 6.19
(d, J ¼ 2.0 Hz, 8-H), 6.26 (d, J ¼ 2.0 Hz, 6-H), 7.22e7.35
(m, 4H, 20-H, 30-H, 50-H and 60-H), 7.41 (d, 2H, J ¼ 8.8 Hz,
200-H and 600-H), 8.10 (d, 2H, J ¼ 8.8 Hz, 300-H and 500-H),
10.11 (s, 1H, NH).
MS: m/z ¼ 404 [M þ 1]. IR (KBr): 3315, 1604 cmꢁ1
.
1H
NMR (DMSO-d6) d: 2.31 (s, 3H, CH3), 2.78e2.85 (m, 1H,
3-H), 3.29e3.34 (m, 1H, 3-H), 3.78 (s, 3H, OCH3), 5.15
(dd, 1H, J ¼ 12.0 Hz, 2.4 Hz, 2-H), 6.85 (d, 1H, J ¼ 8.4 Hz,
8-H), 7.00 (d, 2H, J ¼ 8.0 Hz, 30-H and 50-H), 7.10 (dd, 1H,
J ¼ 8.4 Hz, 1.6 Hz, 7-H), 7.35 (d, 2H, J ¼ 8.0 Hz, 20-H and
60-H), 7.49 (d, 2H, J ¼ 8.8 Hz, 200-H and 600-H), 7.88 (d, 1H,
J ¼ 1.6 Hz, 5-H), 8.14 (d, 2H, J ¼ 8.8 Hz, 300-H and 500-H),
10.30 (s, 1H, NH).
4.1.1.6. 5,7-Dimethoxyl-2-(4-methoxylphenyl)chroman-4-one-
(4-nitrophenyl)hydrozone 6f. Yield: 90%, m.p. 218e219 ꢀC.
1
ESI-MS: m/z ¼ 450 [M þ 1]. IR (KBr): 3314, 1602 cmꢁ1. H
4.1.1.11. 2-Phenylchroman-4-one-[4-(methylsufonyl)phenyl]-
hydrozone 7a. Yield: 92%, m.p. > 250 ꢀC. ESI-MS: m/
NMR (DMSO-d6) d: 2.72e2.79 (m, 1H, 3-H), 3.21e3.26
(m, 1H, 3-H), 3.73 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 5.08 (dd, 1H, J ¼ 12.0 Hz, 2.8 Hz, 2-H), 6.18
(d, 1H, J ¼ 2.4 Hz, 8-H), 6.24 (d, 1H, J ¼ 2.4 Hz, 6-H), 6.97
(d, 2H, J ¼ 8.4 Hz, 30-H and 50-H), 7.27 (brs, 2H, 20-H and
z ¼ 393 [M þ 1]. IR (KBr): 3319, 1597 cmꢁ1
.
1H NMR
(DMSO-d6) d: 2.73e2.80 (m, 1H, 3-H), 3.08 (s, 3H, SO2CH3),
3.29e3.34 (m, 1H, 3-H), 5.23 (dd, 1H, J ¼ 12.0 Hz, 2.8 Hz, 2-
H), 6.95 (d, 1H, J ¼ 8.0 Hz, 8-H), 7.02 (t, 1H, J ¼ 8.0 Hz,

H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
231
7-H), 7.25 (t, 1H, J ¼ 8.0 Hz, 6-H), 7.34e7.45 (m, 5H, 20-H,
30-H, 40-H, 50-H and 60-H), 7.56 (d, 2H, J ¼ 8.8 Hz, 200-H and
600-H), 7.70 (d, 2H, J ¼ 8.8 Hz, 300-H and 500-H), 8.05 (dd, 1H,
J ¼ 8.0 Hz, 2.0 Hz, 5-H), 9.99 (s, 1H, NH).
under reduced pressure. Then alcohol (4 mL) was added
into the residue and refluxed for 30 min. The reaction mix-
ture was diluted with water (10 mL) and extracted thor-
oughly with CH2Cl2 (5 mL ꢃ 3). Then the CH2Cl2 layer
was washed successively with brine (5 mL ꢃ 2), dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
Usual workup afforded the crude product which was further
purified by chromatogram.
4.1.1.12.
2-(4-Methoxyphenyl)-6-methylchroman-4-one-[4-
(methylsufonyl)phenyl]hydrozone 7b. Yield: 93%, m.p. > 250 ꢀC.
ESI-MS: m/z ¼ 437 [M þ 1]. IR (KBr): 3307, 1600 cmꢁ1. H
1
NMR (DMSO-d6) d: 2.31 (s, 3H, CH3), 2.75e2.82 (m, 1H, 3-H),
3.11 (s, 3H, SO2CH3), 3.27e3.32 (m, 1H, 3-H), 3.78 (s, 3H,
OCH3), 5.13 (dd, 1H, J ¼ 12.0 Hz, 2.4 Hz, 2-H), 6.84 (d, 1H,
J ¼ 8.4 Hz, 8-H), 7.00 (d, 2H, J ¼ 8.0 Hz, 20-H and 60-H), 7.07
(dd, 1H, J ¼ 8.4 Hz, 1.6 Hz, 7-H), 7.38 (d, 2H, J ¼ 8.4 Hz, 200-H
and 600-H), 7.49 (d, 2H, J ¼ 8.0 Hz, 30-H and 50-H), 7.73 (d, 2H,
J ¼ 8.4 Hz, 300-H and 500-H), 7.86 (d, 1H, J ¼ 1.6 Hz, 5-H), 9.97
(s, 1H, NH).
