Combination of amino acid/dipeptide with ligustrazine-betulinic acid as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2017.02.036

The lead compound TBA, 3β-Hydroxy-lup-20(29)-ene-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester, which exhibited promising antitumor activity and induced tumor cell apoptosis in various cancer cell lines, had previously been reported. Moreover, reports have revealed that the introduction of amino acid to betulinic acid could improve selective cytotoxicity as well as water solubility. Thus, a series of novel TBA amino acid and dipeptide derivatives were designed, synthesized and screened for selective cytotoxic activity against five cancer cell lines (HepG2, HT-29, Hela, BCG-823 and A549) and the not malignant cell line MDCK by standard MTT assay. Most of the tested TBA-amino acid and dipeptide analogues showed stronger anti-proliferative activity against all tested tumor cell lines than TBA. Among them, BA-25 exhibited the greatest cytotoxic activity on tumor cell lines (mean IC₅₀= 2.31 ± 0.78 μM), that was twofold than the positive drug cisplatin (DDP), while it showed lower cytotoxicity on MDCK cell line than DDP. Further cell apoptosis analyses indicated BA-25-induced apoptosis was associated with loss of mitochondrial membrane potential and increase of intracellular free Ca²⁺concentration.

Full text of DOI:10.1016/j.ejmech.2017.02.036

Accepted Manuscript
Combination of amino acid/dipeptide with ligustrazine-betulinic acid as antitumor
agents
Bing Xu, Wen-Qiang Yan, Xin Xu, Gao-Rong Wu, Chen-Ze Zhang, Yao-Tian Han, Fu-
Hao Chu, Rui Zhao, Peng-Long Wang, Hai-Min Lei
PII:
S0223-5234(17)30101-0
10.1016/j.ejmech.2017.02.036
EJMECH 9232
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 January 2017
Revised Date: 12 February 2017
Accepted Date: 13 February 2017
Please cite this article as: B. Xu, W.-Q. Yan, X. Xu, G.-R. Wu, C.-Z. Zhang, Y.-T. Han, F.-H. Chu, R.
Zhao, P.-L. Wang, H.-M. Lei, Combination of amino acid/dipeptide with ligustrazine-betulinic acid as
antitumor agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.036.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
Graphical Abstract
New series of TBA amino acid/dipeptide derivatives were synthesized and evaluated
for their antitumor activity. BA-25 was found to be the most active one.

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Original article
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Combination of Amino Acid/Dipeptide with
Ligustrazine-Betulinic Acid as Antitumor Agents
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Bing Xua, Wen-Qiang Yana, Xin Xua, Gao-Rong Wua, Chen-Ze Zhanga,
Yao-Tian Hana, Fu-Hao Chu, Rui Zhaoa, Peng-Long Wanga,*, Hai-Min Leia,*
a
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School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing
100102, China
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*To corresponding author: e-mail address: hm_lei@126.com (H. Lei),
wpl581@126.com (P. Wang)
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Abstract: The lead compound TBA, 3β-Hydroxy-lup-20(29)-ene-28-oic acid-3,
5, 6-trimethylpyrazin-2-methyl ester, which exhibited promising antitumor
activity and induced tumor cell apoptosis in various cancer cell lines, had
previously been reported. Moreover, reports have revealed that the introduction
of amino acid to betulinic acid could improve selective cytotoxicity as well as
water solubility. Thus, a series of novel TBA amino acid and dipeptide
derivatives were designed, synthesized and screened for selective cytotoxic
activity against five cancer cell lines (HepG2, HT-29, Hela, BCG-823 and A549)
and the not malignant cell line MDCK by standard MTT assay. Most of the
tested TBA-amino acid and dipeptide analogues showed stronger
anti-proliferative activity against all tested tumor cell lines than TBA. Among
them, BA-25 exhibited the greatest cytotoxic activity on tumor cell lines (mean
IC50 = 2.31+0.78 µM), that was twofold than the positive drug cisplatin (DDP),
while it showed lower cytotoxicity on MDCK cell line than DDP. Further cell
apoptosis analyses indicated BA-25-induced apoptosis was associated with loss
of mitochondrial membrane potential and increase of intracellular free Ca2+
concentration.
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Keywords: Betulinic acid, Amino acids derivatives, Dipeptide derivatives, Selective
cytotoxicity, Structure-activity relationships, Apoptosis;

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1. Introduction
Cancer is one of the major diseases which threats human life and health seriously
[1-2]. The cytotoxic agents are still one of the main clinical treatments for the
malignant tumor. However, these drugs usually have some severe side effects with
poor patients compliance, therefore the discovery of the targeted anti-tumor drugs is
of great significance [3-6]. Because of their strong selective cytotoxicity and potent
apoptosis induction activity, pentacyclic triterpenoids and their derivatives had
become the focus of scientific interest [7-10].
Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), a major effective component of
traditional Chinese medicine Rhizoma Chuanxiong (Ligusticum, chuanxiong Hort),
has been used for the treatment of cardiovascular and cerebrovascular diseases in the
clinic in China for many years [11-13]. Recently, ligustrazine was found to possess
anticancer activity in vivo and in vitro, it could induce cancer cell apoptosis and
reverse multidrug resistance in tumors [14-16]. Meanwhile, recent researches revealed
that the introduction of ligustrazine to the anti-tumor components could increase their
cytotoxicity and selectivity [17, 18]. This has stimulated interest in using ligustrazine
as the scaffold to synthesize new anticancer agents by combination it with other
anti-tumor ingredients [16, 19-21]. In our previous study, we successfully synthesized
a series of novel ligustrazine-triterpenes derivatives and observed that these
derivatives
possessed
potent
selective
cytotoxicity,
of
which,
3β-Hydroxy-lup-20(29)-ene-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester (TBA)
displayed promising selective cytotoxicity (IC50 < 5.23 µM) [17-19, 21, 22]. In
addition, reports have shown that the introduction of amino acid or dipeptide to
triterpenes could improve selective cytotoxicity as well as water solubility [8, 21,
23-25]. Based on the above, we attempted the synthesis of several TBA amino acids
and dipeptide derivatives BA-X by introducing various amino acids or dipeptides to
the C3 of TBA, in order to improve its antitumor activities and tumor targeting. All
newly synthesized compounds were fully characterized by H-NMR, 13C-NMR,
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HRMS and tested for cytotoxic activity against a panel of tumor cell lines and normal
cell line, including HepG2, HT-29, Hela, BGC823, A549 and MDCK. Meanwhile, the

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preliminary anti-tumor mechanisms of the most potent compound were also
investigated by fluorescence staining observation and flow cytometric analysis in
present study. In addition, the structure-activity relationships of these derivatives were
briefly discussed.
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2. Results and discussion
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2.1. Chemistry
The designed derivatives were prepared following the procedures in Scheme 1-4.
The compound TBA (BA-01) was prepared according to our previous study with
some modifications [19]. According to the literature procedure, the intermediate
(3,5,6-trimethylpyrazin-2-yl)methanol (4) was successively obtained [26]. Then the
intermediate 4 was further reacted with tosyl chloride (TsCl) in tetrahydrofuran (THF)
in the presence of triethylamine (TEA) and 4-dimethylaminopyridine (DMAP) to
yield the important intermediate 2-(chloromethyl)-3,5,6-trimethylpyrazine (5)
(Scheme 1). Subsequently, it underwent alkylation reaction in N,
N-dimethylformamide (DMF) with betulinic acid (BA) to afford the compound TBA
(BA-01) (Scheme 2).
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(Insert Scheme 1)
(Insert Scheme 2)
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The TBA amino acid derivatives BA-02---BA-15 (Scheme 3, Table 1) were
obtained by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
mediated esterification from the corresponding protected (N-Boc, N-Cbz) amino acids
and TBA. Deprotection was performed with trifluoroacetic acid (TFA) in dry
dichloromethane (DCM) or by treating the compounds with Pd/C (10%) in methanol
(MeOH). It was worth noting that, to avoid the formation of undesirable by-products,
the hydroxyl groups of Cbz-L-serine and Cbz-L-threonine should be protected with
tert-Butyldimethylsilyl chloride (TBDMSCl) before esterification with TBA in the
synthesis of BA-14 and BA-15. Removal of the TBDMS groups was achieved using

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1M tetrabutylammonium fluoride (TBAF) solution in THF (Scheme 3).
(Insert Scheme 3)
(Insert Table 1)
(Insert Table 1)
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In similar fashions, the TBA dipeptide derivatives BA-16---BA-27 were prepared
according to the procedures in Scheme 4. In brief, the TBA amino acids derivatives
BA-02, BA-04, BA-06 and BA-12 underwent peptide coupling reactions with the
corresponding N-Boc protected amino acids (L-Gly, L-Sar, L-Pro, L-Ala) in the
presence of EDCI, 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine
(DIPEA) in dry DCM to afford the corresponding TBA dipeptide intermediates,
which were further treated with TFA in dry DCM to give the final compounds
BA-16---BA-27 (Table 2). The structures of all target derivatives were confirmed by
spectral (1H-NMR, 13C-NMR and HRMS) analysis.
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(Insert Scheme 4)
(Insert Table 2)
2.2. Biology
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2.2.1 Cytotoxicity Assay
The in vitro antitumor activity of TBA amino acids and dipeptide derivatives was
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evaluated on five tumor cell lines (HepG2, HT-29, Hela, BCG-823, A549) using the
standard MTT assay, and their toxicity was tested using MDCK cells. The IC50 values
were summarized in Table 3. As shown in Table 3, after combination with amino acid
or dipeptide, most of the targeted compounds showed significantly improved
cytotoxicity on all tested tumor cell lines compared to the starting material TBA. The
cytotoxicity detection also revealed that most of TBA amino acids derivatives (such
as BA-02, BA-09, BA-10, BA-12 et al) and nearly all TBA dipeptide derivatives
exhibited better antiproliferative activities than the positive drug DDP, while they
showed lower cytotoxicity than DDP on MDCK cell line. Among the candidates,

