Design, synthesis, and cytotoxic analysis of novel hederagenin–pyrazine derivatives based on partial least squares discriminant analysis

Article abstract of DOI:10.3390/ijms19102994

Hederagenin (He) is a novel triterpene template for the development of new antitumor compounds. In this study, 26 new He–pyrazine derivatives were synthetized in an attempt to develop potent antitumor agents; they were screened for in vitro cytotoxicity against tumor and non-tumor cell lines. The majority of these derivatives showed much stronger cytotoxic activity than He. Remarkably, the most potent was compound 9 (half maximal inhibitory concentration (IC₅₀) was 3.45 ± 0.59 μM), which exhibited similar antitumor activities against A549 (human non-small-cell lung cancer) as the positive drug cisplatin (DDP; IC₅₀ was 3.85 ± 0.63 μM), while it showed lower cytotoxicity on H9c2 (murine heart myoblast; IC₅₀ was 16.69 ± 0.12 μM) cell lines. Compound 9 could induce the early apoptosis and evoke cell-cycle arrest at the synthesis (S) phase of A549 cells. Impressively, we innovatively introduced the method of cluster analysis modeled as partial least squares discriminant analysis (PLS-DA) into the structure–activity relationship (SAR) evaluation, and SAR confirmed that pyrazine had a profound effect on the antitumor activity of He. The present studies highlight the importance of pyrazine derivatives of He in the discovery and development of novel antitumor agents.

Full text of DOI:10.3390/ijms19102994

International Journal of
Molecular Sciences
Article
Design, Synthesis, and Cytotoxic Analysis of Novel
Hederagenin–Pyrazine Derivatives Based on Partial
Least Squares Discriminant Analysis
Kang Fang, Xiao-Hua Zhang, Yao-Tian Han, Gao-Rong Wu, De-Sheng Cai, Nan-Nan Xue,
Wen-Bo Guo, Yu-Qin Yang, Meng Chen, Xin-Yu Zhang, Hui Wang, Tao Ma, Peng-Long Wang *
and Hai-Min Lei *
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
18364166994@163.com (K.F.); xhua_zhang@sina.com (X.-H.Z.); hanyaotiangz@163.com (Y.-T.H.);
gaorongwu09@163.com (G.-R.W.); cai1225366978@163.com (D.-S.C.); 18810612758@163.com (N.-N.X.);
wb_guo@126.com (W.-B.G.); yangyq5@163.com (Y.-Q.Y.); chenmeng_0809@163.com (M.C.);
xinyums@126.com (X.-Y.Z.); 15652387323@163.com (H.W.); mosesmatao@163.com (T.M.)
* Correspondence: wangpenglong@bucm.edu.cn (P.-L.W.); hm_lei@126.com (H.-M.L.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 August 2018; Accepted: 25 September 2018; Published: 30 September 2018
Abstract: Hederagenin (He) is a novel triterpene template for the development of new antitumor
compounds. In this study, 26 new He–pyrazine derivatives were synthetized in an attempt to
develop potent antitumor agents; they were screened for in vitro cytotoxicity against tumor and
non-tumor cell lines. The majority of these derivatives showed much stronger cytotoxic activity than
He. Remarkably, the most potent was compound
3.45 0.59 M), which exhibited similar antitumor activities against A549 (human non-small-cell
lung cancer) as the positive drug cisplatin (DDP; IC50 was 3.85 0.63 M), while it showed lower
cytotoxicity on H9c2 (murine heart myoblast; IC50 was 16.69 0.12 M) cell lines. Compound
9 (half maximal inhibitory concentration (IC50) was
±
µ
±
µ
±
µ
9
could induce the early apoptosis and evoke cell-cycle arrest at the synthesis (S) phase of A549 cells.
Impressively, we innovatively introduced the method of cluster analysis modeled as partial least
squares discriminant analysis (PLS-DA) into the structure–activity relationship (SAR) evaluation,
and SAR confirmed that pyrazine had a profound effect on the antitumor activity of He. The present
studies highlight the importance of pyrazine derivatives of He in the discovery and development of
novel antitumor agents.
Keywords: hederagenin; PLS-DA; antitumor activity; pyrazine; apoptosis
1. Introduction
Natural molecules are an indispensable source of lead compounds for drug discovery [
Pentacyclic triterpenes are widely present in medicinal plants, and they show a unique range of
pharmacological activities, including great antitumor, anti-inflammatory, and antiviral activities [ ].
They attracted the attention of scholars due to their enormous chemopreventive and antineoplastic
potential [ ]. In view of this, the structural modification of pentacyclic triterpenoids is always
a focus in drug research [ 11]. Hederagenin (He, 3b,23-dihydroxyurs-12-ene-28-oic acid; Figure 1) is
a member of the pentacyclic triterpenoid family, which is extracted mainly from the medicinal plant
ivy (Hedera helix L.) and Dipsacus asper [12 13]. A variety of pharmacological activities were ascribed
1–3].
4–6
7,8
9
–
,
to He, such as anticancer [14], anti-inflammatory [15], neuroprotective [16], and antiatherosclerosis
effects [17]. Notably, He exhibits potential antitumor activity in several cancer cell lines. He could
induce apoptotic cell death in LoVo and breast cancer cells through regulation of the mitochondrial
apoptosis pathway [18,19], as well as evoke apoptosis in cisplatin-resistant head and neck cancer
Int. J. Mol. Sci. 2018, 19, 2994; doi:10.3390/ijms19102994
www.mdpi.com/journal/ijms

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cells by inhibiting the nuclear factor erythroid-2-related factor 2/antioxidant responsive element
(Nrf2–ARE) pathway [15].
Numerous derivatization studies performed on He exhibited excellent anticancer activities.
A previous study demonstrated that He derivatives carrying a heterocyclic group at C-28 had strong
cell-growth-inhibitory activity against several cancer cell lines (half maximal inhibitory concentration
(IC50) ranging from 1.1 to 6.5 µM) [20]. Furthermore, He derivatives with aryl-1H-1,2,3-triazol-4-yl-
methyl esters at C-28 have stronger cell-growth-inhibitory activity than the amides against several cancer
cell lines [21]. The above literature studies suggested that He derivatives with heterocyclic group esters
at C-28 might exhibit excellent antitumor activities. However, the number of He derivatives that was
synthesized and investigated with respect to antitumor activity is still quite limited. Therefore, there is
a great interest in the synthesis and evaluation of novel He derivatives in order to develop more potential
antitumor drugs. Pyrazine is an N-heteroaromatic ring, and the incorporation of N-heteroaromatic rings
into organic molecules could improve their antitumor activity and aqueous solubility [10
research group proved that the introduction of ligustrazine to betulinic acid, ursolic acid, glycyrrhetinic
acid, and oleanolic acid could significantly improve their antitumor effects in vitro 24 25]. Among
them (Figure 1), compound 3 -hydroxyolea-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester
(I), 3 -hydroxyurs-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (II), and 3 -hydroxy-lup-
,22,23]. Our
[
,
β
β
β
20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (III) exhibited significant cytotoxicity
against a variety of tumor cell lines. As a consequence of the desirable properties of N-heteroaromatic
rings, N-heteroaromatic rings became very important in the design and development of new drugs.
However, to the best of our knowledge, there is no systematic literature concerning pyrazines with
different methyl numbers and positions in the aforementioned field. In this paper, this issue was
discussed preliminarily. In addition, the length of the carbon chain also had a great influence on
biological activity [26,27]. It could enrich compound diversity and provide more possibilities for the
discovery of lead drugs by introducing carbon chains.
The structure–activity relationship (SAR) evaluation of novel compounds plays a vital role in
discovering new drugs. Thus, finding and applying a rapid and trustworthy method is needed.
Partial least squares discriminant analysis (PLS-DA) is a linear classification method that combines
the discrimination power of a classification technique with the properties of partial least squares
regression. PLS-DA models could classify samples on the basis of calculating class thresholds on the
basis of the Bayes theorem (samples can be unclassified) or class probabilities (samples are assigned
to a class), and it was widely used to judge how to classify research objects [28,29]. Compared with
direct observation, the introduction of PLS-DA into the structure–activity relationship evaluation
could reflect the structure–activity law more intuitively. In this paper, we provided a new reference for
structure–activity relationship analysis by applying cluster analysis to structure–activity analysis.
In line with these fascinating features, we reported 26 He–pyrazine derivatives. Their chemical
structures were ingeniously designed, and they exhibited certain regularity. Based on the SAR,
we preliminarily clarified the law of structure–activity on the antitumor effect of He in vitro, including
pyrazine with different numbers and positions of methyl groups, with a variable length of carbon
chain as a linker, as well as a modification site. All new derivatives were screened for in vitro
cytotoxicity against tumor and non-tumor cell lines including HepG2, A549, MCF-7, H9c2, and MDCK
cells. Moreover, SAR was analyzed by PLS-DA. The result of SAR provided a reference for the
modification of He for subsequent research. Furthermore, fluorescence staining observations and flow
cytometric analyses were performed to investigate the potential mechanism of action of the most active
compound 9 in A549 cells.

