New synthesis method for sultone derivatives: Synthesis, crystal structure and biological evaluation of S-CA

Article abstract of DOI:10.3390/molecules20034307

There has been no remarkable progress in the synthesis of sultones in recent years. To facilitate more detailed studies of this functional group, we found a new method to synthesize the sulfonic acid lactone derivatives and finish its ring-closing reactio

Full text of DOI:10.3390/molecules20034307

Molecules 2015, 20, 4307-4318; doi:10.3390/molecules20034307
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
New Synthesis Method for Sultone Derivatives: Synthesis,
Crystal Structure and Biological Evaluation of S-CA
1
1
1
2
1
1
Bi Li , Wenqiang Yan , Chenze Zhang , Yuzhong Zhang , Miao Liang , Fuhao Chu ,
1
1
1,
1,
Yan Gong , Bing Xu , Penglong Wang * and Haimin Lei *
1
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
E-Mails: libimegan@163.com (B.L.); ywq3226925@163.com (W.Y.);
zcz920418@163.com (C.Z); mimi1111202@sina.com (M.L.); chufhao@163.com (F.C.);
gongyan90@163.com (Y.G.); weichenxubing@126.com (B.X.)
2
School of Basic Medicine, Beijing University of Chinese Medicine, Beijing 100029, China;
E-Mail: zyz100102@126.com
*
Authors to whom correspondence should be addressed; E-Mails: wpl581@126.com (P.W.);
hm_lei@126.com (H.L.); Tel.: +86-10-8473-8640 (H.L.); Fax: +86-10-8268-6933 (H.L.).
Academic Editor: Derek J. McPhee
Received: 25 January 2015 / Accepted: 27 February 2015 / Published: 6 March 2015
Abstract: There has been no remarkable progress in the synthesis of sultones in recent
years. To facilitate more detailed studies of this functional group, we found a new method
to synthesize the sulfonic acid lactone derivatives and finish its ring-closing reaction. A
new sultone derivative, (E)-ethyl 4-oxo-6-styryl-3,4-dihydro-1,2-oxathiine-5-carboxylate
1
13
2
,2-dioxide (S-CA), was synthesized and structurally identified by H-NMR, C-NMR,
HMQC and X-ray single crystal diffraction analysis. The new rapid synthesis extended the
method of ring-closing reaction of sulfonic acid lactone derivatives. The angiogenesis
activities of S-CA were evaluated by the chick chorioallantoic membrane (CAM) model. It
could selectively suppress small angiogenesis in CAM, without influencing either middle
and large angiogenesis. In addition, anticancer efficacy of S-CA was evaluated in vivo
using a murine sarcoma S180 model. Reduction of the tumor weight and tumor HE
staining regions demonstrated that S-CA (10 mg/kg, intraperitoneal injection) had potent
inhibition effects and a 44.71% inhibitory rate in S180 mice. Moreover, an acute toxicity
test showed that the LD50 value of S-CA via intraperitoneal injection was 25.624 mg/kg.

