Amino acid derivatives of ligustrazine-oleanolic acid as new cytotoxic agents

Article abstract of DOI:10.3390/molecules191118215

A series of novel ligustrazine-oleanolic acid (TOA) derivatives were designed, and synthesized by conjugating amino acids to the 3-hydroxy group of TOA by ester bonds. Their cytotoxicity was evaluated on four cancer cell lines (HepG2, HT-29, Hela and BGC-823) by standard MTT assays. The ClogP values were calculated by means of computer simulation, and logP values of both 3β-glycine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6a) and TOA were determined using a shake flask-ultraviolet spectrophotometry method. It was found that 6a and the 3β-L-lysine ester-6g not only displayed good cytotoxicity (IC₅₀ < 3.5 μM) but also possessed better hydrophilicity than TOA. Moreover, 6a (IC₅₀ = 4.884 μM) had lower nephrotoxicity than both 6g (IC50 = 2.310 μM) and cisplatin (CDDP, IC50 = 3.691 μM) on MDCK cells. Combining Giemsa and DAPI staining, it was further verified that 6a could induce HepG2 apoptosis via nuclei fragmentation and had lower nephrotoxicity. In addition, the structure-activity relationships of these derivatives are briefly discussed.

Full text of DOI:10.3390/molecules191118215

Molecules 2014, 19, 18215-18231; doi:10.3390/molecules191118215
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Amino Acid Derivatives of Ligustrazine-Oleanolic Acid as New
Cytotoxic Agents
1
1
1
2
1
1
1
3
Fuhao Chu , Xin Xu , Guoliang Li , Shun Gu , Kuo Xu , Yan Gong , Bing Xu , Mina Wang ,
3
3
1,
1,
Huazheng Zhang , Yuzhong Zhang , Penglong Wang * and Haimin Lei *
1
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
E-Mails: chufhao@163.com (F.C.); xu.xin@vip.126.com (X.X.); liguoliangsx@163.com (G.L.);
xukuoworld@126.com (K.X.); gongyan90@163.com (Y.G.); weichenxubing@126.com (B.X.)
2
Key Laboratory for Neurodegenerative Diseases of Ministry of Education, Xuanwu Hospital of
Capital Medical University, Beijing 100053, China; E-Mail: gszy2003@163.com
3
School of Basic Medicine, Beijing University of Chinese Medicine, Beijing 100029, China;
E-Mails: wangmina2013@126.com (M.W.); zhanghuazheng1109@163.com (H.Z.);
zyz100102@126.com (Y.Z.)
*
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.).
External Editor: Derek J. McPhee
Received: 25 August 2014; in revised form: 27 October 2014 / Accepted: 28 October 2014 /
Published: 7 November 2014
Abstract: A series of novel ligustrazine-oleanolic acid (TOA) derivatives were designed,
and synthesized by conjugating amino acids to the 3-hydroxy group of TOA by ester
bonds. Their cytotoxicity was evaluated on four cancer cell lines (HepG2, HT-29, Hela and
BGC-823) by standard MTT assays. The ClogP values were calculated by means of
computer simulation, and logP values of both 3β-glycine ester olean-12-en-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (6a) and TOA were determined using a shake
flask-ultraviolet spectrophotometry method. It was found that 6a and the 3β-L-lysine
ester-6g not only displayed good cytotoxicity (IC50 < 3.5 μM) but also possessed better
hydrophilicity than TOA. Moreover, 6a (IC50 = 4.884 μM) had lower nephrotoxicity than
both 6g (IC50 = 2.310 μM) and cisplatin (CDDP, IC50 = 3.691 μM) on MDCK cells.
Combining Giemsa and DAPI staining, it was further verified that 6a could induce HepG2
apoptosis via nuclei fragmentation and had lower nephrotoxicity. In addition, the
structure-activity relationships of these derivatives are briefly discussed.

Molecules 2014, 19
18216
Keywords: amino acid; ligustrazine-oleanolic acid; anticancer; ClogP; low toxicity; Giemsa
and DAPI staining
1
. Introduction
Based on the principle of chemical combination, the attempt to discover lead compounds from
Traditional Chinese Medicines (TCMs) has drawn considerable attentions [1–6]. There are potential
advantages in having such agents with complementary pharmacological activities in the form of
a single chemical entity [2]. In the earlier studies in this field, several ligustrazine derivatives had been
designed, synthesized and biologically evaluated [3–6]. Thereafter, a novel anticancer lead compound
3
β-hydroxyolea-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (TOA, C38
was screened [6–11]. TOA was synthesized by conjugating the effective antitumor ingredients ligustrazine
TMP) and oleanolic acid (OA). It exhibited promising anticancer effects in vitro [6] and prevented the
H
58
O
3
N
2
, Figure 1)
(
expression of nuclear transcription factor NF-κB/p65 and COX-2 in S180 mice [7]. In addition, the
acute toxicity tests verified that the LD50 value of TOA exceeded 6.0 g/kg via gavage in mice.
However, the shortcoming of TOA’s poor hydrophilicity limited its oral bioavailability. In our
previous study, solid dispersions and microemulsion systems of TOA were developed to enhance the
bioactivity of TOA, and they displayed favorable pharmacokinetic profiles in vivo. The TOA
microemulsion formulation showed 28-fold and 4-fold higher maximum concentration (Cmax) than the
pure drug and solid dispersion, respectively [10,11].
Figure 1. The structure of the anticancer lead compound TOA.
As essential nutrients of the human body, amino acids have favorable hydrophilicity. Numerous
studies have showed that by introducing amino acid groups into the structures of insoluble drugs, the
hydrophilicity and the bioactivity could be enhanced [12–22]. In addition, some studies have indicated
that low aqueous-solubility drugs conjugated with amino acids or dipeptides could be selectively
identified and taken up by certain transporters, thus significantly improving their membrane
permeability and cytotoxicity [22,23].
In this paper, twelve different amino acids were selected for conjugation to the 3-hydroxy moiety of
TOA by ester bond formation. Their cytotoxicity was then evaluated on four cancer cell lines,
including the human hepatoma cell line HepG2, human colorectal cancer cell line HT-29, human
cervical cancer cell line Hela and human gastric cancer cell lines BGC-823, by standard thiazolyl blue
(MTT) assays. In the study, the antineoplastic drug cisplatin (CDDP) was selected as the positive