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BA-25 was the most active one, which exhibited perfect antiproliferative activities
(mean IC50 = 2.31+0.78 µM) on all tested cancer cell lines. For example, the IC50
values of BA-25 for HT-29, Hela and BGC-823 (1.70+0.34 µM, 1.74+0.99 µM,
1.79+0.28 µM) is much lower than those of the positive drug cisplatin (DDP)
(4.10+1.17 µM, 5.60+0.78 µM, 4.25+0.32 µM). Meanwhile BA-25 exhibited higher
cytotoxic selective towards MDCK cells (IC50 = 10.84+0.27 µM) than DDP. It is
worth noting that, although the compound BA-10, BA-23 and BA-24 exhibited
slightly lower cytotoxicity than BA-25 on the tested cancer cells, their pronounced
cytotoxic selective (therapeutic index (TI)>10) towards MDCK cells distinguished
122 them from this series. Their IC50 value for MDCK cells was more than 40 uM (see
123 Table 3).
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From the obtained results, it was observed that TBA aliphatic amino acids
derivatives exhibited better anti-proliferative activities than those aromatic amino
acids and heterocyclic amino acids derivatives, as exemplified by BA-02, BH-04,
BA-10, BA-12 > BA-03, BA-11; Structure-activity relationship analysis among the
TBA aliphatic amino acids derivatives also revealed that the compounds with small
molecule aliphatic amino acids seemed to be more active than those with high
molecular weight aliphatic amino acids, such as BA-02, BA-04 > BA-07, BA-08. In
addition, it was observed the TBA basic amino acids derivatives seemed to be more
active than those acidic amino acids derivatives, such as BA-10 > BA-05. The result
also revealed that the TBA dipeptide derivatives were more active than the amino
acids derivatives. Potency is an important criterion for assessing leads, but it is not the
only consideration when selecting a lead compound for further optimization into a
drug [27, 28]. The selectivity, solubility and hydrophobicity were also essential to the
perfect drug candidates, because these properties were closely associated with
absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of
the compounds [29-31]. Thus the compound BA-10, BA-23, BA-24 and BA-25 was
selected for further pharmacodynamics and pharmacokinetic evaluation, including the
in vivo antitumor activity and plasma stability, in vivo pharmacokinetics, in the hope
of producing drug candidates for drug development. And the results will be reported
in due course. Meanwhile the most active compound BA-25 (mean IC50 = 2.31+0.78
µM) was selected for further analysis to study its mechanism of growth inhibition on
HepG2 cell line in this study.

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(Insert Table 3.)
2.2.2. Analyses of apoptosis
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2.2.2.1. Morphological detection of apoptosis using Giemsa staining
To investigate the mechanism of HepG2 cell death induced by BA-25, the
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morphologic changes of cells were observed by Giemsa staining. As shown in Fig. 1,
the morphology of HepG2 cells in the negative control was normal; When the cells
treated with BA-25 (1.5, 3, 6 µM) for 72 h, typical signs for apoptosis were found.
Even at low concentration of BA-25 (1.5 µM), cells had showed typical apoptosis
morphological features, such as cell shrinkage, chromatin margination and nuclear
fragmentation. With the increase of the concentration of BA-25, the characteristic of
apoptosis was more and more obvious. When the concentration of BA-25 was raised
up to 6 µM, almost all cells showed typical apoptotic morphological changes such as
nuclear condense, nuclear fragmentation. These results indicated that BA-25
significantly induced HepG2 cells apoptosis.
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(Insert Fig. 1)
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2.2.2.2. Morphological detection of apoptosis using DAPI staining
To further characterize the effects of apoptosis induction of BA-25 on HepG2, the
nucleus morphological changes in BA-25-treated HepG2 were observed by DAPI
staining. After BA-25 treatment for 72 h, HepG2 cells showed nuclear morphological
changes typical of apoptosis in a dose-dependent manner, such as nuclear
condensation, nuclear fragmentation and the formation of apoptotic bodies (Fig. 2).
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(Insert Fig. 2)
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2.2.2.3. Detection of apoptosis using Annexin V-FITC/PI staining
To further perform a comprehensive view on the apoptosis induced by BA-25,
apoptotic rates were analyzed by flow cytometry using an Annexin V-FITC/PI
staining. As shown in Fig. 3, the HepG2 cells demonstrated a remarkable response to
the apoptotic effect of BA-25 in a dose-dependent fashion. The apoptotic effect was
determined by counting the apoptosis ratios (including the early and late apoptosis
ratios). Following different concentrations (2, 3, 4 µM) of BA-25 treatment, the
apoptosis ratios increased from 6.3% of the control to 10.9%, 17.8%, 36.8%,

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respectively. These results indicated that BA-25 had the potential to induce HepG2
cell apoptosis.
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(Insert Fig. 3)
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2.2.2.4. Measurement of mitochondrial membrane potential
Disruption of mitochondrial membrane potential (∆Ψm) is one of the earliest
intracellular events that occur following the onset of apoptosis [32, 33]. To study
whether the mitochondrial events were involved in the apoptosis induced by BA-25,
∆Ψm was measured quantitatively using flow cytometry with cell-permeable cationic
dye Rhodamine 123 (Rh123). Rh123 accumulates in normal mitochondria due to its
high negative charge, and the reduction of ∆Ψm will lead to the release of Rh123 and
reduction of its fluorescence intensity [34]. As shown in Fig. 4, the fluorescent
intensity decreased in a dose-dependent manner. HepG2 cells treated with BA-25 (2
µM, 3 µM, 4 µM) for 72 h exhibited a lower fluorescent intensity (MFI: 6416, 5411,
2147) compared with untreated HepG2 cells (MFI: 26410) (Fig. 4).
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(Insert Fig. 4)
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2.2.2.5. Assessment of intracellular Free Ca2+
A sustained increase in intracellular Ca2+ concentrations is recognized as a factor for
cell death or injury [35]. The membrane-permeable Ca2+-sensitive fluorescent dye
Fluo-3AM was used to evaluate the level change of intracellular free Ca2+ in HepG2
cell line after co-culture with BA-25. The Fluo-3 AM can across the cell membrane
and be cut into Fluo-3 with the cells. The Fluo-3 can specifically combine with the
Ca2+ and has a strong fluorescence with an excitation wavelength of 488 nm [36, 37].
As shown in Fig. 5, with the increase of BA-25 concentration, intracellular free Ca2+
fluorescence increased dramatically (MFI 2743, 3760, 6353) compared with the control
group (MFI 1992). The results indicated that the increase of intracellular Ca2+ was
related with BA-25-induced HepG2 cell apoptosis (Insert Fig. 5).
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3. Conclusions
In this study, we successfully synthesized 27 novel TBA amino acid and dipeptide
derivatives by attaching different amino acids or dipeptides to the C3 of TBA. Their
cytotoxicity was determined by the standard MTT assay. The result indicated that

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most of the synthesized TBA-amino acid and dipeptide analogues showed
significantly improved selective cytotoxicity against all tested tumor cell lines
compared to TBA. From the obtained result, we also observed that the compounds
with small molecule aliphatic amino acids seemed to be more active than those with
high molecular weight aliphatic amino acids, such as BA-02, BA-04 > BA-07, BA-08;
The TBA dipeptide derivatives were more active than the amino acids derivatives.
Among all the new derivatives, compound BA-25 showed the greatest cytotoxicity on
tumor cell lines (IC50 = 2.31+0.78 µM), that was twofold than the positive drug DDP,
while it showed lower cytotoxicity on MDCK cell line than DDP. Further anti-tumor
mechanistic investigation showed that BA-25-induced apoptosis in HepG2 cells was
involved loss of the mitochondria membrane potential and increase of intracellular
free Ca2+ concentration. In addition, although the compound BA-10, BA-23 and
BA-24 exhibited slightly lower cytotoxicity than BA-25 on the tested cancer cells,
their pronounced cytotoxic selective towards MDCK cells distinguished them from
this series. And the compound BA-10, BA-23, BA-24 and BA-25 was selected for
further pharmacodynamics and pharmacokinetic evaluation, including the in vivo
antitumor activity and plasma stability, in vivo pharmacokinetics, in the hope of
producing drug candidates for drug development. These results suggested that the
attempt to discover high selectivity anti-tumor lead compounds by introduction of
amino acid or oligopeptides to cytotoxic agents was viable.
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4. Experimental Section
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4.1. Chemistry
Reagents were bought from commercial suppliers without any further purification.
Melting points were measured at a rate of 5 °C/min using an X-5 micro melting point
apparatus (Beijing, China) and were not corrected. Reactions were monitored by TLC
using silica gel coated aluminum sheets (Qingdao Haiyang Chemical Co., Qingdao,
China). The specific rotation was measured using an Autopol I (Rudolph Instruments)
automatic polarimeter at 25 °C in MeOH. NMR spectra were recorded on a BRUKER
AVANCE 500 NMR spectrometer (Fällanden, Switzerland) with tetramethylsilane
(TMS) as an internal standard; chemical shifts δ were given in ppm and coupling