Molecules 2015, 20
Keywords: sultone derivative; ring-closing reaction; CAM; acute toxicity; S180
4308
1
. Introduction
The importance of the sultone unit in organic chemistry cannot be overstated. Since the term “sultone”
was first introduced into the literature by Erdmann in 1888, it has emerged as valuable heterocyclic
intermediate [1–4]; sultones undergo cleavage of their carbon–oxygen bond in the presence of
nucleophiles and can be used as sulfoalkylating agents in both general organic synthesis and natural
product synthesis [5,6]. Methods used to synthesize sultones and include intramolecular Diels–Alder
reactions, ring-closing metathesis, Pd-catalyzed intramolecular coupling reactions, Rh-catalyzed C–H
insertion, Rh-catalyzed carbene cyclization cycloaddition cascade reactions, nucleophilic addition
reactions etc. [7,8]. The major step in synthesis of sultone is ring-closing reaction which was often
complicated by the need to use special and expensive catalysts such as Rh and Pd [8–10]. However,
the search for a simple and short synthesis of sultones has never been suspended for a moment in
recent years [11]. To facilitate the synthesis of this functional group, in present work, we found a new
method to synthesize sulfonic acid lactone derivatives and perform their ring-closing reactions. The
simple and rapid synthesis method we have found has never been reported previously. The newly
1
13
synthesized compound S-CA was characterized by H-NMR, C-NMR, HMQC and X-ray single crystal
diffraction analysis.
The biological activities of sultones previously included toxicological, skin sensitization, antiviral
(anti-HIV and HCMV) and antitumor activities [12–14]. Some compounds prepared by total synthesis
had antitumor activities due to their sulphonic acid ester structures [15,16]. Therefore, in this article an
angiogenesis evaluation on CAM and antitumor test on murine sarcoma S180 of S-CA were
investigated. Moreover, an acute toxicity test, as a part of safety evaluation of S-CA, was carried out
via intraperitoneal injection in Kunming mice to investigate its potential toxicity.
2
2
2
. Results and Discussion
.1. Chemistry
.1.1. Synthesis of S-CA
S-CA is synthesized from cinnamic acid (CA) via a new, simple and rapid ring-closing reaction
method shown in Scheme 1. The yields were calculated based on the last step of the reaction. The
typical synthetic procedure involved a novel formation of sultone group with acetic anhydride and
concentrated sulfuric acid. Cinnamoyl chloride was synthesized from CA using SOCl (1), and then we
2
obtained 2-cinnamoyl-3-ketobutanoic acid ethyl ester (2) by futher reaction with ethyl acetoacetate
under strong alkaline conditions. During the synthesis of compound 2, we observed that the yield of
the reaction could be further increased by using NaH instead of sodium ethoxide. Finally, S-CA was
obtained by ring closure of compound 2 using acetic anhydride and concentrated sulfuric acid.

Molecules 2015, 20
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Conditions and Regents: (a) SOCl
2
, reflux, 1 h, 96.7%; (b) NaH, ethyl acetoacetate, THF, ice bath, 2 h,
5
4.7%; (c) Ac O, H SO , ice bath , 0.5 h, 35.8%.
2
2
4
Scheme 1. The synthetic route to S-CA.
The most common procedures for the cyclization reaction of sultone derivatives were always
complicated by the need for special catalysts and the complexity of their process steps. In contrast to
previous syntheses of sultone derivatives, we prepare the targets in a more simple and rapid way. Since
no catalysts and cheap reagents were used, this practical approach might be preferred as a relatively
“
green process”. The new synthetic method expands the scope of methods currently available to
introduce the sultone functional group [8]. Meanwhile, this methodology could find applications in the
synthesis of naturally occurring sultones. The synthesis method in this article may serve as a basis for a
further study of deducing the reaction mechanism. A possible mechanism might be reaction of sulfuric
acid with Ac O giving a kind of “mixed anhydride form” followed by nucleophilic attack of the α-C of
2
the enol in compound 2 on this intermediate species. However, a detailed explanation and further
studies on the scope and limitations of this atypical reaction needed to be addressed in future work.
2
.1.2. Crystal Structure of S-CA
A single crystal of S-CA was obtained by recrystallization and its crystal structure was further
confirmed by X-ray single crystal diffraction analysis. The structure is shown in Figure 1.
Crystallographic data and experimental details for structural analyses are summarized in Table 1.
Figure 1. Crystal structure of S-CA.