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2. Result and Discussion
2.1. Chemistry
Based on the structure of He and some reports [30
,
31], we chose available sites for chemical
modification to improve antitumor activity at C-28 and C-23, and we designed 26 compounds by
combining a pyrazine ring (Figure 1). Firstly, hederagenin (He) was isolated from the himalayan teasel
root as detailed in the experimental section.
Figure 1. Structural modification of hederagenin (He).
As shown in Schemes 1–4, all the designed derivatives were synthesized [20,24]. In Scheme 1,
six kinds of chloromethyl pyrazines were produced by N-chlorosuccinimide (NCS) and benzoyl
peroxide (BPO) in carbon tetrachloride (CCl4) under light conditions with nitrogen protection, and
the yield ranged from 40–60%. Then, compounds
1–6 were obtained through a combination of He
and chloromethyl pyrazines in N,N-dimethylformamide (DMF) containing K2CO3 at 85 ◦C, and the
yield was 35–90%. As seen in Scheme 2, compound
in DMF containing K2CO3 at 85 C with 95% yield. Then, compounds
7
was obtained from He with 2-bromoethanol
13 were produced with
◦
8–
20–90% yield through a combination of He and pyrazinic acid with methyl groups using 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP)
in dry dichloromethane (DCM). In addition, pyrazinic acid with methyl groups was obtained from
pyrazine with a methyl group in H2O with KMnO4, and the yield was 70–90%. Interestingly, compounds
20–26 were obtained with 30–90% yield using a similar method to that shown in Scheme 2. In this
reaction, the hydroxyl group of 4-chlorobutanol reacted with the carboxyl group of He instead of methyl
chloride. As described in Scheme 3, the intermediate was obtained by combining pyrazinic acid with
methyl groups with 4-chlorobutanol in EDCI and DMAP in DCM, and the yield was 80–95%; then,
compounds 14
under K2CO3 in DMF at 85 ◦C. Interestingly, according to the original routine, we planned on gaining
compounds 14 19 with the synthesis conditions of Scheme 4; however, compounds 21 26 were obtained
unexpectedly. Based on the data, we redesigned the experimental steps as Scheme 3, and obtained
compounds 14 19 successfully. All reactions were carried out as detailed in the experimental section,
–19 were produced with 30–90% yield through a combination of He and the intermediate
–
–
–
which also describes the spectra for all compounds (spectra data can be found in Supplementary
Materials) [20,32–34].

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Scheme 1. Synthesis of hederagenin (He) derivatives 1–6. Reagents and conditions: (i) N-chlorosuccinimide
(NCS), benzoyl peroxide (BPO), carbon tetra◦chloride (CCl4), N2, 60 ◦C, 8 h, with 60–80% yield;
(ii) N,N-dimethylformamide (DMF), K2CO3, 85 C, 4 h, with 35–90% yield.
Scheme 2. Synthesis of He derivatives
7
–
13. Reagents and conditions: (
i
) 2-bromoethanol, DMF,
◦
◦
K2CO3, 85 C, 4 h, with 95% yield; (ii) KMnO4, H2O, 75 C, 2 h, with 60–85% yield; (iii) CH2Cl2,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine
(DMAP), room temperature (rt), 12 h, with 20–90% yield.

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Scheme 3. Synthesis of He derivatives 14–19. Reagents and conditions: (i
) KMnO4, H2O, 75 ◦C, 2 h,
with 60–85% yield; (ii) CH2Cl2, 4-chlorobutanol, EDCI, DMAP, rt, 12 h, with 75–95% yield; (iii) DMF
◦
K2CO3, 75 C, 4 h, with 30–90% yield.
Scheme 4. Synthesis of He derivatives 20
75 C, 4 h, with 54% yield; (ii) CH2Cl2, EDCI, DMAP, rt, 12 h, with 30–90% yield.
–
26. Reagents and conditions: (
i
) DMF, K2CO3, 4-chlorobutanol,
◦
2.2. Biological Screening
2.2.1. Cytotoxicity Assay
The cytotoxicity of hederagenin derivatives in vitro was evaluated on three tumor cell lines (A549:
human non-small-cell lung cancer; MCF-7: human mammary cancer; HepG2: human hepatocellular
carcinoma) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
In addition, their toxicity evaluations were tested on H9c2 (murine heart myoblast) and MDCK
(Madin–Darby canine kidney) cells. The IC50 values of these compounds are summarized in Table 1.

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Table 1. The in vitro cytotoxicity of compounds against various cell lines. A549—human non-small-
cell lung cancer; MCF-7—human mammary cancer; HepG2—human hepatocellular carcinoma;
MDCK—Madin–Darby canine kidney; H9c2—murine heart myoblast; He—hederagenin; DDP—cisplatin.
Half Maximal Inhibitory Concentration (IC50) Values (µM)
Compound
A549
MCF-7
HepG2
MDCK
H9c2
1
2
3
4
5
6
7
17.62 ± 0.37
20.55 ± 0.29
15.03 ± 0.35
17.07 ± 1.06
24.37 ± 0.37
25.52 ± 0.26
>30
16.05 ± 0.01
15.78 ± 0.22
16.83 ± 9.25
17.31 ± 0.48
>30
18.79 ± 0.04
27.23 ± 1.82
18.93 ± 0.35
25.53 ± 1.40
24.94 ± 0.06
>30
18.06 ± 0.25
26.71 ± 1.64
18.42 ± 3.76
26.12 ± 1.02
23.53 ± 0.67
24.08 ± 0.76
>30
14.73 ± 0.51
25.09 ± 0.43
>30
20.42 ± 2.24
28.46 ± 0.29
>30
>30
>30
>30
>30
8
9
23.92 ± 0.31
3.45 ± 0.59
20.93 ± 0.68
14.12 ± 0.01
10.05 ± 2.47
8.15 ± 0.17
17.60 ± 2.33
17.98 ± 0.04
24.78 ± 0.66
19.57 ± 0.22
16.17 ± 0.15
12.23 ± 0.4
>30
9.29 ± 0.99
10.40 ± 0.43
18.67 ± 0.32
21.97 ± 0.87
>30
>30
>50
3.85 ± 0.63
25.35 ± 0.09
8.73 ± 1.49
27.24 ± 2.09
27.30 ± 0.57
17.28 ± 0.58
18.03 ± 0.18
31.23 ± 0.15
19.77 ± 1.27
28.49 ± 0.28
22.88 ± 0.66
28.29 ± 0.41
27.57 ± 0.52
>30
24.98 ± 0.18
20.53 ± 3.63
21.26 ± 1.28
17.95 ± 2.80
15.63 ± 1.41
18.30 ± 1.16
>50
17.41 ± 0.75
8.71 ± 0.38
28.17 ± 0.21
14.84 ± 0.03
14.42 ± 0.03
13.24 ± 0.29
7.05 ± 0.17
6.71 ± 0.20
6.99 ± 0.63
8.15 ± 0.22
11.91 ± 0.99
9.06 ± 0.32
>30
7.99 ± 0.12
7.15 ± 0.43
7.17 ± 1.09
6.89 ± 0.21
6.42 ± 0.24
7.15 ± 0.85
>50
20.13 ± 0.30
14.11 ± 0.04
25.42 ± 0.28
18.64 ± 0.18
18.64 ± 0.71
23.22 ± 0.01
18.10 ± 0.12
17.12 ± 1.01
20.12 ± 0.06
20.93 ± 3.03
16.55 ± 0.22
16.43 ± 0.04
>30
14.85 ± 0.06
16.34 ± 1.57
19.49 ± 2.47
19.27 ± 1.47
>30
>30
>50
9.97 ± 1.12
21.66 ± 0.86
16.69 ± 0.12
19.60 ± 3.29
14.72 ± 0.16
13.16 ± 0.20
10.51 ± 0.61
15.90 ± 0.22
17.56 ± 1.85
14.99 ± 0.12
22.31 ± 0.30
>30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
He
DDP
16.50 ± 0.22
>30
16.68 ± 0.10
16.26 ± 0.07
23.35 ± 0.55
27.87 ± 0.62
>30
>30
>50
5.31 ± 0.26
5.17 ± 0.28
3.42 ± 0.68
Many of the derivatives displayed more significant growth inhibition effects than He. Remarkably,
as shown in Figure 2, the most promising was compound , which exhibited similar antitumor activities
9
against A549 as the positive drug cisplatin (DDP), while it showed lower cytotoxicity than DDP on
MDCK and H9c2 cell lines.
Figure 2. Comparison of cytotoxicity between compound 9 and cisplatin (DDP).

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2.2.2. Cluster Analysis
Principal Component Analysis (PCA)
A PCA study was designed to verify the adaptability of experimental data to the model. For the
initial overview of the dataset and the detection of trends and outliers, PCA was carried out. Analysis
showed no samples being outside the Hotelling T2 95% confidence ellipse that could influence the
analyses, and high values of explained variation and predictive ability were obtained (Table 2). All data
were analyzed using SIMACA 13.0.
Table 2. The evaluation of explained variation (R2X) and predictive ability (Q2(cum)).
Cell Types
R2X
Q2
(cum)
A549
HepG2
MCF-7
0.761
0.872
0.748
0.159
0.697
0.148
Partial Least Squares Discriminant Analysis (PLS-DA)
To further explore the structure–activity relationship, PLS-DA was performed, comparing all
designed pairs of groups of He derivatives. Analyses revealed an antitumor activity discrimination
between the different types of He derivatives compared in pairs. As Figures 3 and 4 depict, He
derivatives combined with the same pyrazine exhibited similar in vitro antitumor activities, and the
length of the carbon chain at the attachment site caused a difference in activity. In different tumor
cell models, He derivatives linked with one carbon, two carbons, and four carbons were divided into
three groups according to the difference in in vitro antitumor activity (Figure 3). As for the effect of the
structure modification site of He, He derivatives modified at C-28 and C-23 were divided into two
groups according to the difference in in vitro antitumor activity (Figure 4). It is worth noticing that
this put forward a reference point for the selection of structural modification sites for He. Through
data analysis, we found that the number of methyl groups on the pyrazines also showed a certain
degree of regularity in their effect on activity. There was no law between the position of methyl groups
on pyrazines and antitumor activity. Pyrazine derivatives with different numbers of methyl groups
were divided into six groups according to the difference in in vitro antitumor activity. Incredibly, the
analysis results of cluster analysis were consistent with the results of direct observation. For example,
compounds 21
–
26 (similar activities on HepG2, IC50 ranging from 6.42 to 7.99
µM) could be separated
from the others (IC50 > 8
µM on HepG2) by direct observation. Similarly, the results of cluster analysis
of PLS-DA supported this result (Figure 4). The cluster analysis of PLS-DA might provide us with
further directions for further analysis of He derivatives.
Figure 3. Partial least squares discriminant analysis (PLS-DA) of carbon chain length on HepG2 (human
hepatocellular carcinoma). Four carbon I—a carbon chain of four carbon atoms and pyrazine were
combined at the C-28 position; Four carbon II—a carbon chain of four carbon atoms was added at the
C-28 position, but pyrazine was added at the C-23 position.