4.1.2.1.
(3aRS,4RS )2-(2-Chloro-4-nitrophenyl)-3a,4-dieth-
oxyl-4-(4-chlorophenyl)chromane[4,3-d]-D1,9b-1,2,3-thiadia-
zoline 10a. Yield: 58%, m.p. 192e194 ꢀC. ESI-MS: m/
z ¼ 545 [M þ 1]. IR (KBr): 2982, 1609 cmꢁ1
.
1H NMR
(DMSO-d6) d: 0.80 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.88 (t,
3H, J ¼ 7.2 Hz, OCH2CH3), 2.86e2.90 (m, 1H, OCH2CH3),
3.08e3.12 (m, 1H, OCH2CH3), 3.26e3.30 (m, 1H,
OCH2CH3), 3.37e3.41 (m, 1H, OCH2CH3), 7.24e7.31 (m,
2H, 7-H and 8-H), 7.57 (d, 3H, J ¼ 8.8 Hz, 6-H, 20-H and
60-H), 7.65 (d, 2H, J ¼ 8.8 Hz, 30-H and H-50), 7.74 (d, 1H,
J ¼ 9.2 Hz, 600-H), 8.09 (dd, 1H, J ¼ 8.0 Hz, 1.6 Hz, 9-H),
8.21 (dd, 1H, J ¼ 9.2 Hz, 2.8 Hz, 500-H), 8.42 (d, 1H,
J ¼ 2.8 Hz, 300-H).
4.1.1.13. 2-(4-Methoxyphenyl)chroman-4-one-[4-(methylsufo-
nyl)phenyl]hydrozone 7c. Yield: 93%, m.p. > 250 ꢀC. ESI-
MS: m/z ¼ 423 [M þ 1]. IR (KBr): 3307, 1600 cmꢁ1
.
1H
NMR (DMSO-d6) d: 2.76e2.83 (m, 1H, 3-H), 3.08 (s, 3H,
SO2CH3), 3.28e3.33 (m, 1H, 3-H), 3.75 (s, 3H, OCH3),
5.16 (dd, 1H, J ¼ 12.0 Hz, 3.2 Hz, 2-H), 6.92 (d, 1H,
J ¼ 8.0 Hz, 8-H), 6.98 (d, 2H, J ¼ 8.8 Hz, 30-H and 50-H),
7.00 (t, 1H, J ¼ 8.0 Hz, 7-H), 7.24 (t, 1H, J ¼ 8.0 Hz, 6-H),
7.35 (d, 2H, J ¼ 8.8 Hz, 200-H and 600-H), 7.47 (d, 2H,
J ¼ 8.8 Hz, 20-H and 60-H), 7.70 (d, 2H, J ¼ 8.4 Hz, 300-H
and 500-H), 8.05 (d, 1H, J ¼ 8.0 Hz, 5-H), 9.98 (s, 1H, NH).
4.1.2.2.
(3aRS,4RS )2-(2-Chloro-4-nitrophenyl)-3a,4-dieth-
oxyl-4-(methoxylphenyl)chromane[4,3-d]-D1,9b-1,2,3-thiadia-
zoline 10b. Yield: 50%, m.p. 170e172 ꢀC. ESI-MS: m/
z ¼ 541 [M þ 1]. IR (KBr): 2921, 1609 cmꢁ1
.
1H NMR
(DMSO-d6) d: 0.80 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.88 (t,
3H, J ¼ 7.2 Hz, OCH2CH3), 2.86e2.90 (m, 1H, OCH2CH3),
3.08e3.12 (m, 1H, OCH2CH3), 3.26e3.30 (m, 1H,
OCH2CH3), 3.37e3.41 (m, 1H, OCH2CH3), 3.96 (s, 3H,
OCH3), 6.93 (d, 2H, J ¼ 8.8 Hz, 30-H and 50-H), 7.24e
7.31 (m, 2H, 7-H and 8-H), 7.57 (d, 1H, J ¼ 8.8 Hz, 6-
H), 7.61 (d, 2H, J ¼ 8.8 Hz, 20-H and 60-H), 7.71 (d, 1H,
J ¼ 9.2 Hz, 600-H), 8.09 (dd, 1H, J ¼ 8.0 Hz, 1.6 Hz, 9-H),
8.19 (dd, 1H, J ¼ 9.2 Hz, 2.8 Hz, 500-H), 8.29 (d, 1H,
J ¼ 2.8 Hz, 300-H).
4.1.1.14. 5,7-Dimethoxy-2-phenylchroman-4-one-[4-(methyl-
sufonyl)phenyl]hydrozone 7d. Yield: 85%, m.p. 225e227 ꢀC.
