Synthesis and biological evaluation of novel ocotillol-type triterpenoid derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2013.07.041

A novel class of ocotillol-type triterpenoid derivatives have been synthesized and evaluated for their in vitro antibacterial activity against several representative pathogenic bacterial strains. Compounds 20(S)-protopanaxadiol (PPD), 3, 5, 16 and 24 exhibited potent antibacterial activity against Gram-positive bacteria. Compounds 3 and 5 also displayed promising antibacterial activity against a community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA; strain USA300). Furthermore, compounds PPD, 3 and 16 combined with two commercially available antibiotics kanamycin and chloramphenicol showed strong synergistic inhibitory effects at their sub-MIC concentrations against S. aureus USA300 and Bacillus subtilis 168. Additionally, cytotoxic activity assay showed that the compounds tested did not affect cell viability of the human epithelial kidney (HEK-293) and human cervical (HeLa) cells at their MICs.

Full text of DOI:10.1016/j.ejmech.2013.07.041

Accepted Manuscript
Synthesis and biological evaluation of novel ocotillol-type triterpenoid derivatives as
antibacterial agents
Zhiwen Zhou, Cong Ma, Hengyuan Zhang, Yi Bi, Xia Chen, Hua Tian, Xiaoni Xie,
Qingguo Meng, Peter John Lewis, Jinyi Xu
PII:
S0223-5234(13)00490-X
10.1016/j.ejmech.2013.07.041
EJMECH 6329
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 May 2013
Revised Date: 16 July 2013
Accepted Date: 17 July 2013
Please cite this article as: Z. Zhou, C. Ma, H. Zhang, Y. Bi, X. Chen, H. Tian, X. Xie, Q. Meng,
P.J. Lewis, J. Xu, Synthesis and biological evaluation of novel ocotillol-type triterpenoid
derivatives as antibacterial agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.07.041.
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ACCEPTED MANUSCRIPT
Graphic abstract
Synthesis and biological evaluation of novel ocotillol-type
triterpenoid derivatives as antibacterial agents
Zhiwen Zhoua, #, Cong Mab, #, Hengyuan Zhanga, #, Yi Bic, Xia Chena, Hua Tiana,
Xiaoni Xiea, Qingguo Mengc,*, Peter John Lewisb,*, Jinyi Xua, *
Compounds 3 and 16 combined with kanamycin and chloramphenicol showed strong
synergistic inhibitory effects at their sub-MIC concentrations against S.aureus USA
300 and B.subtilis 168.
Corresponding authors. Tel.: +86 025 83271299 (J.Y. Xu); Tel.: +86-535-6706022 (Q. G. Meng);
Tel.: +612 4921 5701 (P. J. Lewis).
E-mail addresses: jinyixu@china.com (J.Y. Xu); qinggmeng@163.com (Q. G. Meng);
Peter.Lewis@newcastle.edu.au (P. J. Lewis).

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel ocotillol-type
triterpenoid derivatives as antibacterial agents
Zhiwen Zhoua, #, Cong Mab, #, Hengyuan Zhanga, #, Yi Bic, Xia Chena, Hua Tiana, Xiaoni
Xiea, Qingguo Mengc,*, Peter John Lewisb,*, Jinyi Xua, *
aState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, PR China
bBiological Sciences, School of Environmental and Life Sciences, University of Newcastle,
Callaghan, NSW 2308, Australia
cSchool of Pharmacy, Yantai University, Yantai, 264005, China
# These authors contributed equally to this work
*Corresponding authors:E-Mails: E-Mails: jinyixu@china.com; Tel.: +86-25-8327-1299 (J.Y. Xu.) ;
qinggmeng@163.com; Tel.: +86-535-6706022 (Q. G. Meng); E-Mail: Peter.Lewis@newcastle.edu.au; Tel.:
+612 4921 5701 (P. J. Lewis)
Abstract
A novel class of ocotillol-type triterpenoid derivatives have been synthesized and
evaluated for their in vitro antibacterial activity against several representative pathogenic
bacterial strains. Compounds 20(S)-protopanaxadiol (PPD), 3, 5, 16 and 24 exhibited
potent antibacterial activity against Gram-positive bacteria. Compounds 3 and 5 also
displayed promising antibacterial activity against a community-associated methicillin-
resistant S. aureus (CA-MRSA; strain USA300). Furthermore, compounds PPD, 3 and 16
combined with two commercially available antibiotics kanamycin and chloramphenicol
showed strong synergistic inhibitory effects at their sub-MIC concentrations against
S.aureus USA 300 and B.subtilis 168. Additionally, cytotoxic activity assay showed that
the compounds tested did not affect cell viability of the human epithelial kidney (HEK-
293) and human cervical (HeLa) cells at their MICs.