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constants J in Hz. HR-MS were acquired using a Thermo Sientific TM LTQ Orbitrap
XL hybrid FTMS instrument (Thermo Technologies, New York, NY, USA). Cellular
morphologies were observed using an inverted fluorescence microscope (Olympus
IX71, Tokyo, Japan).
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(3,5,6-trimethylpyrazin-2-yl)methanol (4). Compound 4 was prepared according to
the method described by Deng et al. [26].
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2-(chloromethyl)-3,5,6-trimethylpyrazine (5). To a solution of the important
intermediate (3,5,6-trimethylpyrazin-2-yl)methanol (4) (10mmol) in dry
tetrahydrofuran (20 mL), TsCl (13mmol), TEA (20mmol) and DMAP (2mmol) were
added. The mixture was allowed to stir at room temperature for 12 h. Then the
solution was evaporated under vacuum and washed with brine. After drying the
organic layer over anhydrous Na2SO4 and evaporating the solvent under vacuum, the
crude product was purified by flash chromatography (silica gel, petroleum ether:
acetone = 10:1).
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3β-Hydroxy-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester (TBA).
Compound 5 (5.0 mmoL) and betulinic acid (5.0 mmoL) were dissolved in 25 mL dry
DMF, then K2CO3 (5.0 mmoL) was added and the mixture was at 25 °C for 12 h.
Then the reaction mixture was poured into ice-water and the crude product was
extracted with ethyl acetate. After drying the organic layer over anhydrous Na2SO4
and evaporating the solvent under vacuum, the crude product was purified by flash
chromatography (silica gel, petroleum ether: acetone = 10:1). White powder, yield
1
54.1%, mp: 184.6–185.4 °C, [α]D = +16 (c 0.50, MeOH); H-NMR (CDCl3) (ppm):
0.78, 0.80, 0.82, 0.96, 0.98, 1.67 (s, 3H, 30-CH3 of BA), 2.51 (s, 3H, -CH3), 2.53 (s,
3H, -CH3), 2.57 (s, 3H, -CH3), 3.02 (m, 1H), 3.19(m, 1H), 4.61, 4.74 (each, brs, 1H,
13
=CH2), 5.20, 5.23 (each, d, J = 12.5 Hz, 1H, -CH2); C-NMR (CDCl3) (ppm): 14.7,
15.4, 15.9, 16.1, 18.3, 19.4, 20.4 (-CH3), 20.9, 21.4 (-CH3), 21.6 (-CH3), 25.5, 27.4,
28.0, 29.7, 30.6, 32.1, 34.4, 36.9, 37.2, 38.1, 38.7, 38.9, 40.7, 42.4, 46.9, 49.5, 50.6,
55.4, 56.7, 64.3 (-CH2), 79.0, 109.6, 145.4, 148.7, 148.9, 150.5, 150.9, 175.5 (-COO-);
HRMS (ESI) m/z: 591.45212 [M + H]+, calcd. for C38H58N2O3 590.44474.
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4.1.1. General Procedure for the Preparation of TBA amino acids derivatives
BA-02---BA-13

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4.1.1.1. General procedure for esterification at carbon C3 (method A)
The compound TBA (1 equiv.) was dissolved in dry DCM (25 mL), DMAP (0.1 equiv.)
and the protected amino acid (1.3 equiv.) were added. After addition of EDCI (1.5
equiv.), the mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with
50 ml CH2Cl2, then successively washed with water and brine (10 mL each), dried over
sodium sulphate and filtered, and the solvent was evaporated. Then the crude product
was purified by flash chromatography (silica gel, dichloromethane: methanol = 40:1).
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4.1.1.2. General procedure for deprotection (method B)
To a solution of the Boc-protected compound in dry DCM (10 mL), TFA (1 mL/10
mL DCM) was added. The mixture was allowed to stir in ice bath for 2 h. After
completion of the reaction (as monitored by TLC), the solution was evaporated and
washed with a saturated sodium carbonate solution (20 mL). The aqueous layer was
extracted with DCM (3 × 25 mL), the combined organic extracts were washed with
brine (20 mL), dried over sodium sulfate, filtrated and evaporated. Purification was
performed by flash chromatography (silica gel, dichloromethane: methanol = 40:1).
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4.1.1.3. General procedure for the deprotection (method C)
Pd/C (10%; 80 mg) was added to a solution of the Cbz-protected compound in 30 mL
MeOH. The mixture was stirred at room temperature for 12 h. After completion of the
reaction (as monitored by TLC), the solvent was filtered to remove Pd/C. The filtrate
was concentrated in vacuum and the residue was purified by flash chromatography
(silica gel, dichloromethane: methanol = 40:1).
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3β-(Glycyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-02). Obtained from TBA by method A and method B as a colorless powder;
Yield: 75.3%; mp: 104.7–105.4 °C, [α]D = +22 (c 0.50, MeOH); 1H-NMR (500 MHz,
CDCl3): δ (ppm) 0.77, 0.83, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.82 (s, each,
6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.53 (s, each, 3H,
3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.44-3.47 (m, 2H,
-CH2NH2), 4.50-4.53 (m, 1H, -OCOCH-), 4.58, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20
13
(d, each, 1H, J = 15Hz, -OCH2-); C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0,
16.3, 16.6, 18.3, 19.5, 20.6 (-CH3), 21.0, 21.5 (-CH3), 21.8 (-CH3), 23.9, 25.6, 28.1,

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30.7, 32.2, 34.4, 37.1, 37.2, 38.0, 38.2, 38.5, 40.8, 42.5 (-CH2NH2), 47.0, 49.6, 50.6,
55.6, 56.8, 64.5 (-CH2), 81.9 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.6,
151.1 (-CH=C-), 173.9 (-CONH2), 175.7 (-COO-); HRMS (ESI) m/z: 648.47040
[M+H]+, calcd. for C40H61N3O4 647.46621.
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304
305
306
307
308
309
310
311
312
313
314
3β-(L-Phenylalanyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-03). Obtained from TBA by method A and method C as a colorless powder;
1
Yield: 65.3%; mp: 82.0–82.7 °C; [α]D = +186 (c 0.50, MeOH); H-NMR (500 MHz,
CDCl3): δ (ppm) 0.76, 0.77, 0.80, 0.82, 0.94 (s, each, 3H, 5×-CH3, methyl of BA),
1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.54 (s, each, 3H, 3×-CH3, methyl of TMP),
2.82-2.86 (m, 1H, -CH2CH-), 2.96-3.02 (m, 1H, -CCHCH2-), 3.15-3.18 (m, 1H,
-CH2CH-), 3.72-3.76 (m, 1H, -CHNH2), 4.49-4.52 (m, 1H, -OCOCH-), 4.59, 4.71 (brs,
each, 1H, =CH2), 5.17, 5.20 (d, each, 1H, J = 15Hz, -OCH2-), 7.19-7.33 (m, 5H,
-C6H5); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.6, 15.9, 16.2, 16.6, 18.1, 19.3, 20.5
(-CH3), 20.9, 21.4 (-CH3), 21.7 (-CH3), 23.7, 25.5, 28.0, 29.6, 30.5, 32.0, 34.2, 36.9,
37.1, 37.9, 38.1, 38.3, 40.7, 41.1, 42.4, 46.9, 49.5, 50.4, 55.4, 56.1, 56.7, 64.4 (-CH2),
81.9, 109.7 (-CH=C-), 126.8, 128.6, 129.3, 137.3, 145.3, 148.8, 150.5, 156.0
(-CH=C-), 174.4, 175.5 (-COO-); HRMS (ESI) m/z: 738.51666 [M+H]+, calcd. for
C47H67N3O4 737.51316.
315
316
317
318
319
320
321
322
323
324
325
3β-(L-Alanyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-04). Obtained from TBA by method A and method B as a colorless powder;
Yield: 73.0%; mp: 126.3–127.2 °C, [α]D = +20 (c 0.50, MeOH); 1H-NMR (500 MHz,
CDCl3): δ (ppm) 0.78, 0.84, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.82 (brs, 6H,
2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.53 (s, each, 3H,
3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.45-3.55 (m, 1H, -CHNH2),
4.48-4.51 (m, 1H, -OCOCH-), 4.58, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each,
1H, J = 15Hz, -OCH2-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7,
18.3, 19.5, 20.6 (-CH3), 20.8, 21.0, 21.5 (-CH3), 21.8 (-CH3), 23.8, 25.6, 28.1, 29.8,
30.7, 31.1, 32.2, 34.4, 37.0, 37.2, 38.1, 38.2, 38.5, 40.8, 42.5, 47.0, 50.5 (-CHNH2),
50.6, 55.5, 56.8, 64.5 (-CH2), 81.6 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0,