1
ESI-MS: m/z ¼ 453 [M þ 1]. IR (KBr): 3317, 1625 cmꢁ1. H
NMR (DMSO-d6) d: 2.74e2.81 (m, 1H, 3-H), 3.04 (s, 3H,
SO2CH3), 3.25e3.30 (m, 1H, 3-H), 3.71 (s, 3H, OCH3),
3.76 (s, 3H, OCH3), 5.49 (dd, 1H, J ¼ 10.0 Hz, 2.8 Hz, 2-H),
6.13 (d, 1H, J ¼ 2.0 Hz, 6-H), 6.20 (d, 1H, J ¼ 2.0 Hz, 8-H),
6.93 (d, 2H, J ¼ 8.8 Hz, 200-H and 600-H), 7.23e7.39 (m, 5H,
20-H, 30-H, 40-H, 50-H and 60-H), 7.65 (d, 2H, J ¼ 8.8 Hz,
300-H and 500-H), 11.42 (s, 1H, NH).
4.1.2.3.
(3aRS,4RS )6-Chloro-2-(2-chloro-4-nitrophenyl)-
4-(4-chlorophenyl)-3a,4-diethoxyl-7,9-dimethoxylchromane[4,3-
d]-D1,9b-1,2,3-thiadiazoline 10c. Yield: 53%, m.p. 219e
221 ꢀC. ESI-MS: m/z ¼ 639 [M þ 1]. IR (KBr): 2924,
4.1.1.15.
6-Methyl-2-phenylchroman-4-one-[4-(methylsufo-
nyl)phenyl]hydrozone 7e. Yield: 86%, m.p. > 250 ꢀC. ESI-
MS: m/z ¼ 407 [M þ 1]. IR (KBr): 3323, 1599 cmꢁ1
.
1H
1601 cmꢁ1
.
1H NMR (CDCl3) d: 0.88 (t, 3H, J ¼ 7.4 Hz,
NMR (DMSO-d6) d: 2.29 (s, 3H, CH3), 2.70e2.77 (m, 1H,
3-H), 3.08 (s, 3H, SO2CH3), 3.31e3.36 (m, 1H, 3-H), 5.18
(dd, J ¼ 12.0 Hz, 2.8 Hz, 2-H), 6.84 (d, 1H, J ¼ 8.4 Hz, 8-
H), 6.93 (d, 2H, J ¼ 8.8 Hz, 200-H and 600-H), 7.06 (d, 1H,
J ¼ 8.4 Hz, 7-H), 7.36 (t, 2H, J ¼ 8.8 Hz, 30-H and 50-H),
7.43 (t, 1H, J ¼ 8.4 Hz, 40-H), 7.54 (d, 2H, J ¼ 8.8 Hz, 20-H
and 60-H), 7.70 (d, 2H, J ¼ 8.8 Hz, 300-H and 500-H), 7.84 (s,
1H, 5-H), 9.94 (s, 1H, NH).
OCH2CH3), 0.97 (t, 3H, J ¼ 7.4 Hz, OCH2CH3), 2.84e
2.91 (m, 1H, OCH2CH3), 3.15e3.22 (m, 1H, OCH2CH3),
3.33e3.40 (m, 1H, OCH2CH3), 3.44e3.51 (m, 1H,
OCH2CH3), 4.03 (s, 3H, OCH3), 4.06 (s, 3H, OCH3), 6.39
(s, 1H, H-8), 7.41 (d, 2H, J ¼ 8.0 Hz, 30-H and 50-H),
7.68 (d, 3H, J ¼ 8.0 Hz, 20-H, 60-H and H-600), 8.10 (dd,
1H, J ¼ 8.0 Hz, 2.4 Hz, 500-H), 8.30 (d, 1H, J ¼ 2.4 Hz,
300-H). 13C NMR (CDCl3) d: 14.7 (q, OCH2CH3), 15.2 (q,
OCH2CH3), 56.6 (q, OCH3), 56.7 (q, OCH3), 59.9 (t,
OCH2CH3), 60.2 (t, OCH2CH3), 90.5 (d, 8-C), 99.0 (s, 6-
C), 102.8 (s, 3a-C), 103.6 (s, 9a-C), 108.0 (s, 4-C), 122.6
(d, 30-C and 50-C), 123.0 (d, 500-C), 123.7 (s, 4-C), 126.6
(d, 300-C), 127.2 (s, 200-C), 128.4 (s, 10-C), 128.6 (d,
600-C), 128.8 (d, 20-C and 60-C), 135.7 (s, 40-C), 135.9 (s,
4.1.2. General procedure for the preparation of 2,4-diaryl-
chromane[4,3-d]-D1,9b-1,2,3-thiadiazolines 10 and 11
A mixture of the 2-arylchroman-4-one-arylhydrazone
(5 mmol) and SOCl2 (4 mL) was refluxed for 30 min under
nitrogen atmosphere. The excess SOCl2 was evaporated

232
H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
400-C), 143.7 (s, 100-C), 144.5 (s, 5a-C), 149.8 (s, 9-C), 151.0
(s, 9b-C), 158.2 (s, 7-C).