Keywords
Triterpenoid; Ocotillol; Antibacterial activity; synergistic effect; cytotoxicity
1. Introduction
Since the discovery of Penicillin, the development of antibiotics has been carried out
for over 80 years. In addition to the β-lactam family antibiotics, that have many structural
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variants, other antibiotics or synthetic antimicrobial agents have also been developed,
including the macrolides, tetracyclines, quinolones and glycopeptides which have
different mechanisms of antibacterial action [1]. However, due to horizontal gene
transfer and unlinked point mutations, more and more bacteria with resistance to
antibiotics were isolated, with methicillin-resistant Staphylococcus aureus (MRSA)
infections representing a particularly serious health problem worldwide [2]. More
recently, infections due to community-associated (CA) MRSA, which is responsible for
diseases such as necrotizing pneumonia, severe sepsis and necrotizing fasciitis, have risen
rapidly, sparking fears of an epidemic [3]. In recent decades, vancomycin-resistant
Staphylococcus aureus (VRSA) strains were also identified [4]. Consequently, novel
antimicrobial agents with distinct structures are urgently needed to help battle emerging
antibiotic-resistant bacterial infections.
Natural products have been the single most productive source of leads for the
development of drugs particularly as anti-cancer agents and anti-infectives [5]. The
triterpenoids, including sterols, steroids, and saponins, are a large and structurally diverse
group of natural products with well-characterized biological activities [6]. Many of them
such as ursolic acid (UA), oleanolic acid (OA), betulinic acid (BA) (Figure 1) show
antimicrobial activities [7]. Previous reports indicated that the weak antimicrobial activity
of butulinic acid as compared to its analogues ursolic acid and oleanolic acid is probably
due to lack of the E ring that is present oleanene [8]. However, surprisingly, 23-
hydroxybetulinic acid (HBA) (Figure 1) demonstrates moderate antimicrobial activity
against Gram-positive bacteria Bacillus subtilis (closely related to Bacillus anthracis)
strain 168 and Staphylococcus aureus strain RN4220 in our preliminary testing (Table 1),
which suggests the E ring of oleanene/oleanane in triterpenoid natural products may not
be the unique critical factor for antimicrobial activity.
Although the antibacterial activity of some natural products may be less than that of
certain marketed drugs, their lower cytotoxicity compared to the traditional antibiotics
was preferred. In addition, natural products usually have an element of structural
complexity which is required for the inhibition of many antibacterial protein targets [9].
Ocotillol (Figure 1), a triterpene isolated from Fouquieria splendens Engelm, is
characterized by bearing a five-membered epoxy ring at C-20 [10]. Pseudo-ginsenoside-
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F11 (Figure 1) which is an ocotillol-type ginsenoside found in Panax quinquefolium
(American ginseng) [11] has been proven to possess a protective effect on myocardial
ischemia injury [12]. Recent research showed plant derived triterpenoid saponins
commonly involve in plant defense system by perturbating membranes of bacteria or
virus [13], as well as increasing membrane permeability of mammalian cells [14]. Thus,
triterpenoid derivatives are potential to be developed as antimicrobial agents by affecting
bacterial cell viability through membrane destruction. So far few studies have been
devoted to the antimicrobial effect of ocotillol-type triterpenoid derivatives; furthermore,
compared to oleanane, tetracyclic triterpenoid compounds are more potent in drug
discovery for their low cytotoxicity [15] and diverse functional groups as well as their
prospects for modification. Therefore, the present study described the synthesis of the
tetracyclic triterpenoid derivatives from 20(S)-protopanaxadiol (PPD) and evaluated for
their in vitro antibacterial, synergistic antibacterial activities and cytotoxicity [16].