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Figure 4. PLS-DA of combining position on HepG2. C-28—pyrazine was added at the C-28 position;
C-23—pyrazine was added at the C-23 position.
Structure–Activity Relationship Analysis
Combining the data analysis from Section 2.2.1 and Section Partial Least Squares Discriminant
Analysis (PLS-DA), we could easily find that the structural modification site of He, the length of
the carbon chain, and the type of pyrazine had an effect on the in vitro antitumor activity of the
He derivatives. In general, as observed for compounds 14–19 and 21–26, structural modification
at positions C-28 and C-23 could improve antitumor biological activity in vitro, while structural
transformation of C-23 might have more potential to enhance cytotoxicity on the same series of tumor
cells; it was confirmed that the IC50 values of compounds 21
compounds 14 19. In general, compounds with chains containing two and four carbons were superior
to those with one carbon for improving the antitumor activity of the compounds. For example,
compounds 14 19 with a chain containing four carbons generally exhibited superior antitumor activity
to others, especially on HepG2. He derivatives and 15 with 2,6-dimethylpiperazine showed better
antitumor activity in vitro than others. In addition, comparing compounds and 20 with others,
we knew that the introduction of the pyrazine structure was essential for improving the in vitro
antitumor activity of He. Notably, compound He combined with 2,6-dimethylpiperazine via a chain
containing two carbon atoms), the drug with the most potential, was also in line with this law. It is
–26 were usually lower than those of
–
–
2, 9
7
9
(
worth mentioning that compounds 14
–
19 and 21
–
26 exhibited good antitumor activity and some
HepG2
cytotoxic selectivity toward HepG2 cells in vitro (the selective inhibition (SI = IC50 MCF-7/IC50
)
value of compound 14 was 4.43). What these compounds had in common was that they were modified
by four-carbon chains. This meant that changes in the length of the carbon chain at C-28 and C-23
might alter the selectivity of compounds for different cell lines; we speculated that this result might be
caused by the combination of the compound and the tumor cell target.
2.2.3. Analyses of Apoptosis
Morphological Detection of Apoptosis Using Giemsa Staining
To characterize the morphological detection of apoptosis induced by compound
9 on A549,
the nuclear and cytoplasmic morphological changes in compound- -treated A549 were observed with
9
Giemsa staining. As shown in Figure 5, the number of A549 cells in the control group was higher than
other groups, and the cells appeared polygonal with a normal cell shape. With an increase in dose,
the phenomenon of cell shrinkage and cell disruption became more obvious. At the dose of 2
µM,
the cells had a lot of puffing and fragmentation. When the concentration was 6
no normal cell morphology.
µM, there was almost

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Figure 5. Morphological detection of apoptosis using Giemsa staining (200
×
) on A549 (human
) 2 M; ( ) 4 M;
non-small-cell lung cancer) cells treated with compound
9: (A) control group; (
B
µ
C
µ
(D) 6 µM.
40,6-Diamidino-2-phenylindole (DAPI) Staining
To characterize the effects of apoptosis inducted by compound
morphological changes in compound- -treated A549 were observed with DAPI staining. After treating
with compound for 72 h, A549 cells showed nuclear morphological changes typical of apoptosis in
9 on A549, the nuclear
9
9
a dose-dependent manner. As pictured in Figure 6, in the control group, the nucleus was intact and
evenly colored. The light-blue fluorescence was diffuse and the cells did not show the characteristics of
apoptosis. When the concentration of the drug increased, nuclear condensation, nuclear fragmentation,
and the formation of apoptotic bodies appeared. When treated at 6 µM, the number of cells decreased
drastically, the shape of the cells became irregular, and nuclear fragmentation occurred.
Detection of Apoptosis Using Annexin V Fluorescein Isothiocyanate (FITC)/Propidium Iodide
(PI) Staining
To further depict the apoptosis induced by compound
cytometry using Annexin V-FITC/PI staining. As depicted in Figure 7, with different concentrations (0,
2, 5, and 10 M) of compound treatment, the percentage of apoptotic cells (including early and late
9, apoptotic rates were analyzed by flow
µ
9
apoptosis ratios) increased from 3.7% of the control to 17.4%, 72.0%, and 91.5%, respectively. The result
indicates that compound 9 had the potential to induce the apoptosis of A549 cells.

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0
Figure 6. Morphological detection of apoptosis using 4 ,6-diamidino-2-phenylindole (DAPI) staining
(100×) on A549 cells treated with compound 9: (A) control group; (B) 2 µM; (C) 4 µM; (D) 6 µM.
Figure 7. Detection of apoptosis using Annexin V fluorescein isothiocyanate (FITC)/propidium iodide
(PI) staining on A549 cells treated with compound 9: (A) control group; (B) 2 µM; (C) 5 µM; (D) 10 µM.
2.2.4. Detection of the Effect of Compound 9 on Cell-Cycle Progression
The remarkable activity of compound against the A549 cell line prompted us to investigate
its effect on the cell cycle of A549 by flow cytometry. As shown in Figure 8, when the concentration
increased (0, 2, 5, and 10 M), the percentage of A549 cells in the synthesis (S) phase overtly increased
9
µ
(from 16.67% to 60.46%) which clearly indicated that compound
a concentration-dependent manner.
9 arrested the cells in the S phase in

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Figure 8. Cell-cycle distribution of A549 cell lines after treatment with compound
concentrations: (A) control group; (B) 2 µM; (C) 5 µM; (D) 10 µM.
9 at different
3. Discussion
A series of different esters of He were designed and synthesized. All these compounds were
screened for cytotoxic activity employing a panel of five cell lines, including HepG2, A549, MCF-7,
H9c2, and MDCK cells, using the MTT assay. From these data analyzed with PLS-DA, it was evident
that almost all derivatives exhibited higher cytotoxicity for all tested cancer cell lines compared to
He. Compound
9 was found to be the most likely drug candidate, showing IC50 values ranging
from 3.22–4.48 M. As shown by Giemsa, DAPI, and Annexin V-FITC/PI staining, it was found that
µ
compound
compound
9
9
mainly acted by inducing early apoptosis. Moreover, the present research found that
could induce cell-cycle arrest at the S phase. After exploring the structure–activity
relationship, we found that the introduction of a 2,6-dimethyl pyrazine structure with a linker
containing 2–4 carbon atoms at position 23 of He might be more effective in enhancing the antitumor
effect of the compound in vitro. Further intensive modifications at C-23 and studies are currently being
performed in our research group, and the results will be reported in due course.
4. Materials and Methods
4.1. General Aspects
Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz. Reagents were bought from
commercial suppliers without any further purification. NMR spectra were recorded on a Bruker-500 or
-400 spectrometer (Bruker, Dresden, Germany) with tetramethylsilane (TMS; TCI, Tokyo, Japan) as

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an internal standard; high-resolution mass spectra (HRMS) were acquired using a Thermo Sientific
TMLTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies, New York, NY, USA), and an X-5
micro melting point apparatus (Beijing, China).
4.1.1. Isolation of Hederagenin (He)
Dried Dipsacus asper Wall.ex Henry was extracted with 70% ethanol; three extractions at refluxed
temperature were carried out, and the solvent was kept in contact with the sample for 3–9 h at
a concentration ratio of 10 to 20 times the amount of ethanol than medicinal herbs. The combined
ethanol extracts were concentrated under reduced pressure in a rotary evaporator. Then, 7%
hydrochloric acid solution in water (20 times the residue) was added to this residue, and the mixture
was heated under reflux temperature for 4–8 h. After this hydrolysis, the residue was filtered off, and
the precipitate was obtained, washed with water to neutrality, and dried at 65 ◦C to obtain a crude
product of He. The crude product was purified by column chromatography on a silica gel with solvent
(DCM:MeOH = 70:1), and the mixture was concentrated and evaporated to dryness. After standing
overnight under 25–50 mL of anhydrous ethanol, the residue was filtered o◦ff, and He was obtained.
Hederagenin (He): yield <10%; white powder, melting point (m.p.) >300 C; HRMS (electrospray
ionization (ESI)) m/z: [M + Cl]− 507.3274, calculated for C30H48ClO4 507.3247. All spectroscopic data
(NMR and MS) were in agreement with the literature.
4.1.2. Preparation of Chloromethylpyrazine
Firstly, 2-methylpyrazine (10.63 mmol), NCS (1.42 g, 10.63 mmol), and BPO (0.26 g, 1.06 mmol)
were added to a solution of CCl4 (40 mL), and the mixture was stirred for 2 h at room temperature
under light conditions with nitrogen protection. The mixture was heated at reflux temperature for
8 h, cooled down, and then stirred in an ice bath for 1 h, before being filtered by suction, washed
with 10 mL of carbon tetrachloride, and distilled under reduced pressure to give an irritating yellow
oily liquid. The product was left untreated. Other methyl pyrazines included 2,3-dimethylpyrazine,
2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2,3,5,6-tetramethyl pyrazine,
carried out as 2-methylpyrazine [24,25].
4.1.3. Preparation of Pyrazinic Acid
Powdered potassium permanganate (3.3 g, 20.90 mmol) was dissolved in water (50 mL), then
dropped into the aqueous solution of methyl pyrazine within 20 min. The mixture was stirred for 2 h at
◦
75 C. The reaction was monitored with thin-layer chromatography (TLC) until the end of the reaction.
The mixture was cooled down, filtered, and washed with 50 mL of water; then, the filtrate was adjusted
to pH 1.5 with nitric acid, slowly warmed to 50 ◦C, and maintained for 10 min, before being cooled
down, extracted with 3
×
20 mL of ethyl acetate, and dried over anhydrous sodium sulfate. The product
was left untreated. Other methyl pyrazines included 2,3-dimethylpyrazine, 2,6-dimethylpyrazine,
2,3,5-trimethylpyrazine and 2,3,5,6-tetramethyl pyrazine, carried out as 2,6-dimethylpyrazine [35,36].
4.1.4. Preparation of Compound 7
He (1.246 g, 2.64 mmol) and K2CO3 (728 mg, 5.28 mmol) were added to a solution of
2-bromoethanol (1.649 g, 13.20 mmol) in DMF (20 mL), and the mixture was stirred for 4 h at 85 ◦C.
The reaction mixture was then diluted with ethyl acetate (30 mL), washed with water and brine
successively, dried over anhydrous sodium sulfate, and purified by column chromatography to yield
the white powder.
2-Hydroxyethyl
hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-4a(2H)-carboxylate
(compound ): yield 95% (after chromatograph with DCM/MeOH 1–2%) as a white powder; m.p.
246.6 C. H NMR (400 MHz, CDCl3) 5.28 (s, 1H, H-12), 4.05 (t, J = 5.9 Hz, 2H, H-31), 3.72 (d,
J = 10.2 Hz, 1H, H-23a), 3.63 (t, J = 7.7 Hz, 1H, H-3), 3.56 (t, J = 6.3 Hz, 2H, H-32), 3.43 (d, J = 9.9 Hz, 1H,
(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-
7
◦
1
δ

