D-ring modified novel isosteviol derivatives: Design, synthesis and cytotoxic activity evaluation

Article abstract of DOI:10.1016/j.bmcl.2012.07.083

A series of polyhydric, amino alcohol and tricyclic derivatives were facilely synthesized by D-ring modification of isosteviol. These compounds were screened for their cytotoxic activities against four human tumor cell lines in vitro. Among them, the 15-α-aminomethyl-16-β-hydroxyl isosteviol 23 exhibits significant cytotoxicity superior to the positive control (cisplatin) against EC9706, PC-3 and HCT-116 cell lines.

Full text of DOI:10.1016/j.bmcl.2012.07.083

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5827–5832
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
D-ring modified novel isosteviol derivatives: Design, synthesis
and cytotoxic activity evaluation
⇑
⇑
Tao Zhang, Li-Hui Lu, Hao Liu, Jun-Wei Wang, Rui-Xue Wang, Yun-Xiao Zhang , Jing-Chao Tao
College of Chemistry and Molecular Engineering, New Drug Research & Development Center, Zhengzhou University, 75 Daxue Road, Zhengzhou 450052, 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 polyhydric, amino alcohol and tricyclic derivatives were facilely synthesized by D-ring mod-
ification of isosteviol. These compounds were screened for their cytotoxic activities against four human
tumor cell lines in vitro. Among them, the 15-a-aminomethyl-16-b-hydroxyl isosteviol 23 exhibits sig-
nificant cytotoxicity superior to the positive control (cisplatin) against EC9706, PC-3 and HCT-116 cell
lines.
Received 20 June 2012
Revised 15 July 2012
Accepted 24 July 2012
Available online 31 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Tetracyclic diterpenoids
Isosteviol
Amino alcohol
Antitumor activity
The investigation aimed at discovering anti-carcinogenic com-
pounds has attracted considerable interest in recent years since
cancer is a leading cause of death worldwide. Natural products
have always played a major role in anticancer medicine1–3 and
the unique metabolites produced by diterpenoids have increas-
ingly become major players in antitumor drug discovery.4–6 Iso-
steviol is a tetracyclic diterpenoid with a beyerane skeleton,
obtained by acid hydrolysis of stevioside.7 More and more re-
searches in recent years showed that isosteviol as well as its deriv-
et al. reported that the cytotoxicity was greatly improved when
amino alcohol fragment was introduced to the ring-A of boswellic
acids (Fig. 1, Compound (B)).28 However, few reports have focused
on the activity relationship of amino alcohol substituted isosteviol.
Moreover, compounds with tricyclic skeleton are widely found in
natural products, and some of them are proved to be good antitu-
mor agents.29 Isosteviol, an ent-beyerane type tetracyclic diter-
pene, can be converted to tricyclic compounds by D-ring opening
reaction and showed excellent inhibition constants.30 On the basis
of these results, we designed and synthesized a series of novel pol-
yhydric, amino alcohol and tricyclic derivatives via D-ring modifi-
cations of isosteviol for the purpose of discovering new antitumor
active compounds.
atives possess
a remarkably broad spectrum of biological
activities.8–15
Previous reports proved that modifications on D-ring of the iso-
steviol skeleton could change its biological activities or lead to new
activities.16 Zhang and co-workers17 reported that exo-methylene
cyclopentanone sub-structure in the D-ring of tetracyclic diterpe-
noids could conduce noteworthy cytotoxicity superior to adriamy-
cin. Nguyen’s group18 found two new symmetric dimers of ent-
kaurane diterpenoid with the connectivity at ring-D from Croton
tonkinensis showed a potent anti-tumor activity. Our previous
study also showed that the C-15 and C-16 functionalized isosteviol
had good cytotoxic activities against B16-F10 melanoma cells.19 In
addition, some work also indicated that biological activities of ter-
penoids would be improved by the introduction of hydroxyl groups
through chemical synthesis20 or microbial transformations.21–23
It is also well known that amino alcohols are important struc-
tural fragments of many bioactive compounds,24–26 such as Nal-
trexone (Fig. 1, Compound (A)) and their derivatives.27 Taneja
Initial synthetic efforts were focused on novel polyhydric iso-
steviol derivatives and the synthetic routes are depicted in
Scheme 1.