Molecules 2014, 19
18217
control. Due to its nephrotoxicity in clinical use, the Madin-Darby canine kidney cell line (MDCK) was
used to test the toxicity of the TOA-amino acid derivatives and CDDP. The ClogP values were
calculated by means of computer simulation. logP values of both 6a and TOA were determined using a
shake flask-ultraviolet spectrophotometry method. Model cellular morphological and nuclear damage
studies were carried out by Giemsa and DAPI staining, respectively. In addition, the structure-activity
relationships of the new TOA-amino acid derivatives are briefly discussed.
2
. Results and Discussion
2
.1. Chemistry
Compound 3 (TMP-Br) was synthesized by the treatment of anhydrous tetramethylpyrazine (TMP) with
N-bromosuccinimide (NBS) [24]. The product was used in the next step without further purification.
Compound 4 (TOA) was obtained through the formation of an ester bond between compound 3 and
OA after stirring for 1.5 h at room temperature under alkaline conditions in dry tetrahydrofuran (THF)
(
Scheme 1).
Scheme 1. Synthesis route to TOA.
4
Reagents and conditions: (a) Benzene, reflux, 10 h, 100.0%; (b) CCl , NBS , hν, reflux, 12 h, 70.0%; (c) OA,
THF, K CO , reflux, 1.5 h, 57.0%.
2
3
To avoid the formation of by-products, the amino acid groups were protected with t-butyloxy-
carbonyl (Boc-) or benzoyloxycarbonyl groups (Cbz-). The general synthesis of compounds 6a–6l
was conducted according to Scheme 2. Ester condensation reactions were catalyzed by
1
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine
DMAP). The removal of the Boc- groups was conducted in 3 M HCl/ethyl acetate solution, and
Cbz- groups were de-protected with H , Pd/C (or Pd(OH) /C). Before esterification with TOA, the
(
2
2
hydroxyl group of Cbz-L-serine was protected with TBDMS-Cl; the TBDMS group of the intermediate
was de-protected with TBAF to produce 6l. The structures of the new compounds that had not been
reported previously were elucidated by HRMS and NMR spectroscopy.
2
.2. Biological Activities
The cytotoxicity of TOA-amino acid derivatives was evaluated on HepG2, HT-29, Hela and
BGC-823 cancer cells using the MTT assay. In addition their toxicity evaluation was carried out on
MDCK cells. As shown in Table 1, most of the synthesized compounds exhibited preferable
cytotoxicity. Compounds 6a, 6b, 6c, 6g and 6l demonstrated much better cytotoxicity (IC50 < 8.0 μM)
than TOA on all cancer cell lines. In particular, 6a and 6g showed much better cytotoxicity (IC50 < 3.5 μM)

Molecules 2014, 19
18218
against the four cell lines than the others. Moreover, 6a (IC50 = 4.884 μM) had lower nephrotoxicity
than both 6g (IC50 = 2.310 μM) and CDDP (IC50 = 3.691 μM) on MDCK cells.
Scheme 2. Synthesis routes to TOA-amino acid derivatives.
Reagents and conditions: (a) EDCI, DAMP, r.t.12 h, 45%–90%; (b) 3 M HCl/ethyl acetate solution, r.t.1 h,
9
0%; (c) 10% Pd/C or Pd(OH)
2 2
/C, H , r.t.12 h, 96%; (d) TBDMS-Cl, imidazole, DMF, r.t.12 h; (e) TBAF,
dry THF, ice-salt bath, 0.5 h.
In addition, it was observed that introducing different types of amino acids on the 3-hydroxy group
of TOA significantly different cytotoxicity resulted. It was worth noting that cytotoxicity showed a
negative correlation with the length of amino acids’ branched carbon chain, in the order 6a > 6b > 6f >
6
e > 6d. The cytotoxicity of cyclic secondary amines was better than that of cyclic acylamino
derivatives, such as 6c > 6j. Increasing the number of amino groups might enhance the activity. For
instance, 6g containing two amino groups had strong cytotoxicity (IC50 < 2.5 μM) on both cancer and
MDCK cells.

Molecules 2014, 19
Table 1. Anti-proliferative effects and ClogP values of TOA-amino acid derivatives.
18219
IC50 Values (μM)
Compound
ClogP
HepG2
2.856
21.45
1.999
2.322
2.391
-
HT-29
Hela
9.838
8.683
3.256
7.690
6.754
-
BGC-823
MDCK
3.691
-
b
CDDP
TOA
6.112
6.819
-
-
a
-
8.125
7.433
7.835
8.590
8.763
8.750
8.479
7.343
8.831
8.346
8.117
8.895
6.860
6
a
2.498
4.770
3.213
-
3.062
4.474
4.976
-
4.884
5.125
5.034
-
6
b
6
c
6
d
6
e
5.969
4.257
2.270
8.216
10.22
5.785
11.44
4.783
9.830
5.956
2.347
9.503
16.85
5.480
-
-
-
-
6
f
-
9.522
2.481
-
9.785
2.310
8.119
15.78
12.62
10.82
3.122
6
g
2.383
-
6
h
6
i
12.61
11.47
-
16.04
10.73
10.01
4.898
6
j
6
k
6
l
4.829
5.217
The derivatives were evaluated on human hepatoma cell lines (HepG2), human colorectal cancer cell lines
HT-29), women cervical cancer cell lines (Hela) and human gastric cancer cell lines (BGC-823) using MTT
(
assay, as well as their toxicity evaluation was carried out on Madin-Darby canine kidney cell lines (MDCK).
-a meant IC50 > 25 μM, the compounds’ effects were considered to be weaker than TOA and not evaluated.
b
Data are derived from three independent experiments. - means the ClogP value was not calculated, due to
structural differences between CDDP and others.
2
.3. Computer Simulation of ClogP and Determination of logP
Lipophilicity (ClogP) is used in rational drug design as a key factor related to the cell
transmembrane transport or other biological processes [25–27]. ClogP values can either be calculated
experimentally [28–30] or predicted by means of commercially available programs [31–35]. In this
work, ClogP values of the TOA-amino acid derivatives were predicted using Sybyl-X 2.0 (Tripos,
Certara Inc., St. Louis, MO, USA) [5]. As shown in Table 1, 6a, 6b, 6g, 6j and 6l possessed lower
values compared to the ClogP value of TOA. It found that smaller amino acids, and polar or basic
amino acids could decrease the derivatives’ ClogP values. To determine the octanol-water partition
coefficient (logP), a shake flask-ultraviolet spectrophotometry method [36] was used to determine
the concentration of 6a and TOA in the water phase and organic phase at pH 6.8 with n-caprylic
alcohol-water as the simulation system. It determined that the logP values of 6a and TOA were 4.30
and 6.11, respectively. This indicated that by conjugating amino acids the logP value could be decreased
and the hydrophilicity improved, which was in accordance with previous studies [12,13,21,22].
However, the better activity connected with the increases of these amino acid conjugates’ solubility is
worthy of further study.
2.4. Changes in Cellular Morphologies [37]
Apoptosis has been considered a major mechanism of chemotherapy-induced cell death, and the
drug cytotoxicity often elicits cellular morphology changes [38], including loss of intercellular