ACCEPTED MANUSCRIPT
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150.6, 151.1 (-CH=C-), 175.7 (-COCH-), 176.3 (-COO-); HRMS (ESI) m/z:
662.48627 [M+H]+, calcd. for C41H63N3O4 661.48186.
328
329
330
331
3β-(L-Aspartyl)-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester
(BA-05). Obtained from TBA by method A and method C as a colorless powder. Yield:
45.6%; mp: > 210 °C, [α]D = +36 (c 0.50, MeOH); HRMS (ESI) m/z: 706.47534
[M+H]+, calcd. for C42H63N3O6 705.47169.
332
333
334
335
336
337
338
339
340
341
342
343
344
345
3β-(L-Prolyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-06). Obtained from TBA by method A and method B as a colorless powder;
Yield: 72.7%; mp: 147.4–148.3 °C, [α]D = +13 (c 0.50, MeOH); 1H-NMR (500 MHz,
CDCl3): δ (ppm) 0.77, 0.83, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.82 (brs, 6H,
2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.53 (s, each, 3H,
3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.22-3.25 (m, 1H,
-CH2CH2-), 4.10-4.13 (m, 1H, -NHCHCH2-), 4.47-4.56 (m, 1H, -OCOCH-), 4.59,
13
4.71 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each, 1H, J = 15Hz, -OCH2-); C-NMR
(125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.2, 19.5, 20.6 (-CH3), 21.0, 21.5
(-CH3), 21.8 (-CH3), 23.8, 24.6, 25.5, 28.2, 29.7, 29.8, 30.6, 32.1, 34.3, 37.0, 37.2,
38.2, 38.4, 40.8, 42.5, 46.4, 47.0, 49.6, 50.6, 55.5, 56.8, 59.7 (-NHCH-), 64.5 (-CH2),
83.5 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 171.5
(-COCH-), 175.7 (-COO-); HRMS (ESI) m/z: 688.50122 [M+H]+, calcd. for
C43H65N3O4 687.49751.
346
347
348
349
350
351
352
353
3β-(L-Leucyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-07). Obtained from TBA by method A and method B as a colorless powder;
Yield: 58.4%; colorless powder; mp: 81.8–82.5 °C, [α]D = +24 (c 0.50, MeOH);
1H-NMR (500 MHz, CDCl3): δ (ppm) 0.78, 0.83 (s, each, 3H, 2×-CH3, methyl of BA),
0.84 (brs, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.54 (s,
each, 3H, 3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.48 (brs, 1H,
-CHNH2), 4.48-4.51 (m, 1H, -OCOCH-), 4.59, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20
13
(d, each, 1H, J = 15Hz, -OCH2-); C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0,

ACCEPTED MANUSCRIPT
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16.3, 16.8, 18.3, 19.5, 20.6 (-CH3), 21.0, 21.5 (-CH3), 21.8 (-CH3), 22.0, 23.1, 23.8,
25.0, 25.6, 28.2, 29.8, 30.7, 32.2, 34.4, 37.1, 37.2, 38.1, 38.2, 38.5, 40.8, 42.5, 47.0,
49.6, 50.6, 55.6, 56.8 (-CHNH2), 64.5 (-CH2), 69.1, 81.9 (-OCOCH-), 109.8
(-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 175.7 (-COO-); HRMS (ESI)
m/z: 704.53546 [M+H]+, calcd. for C44H69N3O4 703.52881.
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360
361
362
363
364
365
366
367
368
369
370
371
3β-(L-Isoleucyl)-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester
(BA-08). Obtained from TBA by method A and method B as a colorless powder;
1
Yield: 60.5%; mp: 88.6–89.4 °C, [α]D = +27 (c 0.50, MeOH); H-NMR (500 MHz,
CDCl3): δ (ppm) 0.78, 0.82 (s, each, 3H, 2×-CH3, methyl of BA), 0.85 (brs, 6H,
2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.54 (s, each, 3H,
3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.48 (brs, 1H, -CHNH2),
4.50-4.53 (m, 1H, -OCOCH-), 4.59, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each,
1H, J = 15Hz, -OCH2-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 11.9, 12.0, 14.8, 15.9,
16.0, 16.3, 16.9, 18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.9, 24.8,
25.6, 26.2, 28.2, 29.8, 30.7, 32.2, 34.4, 37.1, 37.2, 37.9, 38.2, 38.5, 40.8, 42.5, 47.0,
49.6, 55.6, 56.8 (-CH-NH2), 59.3, 64.5 (-CH2), 82.5 (-OCOCH-), 109.8 (-CH=C-),
136.9, 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 175.7 (-COO-); HRMS (ESI) m/z:
704.52814 [M+H]+, calcd. for C44H69N3O4 703.52881.
372
373
374
375
376
377
378
379
380
381
382
3β-(L-Pyroglutamyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-09). Obtained from TBA by method A as a colorless powder; Yield: 52.8%;
1
mp: 118.2–119.1 °C, [α]D = +24 (c 0.50, MeOH); H-NMR (500 MHz, CDCl3): δ
(ppm) 0.78, 0.82, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.83 (brs, 6H, 2×-CH3,
methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.54 (s, each, 3H, 3×-CH3,
methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 4.22-4.24 (m, 1H, -NHCH-),
4.52-4.55 (m, 1H, -OCOCH-), 4.59, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each,
1H, J = 15Hz, -OCH2-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7,
18.3, 18.5, 20.5, 21.0 (-CH3), 21.5 (-CH3), 21.7 (-CH3), 23.8, 25.2, 25.6, 28.2, 29.4,
29.7, 29.8, 30.7, 32.1, 34.3, 37.0, 37.2, 38.1, 38.2, 38.4, 40.8, 42.5, 47.0 (-CH-NH-),
49.6, 50.6, 55.5, 55.8, 56.8, 64.4 (-CH2), 82.7 (-OCOCH-), 109.8 (-CH=C-), 145.6,

ACCEPTED MANUSCRIPT
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148.8, 149.1, 150.6, 151.0 (-CH=C-), 171.7 (-COCH-), 175.7 (-COO-), 177.7
(-CONH-); HRMS (ESI) m/z: 702.48419. [M+H]+, calcd. for C43H63N3O5 701. 47677.
385
386
387
388
389
390
391
392
393
394
395
396
397
3β-(L-Lysyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-10). Obtained from TBA by method A and method C as a colorless powder;
1
Yield: 56.8%; mp: >220 °C, [α]D = +30 (c 0.50, MeOH); H-NMR (500 MHz,
CD3OD): δ (ppm) 0.67, 0.88, 0.89, 0.91, 1.00 (s, each, 3H, 5×-CH3, methyl of BA),
1.67 (s, 3H, 30-CH3 of BA), 2.52, 2.53, 2.60 (s, each, 3H, 3×-CH3, methyl of TMP),
2.93-3.01 (m, 2H, -CH2NH2), 3.01-3.04 (m, 1H, -CCHCH2-), 3.57-3.60 (m, 1H,
-CHNH2), 4.52-4.55 (m, 1H, -OCOCH-), 4.61, 4.71 (brs, each, 1H, =CH2), 5.23, 5.27
(d, each, 1H, J = 20Hz, -OCH2-); 13C-NMR (125 MHz, CD3Cl+ CD3OD): δ (ppm)
14.0, 15.1, 15.5, 15.9, 17.6, 18.5, 19.3 (-CH3), 20.3 (-CH3), 20.4 (-CH3), 21.7, 23.1,
25.0, 26.4, 27.4, 29.1, 30.0, 31.5, 31.6, 33.7, 36.3, 36.6, 37.4, 37.7, 37.9, 38.7, 40.2,
41.9, 46.6, 48.9, 50.0, 52.9, 55.0, 56.3 (-CH-NH2), 63.5 (-CH2), 82.5 (-OCOCH-),
111.5 (-CH=C-), 145.2, 148.6, 149.3, 150.0, 150.8 (-CH=C-), 168.7 (-COCH-), 175.4
(-COO-); HRMS (ESI) m/z: 719.54608. [M+H]+, calcd. for C44H70N4O4 718.53971.
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399
400
401
402
403
404
405
406
407
408
409
410
411
3β-(L-Tryptophanyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-11). Obtained from TBA by method A and method C as a colorless powder;
Yield: 61.2%; mp: 119.8–120.6 °C, [α]D = +30 (c 0.50, MeOH); 1H-NMR (500 MHz,
CDCl3): δ (ppm) 0.78 (s, 6H, 2×-CH3, methyl of BA), 0.83 (s, 6H, 2×-CH3, methyl of
BA), 0.94 (s, 3H, -CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.49, 2.50, 2.54
(s, each, 3H, 3×-CH3, methyl of TMP), 2.95-3.02 (m, 2H, -CH2NH2), 3.35-3.37 (m,
1H, -CCHCH2-), 3.70-3.81 (m, 1H, -CHNH2), 4.45-4.53 (m, 1H, -OCOCH-), 4.68,
4.72 (brs, each, 1H, =CH2), 5.18, 5.21 (d, each, 1H, J = 15Hz, -OCH2-), 7.05-7.20,
7.29-7.37, 7.60-7.63 (m, 4H, -C6H5); 13C-NMR (125 MHz, CDCl3) : δ (ppm) 14.8,
16.0, 16.3, 16.7, 18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.8, 25.6,
28.1, 29.8, 30.1, 30.7, 32.2, 34.4, 37.1, 37.2, 38.1, 38.2, 38.5, 40.8, 42.5, 47.0, 49.6,
50.6, 54.9, 55.5, 56.8 (-CH-NH2), 64.5 (-CH2), 82.5 (-OCOCH-), 109.8 (-CH=C-),
110.3, 111.6, 111.4, 118.8, 119.6, 122.3, 123.8 (-NHCH-), 127.3, 136.5, 145.5, 148.9,
148.9, 150.6, 151.1 (-CH=C-), 175.7 (-CO-); HRMS (ESI) m/z: [M+H]+ 777.53125,