Esterification of compound 1 with CH3CH2Br and KOH in DMSO
afforded the corresponding isosteviol ethyl ester 2 in high yield,
HO
H
O
N
H
OH
HO
O
NH2
A
B
⇑
Corresponding author. Tel./fax: +86 371 67767200.
E-mail address: jctao@zzu.edu.cn (J.-C. Tao).
Figure 1. Structure of Compound A and B.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.083

5828
T. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5827–5832
OH
OH
OH
N
OH
c
O
OH
O
OH
COOEt
COOEt
4
COOEt
6
5
8
HO
c
c
d
f
12
17
11
18
13
O
O
1
N
N
14
16
9
a
e
b
OH
OH
2
3
10
8
15
4
6
7
O
O
5
COOH
COOEt
COOEt
COOEt
19
20
2
3
1
7
c
g
OH
OH
OH
CHO
OH
f
h
OH
N
OH
COOEt
COOEt
COOEt
COOEt
10
11
12
9
Scheme 1. Reagents and conditions: (a) EtBr, KOH, DMSO, rt, 3 h (95%); (b) Ac2O, SeO2, reflux, 6 h (83%); (c) 1.2 equiv. NaBH4, MeOH, 0 °C; (d) 3.0 equiv NaBH4, MeOH, 0 °C to
rt, 2 h (90%); (e) NH2OHÁHCl, NaHCO3, EtOH, reflux; (f) 2.5 equiv. LiAlH4, THF, reflux (80%); (g) HCHO, C2H5ONa, C2H5OH, 60 °C, 3 h (92%); (h) TEMPO, NBS, CH2Cl2/H2O, TBAB,
reflux (90%).
whereas only very low yields of 2 were obtained while using the
classical esterification methods. Compound 2 was converted to 6
easily under NaBH4/EtOH conditions.31 The 15,16-diketone 3 was
obtained as an orange crystal by the oxidation of 2 with selenium
dioxide in acetic anhydride under reflux condition in high yield.
Reduction of 3 with 1.2 equiv. or 3 equiv of NaBH4 in methanol
in ice bath gave uniquely the mono reduced product 4 or the 1,3-
diol 5, respectively. But a mixture of 4 and 5 were obtained when
the amount of NaBH4 was from 1.2 to 3 equiv. Compound 5 could
also be prepared by the reduction of 4 with NaBH4. The absolute
configurations of the two newly formed chiral centers at C-15
and C-16 in compound 5 were identified by X-ray crystallographic
analysis and the two hydroxyl groups located at endo position
unambiguously (Fig. 2).33 The steric effects of C10-CH3, C13-CH3
and ring carbon may be the reason to direct the approach of HÀ
onto the two carbonyls easily from the exo positions.
Figure 3. X-ray structure of compound 9.
It is well known that oximido groups are usually considered as
bioactive elements in biological studies. Therefore, one of the two
carbonyls in compound 3 was designed to be converted to oximido
following the introduction of a hydroxy unit by reduction of the
other carbonyl group for the purpose of investigating the struc-
ture–activity relationship. After treatment of 3 with hydroxyl-
amine hydrochloride and NaHCO3 in ethanol under reflux
condition, only the 15-ketone-16-oxime derivative 7 was obtained
as a yellow crystal. But attempts to obtain the 15-oximed deriva-
tive failed to meet our expectation. Then we decided to convert
the carbonyl group at C-15 and the ester group at C-20 of com-
pound 7 to hydroxyl groups in order to construct multi hydroxyl
derivatives. When excess amount of LiAlH4 in THF was employed
as reducing agent, compound 8 was obtained as a white solid,
whereas NaBH4 could only reduce the C-15 carbonyl to give com-
pound 9 as the unique product in high yield. The NOESY spectrum
of compound 8 suggests that the C-15 hydroxyl group was at endo
configuration. The absolute configuration of compound 9 was con-
firmed by the X-ray structure (Fig. 3),33 indicating an endo direc-
tion of the hydroxyl group at C-15.
Moreover, in our previous work, compound 10 could be suc-
cessfully achieved through a one-pot ‘Aldol-Cannizzaro reaction’
process,32 which promoted us to probe 1,3-diol and its derivatives
for the evaluation of their antitumor activities. Firstly, a clean and
convenient TEMPO catalyzed oxidization of 10 gave 11 in 90%
yield. Followed oximation reaction of 11 with hydroxylamine
hydrochloride in the presence of NaHCO3 in ethanol, compound
12 was furnished (87%).