1,2,3-thiadiazoline 10h. Yield: 53%, m.p. 103e106 ꢀC. ESI-
MS: m/z ¼ 603 [M þ 1]. IR (KBr): 2927, 1587 cmꢁ1
.
1H
NMR (DMSO-d6) d: 0.85 (t, J ¼ 7.2 Hz, 3H, OCH2CH3),
0.92 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 2.85e2.93 (m, 1H,
OCH2CH3), 3.11e3.19 (m, 1H, OCH2CH3), 3.36e3.50 (m,
2H, OCH2CH3), 6.14 (d, 1H, J ¼ 1.2 Hz, OCH2O), 6.17 (d,
1H, J ¼ 1.2 Hz, OCH2O), 7.10 (s, 1H, 30-H), 7.13 (d, 1H,
J ¼ 8.0 Hz, 6-H), 7.22 (s, 1H, 60-H), 7.37 (dd, 1H,
J ¼ 8.0 Hz, 1.6 Hz, 7-H), 7.75 (d, 1H, J ¼ 9.2 Hz, 600-H),
7.87 (d, 1H, J ¼ 1.6 Hz, 9-H), 9.19 (dd, 1H, J ¼ 9.2 Hz,
2.4 Hz, 500-H), 8.41 (d, 1H, J ¼ 2.4 Hz, 300-H).
4.1.2.4. (3aRS,4RS )6-Chloro-2-(2-chloro-4-nitrophenyl)-4-(2-
chlorophenyl)-3a,4-diethoxyl-7,9-dimethoxylchromane[4,3-d]-
D1,9b-1,2,3-thiadiazoline 10d. Yield: 45%, m.p. 222e224 ꢀC.
1
ESI-MS: m/z ¼ 639 [M þ 1]. IR (KBr): 2972, 1603 cmꢁ1. H
NMR (CDCl3) d: 0.91 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 1.01 (t,
3H, J ¼ 7.2 Hz, OCH2CH3), 2.87e2.95 (m, 1H, OCH2CH3),
3.22e3.29 (m, 1H, OCH2CH3), 3.45e3.61 (m, 2H, OCH2CH3),
4.03 (s, 3H, 3H, OCH3), 4.05 (s, 3H, OCH3), 6.39 (s, 1H, 6-H),
7.30e7.35 (m, 2H, 40-H and 50-H), 7.42e7.44 (m, 1H, 60-H),
7.71 (d, 1H, J ¼ 9.2 Hz, 500-H), 7.87e7.69 (m, 1H, 30-H), 8.10
(d, 1H, J ¼ 9.2, 600-H), 8.29 (s, 1H, 300-H).
4.1.2.9. (3aRS,4RS )4-(2-Chloro-4,5-dimethoxyphenyl)-2-(2-
chloro-4-nitrophenyl)-3a,4-diethoxyl-8-methylchromane[4,3-
d]-D1,9b-1,2,3-thiadiazoline 10i. Yield: 37%, m.p. 105e
107 ꢀC. ESI-MS: m/z ¼ 619 [M þ 1]. IR (KBr): 2977,
4.1.2.5.
(3aRS,4RS )6-Chloro-2-(2-chloro-4-nitrophenyl)-
1
4-(4-methylphenyl)-3a,4-diethoxyl-7,9-dimethoxylchromane
[4,3-d]-D1,9b-1,2,3-thiadiazoline 10e. Yield: 58%, m.p. 134e
136 ꢀC. ESI-MS: m/z ¼ 619 [M þ 1]. IR (KBr): 2927,
1582 cmꢁ1. H NMR (DMSO-d6) d: 0.81 (t, 3H, J ¼ 7.2 Hz,
OCH2CH3), 0.89 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.37 (s, 3H,
CH3), 2.80e2.87 (m, 1H, OCH2CH3), 3.05e3.12 (m, 1H,
OCH2CH3), 3.31e3.37 (m, 1H, OCH2CH3), 3.41e3.48 (m,
1H, OCH2CH3), 3.73 (s, 3H, OCH3), 3.78 (s, 3H, OCH3),
6.97 (s, 1H, 30-H), 7.10 (d, 1H, J ¼ 8.4, 6-H), 7.21 (s, 1H,
60-H), 7.32 (dd, 1H, J ¼ 8.0 Hz, 1.6 Hz, 7-H), 7.71 (d, 1H,
J ¼ 9.2 Hz, 600-H), 7.31 (dd, 1H, J ¼ 8.4 Hz, 1.6 Hz, 7-H),
7.83 (d, 1H, J ¼ 1.6 Hz, 9-H), 8.15 (dd, 1H, J ¼ 9.2 Hz,
2.4 Hz, 500-H), 8.28 (d, 1H, J ¼ 2.4 Hz, 300-H).
1
1598 cmꢁ1. H NMR (DMSO-d6) d: 0.82 (t, 3H, J ¼ 7.2 Hz,
OCH2CH3), 0.90 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.43 (s, 3H,
CH3), 2.86e2.90 (m, 1H, OCH2CH3), 3.08e3.12 (m, 1H,
OCH2CH3), 3.26e3.30 (m, 1H, OCH2CH3), 3.37e3.41 (m,
1H, OCH2CH3), 6.41 (s, 1H, 8-H), 7.03 (d, 2H, J ¼ 8.8 Hz,
30-H and 50-H), 7.22 (d, 2H, J ¼ 8.8 Hz, 20-H and 60-H), 7.75
(d, 1H, J ¼ 9.2 Hz, 600-H), 8.19 (dd, 1H, J ¼ 9.2 Hz, 2.8 Hz,
500-H), 8.39 (d, 1H, J ¼ 2.8 Hz, 300-H).