ACCEPTED MANUSCRIPT
412
calcd. for C49H68N4O4 776.52406.
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425
3β-(L-Sarkosyl)-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester
(BA-12). Obtained from TBA by method A and method B as a colorless powder;
Yield: 76.9%; mp: 172.6–173.4 °C, [α]D = +42 (c 0.50, MeOH); 1H-NMR (500 MHz,
CDCl3) : δ (ppm) 0.77, 0.84, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.82 (brs, 6H,
2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.40, 2.50, 2.53 (s, each, 3H,
3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.52 (brs, 2H, -CH2NH-),
4.48-4.51 (m, 1H, -OCOCH-), 4.56, 4.59 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each,
1H, J = 15Hz, -OCH2-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.6,
18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.9, 25.6, 28.2, 29.8, 30.7,
32.2, 34.4, 34.6, 37.0, 37.2, 38.0, 38.2, 38.5, 40.8, 42.5, 47.0, 49.6, 50.6, 50.7, 55.6,
56.8 (-CH-NH2), 64.5 (-CH2), 83.2 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0,
150.6, 151.1 (-CH=C-), 168.6 (-COCH-), 175.7 (-COO-); HRMS (ESI) m/z: [M+H]+
662.48975, calcd. for C41H63N3O4 661.48186.
426
427
428
429
430
431
432
433
434
435
436
437
438
3β-(L-Valyl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-13). Obtained from TBA by method A and method C as a colorless powder;
1
Yield: 52.3%; mp: > 220 °C, [α]D = +20 (c 0.50, MeOH); H-NMR (500 MHz,
CDCl3): δ (ppm) 0.78, 0.82, 0.94 (s, each, 3H, 3×-CH3, methyl of BA), 0.85 (brs, 6H,
2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.54 (s, each, 3H,
3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H, -CCHCH2-), 3.33-3.34 (m, 1H, -CHNH2),
4.49-4.52 (m, 1H, -OCOCH-), 4.59, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each,
1H, J = 15Hz, -OCH2-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.8,
18.3, 17.1, 18.3, 19.5, 19.6, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.9, 25.6,
28.2, 29.8, 30.7, 31.6, 32.2, 34.4, 37.1, 37.2, 38.0, 38.2, 38.5, 40.8, 42.5, 47.0, 49.6,
50.6, 55.6, 56.8 (-CH-NH2), 60.2, 64.5 (-CH2), 82.2 (-OCOCH-), 109.8 (-CH=C-),
145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 175.7 (-COO-); HRMS (ESI) m/z:
[M+H]+ 690.52112, calcd. for C43H67N3O4 689.51316.

ACCEPTED MANUSCRIPT
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4.1.2. General Procedure for the Preparation of TBA amino acids derivatives
BA-14---BA-15
441
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It was worth noting that, to avoid the formation of by-products, the hydroxyl groups
of Cbz-L-serine Cbz-L-threonine should be protected with TBDMSCl before
esterification with TBA in the synthesis of BA-14 and BA-15. To a magnetically
stirred solution of the corresponding N-Cbz-protected amino acid (N-Cbz-L-serine
N-Cbz-L-threonine) (1.0 equiv.) and imidazole (2.0 equiv.) in dry N, N-dimethyl
formamide (10 mL) at 0°C, the TBDMSCl (1.5 equiv.) was added. Then the reaction
mixture was allowed to warm to room temperature and stirred for 24h. After
completion of the reaction (as monitored by TLC), the reaction mixture was poured
into ice-water, and the sediment was filtered and washed with water. Then the residue
was dissolved with DCM, dried over sodium sulfate and evaporated under vacuum,
and the crude product (N-Cbz-TBDMS-L-serine, N-Cbz-TBDMS-L-threonine) was
obtained.
The starting material TBA (1 equiv.) was dissolved in dry DCM (20 mL), DMAP (0.1
equiv.) and the protected amino acid (1.3 equiv.) were added. After addition of EDCI
(1.5 equiv.), the mixture was stirred at 25 °C for 12 h. Then the solution was washed
with brine. After drying the organic layer over anhydrous Na2SO4 and evaporating the
solvent under vacuum, the crude product was purified by flash chromatography (silica
gel, dichloromethane: methanol = 40:1).
The Cbz-protected compound was dissolved in 20 mL MeOH, Pd/C (10%; 80 mg)
was added. The mixture was allowed to stir at room temperature for 12 h and filtered
to remove Pd/C. The filtrate was concentrated in vacuum and the residue was further
reacted with 1.5 equivalents of 1M TBAF in THF for 1.5 h at 25 °C. Then the solvent
was evaporated under vacuum. Purification was performed by flash chromatography
(silica gel, dichloromethane: methanol = 40:1).
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466
467
3β-(L-Threonyl)-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester
(BA-14). Yield: 46.7%; colorless powder; mp: 103.1-103.9 °C, [α]D = -4 (c 0.50,
MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm) 0.78, 0.85, 0.86 (s, each, 3H, 3×-CH3,

ACCEPTED MANUSCRIPT
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methyl of BA), 0.82 (brs, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA),
2.48, 2.50, 2.54 (s, each, 3H, 3×-CH3, methyl of TMP), 2.97-3.00 (m, 1H, -CCHCH2-),
3.39-3.58 (m, 1H, -CHNH2), 3.85-3.98 (m, 1H, -CHOH), 4.54-4.55 (m, 1H,
-OCOCH-), 4.58, 4.71 (brs, each, 1H, =CH2), 5.16, 5.19 (d, each, 1H, J = 15Hz,
-OCH2-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.8, 18.3, 19.5
(CH3CH-), 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.9, 25.5, 27.7, 28.2, 29.0,
29.7, 30.6, 32.1, 34.3, 37.0, 37.2, 38.0, 38.2, 38.5, 40.8, 42.5, 47.0, 49.6, 50.5, 55.6
(-CH-NH2), 56.8, 64.5 (-CH2), 67.2 (-CHOH), 82.6 (-OCOCH-), 95.8, 109.8
(-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 175.75 (-CO-); HRMS (ESI)
m/z: [M+H]+ 692.49957, calcd. for C42H65N3O5 691.49242.
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479
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481
482
483
484
485
486
487
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489
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491
3β-(L-Seryl)-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl
ester
(BA-15). Yield: 47.5%; colorless powder; mp: 133.2-134.1 °C, [α]D = +16 (c 0.50,
MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm) 0.77, 0.82, 0.93 (s, each, 3H, 3×-CH3,
methyl of BA), 0.83 (brs, 6H, 2×-CH3, methyl of BA), 1.66 (s, 3H, 30-CH3 of BA),
2.48, 2.50, 2.53 (s, each, 3H, 3×-CH3, methyl of TMP), 2.97-3.00 (m, 1H, -CCHCH2-),
3.32-3.35 (s, 1H, -CHNH2), 3.63 (brs, 1H, -OH), 3.72-3.75 (m, 1H, -CH2OH),
3.86-3.94 (m, 1H, -CH2OH), 4.52-4.56 (m, 1H, -OCOCH-), 4.58, 4.71 (brs, each, 1H,
13
=CH2), 5.16, 5.19 (d, each, 1H, J = 15Hz,-OCH2-); C-NMR (125 MHz, CDCl3): δ
(ppm) 14.8, 16.0, 16.3, 16.7, 18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3),
23.9, 24.3, 25.5, 28.1, 29.7, 30.6, 32.1, 34.3, 37.0, 37.2, 38.1, 38.2, 38.4, 40.8, 42.5,
47.0, 49.6, 50.5, 55.5 (-CH-NH2), 56.8, 62.5 (-CH2OH), 64.5 (-CH2), 82.4
(-OCOCH-), 82.7, 109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-),
175.7 (-COO-); HRMS (ESI) m/z: [M+H]+ 678.48480, calcd. for C41H63N3O5
677.47677 .
492
493
494
495
496
4.1.3. General Procedure for the Preparation of TBA dipeptide derivatives
BA-16---BA-27
The corresponding TBA amino acid derivatives (BA-02, BA-04, BA-06, BA-12) (1.0
equiv.), DMAP (0.1 equiv.) and N-Boc-protected amino acids (Boc-L-glycine,
Boc-L-alanine, Boc-L-sarcosine, Boc-L-proline) were dissolved in 25 mL dry DCM,