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H-23a), 2.90–2.82 (m, 1H, H-18), 1.12 (s, 3H, –CH3), 0.96–0.88 (m, 12H, 4
×
–CH3), 0.73 (s, 3H, –CH3).
13C NMR (100 MHz, CDCl3)
δ 178.2, 144.1, 122.3, 76.8, 72.1, 66.1, 61.4, 49.8, 47.6, 46.9, 45.8, 41.8, 41.8,
41.5, 39.3, 38.1, 36.9, 33.8, 33.1, 32.5, 32.5, 30.7, 27.6, 26.7, 25.9, 23.6, 23.4, 23.0, 18.5, 17.1, 15.7, 11.4.
HRMS (ESI) m/z: [M−H2O]+ 499.3790, calculated for C32H50O4 499.3782.
4.1.5. General Procedure for the Synthesis of Compounds 1–6
He (282.2 mg, 0.6 mmol), chloromethyl pyrazine (153.6 mg, about 0.6 mmol, prepared as detailed
in Section 4.1.2), an◦d K2CO3 (248.4 mg, 1.8 mmol) were added to DMF (20 mL), and the mixture was
stirred for 4 h at 85 C. The reaction mixture was then diluted with ethyl acetate (20 mL), washed with
water and brine, dried over anhydrous sodium sulfate, and purified by column chromatography to
yield the white powders.
Synthesis of Compound 1
Pyrazin-2-ylmethyl(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-
hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-4a(2H)-carboxylate
(compound
146.8 ◦C. 1H NMR (400 MHz, CDCl3)
5.31 (s, 1H, H-12), 5.22 (q, J = 13.9 Hz, 2H, H-31), 3.71 (d, J = 10.3 Hz, 1H, H-23a), 3.65–3.59 (m, 1H,
H-3), 3.41 (d, J = 10.3 Hz, 1H, H-23b), 2.91 (m, 1H, H-18), 1.12 (s, 3H, –CH3), 0.93–0.89 (m, 9H, 3
–CH3), 0.87 (s, 3H, –CH3), 0.59 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
177.3, 152.2, 144.2, 144.0,
1
): yield 40% (after chromatograph with DCM/MeOH, 1–1.5%) as a white powder; m.p.
δ
8.68 (s, 1H, –N=C–H), 8.53 (d, J = 9.0 Hz, 2H, 2 –N=C–H),
×
×
δ
143.9, 143.6, 122.8, 77.0, 72.2, 64.8, 49.9, 47.7, 47.1, 45.9, 41.9, 41.9, 41.5, 39.4, 38.2, 37.0, 33.9, 33.2, 32.6,
32.6, 30.8, 27.8, 26.9, 26.0, 23.8, 23.5, 23.2, 18.6, 17.0, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 565.4003,
calculated for C35H53N2O4 565.4000.
Synthesis of Compound 2
(6-Methylpyrazin-2-yl)methyl(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-
2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylate (compound
a white powder; m.p. 107.0 C. H NMR (400 MHz, CDCl3)
2
): yield 35% (after chromatograph with DCM/MeOH, 1–1.5%) as
◦
1
δ 8.47 (s, 1H, –N=C–H), 8.39 (s, 1H,
–N=C–H), 5.30 (s, 1H, H-12), 5.18 (q, J = 13.8 Hz, 2H, H-31), 3.71 (d, J = 10.3 Hz, 1H, H-23a), 3.62 (t,
J = 7.9 Hz, 1H, H-3), 3.41 (d, J = 10.3 Hz, 1H, H-23b), 2.91 (m, 1H, H-18), 2.56 (s, 3H, –CH3), 1.12 (s,
3H, –CH3), 0.93–0.90 (m, 9H, 3
×
–CH3), 0.88 (s, 3H, –CH3), 0.58 (s, 3H, –CH3). 13C NMR (100 MHz,
CDCl3) 177.3, 153.4, 150.9, 143.7, 143.6, 140.4, 122.7, 77.0, 72.3, 64.9, 49.9, 47.7, 47.0, 46.0, 41.9, 41.8,
δ
41.5, 39.4, 38.2, 37.0, 33.9, 33.2, 32.6, 32.6, 30.8, 27.8, 27.1, 26.9, 26.1, 23.8, 23.5, 23.2, 21.6, 18.6, 17.0, 15.8,
11.5. HRMS (ESI) m/z: [M + H]+ 579.4142, calculated for C36H55N2O4 579.4156.
Synthesis of Compound 3
(5-Methylpyrazin-2-yl)methyl(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-
2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylate (compou◦nd
3
): yield 60% (after chromatograph with DCM/MeOH, 1–1.5%) as
a white powder; m.p. 170.3 C. 1H NMR (400 MHz, CDCl3)
δ
8.45 (m, 2H, 2 –N=C–H), 5.30 (d,
×
J = 2.7 Hz, 1H, H-12), 3.71 (d, J = 10.3 Hz, 1H, H-23a), 3.41 (d, J = 10.3 Hz, 1H, H-23b), 2.90 (m, 1H,
H-18), 2.57 (d, J = 6.7 Hz, 3H, –CH3), 1.12 (s, 3H, –CH3), 0.91 (d, J = 6.6 Hz, 9H, 3 –CH3), 0.88 (s, 3H,
–CH3), 0.57 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
177.4, 153.1, 148.8, 143.9, 143.7, 142.7, 122.7,
×
δ
77.0, 72.3, 64.7, 49.9, 47.7, 47.0, 45.9, 41.9, 41.8, 41.5, 39.4, 38.2, 37.0, 33.9, 33.2, 32.6, 32.6, 30.8, 27.8, 26.9,
26.1, 23.8, 23.5, 23.2, 21.4, 18.6, 17.0, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 579.4142, calculated for
C36H55N2O4 579.4156.