4.1.2.10. (3aRS,4RS )2-(2-Chloro-4-nitrophenyl)-3a,4-dieth-
4.1.2.6.
(3aRS,4RS )6-Chloro-2-(2-chloro-4-nitrophenyl)-
oxyl-4-(4-methoxylphenyl)-8-methylchromane[4,3-d]-D1,9b
-
4-(4-methoxylphenyl)-3a,4-diethoxyl-7,9-dimethoxylchromane[4,3-
1,2,3-thiadiazoline 10j. Yield: 53%, m.p. 221e224 ꢀC. ESI-
d]-D1,9b-1,2,3-thiadiazoline 10f. Yield: 40%, m.p. 171e173 ꢀC.
MS: m/z ¼ 555 [M þ 1]. IR (KBr): 2927, 1589 cmꢁ1
.
1H
1
ESI-MS: m/z ¼ 635 [M þ 1]. IR (KBr): 2927, 1583 cmꢁ1. H
NMR (CDCl3) d: 0.89 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.99
(t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.85e2.93 (m, 1H,
OCH2CH3), 3.21e3.27 (m, 1H, OCH2CH3), 3.43e3.60 (m,
2H, OCH2CH3), 3.92 (s, 3H, OCH3), 6.93 (d, 2H,
J ¼ 8.8 Hz, 30-H and 50-H), 7.18 (d, 1H, J ¼ 8.4 Hz, 6-H),
7.38 (dd, 1H, J ¼ 8.4 Hz, 1.6 Hz, 7-H), 7.61 (d, 2H,
J ¼ 8.8 Hz, 20-H and 60-H), 7.73 (d, 1H, J ¼ 9.2 Hz, 500-H),
7.89 (d, 1H, J ¼ 1.6 Hz, 9-H), 8.10 (d, 1H, J ¼ 9.2 Hz, 600-
H), 8.29 (s, 1H, 300-H).
NMR (CDCl3) d: 0.88 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 1.00 (t,
3H, J ¼ 7.2 Hz, OCH2CH3), 2.84e2.92 (m, 1H, OCH2CH3),
3.19e3.26 (m, 1H, OCH2CH3), 3.34e3.42 (m, 1H, OCH2CH3),
3.48e3.56 (m, 1H, OCH2CH3), 3.85 (s, 3H, OCH3), 3.98 (s, 3H,
OCH3), 4.02 (s, 3H, OCH3), 6.61 (s, 1H, 8-H), 6.94 (d, 2H,
J ¼ 8.8 Hz, 30-H and 50-H), 7.60 (d, 2H, J ¼ 8.8 Hz, 20-H and 60-
H), 7.69 (d, 1H, J ¼ 9.2 Hz, 600-H), 8.08 (dd, 1H, J ¼ 9.2 Hz,
1.6 Hz, 500-H), 8.30 (d, 1H, J ¼ 1.6 Hz, 300-H).
4.1.2.7. (3aRS,4RS )2-(2-Chloro-4-nitrophenyl)-4-(4-chloro-
phenyl)-3a,4-diethoxyl-8-methylchromane[4,3-d]-D1,9b-1,2,3-
thiadiazoline 10g. Yield: 76%, m.p. 172e174 ꢀC. ESI-MS: m/
4.1.2.11.
(3aRS,4RS )2-[2-Chloro-4-nitrophenyl]-4-phenyl-
3a,4,7,9-tetramethoxylchromane[4,3-d]-D1,9b-1,2,3-thiadiazo-
line 10k. Yield: 52%, m.p. 107e108 ꢀC. ESI-MS: m/z ¼ 611
1
z ¼ 559 [M þ 1]. IR (KBr): 2977, 1582 cmꢁ1
.
1H NMR
[M þ 1]. IR (KBr): 2980, 1597 cmꢁ1. H NMR (CDCl3) d:
(DMSO-d6) d: 0.80 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.89 (t,
3H, J ¼ 7.2 Hz, OCH2CH3), 2.38 (s, 3H, CH3), 2.86e2.90
(m, 1H, OCH2CH3), 3.07e3.11 (m, 1H, OCH2CH3), 3.24e
3.28 (m, 1H, OCH2CH3), 3.35e3.39 (m, 1H, OCH2CH3),
7.18 (d, 1H, J ¼ 8.4 Hz, 6-H), 7.38 (dd, 1H, J ¼ 8.4 Hz,
1.6 Hz, 7-H), 7.56 (d, 2H, J ¼ 8.8 Hz, 30-H and 50-H), 7.64
(d, 2H, J ¼ 8.8 Hz, 20-H and 60-H), 7.74 (d, 1H, J ¼ 9.2 Hz,
600-H), 7.89 (d, 1H, J ¼ 1.6 Hz, 9-H), 8.21 (dd, 1H,
J ¼ 9.2 Hz, 2.8 Hz, 500-H), 8.42 (d, 1H, J ¼ 2.8 Hz, 300-H).