Molecules 2014, 19
18220
contacts, membrane damage and nuclear fragmentation. These characteristic changes are usually
considered as the hallmarks of apoptosis [39].
2
.4.1. Geimsa Staining
To confirm the apoptopic morphological changes induced by 6a, HepG2 cells were treated with
, 1, 2 and 4 μM of 6a for 72 h and then Giemsa staining was performed. The morphology changes
0
observed in treated cells compared to control cells under inverted phase-contrast microscope at a
magnification of 400×, included loss of intercellular contacts, lysis of nuclei, increased cell debris and
membrane damage (marked by arrows in Figure 2). Meanwhile, we could clearly observe that the
morphological changes of HepG2 cells significantly increased with increasing concentration of 6a.
There was no complete cell morphology when cells were treated with 2 and 4 μM of 6a.
Figure 2. Morphological changes of HepG2 cells after treatment with 0, 1, 2 and 4 μM of
6
a was observed under inverted phase-contrast microscope at a magnification of 400×.
Cells showed changes in cell morphology, included loss of intercellular contacts, lysis of
nuclei, increased cell debris and membrane damage (arrows) with increasing doses of 6a
when compared to the control cells. Data were derived from three independent experiments.
To test the agents’ nephrotoxicity, MDCK cells were exposed to 2 μM of compound 6a and CDDP
for 72 h. Then Giemsa staining was performed on model cells. Changes were observed in the
CDDP- treated groups under inverted phase-contrast microscope at a magnification of 200×, including
less density, larger volume, lighter nuclear staining and cellular atypia. However, no significant
morphological changes were observed in cells treated with 6a and control cells (Figure 3). By
combining Giemsa staining, we found that 6a could induce HepG2 apoptosis and had lower
nephrotoxicity than CDDP.

Molecules 2014, 19
18221
Figure 3. Morphology of MDCK cells after treatment with 6a and CDDP was
observed under a microscope in the same multiples (200×). Data were derived from three
independent experiments.
2
.4.2. DAPI Staining
Apoptosis can be differentiated from necrosis by their characteristic nuclear changes. DAPI is a
nuclear stain which is observed as blue fluorescence when excited under fluorescence microscope [39].
In our present study, HepG2 cells were treated with 0, 1, 2 and 4 μM of 6a for 72 h and then DAPI
staining was performed. Control cells (0 μM) showed intact cell bodies with clear round nuclei, while
treated cells clearly showed condensed chromatin, nuclear fragmentation and weak fluorescence
compared to the control cells (Figure 4). Meanwhile, we could clearly observe that nuclear fragmentation
of HepG2 cells increased significantly with increasing concentration of 6a. Thus, DAPI staining
indicated that 6a could induce HepG2 apoptosis via nuclear fragmentation.
Figure 4. Nuclear fragmentation of HepG2 cells induced by compound 6a with different
doses (0, 1, 2 and 4 μM), which was observed using inverted phase-contrast microscope at
a magnification of 400× with an excitation wavelength of 470 nm. Data were derived from
three independent experiments.
3
. Experimental Section
3.1. General Information
Reactions were monitored by TLC using silica gel coated aluminum sheets (Qingdao Haiyang
1
13
Chemical Co., Qingdao, China) and visualized in UV light (254 nm). H-NMR and C-NMR assays

Molecules 2014, 19
18222
were recorded on a Bruker AVANCE 500 NMR spectrometer (Fällanden, Switzerland) and chemical
shifts are reported in form of δ (ppm). Deuterated chloroform or deuterated dimethylsulfoxide was
served as the solvent (Beijing InnoChem Science and Technology Co., Ltd., Beijing, China). HRMS
spectra were performed on High-resolution ESI mass spectrum (Solarix 9.4T, Bruker, Germany).
Samples were freeze-dried using the vacuum freeze dryer (Beijing Boyikang Co., Ltd., Beijing, China).
Melting points were measured at a rate of 5 °C/min using an X-5 micro melting point apparatus (Beijing
Tech Instrument Co., Ltd., Beijing, China). The specific rotation of the synthesized compounds was
measured using P-1020 polarimeter (Jasco, Tokyo, Japan). DAHZ-300 versatile constant temperature
bath oscillator (Taicang experimental plant, Jiangsu, China) and TU-1810 UV-Vis spectrophotometer
(
Beijing Purkinje General Instrument Co., Ltd., Beijing, China) were used in determination of the
oil-water partition coefficients. Cellular morphologies were observed using an inverted fluorescence
microscope (Olympus IX71, Tokyo, Japan). Silica-gel column chromatography was performed using
2
00–300 mesh silica gel. The yields were calculated based on the last step reaction. All solvents and
chemicals used were analytical or high-performance liquid chromatography grade.
3
2
.2. Chemistry
-(Bromomethyl)-3,5,6-Trimethylpyrazine (3)
Compound 3 was prepared according to our previously reported method [24]. The crude product,
with 70% purity, was not purified further because of its strong mucous membrane irritation.
3
β-Hydroxyolea-12-en-28-oic acid-3,5,6-trimethylpyrazine-2-methyl Ester (TOA, 4)
The crude product was obtained according to a reported method [6]. After further purification by
silica-gel column chromatography, the purity was improved to more than 95%, and the product could
be used directly for the later reactions.
3
.2.1. General Synthesis of the TOA-Amino Acid Derivatives 6a–6f
The corresponding t-butyloxycarbonyl-protected amino acids (0.38 mmol), EDCI (0.50 mmol) and
DMAP (0.025 mmol) were dissolved in dry DCM, then compound 4 (0.25 mmol) was added and the
reaction solution was stirred at room temperature overnight. The crude product was extracted with
CH
vacuum, the crude product was purified by flash chromatography with dichloromethane-methanol
20:1) as eluent. The product was stirred in ethyl acetate with 3 M hydrochloric acid for 1 h. The
2
Cl
2
. After drying the organic layer over anhydrous Na
2
SO and evaporating the solvent under
4
(
reaction solution was then evaporated with vacuum at 30 °C. The crude products were neutralized with
saturated sodium bicarbonate solution and extracted with dichloromethane. After separating by flash
chromatography with dichloromethane-methanol (20:1) as eluent, the products were lyophilized.
3
β-Glycine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6a). White solid, m.p.
2
5
1
1
96.0–97.6 °C, [α]
D
= +63.8 (c 1.0, MeOH), yield: 64%. H-NMR (CDCl
, methyl of OA), 0.90 (brs, 6H, 2×-CH
), 1.00–2.50 (23H, methine- and methylene- of OA), 2.49, 2.51, 2.55 (s,
3
) δ (ppm): H-NMR (CDCl
3
)
δ (ppm): 0.55, 0.85, 0.86, 0.89, 1.11 (s, 15H, 5×-CH
3
3
, methyl
of OA), 2.24 (brs, 2H, -NH
2