ACCEPTED MANUSCRIPT
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500
501
502
503
504
505
506
EDCI (0.1 equiv.) was added. The mixture was stirred at room temperature overnight.
Then the solution was washed with brine, dried over sodium sulfate, filtered and the
solvent was evaporated. Purification was performed by flash chromatography (silica
gel, dichloromethane: methanol = 40:1). Then the Boc-protected compounds were
further reacted with TFA (1mL per 10mL DCM) in dry DCM in ice bath for 2 h. After
completion of the reaction (as monitored by TLC), the solution was evaporated and
washed with a saturated sodium carbonate solution (20 mL). The aqueous layer was
extracted with DCM (3 × 25 mL), the combined organic extracts were washed with
brine (20 mL), dried over sodium sulfate, filtrated and evaporated. Purification was
performed by flash chromatography (silica gel, dichloromethane: methanol = 30:1).
507
508
509
510
511
512
513
514
515
516
517
518
519
3β-[(L-Glycyl)-L-Glycyl]-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-16).Yield: 52.8%; colorless powder; mp: 130.8-131.7 °C, [α]D = +15 (c
0.50, MeOH); 1H-NMR (500 MHz, CD3OD): δ (ppm) 0.67, 0.98 (s, each, 3H, 2×-CH3,
methyl of BA), 0.88 (brs, 9H, 3×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA),
2.52, 2.53, 2.60 (s, each, 3H, 3×-CH3, methyl of TMP), 2.98-3.04 (m, 1H, -CCHCH2-),
3.35 (s, 2H, -CH2NH2), 3.98 (s, 2H, -CH2NH-), 4.50-4.53 (m, 1H, -OCOCH-), 4.61,
4.71 (brs, each, 1H, =CH2), 5.23, 5.26 (d, each, 1H, J = 15Hz, -OCH2-), 5.51 (s, 1H,
-NH-). 13C-NMR (125 MHz, CD3OD): δ (ppm) 15.1, 16.4, 16.7, 16.9, 19.2, 19.5, 20.4,
21.2 (-CH3), 21.4 (-CH3), 22.0 (-CH3), 24.6, 26.7, 28.4, 30.7, 31.6, 33.1, 35.4, 37.8,
38.2, 39.0, 39.6, 41.9 (-CH2-NH-), 42.1, 43.5 (-CH2-NH2), 45.6, 50.6, 51.7, 54.8, 56.8,
57.9, 65.0 (-CH2), 83.4 (-OCOCH-), 110.4 (-CH=C-), 147.1, 150.3, 150.7, 151.7,
152.5 (-CH=C-), 160.0, 171.3 (-CONH-), 175.7 (-COCH2-), 177.0 (-COO-); HRMS
(ESI) m/z: [M+H]+ 705.49500, calcd. for C42H64N4O5 704.48767.
520
521
522
523
524
525
3β-[(L-Sarkosyl)-L-Sarkosyl]-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (BA-17). Yield: 62.3%; colorless powder;
1
mp: 118.4-119.2 °C, [α]D = +12 (c 0.50, MeOH); H-NMR (500 MHz, CDCl3): δ
(ppm) 0.76, 0.81, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 1.66 (s, 3H, 30-CH3 of
BA), 2.48, 2.50, 2.53 (s, each, 3H, 3×-CH3, methyl of TMP), 3.04-3.17 (m, 2H,
-CH2NH-), 2.98 (s, 3H, CH3-N-), 2.98-3.04 (m, 1H, -CCHCH2-), 3.96 (s, 3H,

ACCEPTED MANUSCRIPT
526
527
528
529
530
531
532
533
CH3-NH-), 3.96-4.12 (m, 2H, -CH2N-), 4.50-4.51 (m, 1H, -OCOCH-), 4.58, 4.70 (brs,
13
each, 1H, =CH2), 5.16, 5.19 (d, each, 1H, J = 15Hz, -OCH2-); C-NMR (125 MHz,
CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.4, 19.5, 21.0 (-CH3), 21.5 (-CH3), 21.8
(-CH3), 23.8, 25.6, 28.1, 29.7, 30.7, 32.2, 33.4, 34.4, 35.8 (CH3N-), 37.0, 37.3, 38.0,
38.2, 38.8, 39.0, 40.8, 42.5, 47.0, 49.6, 50.6, 51.8, 55.4 (-CH-NH2), 56.8, 64.5 (-CH2),
79.1 (CH3NH-), 82.9 (-OCOCH-), 109.7 (-CH=C-), 145.5, 148.9, 149.0, 150.7, 151.1
(-CH=C-), 163.3, 168.4 (-CH2COO-), 175.7 (-CO-); HRMS (ESI) m/z: [M+H]+
733.52527, calcd. for C44H65N3O5 732.51897.
534
535
536
537
538
539
540
541
542
543
544
545
546
547
3β-[(L-Prolyl)-L-Sarkosyl]-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (BA-18). Yield: 60.7%; colorless powder;
1
mp: 158.5-159.3 °C, [α]D = +52 (c 0.50, MeOH); H-NMR (500 MHz, CDCl3): δ
(ppm) 0.78, 0.81, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.82 (s, 6H, 2×-CH3,
methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.50, 2.53, 2.56 (s, each, 3H, 3×-CH3,
methyl of TMP), 2.95-2.99 (m, 1H, -CCHCH2-), 3.11 (s, 3H, CH3N-), 3.42, 3.46 (brs,
each, 1H, -CH2-NH-), 3.68-3.71 (m, 1H, -CHNH-), 4.51-4.53 (m, 1H, -OCOCH-),
4.59, 4.71 (brs, each, 1H, =CH2), 5.18, 5.21 (d, each, 1H, J = 15Hz, -OCH2-);
13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.3, 19.5, 20.4, 21.0
(-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.9, 25.6, 28.2, 29.4, 29.8, 30.7, 32.1, 34.3, 36.4,
37.0, 37.2, 38.0, 38.2, 38.5, 40.8, 42.5, 47.0, 49.6, 50.4, 50.6, 55.5, 56.8, 58.3, 62.4,
64.4 (-CH2), 83.1 (-OCOCH-), 109.8 (-CH=C-), 145.8, 148.6, 149.3, 150.6, 151.1
(-CH=C-), 163.3, 168.0 (-CH2COO-), 169.3 (-CON-), 175.6 (-COO-); HRMS (ESI)
m/z: [M+H]+ 759.54059, calcd. for C46H70N4O5 758.53462.
548
549
550
551
552
553
554
3β-[(L-Alanyl)-L-Glycyl]-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-19). Yield: 58.9%; colorless powder; mp: 117.8-118.7°C, [α]D = +17 (c 0.50,
MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm) 0.77, 0.83, 0.93 (s, each, 3H, 3×-CH3,
methyl of BA), 0.82 (s, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48,
2.50, 2.53 (s, each, 3H, 3×-CH3, methyl of TMP), 2.87-2.95 (s, each, 2H, -NH2-),
2.96-3.01 (m, 1H, -CCHCH2-), 3.61-3.63 (m, 1H, -CH-), 4.01 (d, 2H, -NHCH2-),
4.50-4.53 (m, 1H, -CHCH3), 4.58, 4.70 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each, 1H,

ACCEPTED MANUSCRIPT
13
555
556
557
558
559
560
561
J = 15HZ, -OCH2-), 7.80 (s, 1H, -NH-CH2-); C-NMR (125 MHz, CDCl3): δ (ppm)
14.8, 16.0, 16.3, 16.6, 18.3, 19.5, 21.0, 20.6 (-CH3), 21.4, 21.5 (-CH3), 21.8 (-CH3),
23.8, 25.6, 28.1, 29.7, 30.7, 32.2, 34.4, 37.0, 37.2, 38.0, 38.2, 38.5, 39.0, 40.8, 41.4
(-NH-CH2-), 42.5, 47.0, 49.6, 50.6 (-CHNH2), 50.7, 55.5, 56.8, 64.5 (-CH2), 82.6,
109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 169.9 (-CH2-COO-),
175.7 (-COO-); HRMS (ESI) m/z: [M+H]+ 719.51080, calcd. for C43H66N4O5
718.50332.
562
563
564
565
566
567
568
569
570
571
572
573
574
575
3β-[(L-Prolyl)-L-Glycyl]-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-20). Yield: 59.3%; colorless powder; mp: 122.6-123.5 °C, [α]D = +4 (c 0.50,
MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm) 0.77, 0.83, 0. 93 (s, each, 3H, 3×-CH3,
methyl of BA), 0.82 (s, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48,
2.50, 2.53 (s, each, 3H, 3×-CH3, methyl of TMP), 2.99-3.03 (m, 2H, -CH2NH-),
3.07-3.12 (m, 1H, -CCHCH2-), 3.94-3.97 (m, 1H, -NHCH-), 4.00-4.02 (m, 2H,
-CH2CH2-), 4.49-4.52 (m, 1H, -OCOCH-), 4.58, 4.71 (brs, each, 1H, =CH2), 5.17,
5.20 (d, each, 1H, J = 15Hz,-OCH2-), 8.19 (s, 1H, -NH-); 13C-NMR (125 MHz,
CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.6, 18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3),
21.8 (-CH3), 23.8, 25.6, 26.1, 28.2, 29.8, 30.7, 30.8, 32.2, 34.4, 37.1, 37.2, 38.0, 38.2,
38.5, 40.8 (-NHCH-), 41.4, 42.5, 47.0, 47.3, 49.6, 50.6, 55.5, 56.8, 60.5, 62.4
(-CHNH-), 64.5 (-CH2), 82.6 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 150.6,
151.1 (-CH=C-), 169.9 (-CONH-), 175.7 (-COOCH-), 175.7 (-COO-); HRMS (ESI)
m/z: [M+H]+ 745.52527, calcd. for C45H68N4O5 744.51897.
576
577
578
579
580
581
582
583
3β-[(L-Sarkosyl)-L-Glycyl]-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (BA-21). Yield: 57.6%; colorless powder;
1
mp: 117.8-118.6 °C, [α]D = +16 (c 0.50, MeOH); H-NMR (500 MHz, CDCl3): δ
(ppm) 0.77 (s, each, 3H, -CH3, methyl of BA), 0.82 (brs, 9H, 3×-CH3, methyl of BA),
0.93 (s, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.53 (s,
each, 3H, 3×-CH3, methyl of TMP), 2.97-3.01 (m, 1H, -CCHCH2-), 3.32 (s, 2H,
-NHCH2-), 3.78-4.03 (m, 2H, -CH2CO-), 4.50-4.53 (m, 1H, -OCOCH-), 4.58, 4.71
13
(brs, each, 1H, =CH2), 5.16, 5.19 (d, each, 1H, J = 15Hz, -OCH2-); C-NMR (125