Molecules 2015, 20
Table 1. Crystallographic data and structure refinement summary for S-CA.
4310
Phase
Molecular formula
Formula weight
T/K
Compound
C
15
H
14
6
O S
322.32
296(2) K
0.71073
Triclinic
P-1
Wavelength/nm
Crystal system
Space group
a/Å
8.5023(2)
9.1314(2)
b/Å
c/Å
10.6477(2)
α (°)
71.512(2)
β (°)
83.356(2)
γ (°)
V (Å )
72.179(2)
3
746.22(3)
Z
2
F(000)
336
1.434
3
D
calc (mg/m )
−
1
Absorption coefficient (mm )
θ range/(°)
0.243
2.02–27.59
Limiting indices
−10 ≤ h ≤ 11, −11 ≤ k ≤ 11, −13 ≤ l ≤ 13
7340/3419 [R(int) = 0.0197]
99.0%
Reflections collected/unique
Completeness to theta
Data/restraints/parameters
Refinement method
3419/0/199
Full-matrix least-squares on F2
R1 = 0.0513, wR2 = 0.1377
R1 = 0.0822, wR2 = 0.1586
1.055
Final R indices[I > 2σ(I)]
R indices (all data)
2
Goodness-of-fit on F
−
3
Largest diff. peak and hole/(e·Å ×10 )
CCDC
0.656 and −0.215
1052338
2
.2. Biological Evaluation
.2.1. Angiogenesis Activity
According to the references, sulfonate esters can inhibit angiogenesis [17]. Analogously, the
2
angiogenesis activities of S-CA were evaluated by a CAM assay (Figure 2 and Table 2). The model
was established according to our previous work [18].
After implantation, the sponge is treated with a stimulator of blood vessel formation in the absence
or presence of an angiogenesis inhibitor. Macroscopic observation shows that, the newly formed blood
vessels grow radially around the gelatin sponge in the blank control group (Figure 2a). The high
survival rate of embryos and normal growth of medium and large vessels (inner diameter > 50 μm)
indicates successful modeling and low toxicity of S-CA in vitro (Table 2). Suppression of small
vessels (inner diameter < 50 μm) were recognized as anti-angiogenesis activity. We found S-CA could
dramatically suppress small angiogenesis in a dose dependent manner on CAM (Figure 2c,d), and the

Molecules 2015, 20
4311
inhibition effect was similarly to that of a positive control (thalidomide, Figure 2b). Based on the
above evidence, S-CA might serve as an antiangiogenic drug that could enhance the treatment efficacy
of cytotoxic chemotherapy.
Figure 2. Microvascular proliferation of S-CA on CAM (×50). (a) Blank control for S-CA
group, (b) Positive control for S-CA group, (c) 10 μg/egg for S-CA group, (d) 40 μg/egg
for S-CA group.
−
Table 2. Effect of S-CA on angiogenesis inhabitation ( X ± S).
Group
n
Dose (μg/egg) Large Vessels Medium Vessels Small Vessels
Control
13
-
8.92 ± 4.80
12.4 ± 4.77
12.5 ± 6.94
12.6 ± 7.68
11.84 ± 3.33
12.1 ± 3.92
10.33 ± 5.25
9.00 ± 4.44
11.6 ± 2.07
4.16 ± 3.04 *
5.1 ± 1.22 *
3.5 ± 2.73 *
Thalidomide 13
20
10
40
S-CA
S-CA
12
12
*
p < 0.05, large vessels (inner diameter > 100 μm), medium vessels (50 μm < inner diameter < 100 μm),
small vessels (inner diameter < 50 μm).
2
.2.2. Acute Toxicity
At doses of 10, 20, 30 and 50 mg/kg administered intraperitoneally (i.p.), S-CA revealed a regular
dose-dependent increase in mortality following acute toxic test. Acute toxicity LD50 of S-CA is shown
in Table 3. According to the results, the LD50 of S-CA is 25.624 mg/kg by Käber assessment. The
mortality rate (0% at 10.0 mg/kg) progressively rose to 100% at the highest dose tested (50.0 mg/kg).
The no-observed-adverse-effect level for the i.p. dose was 10 mg/kg. Symptoms such as slow
movement decrease in aggressiveness, stopping food intake and weight loss (results not shown) were
observed later and at high doses. The acute toxicity data indicated that 10 mg/kg (i.p.) was harmless
for the further in vivo study in mice.
Table 3. Results of S-CA on mortality of acute toxicity test in mice.
Group Mice Number Start/End Dose (mg/kg) Death Rate (%) LD50 (mg/kg)
95% CIs
1
2
3
4
10/10
10/8
10/4
10/0
10
20
30
50
10
20
2
5.624
13.04–50.34
60
100