Molecules 2014, 19
each, 3H, 3×-CH , methyl of TMP), 2.86 (m, 1H, -CH=CCH-), 3.48 (s, 2H,-NH
18223
3
2
CH
2
-), 4.55 (m, 1H,
1
3
-
OCOCH-), 5.12, 5.19 (d, each, J = 10 Hz, 1H, -OCH
ppm): 15.7, 16.8, 16.9, 18.2, 23.0, 23.4, 23.6, 25.8, 27.6, 28.1, 29.7, 30.7, 32.4, 32.6, 33.1, 33.8,
6.9, 37.8, 38.1, 39.2, 41.3, 41.6, 43.7 (-CH NH ), 45.9, 47.5, 55.3, 64.9, 81.9 (-OCOCH-), 122.3
-CH=C-), 143.6 (-CH=C-), 173.4 (-CO-), 177.2 (-CO-), pyrazine ring: 20.6 (-CH
-CH ), 64.9 (-COCH
: 648.4662.
2
-), 5.24 (brs, 1H, -CH=C-). C-NMR (CDCl
3
) δ
(
3
2
2
(
(
3
), 21.4 (-CH
3
), 21.7
+
3
2
-), 145.4, 148.8, 149.2, 151.0. HRMS (ESI) m/z: 648.47410 [M+H] , calcd for
C
40
H
62
N
3
O
4
3
β-L-Alanine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6b). White solid,
2
5
1
m.p. 89.7–92.1 °C, [α]
D
= +30.5 (c 1.0, MeOH), yield: 56%. H-NMR (CDCl
, methyl of OA), 0.87 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H,
, methyl of OA), 1.00–2.50 (25H, methyl of L-alanine, methine- and methylene- of OA), 2.49,
.51, 2.55 (s, each, 3H, 3×-CH , methyl of TMP), 2.74 (brs, 2H, -NH ), 2.86 (m, 1H, -CH=CCH-),
.58 (s, 1H, -NH CH-), 4.53 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each, J = 10 Hz, 1H, -OCH -), 5.24
) δ (ppm): 15.7, 16.7, 16.8, 18.2, 23.0, 23.5, 23.7, 25.8, 27.6,
8.1, 28.2, 30.7, 32.4, 32.6, 33.1, 33.8, 36.9, 37.8, 38.0, 38.1, 39.2, 41.3, 41.6, 45.9, 46.9, 47.5, 55.3,
5.4 (-CHNH ), 81.9 (-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 173.4 (-CO-), 177.2 (-CO-),
3
) δ (ppm): 0.54, 0.89,
1
2
2
3
.11 (s, each, 3H, 3×-CH
3
3
×-CH
3
3
2
2
2
(
brs, 1H, -CH=C-). 13C-NMR (CDCl
3
2
5
2
pyrazine ring: 20.6 (-CH
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-COCH
2
-), 145.4, 148.8, 149.2, 151.0.
+
HRMS (ESI) m/z: 662.48967 [M+H] , calcd for C41
H
64
N
3
O
4
: 662.48186.
3
β-L-Proline ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6c). White solid,
2
5
1
m.p. 89.6–91.3 °C, [α]
D
= +29.0 (c 1.0, MeOH), yield: 51%. H-NMR (CDCl
, methyl of OA), 0.91 (brs, 6H, 2×-CH
.00–2.50 (26H, methylene- of L-proline, methine- and methylene- of OA), 2.49, 2.51, 2.55 (s, each,
H, 3×-CH , methyl of TMP), 2.86 (m, 1H, -CH=CCH-), 2.95, 3.10 (m, 2H, -NHCH -), 3.74 (brs, 1H,
-),
) δ (ppm): 15.7, 16.9, 18.1, 23.0, 23.4, 23.6, 23.7, 25.4, 25.8,
7.6, 28.1, 29.7, 30.5, 30.7, 32.4, 32.6, 33.1, 33.8, 36.9, 37.8, 38.1, 39.2, 41.3, 41.6, 45.9, 46.9, 47.0
-), 47.5, 55.3, 60.0 (-NHCH-), 81.9 (-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 174.7
3
) δ (ppm): 0.54, 0.86,
0
1
3
.87, 0.89, 1.11 (s, each, 3H, 5×-CH
3
3
, methyl of OA),
3
2
-NH-), 3.81 (m, 1H, -NHCH-), 4.55 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each, J = 10.0 Hz, 1H, -OCH
2
1
3
5.24 (brs, 1H, -CH=C-). C-NMR (CDCl
3
2
(-NHCH
2
(
-CO-), 177.2 (-CO-), pyrazine ring: 20.6 (-CH
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-COCH
2
-), 145.4,
+
1
48.8, 149.2, 151.0. HRMS (ESI) m/z: 688.50525[M+H] , calcd for C43
H
66
N
3
O
4
: 688.49751.
3
β-L-Leucine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6d). White solid,
2
5
1
m.p. 89.6–91.6 °C, [α]
D
= +42.0 (c 1.0, MeOH), yield: 49%. H-NMR (CDCl
, methyl of OA), 0.87 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H,
, methyl of OA), 0.96 (m, 6H, 2×-CH , methyl of L-leucine), 1.00–2.50 (27H, methine- and
, methyl of TMP), 2.86 (m, 1H,
), 4.53 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each, J = 10.0 Hz, 1H,
3
) δ (ppm): 0.54, 0.89,
1
2
.10 (s, each, 3H, 3×-CH
3
3
×-CH
3
3
methylene- of L-leucine and OA), 2.49, 2.51, 2.54 (s, each, 3H, 3×-CH
3
-CH=CCH-), 3.51 (m, 1H, -CHNH
2
1
3
-OCH
2
-), 5.24 (brs, 1H, -CH=C-). C-NMR (CDCl
3.5, 23.6, 24.8, 25.8, 27.6, 28.1, 29.7, 30.7, 32.4, 32.6, 33.1, 33.8, 36.9, 37.8, 38.1, 39.2, 41.3, 41.6,
3.7, 45.9, 46.9, 47.5, 53.3 (-CHNH ), 55.3, 81.9 (-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-),
3
) δ (ppm): 15.7, 16.8, 18.2, 21.8, 23.0, 23.1, 23.4,
2
4
2