ACCEPTED MANUSCRIPT
584
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586
587
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589
MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.6, 18.3, 19.4, 20.6, 21.0 (-CH3), 21.5
(-CH3), 21.8 (-CH3), 21.9, 22.0, 23.7, 25.6, 28.0, 29.7, 30.7, 32.2, 34.4, 34.6, 37.0,
37.2, 38.0, 38.2, 38.5, 40.8, 42.5, 47.0, 49.6, 50.6, 55.5 (-NHCH2-), 56.8, 64.5 (-CH2),
82.6 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.1, 151.1 (-CH=C-), 168.6
(-CONH-), 170.8 (-COOCH-), 175.7 (-COO-); HRMS (ESI) m/z: [M+H]+ 719.51044,
calcd. for C43H66N4O5 718.50332.
590
591
592
593
594
595
596
597
598
599
600
601
602
3β-[(L-Alanyl)-L-Alanyl]-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-22). Yield: 53.1%; colorless powder; mp: 118.3-119.2 °C, [α]D = -2 (c 0.50,
1
MeOH); H-NMR (500 MHz, CDCl3): δ (ppm) 0.78, 0.94 (s, each, 3H, 2×-CH3,
methyl of BA), 0.82-0.83 (m, 9H, 3×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of
BA), 2.48, 2.50, 2.54 (s, each, 3H, 3×-CH3, methyl of TMP), 2.96-3.01 (m, 1H,
-CCHCH2-), 3.56-3.60 (m, 1H, NH2CH-), 4.49-4.52 (m, 1H, -OCOCH-), 4.52-4.57
(m, 1H, -NHCH-), 4.58, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20 (d, each, 1H, J = 15Hz,
-OCH2-), 7.75-7.76 (d, 1H, -NH-); 13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0,
16.3, 16.7, 18.3, 18.7, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.8, 25.6,
28.1, 29.8, 30.7, 32.2, 34.4, 37.1, 37.2, 38.1, 38.2, 38.4, 40.8, 41.6, 42.5, 47.0, 48.4
49.6 (NH2CH-), 50.6, 55.5, 56.8 (-NHCH-), 64.5 (-CH2), 82.4 (-OCOCH-), 109.8
(-CH=C-), 145.5, 148.9, 149.0, 150.1, 151.1 (-CH=C-), 172.8 (-COOCH-), 175.7
(-COO-); HRMS (ESI) m/z: [M+H]+ 733.52606, calcd. for C44H68N4O5 732.51897.
603
604
605
606
607
608
609
610
611
612
3β-[(L-Glycyl)-L-Alanyl]-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-23). Yield: 55.3%; colorless powder; mp: 172.4-173.3 °C, [α]D = -2 (c 0.50,
MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm) 0.78, 0.83, 0.94 (s, each, 3H, 3×-CH3,
methyl of BA), 0.82 (s, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48,
2.50, 2.54 (s, each, 3H, 3×-CH3, methyl of TMP), 2.97-3.01 (m, 1H, -CCHCH2-), 3.56
(brs, 2H, -CH2NH2), 4.49-4.52 (m, 1H, -OCOCH-), 4.58 (m, 1H, -NHCH-), 4.58, 4.71
(brs, each, 1H, =CH2), 5.17, 5.20 (d, each, 1H, J = 15Hz, -OCH2-), 7.87 (s, 1H, -NH-);
13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.3, 18.8, 19.5, 20.6,
21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.8, 25.6, 28.1, 29.8, 30.7, 32.2, 34.4, 37.1,
37.2, 38.1, 38.2, 38.4, 40.8, 42.5 (NH2CH2-), 47.0, 48.2, 49.6, 50.6, 50.7, 55.5

ACCEPTED MANUSCRIPT
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615
(-NHCH-), 56.8, 64.5 (-CH2), 82.3 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0,
150.6, 151.1 (-CH=C-), 172.9 (-COCH-), 175.7 (-COO-); HRMS (ESI) m/z: [M+H]+
719.51044, calcd. for C43H66N4O5 718.50332.
616
617
618
619
620
621
622
623
624
625
626
627
628
629
3β-[(L-Prolyl)-L-Alanyl]-lup-20(29)-ene-28-oic acid-3,5,6-trimethylpyrazin-2-methyl
ester (BA-24). Yield: 56.4%; colorless powder; mp: 113.7-114.5 °C, [α]D = -6 (c 0.50,
MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm) 0.77, 0.79, 0.93 (s, each, 3H, 3×-CH3,
methyl of BA), 0.81-0.86 (m, 6H, 2×-CH3, methyl of BA), 1.67 (s, 3H, 30-CH3 of
BA), 2.48, 2.49, 2.53 (s, each, 3H, 3×-CH3, methyl of TMP), 2.65-2.70 (m, 1H,
-NHCH-), 2.91-3.03 (m, 2H, -CH2NH-), 3.04-3.08 (m, 1H, -CCHCH2-), 3.82-3.83 (m,
1H, -CHNH-), 4.47-4.56 (m, 1H, -OCOCH-), 4.70, 4.73 (brs, each, 1H, =CH2), 5.16,
5.19 (d, each, 1H, J = 15Hz, -OCH2-), 8.07-8.09 (d, 1H, -NH-); 13C-NMR (125 MHz,
CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3),
21.8 (-CH3), 23.8, 25.6, 28.1, 29.7, 30.7, 31.0, 32.2, 34.3, 37.0, 37.2, 38.0, 38.2, 38.5,
40.8, 42.5, 47.0, 47.3, 49.6, 50.0, 50.6 (-NHCH-), 55.5, 56.8, 60.5, 62.8, 64.5 (-CH2),
78.9, 82.2 (-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-),
170.8 (-CONH-), 174.7 (-COOCH-), 175.7 (-COO-); HRMS (ESI) m/z: [M+H]+
759.54053, calcd. for C46H70N4O5 758.53462.
630
631
632
633
634
635
636
637
638
639
640
641
3β-[(L-Sarkosyl)-L-Alanyl]-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (BA-25). Yield: 58.7%; colorless powder;
mp: 136.5-137.3 °C, [α]D = -2 (c 0.50, MeOH); 1H-NMR (500 MHz, CDCl3): δ (ppm)
0.77, 0.83, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.82 (s, 6H, 2×-CH3, methyl of
BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.53 (s, each, 3H, 3×-CH3, methyl of
TMP), 2.63-2.73 (m, 1H, -NHCH-) 2.94-3.01 (m, 1H, -CCHCH2-), 3.35-3.44 (m, 2H,
-NHCH2-), 4.48-4.51 (m, 1H, -OCOCH-), 4.58, 4.71 (brs, each, 1H, =CH2), 5.17, 5.20
(d, each, 1H, J = 15Hz, -OCH2-), 7.80-7.82 (d, 1H, -NH-); 13C-NMR (125 MHz,
CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.3, 18.7, 19.5, 20.6, 21.0 (-CH3), 21.5
(-CH3), 21.8 (-CH3), 23.8, 25.6, 28.1, 29.8, 30.7, 32.2, 34.4, 36.2, 37.1, 37.2, 38.1,
38.2, 38.4, 40.8, 42.5, 47.0, 48.2, 49.6, 50.6, 53.7, 55.5, 56.8, 64.5 (-CH2), 82.4
(-OCOCH-), 109.8 (-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 172.7