2.90 (s, 3H, OCH3), 3.13 (s, 3H, OCH3), 4.03 (s, 3H,
OCH3), 4.06 (s, 3H, OCH3), 6.41 (s, 1H, 8-H), 7.43 (d, 2H,
J ¼ 8.4 Hz, 30-H and 50-H), 7.57e7.72 (m, 3H, 20-H, 60-H
and 600-H), 8.10 (d, 1H, J ¼ 9.6 Hz, 500-H), 8.30 (s, 1H, 300-H).
4.1.2.12. (3aRS,4RS )2-[2-Chloro-4-(methylsulfonyl)-phenyl]-
4-(4-chlorophenyl)-3a,4-diethoxylchromane[4,3-d]-D1,9b
-
1,2,3-thiadiazoline 11a. Yield: 78%, m.p. 112e113 ꢀC. ESI-
MS: m/z ¼ 578 [M þ 1]. IR (KBr): 2954, 1608 cmꢁ1
.
1H
NMR (CDCl3) d: 0.90 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.95
(t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.91e2.95 (m, 1H,
OCH2CH3), 3.08 (s, 3H, SO2CH3), 3.27e3.46 (m, 3H,
4.1.2.8. (3aRS,4RS )4-(6-Chlorobenzo[d][1,3]dioxol-5-yl)-2-
(4-nitrophenyl)-3a,4-diethoxyl-8-methylchromane[4,3-d]-D1,9b
-

H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
233
OCH2CH3), 7.14e7.18 (m, 2H, 6-H and 7-H), 7.39 (d, 2H,
J ¼ 8.4 Hz, 30-H and 50-H), 7.48 (m, 1H, 8-H), 7.63e7.66
(m, 3H, 20-H, 60-H and 600-H), 7.79 (dd, 1H, J ¼ 8.8 Hz,
2.0 Hz, 500-H), 7.98 (d, 1H, J ¼ 2.0 Hz, 300-H), 8.05 (dd, 1H,
J ¼ 8.0 Hz, 1.6 Hz, 9-H).
J ¼ 8.4 Hz, 2.4 Hz, 500-H), 7.84 (d, 1H, J ¼ 2.0 Hz, 9-H),
7.98 (d, 1H, J ¼ 2.4 Hz, H-300).
4.1.3. General procedure for the preparation of 4-aryl-2-[2-
chloro-4-(acetamide)phenyl]-3a,4-diethoxychromane[4,3-
d]-D1,9b-1,2,3-thiadiazolines 12
A mixture of 10 (2 mmol) and Raney-Ni (1.0 g) in EtOH
(6 mL) was stirred at r.t. under H2 for 12 h. The mixture was fil-
tered, and the filtrate concentrated. Ac2O (0.6 mL) and pyridine
(0.06 mL) were added into the residue and stirred for 12 h. Then
the mixture was diluted with water and extracted thoroughly
with CH2Cl2 and then the CH2Cl2 layer washed successively
with aqueous sodium bicarbonate and brine, dried over anhy-
drous Na2SO4. After removal of the solvent, and the crude prod-
uct was further purified by silica gel column chromatography.
4.1.2.13. (3aRS,4RS )2-[2-Chloro-4-(methylsulfonyl)phenyl]-4-
(4-methoxylphenyl)-3a,4-diethoxyl-8-methylchromane[4,3-d]-
D1,9b-1,2,3-thiadiazoline 11b. Yield: 69%, m.p. 131e133 ꢀC.
1
ESI-MS: m/z ¼ 588 [M þ 1]. IR (KBr): 2936, 1608 cmꢁ1. H
NMR (CDCl3) d: 0.90 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.95 (t,
3H, J ¼ 7.2 Hz, OCH2CH3), 2.39 (s, 3H, CH3), 3.07 (s, 3H,
SO2CH3), 2.89e2.96 (m, 1H, OCH2CH3), 3.22e3.45 (m, 3H,
OCH2CH3), 3.87 (s, 3H, OCH3), 6.95 (d, 2H, J ¼ 8.8 Hz, 30-H
and 50-H), 7.06 (d, 1H, J ¼ 8.4 Hz, 6-H), 7.25 (dd, 1H,
J ¼ 8.4 Hz, 2.0 Hz, 7-H), 7.60 (d, 2H, J ¼ 8.8 Hz, 20-H, 60-H),
7.66 (d, 1H, J ¼ 8.4 Hz, 600-H), 7.78 (dd, 1H, J ¼ 8.4 Hz,
2.4 Hz, 500-H), 7.85 (d, 1H, J ¼ 2.0 Hz, 9-H), 7.97 (d, 1H,
J ¼ 2.4 Hz, 300-H).