The following work was focused on the introduction of amino
and related functional groups onto the D-ring of isosteviol. A series
Figure 2. X-ray structure of compound 5.

T. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5827–5832
5829
S
R
S
R
NH
NH
N
H
N
H
19a,
19b,
19c,
R = phenyl
R = naphthyl
R = 3,5-Bis(trifu-
b
b
O
OH
or omethyl)phenyl
COOEt
e
COOEt
e
20a, R = phenyl
20b, R = naphthyl
20c, R = 3,5-Bis(trifu-
or omethyl)phenyl
NOH
a
NH2
NH2
OH
O
O
COOEt
13
COOEt
COOEt
14
7
a
c
c
O
O
H
H
N
NOH
N
b
N
N
OH
OH
O
O
Boc
Boc
COOEt
COOEt
d
COOEt
15
d
16
9
O
O
H
N
H
N
b
HN
HN
OH
18
COOEt
COOEt
17
Scheme 2. Reagents and conditions: (a) Raney Ni/H2 50 psi, 50 °C, THF, 5 h (95%); (b) NaBH4, MeOH, 0 °C to reflux (80–85%); (c) N-Boc-
L
-proline, DCC, DMAP, DCM, rt, 6 h (90–
92%); (d) CF3COOH, DCM, 3 h, rt, (87–90%); (e) isothiocyanate, DCM, rt, 2 h (>97%).
of b-amino alcohol derivatives were firstly synthesized via stereo-
selective reduction of carbonyl and oximido groups in compound
7. The synthetic approach employed to prepare 1,2-amino alcohol
and their derivatives were outlined in Scheme 2.
of 13 or 14 with N-Boc-L-proline and DCC in the presence of DMAP
in CH2Cl2 furnished 15 and 16, ‘respectively’. Then treatment of 15
and 16 in TFA/DCM system generated the corresponding deprotec-
ted prolinamide 17 and 18. thiourea derivatives were synthesized.
In addition, reaction of 13 (14) with isothiocyanate in DCM at room
temperature provided corresponding thiourea derivatives 19 (20)
in almost quantitive yield. The 15-carbonyl could be stereoselec-
tively converted to hydroxyl group by reduction with NaBH4 in
methanol (15–16, 17–18, and 19–20).
Although the attempts to get the single crystals of 14 or its
derivatives were failed, the S-configuration of the amino group at
C-16 can be determined by the X-ray diffraction of 17 (Fig. 4).33
The presence of NOE correlation of C-15-H and C-16-H indicating
that the two hydrogen atoms were at the same direction and the
hydroxyl and the amino groups were both at endo position
(Fig. 5). The configuration of C-15 and C-16 were further confirmed
by the observation of NOE effect between C-14-H and C-15-H (C-
14-H and C-16-H, C-16-H and C-17-H). The structures suggested
that the steric effect of C-10-CH3, C-13-CH3 and ring carbon make
for an easy approach of nucleophile to C-15 and C-16 from exo po-
sition, while hard from endo.
Catalytic hydrogenation of the 15-ketone-16-oxime isosteviol
derivative 7 with Raney Ni/H2 in ethanol or isopropanol gave com-
pound 13 in 95% yield. Reduction of 13 with NaBH4 in anhydrous
methanol generated amino alcohol 14 stereoselcetively as a white
powder. Interestingly, when compound 7 was reduced to com-
pound 9 by NaBH4 firstly, then hydrogenation of 9 with the Ni cat-
alyzed hydrogenation condition, the corresponding 1,2-amino
alcohol 14 was not furnished, but unexpectedly, compound 13
was isolated in 96% yield. The conversion of 9–13 must undergo
an imine rearrangement process.
Succeedingly, in order to further screen the structure-bioactiv-
ity relationship of the amino derivatives, we decided to convert
the 16C-amine to its corresponding amide or thiourea. Reaction
H
H
H
17
H
16
H
H
14
15
NH2
H
OH
COOEt
Figure 5. NOE correlations for compound 14.
Figure 4. X-ray structure of compound 17.