ACCEPTED MANUSCRIPT
642
643
(-CONH-), 175.7 (-COO-); HRMS (ESI) m/z: [M+H]+ 733.52521, calcd. for
C44H68N4O5 732.51897.
644
645
646
647
648
649
650
651
652
653
654
655
656
3β-[(L-Glycyl)-L-Sarkosyl]-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (BA-26). Yield: 78.5%; colorless powder;
1
mp: 117.2-118.1 °C, [α]D = +14 (c 0.50, MeOH); H-NMR (500 MHz, CDCl3): δ
(ppm) 0.77, 0.79, 0.93 (s, each, 3H, 3×-CH3, methyl of BA), 0.81 (s, 6H, 2×-CH3,
methyl of BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.50, 2.53 (s, each, 3H, 3×-CH3,
methyl of TMP), 2.97-3.01 (m, 1H, -CCHCH2-), 3.00-3.06 (m, 2H, -CH2NH2),
3.95-4.13 (m, 3H, CH3NH-), 4.48-4.56 (m, 2H, -CH2-), 4.58, 4.70 (brs, each, 1H,
13
=CH2), 5.16, 5.20 (d, each, 1H, J = 15Hz, -OCH2-); C-NMR (125 MHz, CDCl3): δ
(ppm) 14.8, 15.5, 16.0, 16.3, 16.7, 18.3, 19.5, 20.6, 21.0 (-CH3), 21.5 (-CH3), 21.8
(-CH3), 25.6, 28.1, 28.2, 29.8, 30.7, 31.1, 32.2, 34.4, 37.1, 37.2, 37.3, 38.0, 38.2, 38.5,
40.8, 42.5 (-CH2NH2), 47.0, 49.6, 50.6, 55.5, 56.8 (-NCH2-), 64.5 (-CH2), 79.1, 109.8
(-CH=C-), 145.5, 148.9, 149.0, 150.6, 151.1 (-CH=C-), 175.7 (-COOCH2-); HRMS
(ESI) m/z: [M+H]+ 719.51044, calcd. for C43H66N4O5 718.50332.
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3β-[(L-Alanyl)-L-Sarkosyl]-lup-20(29)-ene-28-oic
acid-3,5,6-trimethylpyrazin-2-methyl ester (BA-27). Yield: 79.8%; colorless powder;
1
mp: 136.2-137.1 °C, [α]D = +34 (c 0.50, MeOH); H-NMR (500 MHz, CDCl3): δ
(ppm) 0.78, 0.93 (s, each, 3H, 2×-CH3, methyl of BA), 0.81 (s, 9H, 3×-CH3, methyl of
BA), 1.67 (s, 3H, 30-CH3 of BA), 2.48, 2.49, 2.53 (s, each, 3H, 3×-CH3, methyl of
TMP), 2.87, 2.95 (s, each, 2H, -CH2-), 4.01 (brs, 2H, -CNH2), 4.06 (brs, 3H, -NCH3),
4.58, 4.70 (brs, each, 1H, =CH2), 5.16, 5.20 (d, each, 1H, J = 15Hz, -OCH2-);
13C-NMR (125 MHz, CDCl3): δ (ppm) 14.8, 16.0, 16.3, 16.7, 18.3, 19.5, 19.6, 20.6,
21.0 (-CH3), 21.5 (-CH3), 21.8 (-CH3), 23.8, 25.6, 28.2, 29.8, 30.7, 31.6, 32.2, 34.4,
36.6 (-NCH3), 37.0, 37.2, 38.0, 38.2, 38.5, 40.8, 41.6, 42.5, 47.0, 49.6, 50.2, 50.6,
55.5, 56.8 (-NCH2-), 64.5 (-CH2), 82.7, 109.81 (-CH=C-), 145.5, 148.9, 149.0, 150.6,
151.1 (-CH=C-), 162.7 (-CH2-COO-), 175.6 (-COOCH2-); HRMS (ESI) m/z: [M+H]+
733.52545, calcd. for C44H68N4O5 732.51897.

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4.2. Bio-Evaluation Methods
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4.2.1. Cell Culture
The human hepatocellular carcinoma cell line (HepG2), human colon carcinoma cell
line (HT-29), human cervical cancer cell line (Hela), human lung cancer cell line
(A549), human gastric cancer cell line (BGC-823) and Madin-Darby canine kidney
cell line (MDCK) were obtained from the Chinese Academy of Medical Sciences &
Peking Union Medical College. The cultures of the cells were maintained as
monolayer in RPMI 1640 supplemented with 10% (v/v) heat inactivated fetal bovine
serum and 1% (v/v) enicillin/streptomycin (Thermo Technologies, New York, NY,
USA) and incubated at 37 °C in a humidified atmosphere with 5% CO2. The TBA
derivatives under study were dissolved in DMSO (Sigma, St. Louis, MO, USA) and
added at required concentrations to the cell culture.
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4.2.2. Cytotoxicity Assay
The cytotoxicity of the compounds was evaluated in vitro via the MTT method
against HepG2, HT-29, Hela, BGC-823, A-549 cell lines with cisplatin as the positive
control. Tumor cells growing in the logarithmic phase were seeded in 96-well plates
at a density 3 × 103 cells/well and incubated overnight. The following day, cells were
then treated with serial dilutions of the tested compounds for 72h. At the end of this
incubation, 20µL of 5 mg/mL methylthiazol tetrazolium (MTT) was added to each
well and incubation proceeded at 37°C for another 4 h. After the supernatant medium
was thrown away, 150 µL dimethylsulphoxide (DMSO) were added to each well and
absorbance was measured at 490 nm using a plate reader (BIORAD 550
spectrophotometer, Bio-rad Life Science Development Ltd., Beijing, China).
Experiments were performed in triplicates and the values were the average of three (n
= 3) independent experiments. The concentration of the compound which gives 50%
growth inhibition corresponds to the IC50. Tumor cell growth inhibitory rate was
calculated in the following Equation (1):
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% inhibition = (1 − Sample group OD/Control group OD) × 100% (1)
4.2.3. Morphological detection of apoptosis using Giemsa staining
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To observe the changes in cell morphology after treatment with BA-25, Giemsa
staining was performed as previously described [19]. Briefly, exponentially growing

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HepG2 cells (4 × 103 cells/well) were cultured in 6-well plates overnight and then
treated with various concentrations of BA-25 (1.5, 3, 6 µM) for an additional 72 h.
Following twice washes with PBS and fixing with cold methanol, cells were stained
with 6% Giemsa (Giemsa, Molecular Probes/Invitrogen Life Technologies, Carlsbad,
CA, USA) solution for 5 min, washed with water and dried. The cell morphological
changes were observed under a fluorescent microscope [38].
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4.2.4. Morphological detection of apoptosis using DAPI staining
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Morphological observation of nuclear changes was performed by DAPI staining in
this assay. HepG2 cells were seeded in the six well plates at a density of 4 × 103
cells/well and were allowed to grow for 12 h. Then the cells were treated with
different concentration ranges of BA-25 (1.5, 3, 6 µM) for 72 hours. After the
treatment period, cells were washed with PBS and fixed with 4% paraformaldehyde.
Then the liquid was removed and the cells were stained with 1 mg/mL DAPI (DAPI,
Molecular Probes/Invitrogen Life Technologies, Carlsbad, CA, USA) for 1 min in
dark. After staining, cells were visualised under a fluorescence microscope [39].
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4.2.5. Detection of apoptosis using Annexin V-FITC/PI staining
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To determine early apoptosis and secondary necrosis, HepG2 cells were stained with
annexin-V FITC apoptosis detection kit (Beijing BioDee Biotech. Co., Ltd., Beijing,
China) as per manufacturer instruction. After exposure to BA-25 (2, 3, 4 µM) for 72
h, HepG2 cells were collected, washed twice with cold PBS and centrifuged at 1000
rpm for 5 min. The resulting pellet was mixed with 200 µL binding buffer of the
Annexin V-FITC kit, then 10 µL FITC labeled annexin V was added and mixed
gently. After incubation at 4°C for 15min in the dark, the cells were washed twice and
resuspended in 300 µL binding buffer containing 10 µL PI. Then the cells were
immediately analyzed with a flow cytometry [40].
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4.2.6. Measurement of mitochondrial membrane potential
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Evaluation of mitochondrial membrane potential in HepG2 cells was performed by
flow cytometry using the rhodamine 123 (Rh123) staining. HepG2 cells (1 × 105 cells/
well) in logarithmic growth phase were incubated in 6-well culture plate for 24 h.
Then the cells were exposed to 2, 3 and 4 µM BA-25 for 72h and harvested by

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trypsinnization. After twice washes with cold PBS, the cells were incubated at 37°C
with 10 µg/mL Rh123 (Rh123, Beijing BioDee Biotech. Co., Ltd., Beijing, China) for
30 min. Following twice washes with PBS, fluorescent intensities were determined by
flow cytometry with excitation and emission wavelength set at 488 nm and 530 nm,
respectively [33, 36].
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4.2.7. Assessment of intracellular Free Ca2+
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For the measurement of intracellular free Ca2+, HepG2 cells were seeded in 6-well
plate at 1 × 105 cells/well and were allowed to grow for 24h. After BA-25 (2, 3, 4 µM)
treatment for 72h, the cells were harvested and washed twice with cold PBS, then
resuspended in HBSS buffer with 10 µM Fluo-3AM (Fluo-3AM, Shanghai Beyotime
Biotech. Co., Ltd., Shanghai, China), and incubated for 30 min at 37 °C in the dark.
The cells were then subjected to flow cytometric analysis at 488 nm excitation
wavelength [36, 37].
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Acknowledgments
This study was supported by the National Natural Science Foundation of China (No.
81173519); 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 young teachers’ scientific research
project of Beijing University of Chinese Medicine (No. 2015-JYB JSMS023).
Author Contributions
Ideas and experiment design: Bing Xu, Peng-Long Wang and Hai-Min Lei; Chemistry
and Biology: Bing Xu, Wen-Qiang Yan, Xin Xu, Yao-Tian Han; Analysis and
interpretation of data: Rui Zhao, Fu-Hao Chu, Gao-Rong Wu; Writing and review of
the manuscript: All the authors; Study supervision: Haimin Lei, Penglong Wang.
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Conflicts of Interest
The authors declare no conflict of interest.
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References

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