4.1.3.1. (3aRS,4RS )2-[2-Chloro-4-(acetamide)phenyl]-4-(4-
chlorophenyl)-3a,4-diethoxylchromane[4,3-d]-D1,9b-1,2,3-thia-
diazoline 12a. Yield: 90%, m.p. 150e152 ꢀC. ESI-MS: m/
z ¼ 558 [M þ 1]. IR (KBr): 3413, 2980, 1703, 1681,
1597 cmꢁ1
.
1H NMR (CDCl3) d: 0.82 (t, 3H, J ¼ 7.2 Hz,
4.1.2.14. (3aRS,4RS )2-[2-Chloro-4-(methylsulfonyl)phenyl]-4-
(4-methoxylphenyl)-3a,4-diethoxylchromane[4,3-d]-D1,9b-1,2,
3-thiadiazoline 11c. Yield: 66%, m.p. 195e197 ꢀC. ESI-MS:
OCH2CH3), 0.90 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.86e2.90
(m, 1H, OCH2CH3), 3.08e3.12 (m, 1H, OCH2CH3), 3.26e
3.30 (m, 1H, OCH2CH3), 3.37e3.41 (m, 1H, OCH2CH3),
7.24e7.31 (m, 2H, 6-H and 7-H), 7.35e7.42 (brs, 4H, 30-H,
50-H, 500-H and 600-H), 7.59 (m, 2H, 8-H and 300-H), 7.62 (d,
2H, J ¼ 8.0 Hz, 20-H and 60-H), 8.07 (dd, 1H, J ¼ 8.0 Hz,
1.6 Hz, 9-H).
m/z ¼ 574 [M þ 1]. IR (KBr): 2936, 1608 cmꢁ1
.
1H NMR
(CDCl3) d: 0.88 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 0.93 (t, 3H,
J ¼ 7.2 Hz, OCH2CH3), 2.89e2.96 (m, 1H, OCH2CH3), 3.07
(s, 3H, SO2CH3), 3.24e3.45 (m, 3H, OCH2CH3), 3.86
(s, 3H), 6.92 (d, 2H, J ¼ 8.8 Hz, 30-H and 50-H), 7.14e7.18
(m, 2H, 6-H and 7-H), 7.60 (d, 2H, J ¼ 8.8 Hz, 20-H and 60-
H), 7.66 (d, 1H, J ¼ 8.4 Hz, 600-H), 7.78 (dd, 1H, J ¼ 8.4 Hz,
2.4 Hz, 500-H), 7.97 (d, 1H, J ¼ 2.4 Hz, 300-H), 8.05 (dd, 1H,
J ¼ 8.0 Hz, 1.6 Hz, 9-H).
4.1.3.2. (3aRS,4RS )2-[2-Chloro-4-(acetamide)phenyl]-4-(4-
chlorophenyl)-3a,4-diethoxyl-8-methylchromane[4,3-d]-D1,9b
-
1,2,3-thiadiazoline 12b. Yield: 85%, m.p. 187e189 ꢀC. ESI-
MS: m/z ¼ 572 [M þ 1]. IR (KBr): 3417, 2979, 1698,
1595 cmꢁ1
.
1H NMR (CDCl3) d: 0.89 (t, 3H, J ¼ 7.2 Hz,
4.1.2.15. (3aRS,4RS )6-Chloro-2-[2-chloro-4-(methylsulfonyl)-
phenyl]-4-(4-chlorophenyl)-3a,4-diethoxyl-7,9-dimethoxylchro-
mane[4,3-d]-D1,9b-1,2,3-thiadiazoline 11d. Yield: 55%, m.p.
112e113 ꢀC. ESI-MS: m/z ¼ 672 [M þ 1]. IR (KBr): 2936,
OCH2CH3), 0.97 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.18 (s, 3H,
COCH3), 2.38 (s, 3H, CH3), 2.92e2.99 (m, 1H, OCH2CH3),
3.29e4.44 (m, 3H, OCH2CH3), 7.04 (d, 1H, J ¼ 8.4 Hz, 6-
H), 7.19e7.22 (m, 1H, 7-H), 7.31e7.37 (m, 4H, 30-H, 50-H,
500-H and 600-H), 7.62 (d, 2H, J ¼ 8.0 Hz, 20-H and 60-H),
7.69 (s, 1H, 300-H), 7.82 (d, 1H, J ¼ 2.0 Hz, 9-H).
1608 cmꢁ1
.