5830
T. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5827–5832
OH
N
b
a
CHO
CHO
CHO
COOEt
11
COOEt
21
COOEt
22
OH
OH
OH
OAc
e
c
d
NOH
NHAc
NH2
NHAc
COOEt
COOEt
12
COOEt
COOEt
24
23
25
Scheme 3. Reagents and conditions: (a) DBU, pyridine (83%); (c) pyrrolidine, NaH, TBAB, DMF, rt (70%); (c) Raney Ni/H2, 50 psi, 50 °C,THF, 5 h (80%); (d) Ac2O, pyridine, rt, 3 h
(90%); (g) methanol, K2CO3, rt (92%).
pared from 3 through Baeyer–Villiger oxidation with excessive m-
CPBA in high yield. Acid hydrolysis of 27 generated dicarboxylic
acid 28. As Beckmann fragmentation occur on ketoxime35 and
a-
hydroxyl oxime,36 the expected tricyclic compounds could be ob-
tained from 7 and 9 through this reaction. Treatment of compound
7 in acetone with catalytic amount of concentrated sulfuric acid
under reflux afforded compound 29 conveniently. Alternatively,
treatment of compound 7 with TsCl/NaOH gave 30, which was con-
verted to 28 through hydrolysis of the cyano group. For a-hydroxyl
oxime 9, fragmentation in thionyl chloride provided 31 efficiently.
The MTT cell proliferation assay was used to evaluate cytotoxic
activities of these new synthesized compounds37 against four hu-
man cancer cell lines in vitro, including two esophageal carcinoma
cell lines (Eca109 and EC9706), prostatic carcinoma (PC-3) and col-
otectal carcinoma (HCT-116). Cisplatin (DDP) was used as positive
control. From the IC50 values summarized in Table 138, the 1,3-ami-
no alcohol compound 23 showed significant cytotoxicity.
Figure 6. X-ray structure of compound 22. Some hydrogen atoms were omitted for
clarity.
Table 1 revealed that esophageal carcinoma cells were more
sensitive to the tested compounds with the IC50 low to 4.01 lM.
The carbonyl compounds 1, 2, 3 and 26 without hydroxyl or amino
group showed no inhibition to all cancer cell lines, but certain
activities against Eca109 and EC9706 were observed when car-
bonyl groups were converted to hydroxyl group (5, 10, 11 and
12). 16-Oximido group did not improve inhibitory activity (7, 8,
and 9). Noteworthily, introduction of hydroxymethyl, formyl or
oximido to C-15 was beneficial for the cytotoxicity (10, 11 and
12). Compound 14 with 1,2-amino alcohol subunit exhibited note-
worthy activities to the four tested cells lines whereas 15-car-
bonyl-16-amino derivative 13 gave poor results. The results
indicated that the conversion of carbonyl at C-15 and C-16 to hy-
droxyl was important to their bioactivities (4 vs 5, 13 vs 14, 17
vs 18, 19c vs 20c). Formation of phenyl and naphthyl thiourea
and acylation of amino group by proline decreased cytotoxic activ-
ities greatly (14 vs 18, 19a, 19b, 20a, 20b). Nevertheless, 3,5-
Bis(trifluoromethyl) phenyl thiourea (20c) showed certain effect
After the 1,2-amino alcohol derivatives had been successfully
achieved, the following efforts were focused on 1,3-amino alcohol
derivatives. The synthetic routes were outlined in Scheme 3. Com-
pound 21, containing an
a,b-unsaturated aldehyde fragment, was
obtained by eliminating monomolecular H2O from compound 11
in DBU/pyridine system. From the X-ray structure of 22 (Fig. 6)33
we concluded that the nucleophile (pyrrolidine) could only attack
the Michael acceptor (C-16) from the endo side and gives 22 stere-
oselectively. Noteworthily, this aza-Michael reaction needed a
strong base (NaH), and the addition of tetrabutylammonium bro-
mide (TBAB) could improve the yield up to 70% (20% without
TBAB). Compound 12, obtained by oximation of 11, was converted
to 1,3-amino alcohol 23 through hydrogenation catalyzed by Raney
Ni in THF, and then its N-acetyl derivative and N-,O-diacetyl deriv-
ative were prepared. The diacetyl derivative 24 could be converted
to 25 easily under a mild condition in good yield.
As the tricyclic compounds may have some kinds of bioactivi-
ties, we attempted to reconstruct the isosteviol skeleton to the cor-
responding tricyclic derivatives via D-ring opening reaction.