2. Synthetic chemistry
We chose PPD, one of the main ginsengenin components of Panax ginseng, as our
starting material (Scheme 1), as it can also be isolated from other natural sources [17]. As
previously described [16], the 3, 12-diol of PPD was protected using acetic anhydride in
the presence of DMAP; epoxidation of the resulting acetate 1 followed by nucleophilic
addition in situ furnished a mixture of tetrahydrofuran 2. After base treatment, two
stereoisomeric triols 3 and 4 were obtained with desired yields. Triol 3 was then oxidized
by 3 molar equivalents of pyridinium chlorochromate to provide dione 5, whereas 1
molar equivalent of PCC only oxidized 3-hydroxyl of triol 3 to furnish ketone 6; the same
procedure applied to triol 4 formed dione 7 and ketone 8, respectively (Scheme 2).
Regioselective protection of 3-hydroxyl group of triol 3 as mono acetate, followed by
oxidation and/or esterification of the 12-ol and deprotection of the acetic group gave the
derivatives 15-17. Additionally, treatment of triol 3 with excess acetic anhydride
provided diacetate, which was hydrolyzed in basic media to produce 14. Target
compounds 22-24 were also obtained with the same method (Scheme 2, Scheme 3).
3. Results and Discussion
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We have designed and synthesized a series of ocotillol-type triterpenoid derivatives
based on PPD. The minimum inhibitory concentration (MIC) of the synthesized
compounds was determined against several bacterial strains using a standard LB medium
dilution technique. Initial MIC screening results are presented in Table 1. The data
demonstrated that PPD and the ocotillol-type triterpenoid derivatives 3, 5, 6, 16 and 24
inhibited the growth of Gram-positive bacteria in vitro with MIC values about 4-32
ꢀg/mL. Among the synthesized compounds, 16 was found to be the most active with
MIC value of 4 ꢀg/mL against S. aureus. Meanwhile, stereoisomers 3 and 4 displayed
dramatically dissimilar antibacterial activity against Gram-positive bacteria, which
suggest the stereochemistry of the isopropanol group at C-24 may play a key role for the
binding of the pharmacologically active compounds with the target receptor in bacteria.
However, the activity of compound 5 illustrated that the reverse stereochemistry of C-24
is preferred when the hydroxyl groups at C-3 and C-12 are both oxidized, and compound
6 could only mildly inhibit the growth of B. subtilis when the hydroxyl group at C-3 is
oxidized. With a phthalic acid mono ester at C-12 position, both compounds 16 and 24
displayed good activity against Gram-positive bacteria, B. subtilis with MIC values of 16
ꢀg/mL, Again, their activity against S. aureus confirmed the significance of
stereochemistry, with MICs of 4 and 64 ꢀg/mL, respectively. Surprisingly, 15 and 22
exhibited mild activity against both Gram-positive and Gram-negative bacteria, S. aureus
and E. coli, while 14 and 17 showed activity only against Gram-negative bacteria, which
illustrated ocotillol-type derivatives’ potential to somehow selectively inhibit Gram
positive or negative bacteria.
As shown in Table 2, the bioactive compounds against Gram-(+) bacteria were
chosen for testing against a significant highly pathogenic community-associated
methicillin resistant strain S. aureus USA300 [18]. The results showed compounds 3 and
5 also possess good antibacterial activity against MRSA USA300. However, compound
16 could only exhibited moderate activity against this pathogen. The testing of minimum
bactericidal concentration (MBC) was also carried out. Only 16 and 24 showed direct
bactericidal activity against Gram positive bacteria, including CA-MRSA strain for 16
(16: 64 ꢀg/mL against S. aureus RN 4220 and B. subtilis 168; 128 ꢀg/mL against S.
aureus USA300; 24: 128 ꢀg/mL against B. subtilis 168). The bactericidal effects revealed
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the ocotillol-type triterpenoid derivatives not only can affect bacterial cell viability, but
also cause cell death, which is a common effect by increasing cell membrane
permeability.