1H NMR (CDCl3) d: 0.87 (t, 3H, J ¼ 7.2 Hz,
OCH2CH3), 0.96 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.84e2.90
(m, 1H, OCH2CH3), 3.07 (s, 3H, SO2CH3), 3.18e3.25 (m,
1H, OCH2CH3), 3.32e3.40 (m, 1H, OCH2CH3), 3.43e3.51
(m, 1H, OCH2CH3), 4.03 (s, 3H, OCH3), 4.04 (s, 3H,
OCH3), 6.39 (s, 1H, 8-H), 7.40 (d, 2H, J ¼ 8.4 Hz, 20-H and
60-H), 7.6e7.69 (m, 3H, 30-H, 50-H and 600-H), 7.77 (dd, 1H,
J ¼ 8.4 Hz, 2.4 Hz, 500-H), 7.86 (d, 1H, J ¼ 2.4 Hz, 300-H).
4.2. Biology
The tumor cell lines (Bel-72, ECA-109, PC-3, MCF-7, HL-
60, and A-549, S180) were obtained from Shanghai Institute
of Pharmaceutical Industry.
4.1.2.16. (3aRS,4RS )2-[2-Chloro-4-(methylsulfonyl)phenyl]-4-
(4-chlorophenyl)-3a,4-diethoxyl-8-methylchromane[4,3-d]-D1,9b
-
1,2,3-thiadiazoline 11e. Yield: 72%, m.p. 130e132 ꢀC.
4.2.1. Cytotoxic assay
ESI-MS: m/z ¼ 592 [M þ 1]. IR (KBr): 2979, 1609 cmꢁ1
.
The cytotoxic activity in vitro was measured using the MTT
assay [16]. The MTT solution (10.0 ml/well) in RPMI-1640
(Sigma, St. Louis, MO) was added after cells were treated
with the drug for 72 h, and cells were incubated for further
4 h at 37 ꢀC. The purple formazan crystals were dissolved in
100.0 ml DMSO. After 5 min, the plates were read on an auto-
mated microplate spectrophotometer (Bio-Tek Instruments,
Winooski, VT) at 570 nm. Assays were performed in triplicate
1H NMR (CDCl3) d: 0.90 (t, 3H, J ¼ 7.2 Hz, OCH2CH3),
0.95 (t, 3H, J ¼ 7.2 Hz, OCH2CH3), 2.40 (s, 3H, CH3),
2.89e2.96 (m, 1H, OCH2CH3), 3.06 (s, 3H, SO2CH3),
3.25e3.46 (m, 3H, OCH2CH3), 7.06 (d, 1H, J ¼ 8.4 Hz,
6-H), 7.25 (dd, 1H, J ¼ 8.4 Hz, 2.0 Hz, 7-H), 7.38 (d, 2H,
J ¼ 8.8 Hz, 30-H and 50-H), 7.63 (d, 2H, J ¼ 8.8 Hz, 20-H
and 60-H), 7.66 (d, 1H, J ¼ 8.4 Hz, 600-H), 7.78 (dd, 1H,

234
H. Ying et al. / European Journal of Medicinal Chemistry 42 (2007) 226e234
on three independent experiments. The concentration required
for 50% inhibition of cell viability (IC50) was calculated using
the software ‘‘Dose-Effect Analysis with Microcomputers’’.
The tumor cell lines panel consisted of Bel-7402, ECA-109,
PC-3, MCF-7, HL-60 and A-549. In all of these experiments,
three replicate wells were used to determine each point.
4.2.4. Flow cytometric analysis of apoptosis
For flow cytometry analysis of DNA content, HL-60 cells
in exponential growth were treated with graded concentrations
of 11a (0.8e3.2 mM) for 48 h. Cells were washed twice with
PBS and fixed in 70% ethanol at ꢁ20 ꢀC. The cell pellet
was resuspended in 100.0 ml of PBS containing 50.0 mg/ml
RNase (Amersco, Solon, OH) and then incubated at 37 ꢀC for
1 h. After incubation, the cells were stained with 200.0 mg/ml
propidium iodide (PI, Sigma, St. Louis, MO) at 4 ꢀC for
30 min. The fluorescence of 2 ꢃ 104 cell was measured with
FACSCalibur (Becton Dickinson, Lincoln Park, NJ) [18].
4.2.2. Antitumor activity in ICR mice bearing sarcoma 180
cells
Tumor cells of S180 sarcoma were inoculated to mice. After
seven days, tumors were taken out and cells were harvested. Vi-
able tumor cells (2.5 ꢃ 106 cells/mouse) were inoculated to the
armpit of mice by subcutaneous injection. The compound 11a
and CTX were injected intraperitoneally (i.p.) to different
groups of mice (negative control group containing 20 female
mice, other groups containing 10 female mice) 24 h after the in-
oculation once a day for consecutive seven days. Cytophos-
phane (CTX) of 50.0 mg kgꢁ1 was used as a positive control
and physiological saline as negative control. Tumors were dis-
sected and weighed, and inhibition rates were calculated at
day 8. The inhibition rate was calculated as follows: C ꢁ T/
C ꢃ 100, T: average tumor weight of treated group; C: average
tumor weight of negative control group.
Acknowledgements
We thank Department of Chemistry and Pharmaceutical of
1
Informatics Institute of Zhejiang University for H NMR, 13C
NMR and ESI-MS spectra. We are also grateful to other staff
of ZJU-ENS Joint Medicinal Chemistry Laboratory.
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