Molecules 2015, 20
.2.3. S-CA’s Antitumor Activity in Vivo
Anti-angiogenesis as a way of treating primary tumors and reducing their metastases had been
4312
2
proposed by Folkman in 1971 [19]. Administration of 40 μg/egg S-CA led to a significant growth
inhibition of the angiogenesis in the CAM model. In this investigation, we screened the S-CA
antitumor effect in a murine sarcoma S180 model. Treatment with the S-CA resulted in marked
suppression of tumor weight (Table 4). Compared with the model group, the inhibitory rate of S-CA
was 44.71% at the doses of 10 mg/kg; meanwhile cyclophosphamide (CTX) caused 63.52% inhibition.
The growth of implanted sarcoma S180 tumor in mice could be significantly inhibited by the S-CA
group (p < 0.05). Moreover, HE staining also directly showed that both S-CA group and CTX groups’
tumor necrotic regions significantly increased over the model group (p < 0.05) (Figure 3). The tumor
cells of the model group exhibited a high mitotic index, obvious atypia, whereas tumor cells
proliferation of the treated groups were blockaded in some way (Figure 3). Furthermore, the liver
indices, which were similar in the normal and S-CA treated groups (p > 0.05), indicated that S-CA did
not cause serious toxic effects on the liver system [20]. Spleen is one of the main immune organs; it is
responsible for initiating immune reactions in the body. Thus the spleen index directly reflects the
status of the immune system [21]. Spleen indices of S-CA treated groups were higher than both the
normal and CTX groups’. Increases in spleen index suggested that S-CA could regulate the immune
system of S180 mice [22].
Table 4. Antitumor Effects of S-CA in S180 Mice (Mean ± S.D.).
Dose
Mice Number Tumor Weight Inhibitory
Liver Index
(100× g/g)
4.24 ± 0.54
4.51 ± 0.53
4.98 ± 0.26
Spleen Index
(mg/g)
Group
(
mg/kg)
Start/End
10/10
(g)
Rate (%)
Normal
Model
CTX
-
-
-
4.27 ± 0.75
5.25 ± 0.94
4.50 ± 1.39
6.33 ± 1.51 *
-
10/10
0.684 ± 0.416
-
#
20
10
10/10
0.249 ± 0.142
0.342 ± 0.291
63.52%
44.71%
#
S-CA
10/10
4.81 ± 0.32
#
#
*
compared with normal group: * p < 0.05; compared with model group: p < 0.05.
Figure 3. Histopathology of the mice bearing S180 xenografts after the treatment for 15 days.
#
compared with model group: p < 0.05; ## compared with model group: ## p < 0.01. (a)
Model group (400×). (b) Model group (100×). (c) CTX group (100×). (d) S-CA group (100×).
e) Tumor necrotic regions with HE staining of model and treatment groups of S180 mice.
#
(