Scheme 4 illustrated the synthesis of some tricyclic derivatives.
The 15,16-diketone 3 could also be prepared through a bromina-
tion-oxidation process in order to avoid employing the toxic oxi-
dant SeO2 as aforementioned. Compound 26 was firstly prepared
by a one-pot esterification-bromination from isosteviol. As ex-
pected, 3 was prepared by oxidation of 26 in DMSO/air system un-
der reflux condition.34 Moreover, white solid 27 was isolated from
this reaction as a by-product in 20% yield, which could also be pre-
to the cell lines.
cytotoxic against PC-3 cell while 16-pyrrolidine substituted com-
pounds 22 inhibited Eca109 selectively with IC50 value 30.30 M.
Importantly, 1,3-amino alcohol 23 displayed the most potent anti-
cancer agent with IC50 value 4.01 M, 5.02 M, 15.31 M, and
12.25 M against EC9706, Eca109, PC-3 and HCT-116, ‘respec-
a,b-Unsaturated aldehyde 21 only had moderately
l
l
l
l
l
tively’. Interestingly, the anti-tumor activities of 23 were superior
to Cisplatin against EC9706, PC-3 and HCT-116. In addition, acyla-
tion of the 1,3-amino alcohol of 23 led to the lost of its activity (23
vs 24). Selective acylation of amino group caused weaker activities
to the two esophageal carcinoma cells while it was inactive to PC-3
and HCT-116 cell lines (23 vs 25). No activity was observed for the

T. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5827–5832
5831
O
O
O
b
a
O
+
1
Br
O
O
c
COOEt
COOEt
COOEt
27
26
3
d
NOH
NOH
COOH
COOH
OH
O
COOEt
COOEt
COOEt
9
7
28
f
h
e
g
CN
CHO
CONH2
COOH
CN
COOH
COOEt
COOEt
COOEt
30
31
29
Scheme 4. Reagents and conditions: (a) EtBr, DMSO, KOH, 80 °C, 3 h (93%); (b) air, DMSO, reflux, 16 h, 70% of 3 and 20% of 27; (c) m-CPBA, DCM, rt, 48 h (99%); (d) 10% NaOH,
then HCl, (90%); (e) H2SO4, acetone, reflux (98%); (f) TsCl, NaOH, H2O, 90 °C (90%); (g) H2SO4, H2O, 2 h, reflux (98%); (h) SOCl2, rt, 2 h (84%).
Table 1
In vitro anti-tumor activities of isosteviol derivatives against three human cancer cell lines
a
a
Compd
IC50
(l
M)
Compd
IC50 (lM)
EC9706
Eca109
PC-3
HCT-116
EC9706
Eca109
PC-3
HCT-116
1
2
3
4
5
6
7
8
>100b
>100
>100
>100
52.08
>100
>100
>100
>100
91.63
24.42
56.64
97.23
19.33
>100
60.67
>100b
>100
>100
>100
>100
90.26
>100
>100
>100
>100
43.33
26.52
17.87
>100
41.32
>100
69.55
>100
>100
>100
>100
>100
87.56
>100
>100
>100
>100
>100
>100
>100
>100
25.38
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
20.13
>100
>100
>100
19b
19c
20a
20b
20c
21
22
23
24
25
26
27
28
29
30
31
Cisplatin
>100
>100
>100
>100
24.07
>100
>100
4.01
>100
21.23
>100
>100
>100
>100
>100
>100
6.12
>100
>100
72.28
90.88
45.82
>100
30.30
5.02
>100
13.28
>100
>100
>100
>100
>100
>100
4.20
>100
>100
>100
>100
20.63
40.34
>100
15.31
>100
>100
>100
>100
>100
>100
>100
>100b
21.02
>100
>100
>100
>100
14.35
>100
>100
12.25
>100
>100
>100
>100
>100
>100
>100
>100
62.03
9
10
11
12
13
14
17
18
19a
a
Inhibitory activity was assayed by exposure for 72 h.
IC50 Values greater than 100 lM were considered as inactive and omitted here.
b
amide, cyano, aldehyde, carboxyl compounds with tricyclic scaf-
fold (28–31).
oratory of Natural Medicine and Immune-engineering of Henan
Province (Henan University) for the cytotoxic activity
measurements.