Mechanistically, kanamycin is known as a bactericidal agent targeting the 30S of
bacterial ribosome to cause mistranslation and inhibiting translocation during protein
synthesis, while as a bacteriostatic drug, chloramphenicol inhibits the peptidyl transferase
activity of bacterial ribosome, and then stop the bacterial growth by inhibiting protein
synthesis. Like all of the other antibiotics, both of these two drugs have to cross the cell
membrance and enter the cytoplasm for their antibacterial functions. As a result, reducing
membrane permeability is one of the three mechanisms that bacteria generate resistance
to antibiotics including chloramphenicol [19]. Conversely, an antibacterial agent targeting
cell membrane could synergistically strengthen the sensitivity of bacteria to these
antibiotics. The synergistic effects of compounds PPD, 3 and 16 were then investigated at
their sub-MIC concentrations with above two conventional antibiotics against CA-
MRSA S. aureus USA300 and B. subtilis 168 to verify our hypothesis on the effect of
membrane perturbation as the mechanism of action, and the effects were evaluated by
calculating the Fractional Inhibitory Concentration Index (FICI) using fractional
inhibitory concentration (FIC) [20]. The results of the combination assay are listed in
Table 3. As shown in Figure 2, at a specified concentration, most of the combinations
showed effectiveness. Compounds PPD and 3 could reduce the MICs of kanamycin
against S. aureus USA300 from 1 µg/mL to 0.25 and 0.125 µg/mL, respectively
(FICI=0.26, 0.14). Synergistic activity against B.subtilis 168 were also observed when
combination of PPD with kanamycin (FICI=0.25). Furthermore, the MICs of kanamycin
and chloramphenicol were dramatically reduced in combination with compound 16. The
FICIs of 16 and kanamycin or chloramphenicol were calculated to be between 0.004 and
0.008, which was significant smaller than 0.5, suggesting that they acted synergistically
to inhibit the growth of both strains. An additive effect (FICI=0.53) was observed when
PPD was combined with chloramphenicol against B.subtilis 168 and the others’
combinational activities were exhibited as indifferent interactions (1<FICI<2).
In contrast, when PPD, 3 and 16 were combined with kanamycin, the bactericidal
activities of kanamycin against S. aureus USA300 were significantly enhanced from 4
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µg/mL to 2, 1, and 2 µg/mL, respectively. For B.subtilis 168, potent bactericidal effects
were also observed when combination of kanamycin with PPD and 16. Surprisingly,
chloramphenicol alone was bacteriostatic against B.subtilis 168, but exhibited significant
bactericidal avtivity when combined with 16 with MBC value of less than 0.0078.
Reports shown that kanamycin crosses bacterial membrane through lipid-mediated
transportation [21], and chloramphenicol can use both lipid and porin pathways to enter
the bacterial cytoplasm [22]. Compounds PPD, 3 and 16 synergistically enhanced above
two drugs’ antibacterial activity against Gram positive bacteria, which suggest ocotillol-
type derivatives target the phospholipid-mediated pathway, as affecting specifically porin
transportation will not enhance the activity of drugs such as kanamycin. Furthermore, the
outer membrane of Gram negative bacteria consisting lipopolysaccharides (LPS), which
are the essential difference from the membrane structure of Gram positive bacteria, may
explain the ineffectiveness of PPD, 3 and 16 against Gram negative bacteria. However,
as shown in table 1, compounds 14 and 17 displayed specific activity against Gram-
negative bacteria, which could provide us some information for further investigation.