Molecules 2015, 20
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3. Experimental Section
3.1. General Information
Murine sarcoma S180 cells provided by the 302 Military Hospital of China were maintained in
RPMI-1640 medium, supplemented with 10% fetal bovine serum (Hyclone, Aurora, ON, Canada),
00 IU/mL Penicillin-Streptomycin (Hyclone), and Non-Essential Amino Acids (Sigma, St.Louis,
MO, USA), at 37 °C under humidified air with 5% CO . Male Kunming mice (Beijing Vital River
1
2
Laboratory Animal Technology Company Limited, Beijing, China) were kept under standard
laboratory conditions (tap water, constant room temperature 22 °C). Principles of laboratory animal
care were followed and all experiments were carried out in accordance with the “Regulation for the
Administration of Affairs Concerning Experimental Animals” [21].
The S-CA was synthesized in our laboratory. The purity of S-CA was > 98% by HPLC analysis.
HPLC-grade methanol and acetonitrile were purchased from Xinkeao Scientific & Technology Co. Ltd
(
Beijing, China). Other chemicals and reagents were analytical grade and commercially available, and
unless otherwise mentioned, used without further purification. Reactions were monitored by TLC
using silica gel coated aluminum sheets (Qingdao Haiyang Chemical Co., Qingdao, China) and
1
13
visualized in UV light (254 nm). H-NMR and C-NMR assays were recorded on a Bruker AVANCE
00 NMR spectrometer (Fällanden, Switzerland) and chemical shifts are reported in (ppm). X-ray
single crystal diffraction was obtained by using MM007HF Saturn724+ X single crystal diffractometer
Rigaku, Takatsuki-shi, Japan). Melting points are taken with an X-5 micro melting point apparatus
and were uncorrected.
5
(
3
.2. Chemistry
3
.2.1. Synthesis of S-CA
Preparation of cinnamoyl chloride (1). The mixture of CA (7.4 g, 0.05 mol) and thionyl chloride
SOCl , 10.0 mL) was heated under reflux and stirred for 1 h. The progress of the reaction was
monitored by TLC. On completion of the reaction, the extra solvent (SOCl ) was removed by reduced
pressure distillation, and then the faint yellow solid (8.0 g) was obtained finally. Yield: 96.7%, m.p.
3.8–35.1 °C.
(
2
2
3
Preparation of (2Z,4E)-ethyl 2-(1-hydroxyethylidene)-3-oxo-5-phenylpent-4-enoate (2). NaH (0.24 g,
.10 mol) was added to ethyl acetoacetate (5.7 mL, 0.04 mol) was added and the mixture was stirred
0
under ice bath. After air bubbles production ceased, cinnamoyl chloride (6.6 g, 0.04 mol) dissolved in
THF was added drop wise and the reaction was kept under ice bath for 2 h. The reaction mixture was
acid treated with 5% HCl and then extracted by ethyl acetate. After drying the organic layer over
anhydrous Na
2
SO
4
and evaporating the solvent under vacuum, the title compound was finally obtained
1
as a semi-solid (5.7 g, 0.022 mol). Yield: 54.7%. H-NMR (CDCl
3
) (ppm): 1.41 (t, 3H, J = 14.0 Hz,
-
CH
3
), 2.43 (s, 3H, CH
3
), 4.36 (q, 2H, J = 14 Hz, -CH
2
-), 7.43 (d, 1H, J = 15.5 Hz, =CH-), 7.44–7.46
1
3
(
m, 3H, Ar-H), 7.56–7.57 (m, 2H, Ar-H), 7.82 (d, 1H, J = 15.5 Hz, -CH=). C-NMR (CDCl
3
) (ppm):