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Synthesis of Compound 4
(3-Methylpyrazin-2-yl)methyl(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-
2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylate (compound
a white powder; m.p. 170.4 C. H NMR (400 MHz, CDCl3)
4
): yield 36% (after chromatograph with DCM/MeOH, 1–1.5%) as
◦
1
δ 8.41 (s, 1H, –N=C–H), 8.38 (s, 1H,
–N=C–H), 5.22 (m, 3H, H-12, H-31a, H-32b), 3.71 (d, J = 10.3 Hz, 1H, H-23a), 3.64–3.59 (m, 1H, H-3),
3.41 (d, J = 10.3 Hz, 1H, H-23b), 2.85 (m, 1H, H-18), 2.63 (s, 3H, –CH3), 1.10 (s, 3H, –CH3), 0.93–0.88 (m,
12H, 4
×
–CH3), 0.57 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
δ 177.4, 153.1, 148.9, 143.9, 143.7,
142.7, 122.7, 77.0, 72.3, 64.7, 49.9, 47.7, 47.0, 45.93, 41.9, 41.8, 41.5, 39.4, 38.2, 37.0, 33.9, 33.2, 32.6, 32.6,
30.8, 27.8, 26.9, 26.1, 23.8, 23.5, 23.2, 21.4, 18.6, 17.0, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 579.4145,
calculated for C36H55N2O4 579.4156.
Synthesis of Compound 5
(5,6-Dimethylpyrazin-2-yl)methyl(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-
2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylate (compound
1–1.5%) as a white solid; m.p. > 200 C. H NMR (400 MHz, CDCl3)
–N=C–H), 5.22–5.09 (m, 3H, H-12, H-31), 3.67 (d, J = 10.1 Hz, 1H, H-23a), 3.62–3.57 (m, 1H, H-3),
3.37 (d, J = 10.1 Hz, 1H, H-23b), 2.83 (m, 1H, H-18), 2.59–2.49 (m, 6H, 2 –CH3), 1.07 (s, 3H, –CH3),
0.90–0.85 (m, 12H, 4 177.3,
–CH3), 0.52 (d, J = 5.4 Hz, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
5): yield approximately 40% (after chromatograph with DCM/MeOH,
◦
1
δ
8.24 (d, J = 13.0 Hz, 1H,
×
×
δ
152.4, 149.9, 146.3, 143.5, 141.2, 122.6, 76.8, 72.0, 65.0, 49.9, 47.6, 47.0, 45.9, 41.8, 41.7, 41.4, 39.3, 38.2, 37.0,
33.9, 33.1, 32.6, 32.5, 30.8, 27.7, 26.6, 26.0, 23.7, 23.4, 23.1, 21.4, 21.2, 18.5, 16.9, 15.7, 11.6. HRMS (ESI)
m/z: [M + H]+ 593.4295, calculated for C37H57N2O4 593.4313.
Synthesis of Compound 6
(3,5,6-Trimethylpyrazin-2-yl)methyl(4aS,6aS,6bR,9R,10S,12aR)-10-hydroxy-9-(hydroxymethyl)-
2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylate (compound
a white powder; m.p. 127.8 ◦C. 1H NMR (400 MHz, CDCl3)
3.72 (d, J = 10.2 Hz, 1H, H-23a), 3.62 (t, J = 7.8 Hz, 1H, H-3), 3.41 (d, J = 10.3 Hz, 1H, H-23b), 2.85 (m,
1H, H-18), 2.55–2.48 (m, 9H, 3 –CH3), 1.09 (s, 3H, –CH3), 0.93–0.88 (m, 12H, 4 –CH3), 0.53 (s, 3H,
–CH3). 13C NMR (100 MHz, CDCl3)
177.3, 151.1, 149.3, 148.9, 145.5, 143.7, 122.5, 77.0, 72.3, 65.0, 50.0,
6): yield 90% (after chromatograph with DCM/MeOH, 1–1.5%) as
δ
5.24–5.08 (m, 3H, H-12, H-31a, H-31b),
×
×
δ
47.7, 47.0, 46.0, 41.9, 41.8, 41.4, 39.4, 38.2, 37.0, 34.0, 33.2, 32.6, 32.5, 30.8, 27.7, 26.9, 26.0, 23.8, 23.5, 23.2,
21.8, 21.5, 20.7, 18.6, 17.0, 15.8, 11.6. HRMS (ESI) m/z: [M + H]+ 607.4459, calculated for C38H59N2O4
607.4469.
4.1.6. General Procedure for the Synthesis of Compounds 8–13
Pyrazinic acid (0.27 mmol, prepared as detailed in Section 4.1.3), EDCI (0.35 mmol), and DMAP
(0.027 mmol) were added to dried dichloromethane (20 mL). Then, compound
7 (140 mg, 0.27 mmol)
dissolved in dichloromethane (20 mL) was dropped into the aqueous solution of methyl pyrazine
within 20 min, and the mixture was stirred for 12 h at room temperature. The reaction mixture was
then diluted with dichloromethane (20 mL), washed with water and brine, dried over anhydrous
sodium sulfate, concentrated, and purified by column chromatography to yield the white powders.
Synthesis of Compound 8
2-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)ethylpyrazine-
2-carboxylate (comp◦ound
8
): yield 20% (after chromatograph with DCM/MeOH, 1–2%) as a white
powder; m.p. 154.7 C. 1H NMR (400 MHz, CDCl3)
δ
9.29 (d, J = 9.6 Hz, 1H, –N=C–H), 8.80–8.70

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(m, 2H, 2
–CH3), 0.91 (d, J = 10.4 Hz, 9H, 3
163.9, 148.0, 146.2, 144.7, 144.3, 143.4, 122.4, 77.4, 71.9, 66.2, 61.6, 49.6, 48.0, 47.1, 45.9, 42.3, 41.9, 41.7,
39.5, 38.3, 37.0, 34.0, 33.2, 32.6, 30.8, 27.7, 26.4, 26.1, 25.8, 23.7, 23.5, 23.1, 18.8, 17.2, 15.7, 12.3. HRMS
(ESI) m/z: [M + H]+ 623.4045, calculated for C37H55N2O6 623.4055.
×
–N=C–H), 5.30 (s, 1H, H-12), 3.62 (t, J = 7.5 Hz, 1H, H-3), 2.88 (m, 1H, H-18), 0.98 (s, 3H,
–CH3), 0.77 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
178.3,
×
δ
Synthesis of Compound 9
2-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)ethyl-6-
methylpyrazine-2-carboxylate (compound
1–2%) as a white powder; m.p. 122.9 C. H NMR (400 MHz, CDCl3)
9
): yield 35% (after chromatograph with DCM/MeOH,
◦
1
δ 9.06 (d, J = 21.2 Hz, 1H,
–N=C–H), 8.63 (d, J = 5.4 Hz, 1H, –N=C–H), 5.31 (s, 1H, H-12), 4.25–3.99 (m, 2H), 2.87 (m, 1H, H-18),
2.67 (d, J = 9.0 Hz, 3H, –CH3), 1.11 (s, 3H, –CH3), 0.92 (m, 12H, 4
–CH3), 0.77 (s, 3H, –CH3). 13C NMR
(100 MHz, CDCl3) 178.3, 164.2, 154.4, 148.1 144.2, 143.0, 142.2, 122.4, 74.1, 72.0, 66.2, 61.6, 49.7, 48.0,
×
δ
47.1, 45.9, 42.3, 41.9, 41.7, 39.4, 38.3, 37.0, 33.9, 33.2, 32.6, 32.7, 30.8, 27.7, 26.3, 26.1, 25.9, 23.5, 23.1, 21.9,
18.8, 17.2, 15.7, 12.3. HRMS (ESI) m/z: [M +H]+ 637.4219, calculated for C38H57N2O6 637.4211.
Synthesis of Compound 10
2-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)ethyl-5-
methylpyrazine-2-carboxylate (compound 10): yield 55% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 192.6 ◦C. 1H NMR (400 MHz, CDCl3)
δ 9.17 (s, 1H, –N=C–H), 8.59 (s,
1H, –N=C–H), 5.22 (s, 1H, H-12), 4.68–4.59 (m, 2H, H-31), 4.46–4.31 (m, 2H, H-32), 3.70 (d, J = 10.3 Hz,
1H, H-23a), 3.61 (t, J = 7.8 Hz, 1H, H-3), 3.40 (d, J = 10.3 Hz, 1H, H-23b), 2.84 (m, 1H, H-18), 2.67 (s,
3H, –CH3), 1.08 (s, 3H), 0.87 (s, 12H, 4
×
–CH3), 0.65 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
δ
177.6, 163.9, 158.1, 145.5, 144.6, 143.6, 140.5, 122.6, 77.0, 72.2, 63.6, 62.0, 49.9, 47.7, 46.9, 45.9, 41.9, 41.8,
41.3, 39.4, 38.2, 37.0, 33.9, 33.2, 32.5, 32.5, 30.8, 27.7, 26.9, 26.0, 23.7, 23.4, 23.0, 22.1, 18.6, 17.0, 15.7, 11.5.
HRMS (ESI) m/z: [M + H]+ 637.4208, calculated for C38H57N2O6 637.4211.
Synthesis of Compound 11
2-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)ethyl-3-
methylpyrazine-2-carboxylate (compound 11): yield 35% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 146.8 ◦C. 1H NMR (400 MHz, CDCl3)
δ
8.62 (s, 1H, –N=C–H), 8.48
(s, 1H, –N=C–H), 5.31 (s, 1H, H-12), 4.59 (d, J = 10.8 Hz, 1H, H-23a), 4.04 (d, J = 10.8 Hz, 1H, H-23b),
3.67–3.58 (m, 1H, H-3), 2.87 (s, 4H, H-18, –CH3), 1.13 (s, 3H, –CH3), 0.94 (m, 12H, 4 –CH3), 0.77 (s,
3H, –CH3). 13C NMR (100 MHz, CDCl3)
178.3, 165.0, 155.7, 146.5, 144.2, 142.5, 141.7, 122.4, 74.8,
×
δ
73.7, 66.2, 61.6, 50.0, 47.9, 47.1, 45.9, 42.1, 42.0, 41.7, 39.4, 38.2, 37.0, 34.0, 33.2, 32.6, 32.6, 30.8, 27.8, 26.3,
25.9, 23.7, 23.5, 23.3, 23.2, 19.0, 17.2, 15.6, 12.36. HRMS (ESI) m/z: [M + H]+ 637.4222, calculated for
C38H57N2O6 637.4211.
Synthesis of Compound 12
2-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)ethyl-5,6-
dimethylpyrazine-2-carboxylate (compound 12): yield 25% (after chromatograph with DCM/MeOH,
◦
1
1–2%) as a white powder; m.p. 146.2 C. H NMR (400 MHz, CDCl3)
–N=C–H), 5.31 (s, 1H, H-12), 4.59 (d, J = 10.5 Hz, 1H, H-23a), 4.00 (d, J = 10.7 Hz, 1H, H-23b), 2.83 (m,
4H, H-18, –CH3), 2.58 (d, J = 6.8 Hz, 3H, –CH3), 1.13 (s, 3H, –CH3), 0.97–0.90 (m, 12H, 4 –CH3), 0.76
(s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
178.3, 165.2, 154.8, 151.0, 146.5, 144.2, 141.5, 122.4, 75.0,
73.8, 66.2, 61.6, 50.2, 47.8, 47.1, 45.9, 42.1, 42.0, 41.6, 39.4, 38.2, 37.0, 33.9, 33.2, 32.6, 30.8,27.8, 26.2, 25.9,
δ 8.41 (d, J = 60.1 Hz, 1H,
×
δ