Molecules 2014, 19
18224
1
1
73.4(-CO-), 177.2 (-CO-), pyrazine ring: 20.6 (-CH
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-COCH
2
-),
+
45.4, 148.8, 149.2, 151.0. HRMS (ESI) m/z: 704.53694 [M+H] , calcd for C44
H
70
N
3
O
4
: 704.52881.
3
β-L-Isoleucine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6e). White solid,
2
5
1
m.p. 89.8–90.7 °C, [α]
D
= +44.8 (c 1.0, MeOH), yield: 49%. H-NMR (CDCl
, methyl of OA), 0.88 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H,
, methyl of OA), 1.01 (m, 3H, -CH , methyl of L-isoleucine), 1.00–2.50 (30H, methyl-,
3
) δ (ppm): 0.55, 0.89,
1
2
.11 (s, each, 3H, 3×-CH
3
3
×-CH
3
3
methine- and methylene- of L-isoleucine, methine- and methylene- of OA), 2.49, 2.51, 2.54 (s, each,
H, 3×-CH , methyl of TMP), 2.87 (m, 1H, -CH=CCH-), 3.42 (m, 1H, -CHNH ), 4.53 (m, 1H,
OCOCH-), 5.12, 5.19 (d, each, J = 10.0 Hz, 1H, -OCH
δ (ppm): 11.7, 15.3, 15.9, 16.8, 16.9, 18.2, 23.1, 23.4, 23.6, 23.8, 24.4, 25.8, 27.6, 28.1, 30.7, 32.4,
2.6, 33.1, 33.8, 36.9, 37.7, 38.1, 38.7, 39.2, 41.3, 41.6, 45.9, 46.9, 47.5, 55.3, 59.7 (-CHNH ), 81.9
-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 173.5 (-CO-), 177.2 (-CO-), pyrazine ring: 20.6
-CH ), 21.4 (-CH ), 21.7 (-CH ), 64.9 (-COCH -), 145.4, 148.8, 149.4, 151.1. HRMS (ESI) m/z:
: 704.52881.
3
3
2
1
3
-
2
-), 5.24 (brs, 1H, -CH=C-). C-NMR (CDCl )
3
3
2
(
(
3
3
3
2
+
7
04.53668 [M+H] , calcd for C44
H
70
N
3
O
4
3
β-L-Valine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6f). White solid, m.p.
2
5
1
89.5–91.0 °C, [α]
D
= +29.3 (c 1.0, MeOH), yield: 57%. H-NMR (CDCl
, methyl of OA), 0.88 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H, 2×-CH
methyl of OA), 1.00–2.50 (31H, methyl- and methine- of L-valine, methine- and methylene- of OA),
.49, 2.51, 2.54 (s, each, 3H, 3×-CH , methyl of TMP), 2.87 (m, 1H, -CH=CCH-), 3.31 (m, 1H,
CHNH ), 4.53 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each, J = 10.0 Hz, 1H, -OCH -), 5.24 (brs, 1H,
CH=C-). C-NMR (CDCl ) δ (ppm): 15.3, 16.7, 16.8, 16.9, 18.2, 19.7, 23.1, 23.4, 23.6, 23.7, 25.8,
7.6, 28.1, 30.7, 31.7, 32.4, 32.6, 33.1, 33.8, 36.9, 37.7, 38.1, 39.2, 41.3, 41.6, 45.9, 46.9, 47.5,
5.3, 60.4(-CHNH ), 81.9 (-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 173.5(-CO-), 177.2 (-CO-),
3
) δ (ppm): 0.55, 0.89, 1.11
(
s, each, 3H, 3×-CH
3
3
3
,
2
3
-
-
2
2
1
3
3
2
5
2
pyrazine ring: 20.6 (-CH
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-COCH
2
-), 145.4, 148.8, 149.4, 151.1.
+
HRMS (ESI) m/z: 690.52101 [M+H] , calcd for C43
H
68
N
3
O
4
: 690.51316.
3
.2.2. General Synthesis of the TOA-Amino Acid Derivatives 6g–6k
The corresponding benzoyloxycarbonyl-protected amino acids (0.38 mmol), EDCI (0.50 mmol) and
DMAP (0.025 mmol) were dissolved in dry DCM, then compound 4 (0.25 mmol) was added and the
reaction solution was stirred at room temperature overnight. The crude product was extracted with
CH
vacuum, the crude product was purified by flash chromatography with dichloromethane-methanol
20:1) as eluent. The crude product and a little 10% Pd/C or Pd(OH) /C were stirred in methanol under
2
Cl
2
. After drying the organic layer over anhydrous Na
2
SO
4
and evaporating the solvent under
(
2
a hydrogen atmosphere overnight. The reaction solution was filtered and evaporated with vacuum.
After separation by flash chromatography with dichloromethane-methanol (20:1) as eluent, the product
was lyophilized.
3
β-L-Lysine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6g). White solid, m.p.
2
5
1
87.5–88.2 °C, [α]
D
= +156.0 (c 1.0, MeOH), yield: 69%. H-NMR (CDCl
3
) δ (ppm): 0.55, 0.89, 1.11
(
s, each, 3H, 3×-CH
3
, methyl of OA), 0.88 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H, 2×-CH ,
3
3