Molecules 2015, 20
4314
1
4.3 (-CH
3
), 27.4 (-CH
3
2
), 60.4 (-CH ), 108.5, 120.9, 128.5, 128.9, 130.5, 135.0, 143.1, 167.3 (-COO-),
182.0 (-C=O), 199.7 (HO-C=).
Preparation of (E)-ethyl 4-oxo-6-styryl-3,4-dihydro-1,2-oxathiine-5-carboxylate 2,2-dioxide (3).
Compound 2 (5.0 g, 0.02 mol) was dissolved in acetic anhydride (25 mL) under ice bath cooling, and
concentrated sulfuric acid (0.6 mL) was added dropwise over 0.5 h. After completion of this reaction,
the reaction mixture was poured into ice-water and the orange-red precipitated solid was obtained. The
crude product was recrystallized from ethanol to afford the yellow crystal (1.4 g). The purity of
1
13
synthesized S-CA was more than 98%. The structure of S-CA was determined by H-NMR, C-NMR,
1
HMQC, and X-ray single crystal diffraction analysis. Yield: 35.8%, m.p. 128.7–129.1 °C. H-NMR
(CDCl
3
) (ppm): 1.43 (td, 3H, J = 1.5, 14.0 Hz, -CH
3
), 4.42 (s, 2H, -CH
2
-), 4.46 (qd, 2H, J = 1.5, 14 Hz,
-
CH -), 7.05 (dd, 1H, J = 3.5, 15.5 Hz, -CH=), 7.46–7.48 (m, 2H, Ar-H), 7.59–7.60 (m, 3H, Ar-H),
2
13
7
.73 (dd, 1H, J = 3.5, 15.5 Hz, =CH-). C-NMR (CDCl
3
) (ppm): 14.2 (-CH
3
), 59.2 (-CH
2
), 62.7 (-CH ),
2
1
15.4, 116.0 (-CH=), 128.9 (Ar-C), 129.2 (Ar-C), 131.7 (Ar-C), 134.0, 144.4 (=CH-), 162.6, 163.0, 179.8.
.2.2. Crystal Structure
3
The crystal and molecular structure has been determined by X-ray single crystal diffraction. The
coordinate and anisotropy parameters of non-hydrogen atoms were refined with a full-matrix least-squares
−3
−3
−3
−3
procedure to R
1
= 0.0513, wR = 0.1377, (∆p) max = 0.656 × 10 e nm , (∆p) min= −0.215 × 10 e Å .
2
The structure was solved by using the program SHELXS-97 and Fourier difference techniques and
2
refined by full-matrix least-squares method on F using SHELXL-97. Details of the data collection and
refinements of the title compound are given in Table 1 and Figure 1. All non-hydrogen atoms were
refined anisotropically, whereas the hydrogen atoms were generated geometrically. Parameters in CIF
format are available as Electronic Supplementary Publication from Cambridge Crystallographic
Data Centre. CCDC 1052338 contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html.
3
.3. Bio-Evaluation Methods
3
.3.1. Angiogenesis Assay
Fertilized White Leghorn chicken eggs (50–65 g), provided by the Chinese Academy of
Agricultural Sciences, were placed in an incubator as soon as embryogenesis started and were kept
under constant humidity of 65% at 37 °C. Here we present a method for the antiangiogenesis in the
chick embryo chorioallantoic membrane (CAM) based on the implantation of a gelatin sponge on the
top of the growing CAM on day 7 of development. On day 7, under sterile conditions, a square
window was opened on the shell and physiological saline (0.1 mL) was injected in to detach the shell
membrane. Gelatin sponges were implanted, respectively. The control group was treated with acetone
(
10 μL). Moreover, 1 mm sterilized gelatin sponges carrying the S-CA dissolved in acetone at 10 and
0 μg/egg were implanted on the smaller vessels part of CAM. The window was sealed with sterile
4
adhesive and the eggs were returned to the incubator for a further 48 h. Then, the tapes were removed
and the entire CAM was detached after tissue fixation with methanol/acetone (1:1, v/v). We use

Molecules 2015, 20
4315
computer-assisted tracking of images to obtain absolute values for the number of microvessels.
Quantitative evaluation of the angiogenic response, expressed as microvessel density, can be obtained
by applying a morphometric method of “point counting” on histological CAM sections. Data was
analyzed using t-test of statistics analysis system, the values were expressed as mean ± sd of
6
observations and p < 0.05 was considered significant.
3
.3.2. Acute Toxicity Test
S-CA was further investigated for its approximate LD50 and 95% confidence interval in mice. Male
Kunming mice (Beijing Vital River Laboratory Animal Technology Company Limited, Beijing,
China) weighing 19.2–21.2 g, were randomly divided into five groups per ten individuals matched in
weight and size. In the models, group I received solvent (bean oil) and this group was used as controls.
Then the other four groups were given lumbar injection of S-CA in doses of 10, 20, 30, 50 mg/kg
respectively. The general behavior of the mice was observed continuously for 1 h after the treatment
and then intermittently for 4 h and thereafter over a period of 24 h. At the end of this period mortality
was recorded for each group. The acute toxicity was evaluated by the median lethal dose (LD50) and
9
5% confidence interval which were calculated by Käber assessment. The summary of S-CA acute
toxicity expressed as LD50 is shown in Table 2. The mice were further observed for up to 7 days
following treatment for any signs of toxicity and deaths and the latency of death.
3
.3.3. Antitumor Activity in Vivo
S180 cells were harvested and washed three times with RPMI-1640 medium. The cells were
pelleted by brief centrifugation at 800 r/min for 10 min. The supernatant was aspirated, and the cells
7
were resuspended in normal saline at a density of 2 × 10 cells/mL. Male Kunming mice were
6
subcutaneously implanted with 2 × 10 cells/mouse on the left flank (day 0). Twenty-four hours after
inoculation, 30 tumor bearing mice were randomly divided into model group, CTX group, S-CA group
(
n = 10), respectively. Another 10 mice without any treatment were set as normal group. S-CA was
continuously administrated via intraperitoneal injection, every other day for 15 days. S-CA groups
received 10 mg/kg of body weight, the CTX group received CTX (20 mg/kg), model and normal
groups were treated with bean oil, respectively. Twenty-four hours after the last administration, the
mice were sacrificed and the solid tumors, livers and spleens were excised and weighed. The inhibition
rate (IR) of tumor growth was calculated by the following formula: IR (%) = [(A − B)/A]/100, where
A is the average tumor weight of the model group, and B is that of the treatment group mice. The
index of livers was calculated as W
l
/W
was the average mouse body weight (g) of each group. The index of spleen was calculated as
/W , where W was the average spleen weight (mg) of each group and W was the average mouse
m
, where W was the average livers weight (g) of each group and
l
W
W
m
s
m
s
m
body weights (g) of each group.
At the end of the experiments, a thorough necropsy was carried out immediately after the mice were
sacrificed. Samples of the tumors and the following organs, liver and spleen were fixed in 10% neutral
buffered formalin, embedded in paraffin, sectioned at 4 mm, and processed according to the
hematoxylin and eosin (HE) staining protocol. The stained tissues were observed under a light
microscope. Quantitative analysis was made in a blinded manner under a light microscope.