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23.7, 23.5, 23.2, 22.7, 22.0, 21.2, 19.0, 17.2, 15.6, 12.4. HRMS (ESI) m/z: [M + H]+ 651.4375, calculated for
C39H59N2O6 651.4368.
Synthesis of Compound 13
2-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)ethyl-3,5,6-
trimethylpyrazine-2-carboxylate (compound 13): yield 90% (after chromatograph with DCM/MeOH,
◦
1–2%) as a white powder; m.p. 150.6 C. 1H NMR (400 MHz, CDCl3)
δ
5.31 (s, 1H, H-12), 4.60 (d,
J = 10.7 Hz, 1H, H-23a), 3.94 (d, J = 10.7 Hz, 1H, H-23b), 3.66–3.59 (m, 1H, H-3), 2.87 (m, 1H, H-18), 2.79
(s, 3H, –CH3), 2.57 (m, 9H, 3 –CH3), 1.13 (s, 3H, –CH3), 0.99–0.89 (m, 15H, 5 –CH3), 0.77 (s, 3H,
–CH3). 13C NMR (100 MHz, CDCl3)
178.3, 165.3, 155.4, 152.2, 149.6, 144.2, 138.1, 122.4, 75.4, 74.6,
×
×
δ
66.2, 61.6, 50.7, 47.8, 47.1, 45.9, 42.0, 42.0, 41.6, 39.4, 38.2, 36.98, 34.0, 33.20, 32.60, 30.8, 29.8, 27.8, 26.2,
26.0, 23.8, 23.5, 23.2, 22.7, 22.4, 22.3, 21.8, 19.1, 17.2, 15.5, 12.5. HRMS (ESI) m/z: [M + H]+ 665.4531,
calculated for C40H61N2O6 665.4524.
4.1.7. General Procedure for the Synthesis of Compounds 14–19
Pyrazinic acid (248 mg, about 2 mmol, prepared as detailed in Section 4.1.3), 4-chloro-1-butanol
(2.4 mmol), EDCI (2.4 mmol), and DMAP (0.2 mmol) were added into dried dichloromethane (20 mL).
The mixture was stirred for 12 h at room temperature, then diluted with dichloromethane (20 mL),
washed with water and brine, dried over anhydrous sodium sulfate, concentrated and purified by
column chromatography to yield the oily liquid. Next, He (236 mg, 0.5 mmol), K2CO3 (207 mg,
◦
1.5 mmol), and the oily liquid were added to DMF (20 mL), and the mixture was stirred for 4 h at 85 C.
The reaction mixture was then diluted with ethyl acetate (20 mL), washed with water and brine, dried
over anhydrous sodium sulfate, concentrated and purified by column chromatography to yield the
white powders.
Synthesis of Compound 14
4-(((4aS,6aS,6bR,9R,10S,12aR)-10-Gydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)butyl-pyrazine-
2-carboxylate (compound 14): yield 30% (after chromatograph with DCM/MeOH, 1–2%) as a white
powder; m.p. 127 ◦C. 1H NMR (400 MHz, CDCl3)
δ 9.31 (s, 1H, –N=C–H), 8.77 (s, 1H, –N=C–H), 8.74
(s, 1H, –N=C–H), 5.27 (s, 1H, H-12), 4.48 (t, J = 6.3 Hz, 2H, H-34), 4.08 (t, J = 5.7 Hz, 2H, H-31), 3.72 (d,
J = 10.3 Hz, 1H, H-23a), 3.62 (t, J = 7.5 Hz, 1H, H-3), 3.42 (d, J = 10.1 Hz, 1H, H-23b), 2.86 (m, 1H, H-18),
1.12 (s, 3H, –CH3), 0.93–0.88 (m, 12H, 4
×
–CH3), 0.71 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
δ
177.8, 164.0, 147.8, 146.4, 144.6, 143.9, 143.6, 122.5, 77.0, 72.3, 66.0, 63.7, 49.9, 47.7, 46.9, 46.0, 41.9, 41.9,
41.5, 39.5, 38.2, 37.0, 34.0, 33.2, 32.7, 32.6, 30.8, 27.8, 26.9, 26.0, 25.7, 25.4, 23.8, 23, 23.1, 18.6, 17.2, 15.8,
11.5. HRMS (ESI) m/z: [M + H]+ 651.4375, calculated for C39H59N2O6 651.4368.
Synthesis of Compound 15
4-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)butyl-6-
methylpyrazine-2-carboxylate (compound 15): yield 35% (after chromatograph with DCM/MeOH,
◦
1–2%) as a white powder; m.p. 128 C. 1H NMR (400 MHz, CDCl3)
δ 9.09 (s, 1H, –N=C–H), 8.63 (s, 1H,
–N=C–H), 5.27 (s, 1H, H-12), 4.47 (t, J = 6.3 Hz, 2H, H-34), 4.08 (t, J = 5.8 Hz, 2H, H-31), 3.71 (d, J = 10.2
Hz, 1H, H-23a), 3.62 (t, J = 7.5 Hz, 1H, H-3), 3.42 (d, J = 10.2 Hz, 1H, H-23b), 2.86 (m, 1H, H-18), 2.68 (s,
3H, –CH3), 1.11 (s, 3H, –CH3), 0.92–0.87 (m, 12H, 4 × –CH3), 0.71 (s, 3H, –CH3). 13C NMR (100 MHz,
CDCl3) δ 177.8, 164.3, 154.4, 147.8, 143.9, 143.2, 142.6, 122.5, 77.0, 72.3, 65.8, 63.7, 49.9, 47.7, 46.9, 46.0,
41.9, 41.9, 41.5, 39.5, 38.2, 37.0, 34.0, 33.2, 32.7, 32.6, 30.8, 27.8, 26.9, 26.0, 25.7, 25.4, 23.8, 23.5, 23.1, 21.9,
18.6, 17.2, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 665.4527, calculated for C40H61N2O6 665.4524.

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Synthesis of Compound 16
4-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)butyl-5-
methylpyrazine-2-carboxylate (compound 16): yield 26% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 166.1 ◦C. 1H NMR (400 MHz, CDCl3)
δ 9.17 (s, 1H, –N=C–H), 8.58 (s,
1H, –N=C–H), 5.27 (s, 1H, H-12), 4.46 (t, J = 6.4 Hz, 2H, H-34), 4.08 (t, J = 5.9 Hz, 2H, H-31), 3.71 (d,
J = 10.2 Hz, 1H, H-23a), 3.62 (t, J = 7.5 Hz, 1H, H-3), 3.42 (d, J = 10.2 Hz, 1H, H-23b), 2.85 (m, 1H, H-18),
2.67 (s, 3H, –CH3), 1.11 (s, 3H, –CH3), 0.92–0.88 (m, 12H, 4
×
–CH3), 0.71 (s, 3H, –CH3). 13C NMR
(100 MHz, CDCl3) 177.8, 164.3, 158.0, 145.5, 144.5, 143.9, 140.8, 122.5, 77.0, 72.3, 65.7, 63.7, 49.9, 47.7,
δ
46.9, 46.0, 41.9, 41.9, 41.5, 39.5, 38.2, 37.0, 34.0, 33.2, 32.7, 32.6, 30.8, 27.8, 26.9, 26.0, 25.7, 25.4, 23.8,
23.5, 23.1, 22.1, 18.6, 17.2, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 665.4538, calculated for C40H61N2O6
665.4524.
Synthesis of Compound 17
4-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)butyl-3-
methylpyrazine-2-carboxylate (compound 17): yield 31% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 145.2 ◦C. 1H NMR (400 MHz, CDCl3)
δ 8.61 (s, 1H, –N=C–H), 8.52 (s,
1H, –N=C–H), 5.29 (s, 1H, H-12), 4.44 (t, J = 6.5 Hz, 2H, H-34), 4.07 (t, J = 6.0 Hz, 2H, H-31), 3.71 (d,
J = 10.1 Hz, 1H, H-23a), 3.62 (t, J = 7.6 Hz, 1H, H-3), 3.42 (d, J = 10.2 Hz, 1H, H-23b), 2.84 (s, 3H, –CH3),
1.11 (s, 3H, –CH3), 0.92–0.87 (m, 12H, 4
×
–CH3), 0.71 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
δ
177.8, 165.4, 155.3, 146.0, 143.9, 143.3, 141.6, 122.5, 77.0, 72.3, 65.8, 63.7, 49.9, 47.71, 46.9, 46.0, 41.9, 41.9,
41.5, 39.5, 38.2, 37.0, 34.0, 33.2, 32.7, 32.6, 30.8, 27.8, 26.9, 26.0, 25.7, 25.4, 23.8, 23.5, 23.4, 23.1, 18.6, 17.2,
15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 665.4542, calculated for C40H61N2O6 665.4524.
Synthesis of Compound 18
4-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)butyl-5,6-
dimethylpyrazine-2-carboxylate (compound 18): yield 27% (after chromatograph with DCM/MeOH,
◦
1
1–2%) as a white powder; m.p. 134.7 C. H NMR (400 MHz, CDCl3)
δ 8.43 (d, J = 32.0 Hz, 1H,
–N=C–H), 5.27 (s, 1H, H-12), 4.43 (d, J = 6.2 Hz, 2H, H-34), 4.07 (d, J = 3.0 Hz, 2H, H-34), 3.72 (d,
J = 10.1 Hz, 1H, H-23a), 3.62 (t, J = 7.6 Hz, 1H, H-3), 3.42 (d, J = 10.2 Hz, 1H, H-23b), 2.88–2.59 (m,
7H, H-18, 2
(100 MHz, CDCl3)
×
–CH3), 1.11 (s, 3H, –CH3), 0.93–0.87 (m, 12H, 4
×
–CH3), 0.71 (s, 3H, –CH3). 13C NMR
δ
177.8, 155.9, 154.5, 145.8, 143.9, 142.8, 141.4, 122.5, 77.0, 72.3, 65.6, 63.8, 49.9, 47.7,
46.9, 46.0, 41.9, 41.9, 41.5, 39.5, 38.2, 37.0, 34.0, 33.2, 32.6, 30.8, 27.8, 26.9, 26.0, 25.7, 25.4, 23.8, 23.5, 23.1,
22.6, 21.9, 21.3, 18.6, 17.2, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 679.4682, calculated for C41H63N2O6
679.4681.
Synthesis of Compound 19
4-(((4aS,6aS,6bR,9R,10S,12aR)-10-Hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carbonyl)oxy)butyl-3,5,6-
trimethylpyrazine-2-carboxylate (compound 19): yield 90.4% (after chromatograph with DCM/MeOH,
◦
1–2%) as a white powder; m.p. 110.1 C. 1H NMR (400 MHz, CDCl3)
δ
5.27 (s, 1H, H-12), 4.42 (t, J = 6.3
Hz, 2H, H-34), 4.08 (t, J = 5.7 Hz, 2H, H-31), 3.72 (d, J = 10.3 Hz, 1H, H-23a), 3.62 (t, J = 7.5 Hz, 1H,
H-3), 3.42 (d, J = 10.2 Hz, 1H, H-23b), 2.86 (m, 1H, H-18), 2.74 (s, 3H, –CH3), 2.57 (s, 6H, 2 –CH3),
1.12 (s, 3H, –CH3), 0.92–0.87 (m, 12H, 4
–CH3), 0.71 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
×
×
δ
177.8, 166.1, 154.5, 151.1, 149.5, 143.9, 139.8, 122.5, 77.0, 72.3, 65.5, 63.8, 49.9, 47.70, 46.8, 46.0, 41.9, 41.8,
41.5, 39.4, 38.2, 37.0, 34.0, 33.2, 32.6, 32.6, 30.8, 27.8, 26.9, 26.0, 25.7, 25.5, 23.7, 23.5, 23.1, 22.7, 22.3, 21.7,
18.6, 17.2, 15.8, 11.5. HRMS (ESI) m/z: [M + H]+ 693.4849, calculated for C42H65N2O6 693.4837.