Molecules 2014, 19
18225
methyl of OA), 1.00–2.50 (32H, methylene- of L-lysine, methine- and methylene- of OA), 2.49, 2.51,
.54 (s, each, 3H, 3×-CH , methyl of TMP), 2.75 (m, 2H, -CH NH ), 2.87 (m, 1H, -CH=CCH-), 3.44
m, 1H, -CHNH ), 4.52 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each, J = 10.0 Hz, 1H, -OCH -), 5.24 (brs,
) δ (ppm): 15.3, 16.8, 16.9, 18.2, 22.9, 23.1, 23.4, 23.5, 23.6, 25.8,
7.6, 28.1, 30.7, 31.0, 32.4, 32.6, 33.1, 33.8, 34.4, 36.9, 37.7, 38.1, 39.2, 41.4, 41.6, 41.8, 45.9, 46.9,
7.5, 54.6 (-CHNH ), 55.3, 81.9 (-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 175.3 (-CO-), 177.2
2
3
2
2
(
2
2
1
3
1
2
4
H, -CH=C-). C-NMR (CDCl
3
2
(
-CO-), pyrazine ring: 20.6 (-CH
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-OCOCH
2
-), 145.4, 148.8, 149.2,
+
1
51.0. HRMS (ESI) m/z: 719.54488 [M+H] , calcd for C44
H
71
N
4
O
4
: 719.53971.
3
β-L-Phenylalanine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6h). White
2
5
1
solid, m.p. 82.4–83.8 °C, [α]
D
= +63.3 (c 1.0, MeOH), yield: 60%. H-NMR (CDCl
, methyl of OA), 1.00–2.50 (24H, methine- and
methylene- of OA), 2.49, 2.51, 2.54 (s, each, 3H, 3×-CH , methyl of TMP), 2.87 (m, 1H, -CH=CCH-),
.16 (m, 2H, -CH CHNH ), 3.76 (m, 1H, -CHNH ), 4.53 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each,
J = 10.0 Hz, 1H, -OCH -), 5.24 (brs, 1H, -CH=C-), 7.22, 7.23, 7.29, 7.30, 7.31 (m, 5H, -C ).
13C-NMR (CDCl
) δ (ppm): 15.3, 16.8, 16.9, 18.2, 23.1, 23.4, 23.5, 23.6, 25.9, 27.6, 28.1, 30.7, 32.4,
2.6, 33.1, 33.8, 36.9, 37.7, 38.1, 39.2, 41.0, 41.3, 41.7, 45.9, 46.9, 47.5, 55.3, 56.1 (-CHNH ), 81.6
-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 175.3 (-CO-), 177.2 (-CO-), benzene ring: 126.8, 128.5,
3
) δ (ppm): 0.54,
0
.79, 0.84, 0.89, 0.90, 0.91, 1.11 (s, each, 3H, 7×-CH
3
3
3
2
2
2
2
6
H
5
3
3
2
(
1
1
28.6, 129.3, 129.4, 137.2, pyrazine ring: 20.6 (-CH
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-OCOCH
2
-),
+
45.4, 148.8, 149.2, 151.0. HRMS (ESI) m/z: 738.52094 [M+H] , calcd for C47
H
68
N O
3 4
: 738.51316.
3
β-L-Sarcosine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6i). White solid,
2
5
1
m.p. 81.9–82.5 °C, [α]
D
= +48.8 (c 1.0, MeOH), yield: 55%. H-NMR (CDCl
, methyl of OA), 0.91 (brs, 6H, 2×-CH , methyl of OA), 2.45 (s,
), 1.00–2.50 (23H, methine- and methylene- of OA), 2.49, 2.51, 2.54 (s, each, 3H, 3×-CH
3
) δ (ppm): 0.55, 0.86,
0
.87, 0.89, 1.11 (s, each, 3H, 5×-CH
H, -NHCH
3
3
3
3
3
,
methyl of TMP), 2.87 (m, 1H, -CH=CCH-), 3.37 (s, 2H, -CH NH-), 4.57 (m, 1H, -OCOCH-), 5.12,
2
1
3
5
1
3
.19 (d, each, J = 10.0 Hz, 1H, -OCH
2
-), 5.24 (brs, 1H, -CH=C-). C-NMR (CDCl
3
) δ (ppm): 15.4,
6.7, 16.8, 18.3, 23.1, 23.4, 23.6, 23.6, 25.8, 27.6, 28.1, 30.7, 32.4, 32.6, 33.1, 33.9, 36.0 (-NHCH
3
),
6.9, 37.7, 38.1, 39.2, 41.3, 41.6, 45.9, 46.9, 47.5, 52.9 (-CH NH-), 55.3, 81.5 (-OCOCH-), 122.3
2
(
(
-CH=C-), 143.6 (-CH=C-), 172.0(-CO-), 177.2 (-CO-), pyrazine ring: 20.6 (-CH
-CH ), 64.9 (-OCOCH
: 662.48186.
3
), 21.4 (-CH
3
), 21.7
+
3
2
-), 145.4, 148.8, 149.2, 151.0. HRMS (ESI) m/z: 662.48967 [M+H] , calcd for
C
41
H
64
N
3
O
4
3
β-L-Pyroglutamate ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6j). White
2
5
1
solid, m.p. 124.6–125.8 °C, [α]
D
= +35.6 (c 1.0, MeOH), yield: 45%. H-NMR (CDCl
, methyl of OA), 0.88 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H,
, methyl of OA), 1.00–2.50 (27H, methylene- of L-pyroglutamate, methine- and methylene- of
, methyl of TMP), 2.87 (m, 1H, -CH=CCH-), 4.25 (m, 1H,
CHNH-), 4.57 (m, 1H, -OCOCH-), 5.12, 5.19 (d, each, J = 10.0 Hz, 1H, -OCH -), 5.24 (brs, 1H,
) δ (ppm): 15.3, 16.8, 16.8, 18.2, 23.1, 23.4, 23.5, 23.8, 25.1, 25.9, 27.7,
8.1, 29.3, 30.7, 32.4, 32.6, 33.1, 33.9, 36.9, 37.9, 38.1, 39.2, 41.3, 41.9, 45.9, 46.9, 47.5, 55.3, 55.7
-CHNH-), 82.5 (-OCOCH-), 122.3 (-CH=C-), 143.6 (-CH=C-), 171.7 (-CO-), 177.2 (-CO-), 177.7
3
) δ (ppm): 0.55,
0
2
.89, 1.11 (s, each, 3H, 3×-CH
3
3
×-CH
3
OA), 2.50, 2.52, 2.56 (s, each, 3H, 3×-CH
3
-
-
2
1
3
CH=C-). C-NMR (CDCl
3
2
(