In summary, a series of polyhydric, amino alcohol and tricyclic
derivatives of isosteviol were facilely synthesized and their anti-tu-
mor activities were evaluated in vitro. The results revealed that
introduction of hydroxyl and amino alcohol fragments to isosteviol
were beneficial to anticancer activities while D-ring opened com-
pounds were inactive. Esophageal carcinoma cells were more sensi-
tive to these compounds. Especially, 1,3-amino alcohol 23 exhibited
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37. Selected spectral data for compounds 14 and 23: Compound 14: white solid, FT-
IR(KBr):
m
3406, 3342, 3224, 2937, 2850, 1718, 1462, 1232, 1179, 1151, 1096,
1034, 779, 629 cmÀ1
;
1H NMR (400 MHz, CDCl3, ppm): d 4.08–4.10 (m, 2H),
3.54–3.56 (d, 1H, J = 8 Hz), 2.96–2.98 (d, 1H, J = 8.0 Hz), 2.16–2.20 (m, 3H),
1.77–1.91 (m, 3H), 1.70–1.73 (d, 1H, J = 12.0 Hz), 1.60–1.64 (m, 1H), 1.50 (m,
2H), 1.35–1.43 (m, 2H), 1.24–1.28 (m, 6H), 1.17 (s, 3H), 1.10–1.12 (m, 1H),
0.97–1.04 (m, 3H), 0.94 (s, 3H), 0.88 (s, 3H), 0.84–0.80 (m, 1H); 13C NMR
(100 MHz, CDCl3, ppm): d 177.9, 79.5, 59.9, 59.7, 58.4, 57.8, 53.2, 47.5, 43.6,
42.6, 41.3, 40.9, 38.2, 37.7, 34.3, 28.9, 26.3, 22.0, 20.6, 19.7, 14.3, 14.2; HRMS
(ESI, m/z) Calcd for C22H38NO3 [M+H]+364.2846. Found: 364.2848. Compound
23: white solid, FT-IR(KBr, cmÀ1):
m 3432, 2944, 2849, 1718, 1631, 1558, 1460,
1381, 1327, 1233, 1156, 1063, 626. 1H NMR (400 MHz, CDCl3, ppm) d 4.08–
4.10(m, 2H), 3.53–3.54(d, 1H, J = 4.0 Hz), 3.17–3.20 (d, 1H, J = 9.0 Hz), 2.68–
2.74 (t, 1H), 2.14–2.17 (d, 1H, J = 9.0), 2.04–2.08 (m, 1H), 1.91 (s, 1H), 1.69–1.81
(m, 4H), 1.55–1.65 (m, 4H), 1.32–1.42 (m, 2H), 1.24–1.27 (m, 5H), 1,15 (m, 4H),
1.04–1.04 (m, 2H), 0.95–0.98 (m, 3H), 0.90 (m, 4H), 0.76 (s, 3H). 13C NMR
(100 MHz, CDCl3) d 177.3, 85.9, 60.1, 57.7, 57.0, 53.6, 47.0, 43.6, 42.7, 41.3,
39.6, 38.1, 37.9, 35.0, 33.1, 29.7, 29.0, 24.9, 21.9, 19.3, 18.9, 14.1, 13.3. HRMS
(ESI, m/z) Calcd for C23H39NO3Na [M+Na]+ m/z 400.2828. Found:400.2828.
38. In vitro cytotoxicity study: EC9706, Eca109, PC-3 and HCT-116 cells were
cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL
penicillin, and 100 lg/mL streptomycin at 37 °C in humidified air atmosphere
of 5% CO2. Cell cytotoxicity was assessed by MTT assay. Briefly, cells were
plated into 96-well flat-bottomed plates (5 Â 103 cells/well). After 24 h
incubation at 37 °C, removed the culture medium and replaced with fresh
medium containing the studied compounds in different concentrations to the
wells, and the cells were incubated for another 72 h. Afterwards, 0.5 mg/mL
MTT in PBS was added and cells were incubated for a further 4 h. Two hundred
microliters of DMSO was added to each culture to dissolve the reduced MTT
crystals. The MTT-formazan product dissolved in DMSO was estimated by
measuring absorbance at 562 nm with a microplate reader. Then the inhibitory
percentage of each compound at various concentrations was calculated, and
the IC50 value was determined.
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