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4.1.8. Procedure for the Synthesis of Compound 20
He (1.236 g, 2.64 mmol) and K2CO3 (730 mg, 5.28 mmol) were added to a solution of
4-chlorobutanol (1.43 g, 13.20 mmol) in DMF (20 mL), and the mixture was stirred for 4h at
◦
85 C. The reaction mixture was then diluted with ethyl acetate (30 mL), washed with water and
brine successively, dried over anhydrous sodium sulfate, concentrated, and purified by column
chromatography to yield the white powder. Yield 54% (after chromatograph with DCM/MeOH, 1–2%)
◦
as a white powder, m.p. 170.2 C. 1H NMR (400 MHz, CDCl3)
δ
5.28 (s, 1H, H-12), 3.72 (d, J = 10.2 Hz,
1H, H-23a), 3.63 (t, J = 7.6 Hz, 1H, H-3), 3.42 (d, J = 10.2 Hz, 1H, H-23b), 2.89–2.80 (m, 1H, H-18), 1.12 (s,
3H, –CH3), 0.95 (s, 3H, –CH3), 0.93–0.88 (m, 9H, 3
–CH3), 0.73 (d, J = 8.5 Hz, 3H, –CH3). 13C NMR
(100 MHz, CDCl3) 177.7, 143.8, 122.4, 76.9, 72.2, 63.3, 49.8, 47.6, 46.7, 45.9, 44.5, 41.8, 41.7, 41.3, 39.3,
×
δ
38.1, 36.9, 33.9, 33.1, 32.5, 32.5, 30.7, 29.4, 27.6, 26.8, 26.1, 25.9, 23.6, 23.4, 23.0, 18.5, 17.1, 15.7, 11.4.
HRMS (ESI) m/z: [M–H2O + H]+ 545.3752, calculated for C34H54ClO3 545.3756.
4.1.9. General Procedure for the Synthesis of Compounds 21–26
Pyrazinic acid (about 0.27 mmol, prepared as detailed in Section 4.1.3), EDCI (0.35 mmol), and
DMAP (0.027 mmol) were added to dried dichloromethane (20 mL); then, compound 20 (152 mg,
0.27 mmol) dissolved in dichloromethane (20 mL) was dropped into the aqueous solution of methyl
pyrazine within 20 min, and the mixture was stirred for 12 h at room temperature. The reaction mixture
was then diluted with dichloromethane (20 mL), washed with water and brine, dried over anhydrous
sodium sulfate, concentrated, and purified by column chromatography to yield the white powders.
Synthesis of Compound 21
((3S,4R,6aR,6bS,8aS,14bR)-8a-((4-Chlorobutoxy)carbonyl)-3-hydroxy-4,6a,6b,11,11,14b-
hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-4-yl)methyl-pyrazine-
2-carboxylate (compound 21): yield 31% (after chromatograph with DCM/MeOH, 1–2%) as a white
powder; m.p. 117.1 ◦C. 1H NMR (400 MHz, CDCl3)
2H, 2
–CH3), 0.75 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
δ 9.28 (d, J = 9.3 Hz, 1H, –N=C–H), 8.85–8.59 (m,
×
–N=C–H), 5.28 (s, 1H, H-12), 2.86 (m, 1H, H-18), 1.09 (s, 3H, –CH3), 1.02–0.83 (m, 12H, 4
×
δ
177.8, 163.9, 148.0, 146.4, 146.2, 144.7, 143.9,
122.4, 74.0, 71.9, 63.5, 49.6, 48.0, 46.9, 45.9, 44.6, 42.3, 41.8, 41.5, 39.5, 38.3, 37.0, 34.0, 33.2, 32.6, 30.8, 29.5,
27.7, 26.4, 26.2, 26.1, 25.8, 23.7, 23.5, 23.1, 18.8, 17.2, 15.7, 12.3. HRMS (ESI) m/z: [M + H]+ 669.4045,
calculated for C39H58ClN2O5 669.4029.
Synthesis of Compound 22
((3S,4R,6aR,6bS,8aS,14bR)-8a-((4-Chlorobutoxy)carbonyl)-3-hydroxy-4,6a,6b,11,11,14b-
hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-4-yl)methyl
6-methylpyrazine-2-carboxylate (compound 22): yield 35% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 126.9 ◦C. 1H NMR (400 MHz, CDCl3)
δ
9.07 (s, 1H, –N=C–H), 8.63 (s,
1H, –N=C–H), 5.27 (s, 1H, H-12), 2.84 (m, 1H, H-18), 2.64 (s, 3H, –CH3), 1.09 (s, 3H, –CH3), 0.96 (s, 3H,
–CH3), 0.89 (m, 9H, 3 177.7, 164.6, 154.4,
–CH3), 0.74 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
×
δ
148.0, 143.8, 142.9, 142.2, 122.4, 74.0, 71.8, 63.4, 49.6, 47.9, 46.8, 45.9, 44.6, 42.2, 41.8, 41.5, 39.4, 38.3, 36.9,
34.0, 33.2, 32.7, 32.6, 30.8, 29.5, 27.7, 26.3, 26.2, 25.8, 23.7, 23.5, 23.1, 21.8, 18.8, 17.2, 15.6, 12.3. HRMS
(ESI) m/z: [M + H]+ 683.4185, calculated for C40H60ClN2O5 683.4185.
Synthesis of Compound 23
((3S,4R,6aR,6bS,8aS,14bR)-8a-((4-Chlorobutoxy)carbonyl)-3-hydroxy-4,6a,6b,11,11,14b-
hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-4-yl)methyl
5-methylpyrazine-2-carboxylate (compound 23): yield 40% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 175.8 ◦C. 1H NMR (400 MHz, CDCl3)
δ
9.17 (s, 1H, –N=C–H), 8.56 (s,
1H, –N=C–H), 5.29 (s, 1H, H-12), 2.87 (m, 1H, H-18), 2.66 (d, J = 5.3 Hz, 3H, –CH3), 1.10 (s, 3H, –CH3),