Molecules 2014, 19
-CONH-), pyrazine ring: 20.6 (-CH
18226
(
3
), 21.4 (-CH
3
), 21.7 (-CH
3
), 64.9 (-OCOCH
2
-), 145.4, 148.8, 149.2,
+
1
50.9. HRMS (ESI) m/z: 702.48461 [M+H] , calcd for C43
H
64
N
3
O
5
: 702.47677.
3
β-L-Tryptophan ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6k). White
2
5
1
solid, m.p. 206.9–207.7 °C, [α]
D
= +39.3 (c 1.0, MeOH), yield: 43%. H-NMR (DMSO-d
, methyl of OA), 0.87 (brs, 6H, 2×-CH , methyl of
, methyl of TMP), 1.00–2.50 (24H, methine- and
methylene- of OA). 2.76 (m, 1H, -CH=CCH-), 2.96, 3.12 (m, 2H, -CH CHNH ), 3.75 (m, 1H,
), 4.35 (m, 1H, -OCOCH-), 5.09, 5.10, (d, each, J = 10.0 Hz, 1H, -OCH -), 5.11 (brs, 1H,
CH=C-), indole ring: 6.97, 7.06 (m, each, 1H), 7.14 (brs, -CHNH-), 7.34, 7.53 (d, each, J = 10.0 Hz,
6
) δ (ppm):
0.35, 0.60, 0.69, 0.81, 1.06 (s, each, 3H, 5×-CH
3
3
OA), 2.40, 2.42, 2.46 (s, each, 3H, 3×-CH
3
2
2
-CHNH
2
2
-
1
3
1
2
5
H), 10.9 (s, 1H, -NH). C-NMR (DMSO-d
5.4, 26.9, 27.4, 30.1, 30.3, 32.0, 32.1, 32.7, 33.1, 36.3, 37.1, 37.4, 38.7, 40.9, 41.2, 45.4, 46.2, 46.7,
4.4, 54.9 (-CHNH ), 80.6 (-OCOCH-), 121.8 (-CH=C-), 143.2 (-CH=C-), 173.3 (-CO-), 176.1 (-CO-),
), 20.9
-), 145.0, 148.2, 148.7, 150.6. HRMS (ESI) m/z: 777.53162
: 777.52406.
6
) δ (ppm): 14.9, 16.3, 16.4, 17.6, 22.6, 22.8, 23.0, 23.3,
2
indole ring: 109.2, 111.4, 118.2, 118.3, 120.9, 123.8, 127.1, 136.2, pyrazine ring: 20.1 (-CH
3
(
[
-CH
3
), 21.2 (-CH
+
3
), 64.2 (-OCOCH
2
M+H] , calcd for C49
H
69
N
4
O
4
3
.2.3. Synthesis of the TOA-Amino Acid Derivative 6l
Cbz-L-serine (1.0 mmol), TBDMS-Cl (1.2 mmol) and imidazole (2.0 mmol) were stirred in DMF at
room temperature overnight. The crude product was extracted with CH Cl . After drying the organic
layer over anhydrous Na SO and evaporating the solvent under vacuum, the crude product was
purified by flash chromatography with dichloromethane-methanol (20:1) as eluent. The crude product
0.50 mmol), EDCI (0.50 mmol) and DMAP (0.025 mmol) were dissolved in dry DCM, then
compound 4 (0.25 mmol) was added and the reaction solution was stirred at room temperature overnight.
The crude product was extracted with CH Cl . After drying the organic layer over anhydrous Na SO
2
2
2
4
(
2
2
2
4
and evaporating the solvent under vacuum, the crude product was purified by flash chromatography
with dichloromethane-methanol (20:1) as eluent. The crude product and 1.5 equivalents of TBAF were
stirred in THF for about 0.5 h in an ice-bath. The crude product was extracted with CH
drying the organic layer over anhydrous Na SO and evaporating the solvent under vacuum, the crude
product was purified by flash chromatography with dichloromethane-methanol (20:1) as eluent. The
crude product and a little 10% Pd/C or Pd(OH) /C were stirred in methanol under a hydrogen
atmosphere overnight. The crude product was extracted with CH Cl . After drying the organic layer
over anhydrous Na SO and evaporating the solvent under vacuum, the crude product was purified by
flash chromatography with dichloromethane-methanol (20:1) as eluent. The product was lyophilized.
2
Cl
2
. After
2
4
2
2
2
2
4
3
β-L-Serine ester olean-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (6l). White solid, m.p.
2
5
1
107.1–108.5 °C, [α]
D
= +55.5 (c 1.0, MeOH), yield: 36%. H-NMR (CDCl
), 0.87 (brs, 6H, 2×-CH , methyl of OA), 0.91 (brs, 6H, 2×-CH
.17 (s, 1H, -OH), 1.00–2.50 (25H, methine- and methylene- of OA), 2.49, 2.51, 2.54 (s, each, 3H,
×-CH , methyl of TMP), 2.71 (m, 2H, -CH OH), 2.87 (m, 1H, -CH=CCH-), 4.54 (m, 1H, -OCOCH-),
.12, 5.19 (d, each, J = 10.0 Hz, 1H, -OCH
5.7, 16.8, 16.9, 18.2, 23.0, 23.5, 23.7, 23.8, 25.8, 27.6, 28.1, 30.7, 32.4, 32.6, 33.1, 33.8, 36.9, 37.8,
3
) δ (ppm): 0.55, 0.89, 1.11
(s, each, 3H, 3×-CH
3
3
3
, methyl of OA),
2
3
5
1
3
2
1
3
2
-), 5.24 (brs, 1H, -CH=C-). C-NMR (CDCl ) δ (ppm):
3