Molecules 2015, 20
4316
Computer-assisted image tracking was used to calculate the positive tumor areas. The results were
regarded as the mean ± SD of eight different sections.
4
. Conclusions
In summary, a simple method for the synthesis of the sultone derivative S-CA and its
characterization by H-NMR, 13C-NMR, HMQC and X-ray single crystal diffraction analysis are
reported. In addition, S-CA exhibited selective antiangiogenesis on CAM and potent antitumor effect
against S180 model. While there have been no major breakthrough in recent years in synthesis routes
to this functional group, our study can be regarded as a simple method for the synthesis of the sultone
derivative S-CA. Moreover, sultone derivatives are used widely in many other fields, including
industrial applications, for their biological properties and the current mechanistic research into the role
of β-sultones in olefin sulfonation. Many materials containing nucleophilic functionalities have been
modified by propane-sultone, producing fungicides, fire resistant polymers, lubricating oil additives
and emulsifying agents. 1,3-Propanesultone is used as starting material in the synthesis of many
electroplating intermediates and also used for the synthesis of a variety of sensitizing dyes and
surfactants. The new synthesis route extends the ring closing reaction of sulfonic acid lactone
derivatives method and has potential applications in general organic synthesis, natural product
synthesis and some other research fields as well.
1
Based on the current study, S-CA represents a new hit for the biological activities of sulfonate units
and should be further studied for antiangiogenesis in anticancer drug discovery. The difference of
toxicity in the biological evaluation on CAM and an acute toxicity test confirmed the potentially
pharmacologically interesting properties of S-CA. Our completed work lays the foundation for further
research on the anti-tumor mechanism of sultone derivatives. A further study to acquire information
concerning its target organs and pharmacological activity is still in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/03/4307/s1.
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (No.
8
1173519) and the Innovation Team Project Foundation of Beijing University of Chinese Medicine
(
Lead Compound Discovering and Developing Innovation Team Project Foundation).
Author Contributions
Bi Li performed chemistry experiments and wrote the paper; Bi Li, Wenqiang Yan and Chenze Zhang
performed the biological evaluation experiments; Yuzhong Zhang analyzed pharmacological data and
elaborated cell morphology; Miao Liang and Fuhao Chu did the data analyses; Yan Gong and Bing Xu
helped perform the analysis with constructive discussions; Penglong Wang designed the study and
prepared the manuscript; Haimin Lei was principle investigator of the project and provided the
research funding.

Molecules 2015, 20
4317
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Sample of the compound S-CA is available from the authors.
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