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0.98 (s, 3H, –CH3), 0.91 (m, 9H, 3
×
–CH3), 0.75 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
δ
177.8,
164.3, 158.2, 145.2, 144.6, 143.9, 140.5, 122.5, 74.1, 71.8, 63.5, 49.7, 48.0, 46.9, 46.0, 44.6, 42.3, 41.8, 41.5,
39.5, 38.3, 37.0, 34.0, 33.3, 32.7, 32.6, 30.8, 29.5, 27.7, 27.1, 26.3, 25.9, 23.7, 23.5, 23.1, 22.1, 18.8, 17.2, 15.7,
12.3. HRMS (ESI) m/z: [M + H]+ 683.4183, calculated for C40H60ClN2O5 683.4185.
Synthesis of Compound 24
((3S,4R,6aR,6bS,8aS,14bR)-8a-((4-Chlorobutoxy)carbonyl)-3-hydroxy-4,6a,6b,11,11,14b-
hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-4-yl)methyl
3-methylpyrazine-2-carboxylate (compound 24): yield 36% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 106.1 ◦C. 1H NMR (400 MHz, CDCl3)
δ
8.62 (s, 1H, –N=C–H), 8.49 (s,
1H, –N=C–H), 5.29 (s, 1H, H-12), 3.65–3.59 (m, 1H, H-3), 2.86 (m, 4H, H-18, –CH3), 1.12 (s, 3H, –CH3),
0.93 (m, 12H, 3 177.8, 165.0, 155.7, 146.4,
–CH3), 0.75 (s, 3H, –CH3). 13C NMR (100 MHz, CDCl3)
×
δ
143.9, 142.5, 141.7, 122.5, 74.9, 73.7, 63.5, 50.1, 47.9, 46.9, 46.0, 44.6, 42.1, 41.9, 41.5, 39.5, 38.2, 37.0, 34.0,
33.3, 32.6, 32.6, 30.8, 29.5, 27.8, 26.3, 26.3, 25.9, 23.7, 23.5, 23.3, 23.1, 19.0, 17.2, 15.6, 12.4. HRMS (ESI)
m/z: [M + H]+ 683.4175, calculated for C40H60ClN2O5 683.4185.
Synthesis of Compound 25
((3S,4R,6aR,6bS,8aS,14bR)-8a-((4-Chlorobutoxy)carbonyl)-3-hydroxy-4,6a,6b,11,11,14b-
hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-4-yl)methyl
5,6-dimethylpyrazine-2-carboxylate (compound 25): yield 36% (after chromatograph with
◦
1
DCM/MeOH, 1–2%) as a white powder; m.p. 123.6 C. H NMR (400 MHz, CDCl3)
J = 59.1 Hz, 1H, –N=C–H), 5.29 (s, 1H, H-12), 2.83 (d, J = 8.1 Hz, 3H, –CH3), 2.59 (d, J = 6.8 Hz, 3H,
–CH3), 1.12 (s, 3H, –CH3), 0.99–0.89 (m, 12H, 4
–CH3), 0.75 (s, 3H, –CH3). 13C NMR (100 MHz,
CDCl3) 177.8, 165.2, 156.4, 154.8, 146.4, 143.9, 141.5, 122.5, 75.0, 73.9, 63.5, 50.3, 47.9, 46.9, 46.0, 44.6,
δ 8.42 (d,
×
δ
42.1, 41.9, 41.5, 39.4, 38.2, 37.0, 34.0, 33.2, 32.6, 30.8, 29.5, 27.8, 26.3, 25.9, 23.7, 23.5, 23.3, 23.1, 22.0, 21.2,
19.0, 17.3, 15.6, 12.4, 12.4. HRMS (ESI) m/z: [M + H]+ 697.4343, calculated for C41H62ClN2O5 697.4342.
Synthesis of Compound 26
((3S,4R,6aR,6bS,8aS,14bR)-8a-((4-Chlorobutoxy)carbonyl)-3-hydroxy-4,6a,6b,11,11,14b-
hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-4-yl)methyl 3,5,6-
trimethylpyrazine-2-carboxylate (compound 26): yield 90% (after chromatograph with DCM/MeOH,
1–2%) as a white powder; m.p. 168.1 ◦C. 1H NMR (400 MHz, CDCl3)
H-18), 2.79 (s, 3H, –CH3), 2.55 (d, J = 6.2 Hz, 6H, 2 –CH3), 0.99–0.86 (m, 15H, 5
–CH3). 13C NMR (100 MHz, CDCl3)
177.8, 165.3, 155.4, 152.2, 149.6, 143.9, 138.1, 122.5, 75.3, 74.6, 63.5,
δ
5.28 (s, 1H, H-12), 2.86 (m, 1H,
–CH3), 0.75 (s, 3H,
×
×
δ
50.7, 47.8, 46.9, 46.0, 44.6, 42.0, 42.0, 41.5, 39.4, 38.2, 37.0, 34.0, 33.2, 33.0, 30.8, 29.5, 27.8, 27.0, 26.2, 26.0,
23.7, 23.5, 23.1, 22.7, 22.3, 21.8, 19.1, 17.2, 15.5, 12.5. HRMS (ESI) m/z: [M + H]+ 711.4497, calculated for
C42H64ClN2O5 711.4498.
4.2. Biology Evaluation
MCF-7, A549, HepG2, H9c2, and MDCK cells were obtained from the Chinese Academy of
Medical Sciences and Peking Union Medical College. All of the cell lines were maintained in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 1% (v/v) penicillin/streptomycin and 10% (v/v)
fetal bovine serum (FBS;◦Thermo Technologies, New York, NY, USA) under a humidified atmosphere
containing 5% CO2 at 37 C. The stock solutions of He derivatives were dissolved in dimethyl sulfoxide
(DMSO; Sigma, St. Louis, MO, USA) and added at various concentrations to the cell culture. Cellular
morphologies were observed using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan),
a plate reader (BIORAD 550 spectrophotometer, Bio-rad Life Science Development Ltd., Beijing, China),
and a Canton 2 flow cytometer (BD, New York, NY, USA).

Int. J. Mol. Sci. 2018, 19, 2994
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4.2.1. Antitumor Activity
The antitumor activity of these compounds were evaluated on MCF-7, A549, HepG2, MDCK,
and H9c2 cell lines using the MTT assay. Cells at a density of 3
103 cells/well were plated in
a 96-multiwell plate in DMEM containing 10% FBS for 24 h at 37 ◦C with 5% CO2. Then, cells were
treated for 72 h with the required concentrations (3.125, 6.25, 12.5, 25, or 50 M) of the compounds and
reference drugs (He and DDP) dissolved with the vehicle DMSO. After that, the culture substrate was
replaced with 200 L of DMEM containing 20 L of MTT solution at 5 mg/mL and incubated for 4 h.
The supernatant was removed and the dissolved formazans precipitated with 120 L of DMSO. Wells
×
µ
µ
µ
µ
without drugs were used as blanks. A plate reader was used to determine the absorbance at 490 nm.
The above steps were repeated three times. The IC50 values were defined as the concentration of the
compound which gave 50% growth inhibition, and they were calculated using Graph Pad Prism 5.
The inhibition rate was calculated using the following formula, where OD is the optical density:
% inhibition = 1 − (Sample group OD/Control group OD) × 100%.
4.2.2. Giemsa Staining
(1)
A549 cells in the logarithmic growth phase were seeded in 12-well plates (1.2
×
104 cells/well).
◦
After incubation for 24 h at 37 C with 5% CO2, various concentrations (0, 2, 4, or 6
µM) of compound
9
were added to the cultures, and the plate was incubated for further 72 h. Then, we discarded the
cell culture fluid, washed twice with phosphate-buffered saline (PBS), and fixed the cells with cold
methanol. The A549 cells were stained with 6% Giemsa solution for 5 min, washed with water, and
dried. The cell morphological changes were observed under an inverted microscope.
4.2.3. DAPI Staining
A549 cells in the logarithmic growth phase were seeded in 12-well plates (1.2
×
104 cells/well).
M) of compound
◦
After incubation for 24 h at 37 C with 5% CO2, various concentrations (0, 2, 4, or 6
µ
9
were added to the cultures, and the plate was incubated for further 72 h. Then, we discarded the
cell culture fluid, washed twice with PBS, and fixed the cells with 4% paraformaldehyde for 10 min.
After that, we added the appropriate amount of DAPI (1 mg/mL; Molecular Probes/Invitrogen Life
Technologies, Carlsbad, CA, USA) to cover the bottom of the board, after staining for 5 min in the dark.
Nuclear fragments were observed at 450 nm under an inverted fluorescence microscope (Olympus
IX71, Tokyo, Japan).
4.2.4. Annexin V-FITC/PI Staining
According to instructions, A549 cells were stained with an annexin-V FITC apoptosis detection
kit (Beijing BioDee Biotech. Co., Ltd., Beijing, China). A549 cells in the logarithmic growth phase
were seeded in six-well plates (4.8
various concentrations (0, 2, 5, or 10
incubated for a further 72 h. A549 cells were collected, washed twice with cold PBS, and centrifuged at
2400 rpm for 10 min. The resulting pellet was mixed with 200 L of binding buffer of the Annexin
L of FITC-labeled annexin V was added and mixed gently. After incubation at
×
104 cells/well). After incubation for 24 h at 37 ◦C with 5% CO2,
M) of compound 9 were added to the cultures, and the plate was
µ
µ
V-FITC kit; then, 5
µ
4 ◦C for 10 min in the dark, 5
µL of PI was added. Then, the cells were immediately analyzed with
a flow cytometer at 488 nm.
4.2.5. Cell Cycle
According to instructions, A549 cells were stained with the Cell Cycle and Apoptosis Analysis
Kit (Beyotime Biotechnology, Shanghai, China). A549 cells in the logarithmic growth phase were
◦
seeded in six-well plates (4.8
various concentrations (0, 2, 5, or 10
×
104 cells/well). After incubation for 24 h at 37 C with 5% CO2,
µ
M) of compound 9 were added to the cultures, and the plate was

Int. J. Mol. Sci. 2018, 19, 2994
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incubated for a further 72 h. A549 cells were collected, washed twice with cold PBS, and centrifuged
at 2400 rpm for 10 min. The resulting pellet was mixed with 1 mL of 70% ethanol, and retained for
◦
12 h at 4 C, before being centrifuged at 2400 rpm for 10 min. The supernatant was discarded, and the
resulting pellet was mixed with 1mL of cold PBS and centrifuged at 2400 rpm for 10 min. Then, 0.5 mL
of PI staining solution was added and mixed gently. After incubation at 37 ◦C for 30 min, the cells
were immediately analyzed with a flow cytometer at 488 nm.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/
2994/s1.
Author Contributions: K.F. synthesized and characterized the compounds; Y.-T.H., G.-R.W., D.-S.C., and N.-N.X.
determined cytotoxicity values; W.-B.G., Y.-Q.Y., and M.C. recorded and analyzed the experimental data. K.F.
and X.-H.Z. wrote the article under the supervision of P.-L.W. and H.-M.L., X.-Y.Z., H.W., and T.M. perfected the
article. All authors gave approval to the final version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (No. 81173519), the
Fundamental Research Funds for the Central Universities (BUCM-2019-JCRC002), the Innovation Team Project
Foundation of Beijing University of Chinese Medicine (Lead Compound Discovering and Developing Innovation
Team Project Foundation, No. 2011-CXTD-15), Beijing Key Laboratory for Basic and Development Research on
Chinese Medicine, and the young teachers’ scientific research project of Beijing University of Chinese Medicine
(No. 2015-JYB JSMS023).
Acknowledgments: This study was supported by Chinese Academy of Inspection and Quarantine, Beijing
Institute of Technology and Capital Normal University.
Conflicts of Interest: The authors declare no conflicts of interest.
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