Molecules 2014, 19
8.0, 39.2, 41.3, 41.6, 45.9, 46.9, 47.5, 55.3, 56.1 (-CHNH
18227
3
2
), 63.7 (-CH
2
OH), 81.9 (-OCOCH-), 122.3
(
-CH=C-), 143.6 (-CH=C-), 173.4 (-CO-), 177.2 (-CO-), pyrazine ring: 20.6 (-CH
-CH ), 64.9 (-OCOCH
: 678.47677.
3
), 21.4 (-CH
3
), 21.7
+
(
3
2
-), 145.4, 148.8, 149.2, 151.0. HRMS (ESI) m/z: 678.48175 [M+H] , calcd for
C
41
H
64
N
3
O
5
3
.3. Bio-Evaluation Methods
3.3.1. Cytotoxicity Evaluation
The cytotoxicity of these compounds was tested on five cells lines by the standard MTT assay. The
human cancer cells lines (HepG2, HT-29, Hela, BGC-823) and MDCK cells were provided by the
Chinese Academy of Medical Sciences Peking Union Medical College. The tumor cells were cultured in
RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)
penicillin-streptomycin (Thermo Technologies, New York, NY, USA) at 37 °C in a humidified
3
atmosphere with 5% CO
2
. The growing tumor cells at a density 3 × 10 cells/mL were exposed to
various concentrations of the tested drugs and incubated in a 96-well microtiter plate for 72 h (37 °C,
% CO ). After MTT solution (20 μL, 5 mg/mL) was added to each well, the plate was incubated for a
5
2
further 4 h. Then the media was removed. Formazan crystals were dissolved with DMSO (150 μL). After
mixing well, the absorbance was quantified at 490 nm with a BIORAD 550 spectrophotometer (Bio-rad
Life Science Development Ltd., Beijing, China). Wells containing no drugs were used to be blanks.
The IC50 values were defined as the concentration of compounds that produced a 50% reduction of
surviving cells and calculated using the Logit-method. Tumor cell growth inhibitory rate was
calculated in the following Equation (1):
%
inhibition = (1 − Sample group OD/Control group OD) × 100%
(1)
3
.3.2. Giemsa Staining [37]
HepG2 cells in logarithmic growth phase were cultured in 6-well plates for 24 h at 37 °C in a
humidified atmosphere with 5% CO . And then each group was treated with different concentrations of
2
compound 6a for 72 h. The cell culture medium was discarded. And the cells were washed twice with
PBS, kept in PBS/ethanol (1:1) for 2 min, fixated in ethanol for 10 min. After removing the ethanol,
the cells were stained with Giemsa stain for 2 min and observed under inverted phase-contrast
microscope at a magnification of 400×. MDCK cells in logarithmic growth phase were cultured in
6
2
-well plates for 24 h at 37 °C in a humidified atmosphere with 5% CO
2
. The cells were treated with
μM of compound 6a and CDDP for 72 h. The same operation was experimented as described above,
and the cells were observed under inverted phase-contrast microscope at a magnification of 200×.
3
.3.3. DAPI Staining [40,41]
HepG2 cells in logarithmic growth phase were allowed to grow in 6-well plates for 24 h at 37 °C in
a humidified atmosphere with 5% CO . And each group was treated with different concentrations of
2
compound 6a for 72 h. Cell culture medium was discarded and the cells were washed twice with PBS.
The cells were fixed with 4% paraformaldehyde (pH 7.4) for 15 min and then washed twice with PBS.

Molecules 2014, 19
18228
With an excitation wavelength of 470 nm, 4',6-diamidino-2-phenylindole (DAPI, 600 nM, Molecular
Probes/Invitrogen Life Technologies, Carlsbad, CA, USA) staining was then performed for 2 min and
nuclear fragments were observed using inverted phase-contrast microscope at a magnification of 400×.
4
. Conclusions
In summary, a series of novel TOA-amino acid derivatives were synthesized by conjugating amino
acids to the 3-hydroxy group of TOA by ester bonds. The target products were characterized by
spectroscopic data. Their cytotoxicity was evaluated on four cancer cell lines (HepG2, HT-29, Hela and
BGC-823) by the standard MTT assay, and their nephrotoxicity was tested using MDCK cells. The
ClogP values were calculated by means of computer simulation, and logP values of both 6a and TOA
were determined using a shake flask-ultraviolet spectrophotometry method. Among the active
compounds, 6a exhibited promising cytotoxicity, lower toxicity and better hydrophilicity. Combining
Giemsa and DAPI staining, it was further verified that 6a could induce HepG2 apoptosis via nuclei
fragmentation and had lower nephrotoxicity than CDDP. The results suggested that the attempt to
introduce small, polar or basic amino acids into the hydroxy group of TOA could effectively enhance
the hydrophilicity and biological activity. However, it is worthy of further study whether the better
activity of TOA-amino acid derivatives is connected with a hydrophilicity increase of the amino acid
conjugates, the positive charge, selective identification by membrane surface protein receptors or other
ways. Our completed work lays the foundation for further research on the cytotoxicity mechanism,
transmembrane transport and bioavailability of TOA-amino acid derivatives.
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (No.
8
1173519 and No.81073017), the Innovation Team Project Foundation of Beijing University of
Chinese Medicine (Lead Compound Discovering and Developing Innovation Team Project
Foundation, 2011-CXTD-15) and the Graduate Independent Topics of Beijing University of Chinese
Medicine (2014-JYBZZ-XS-118).
Author Contributions
F. Chu, X. Xu, P. Wang and H. Lei designed research. F. Chu, G. Li and K. Xu performed
chemistry experiments and analyzed the data. F. Chu, Y. Gong, M. Wang and H. Zhang performed
pharmacological experiments and analyzed the data. Y. Zhang analyzed pharmacological data and
elaborated cell morphology. B. Xu predicted the ClogP values of the compounds on Computer
Simulation. S. Gu measured specific rotation values. F. Chu and P. Wang wrote the paper. All authors
read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2014, 19
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Sample Availability: Samples of the compounds aren’t available from the authors.
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