Additionally, as described above, in the presence of 16 at sub-MIC concentration,
chloramphenicol turned from bacteriostatic to bactericidal agent, this interesting result
revealed ocotillol-type derivatives are suitable for combination with other antibiotics,
inculding those bacteriostatics to cause bacterial cell death. This discovery is very
significant and highly potential for the development of new drugs in clinic to reduce the
toxicity of antibiotics, specially the ones like kanamycin and chloramphenicol, which are
currently second-line drugs limited to last resort because of their toxicity.
The cytotoxicity of compounds 3, 5 and 16 were then evaluated by MTT assay. The IC50
and selectivity index values (SI, rate between IC50 against human cervical (HeLa) or
human epithelial kidney (HEK 293) cells and MIC against S. aureus RN4220 and CA-
MRSA USA 300) are presented in Table 4 to determine if their observed antibacterial
activity was caused by selective toxicity towards the bacterial cells. The high SI values
indicate low toxicity of the compounds. As shown in Table 4, compounds 3, 5 and 16
showed much higher IC50 values against both HeLa (~50 ꢀg/mL) and HEK-293 (>150
ꢀg/mL) cells than that of the positive control 5-fluorouracil (~0.8 ꢀg/mL), and thus they
will not affect cell viability at their antibacterial MICs (8, 16, 4 µg/mL). The tested
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compounds 3, 5 and 16 provided a better selectivity index on HEK-293 cell with high
values of 19.3, 10.6 and 43.8 against S. aureus RN4220 and 19.3, 10.6 and 5.5 against
CA-MRSA USA 300, respectively. And for HeLa cell, compounds 3 and 16 have also
exhibited low toxicity (compound 3, SI: 6 against S. aureus RN4220 and CA-MRSA
USA 300; compound 16, SI: 13.6 against S. aureus RN4220 and 1.7 against CA-MRSA
USA 300). These results suggested that compounds 3, 5 and 16 could exhibit in vitro
antibacterial activity at non-cytotoxic concentrations.
Based on the antibacterial activity of ocotillol-type triterpenoid derivatives, a
plausible structure-activity relationships (SARs) can be reasoned out as shown in Figure
3, hydrogen donors at C-3 and C-12 are preferred to maintain the activity against Gram-
positive bacteria; on the contrary, decreased activity was observed when the functional
groups at C-3 and C-12 turned to ketone as hydrogen accepter; However, substitution at
C-12 as non-hydrogen donor ester resulted in mild activity against Gram-negative
bacteria, especially compounds 14 and 17 with 24(S)-configuration displayed specific
activity against Gram-negative bacteria; Furthermore, the stereochemistry at C-24
dramatically affects the antibacterial activity with S-configuration preferred, probably due
to the diferent interaction between the substitution of C-12 and the tetrafuran ring.
4. Conclusions
In this paper we have designed and synthesized a series of ocotillol-type triterpenoid
derivatives based on PPD, in which compounds 3, 5, 16 and 24 showed good
antibacterial activity particularly against Gram-positive bacteria, and compounds 14 and
17 exhibited notably activity against Gram negative bacteria. Additional testing against
CA-MRSA strain USA300 demonstrated compounds 3 and 5 also possess potent
antibacterial activity. The subsequent synergistic antibacterial assay revealed that the
ocotillol-type compounds enhanced the susceptibility of S.aureus USA300 and B.subtilis
168 to kanamycin and chloramphenicol (FICI<0.5). Compounds 3, 5 and 16 were also
evaluated for their cytotoxicity and exhibited no significant influence on cell viability on
both HEK-293 and Hela cells at their MICs. These results suggested that ocotillol-type
triterpenoid derivatives represent as potential leads for the development of antibacterial
agents against antibiotic-resistant superbugs. Further investigation is required to
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determine how those organisms can be targeted with our compounds, and the related
experiments are being carried on in our laboratories and will be reported in due course.
5. Experimental
5.1 Chemistry
Most chemicals and solvents were analytical grade and, when necessary, were purified
and dried with standard methods. Melting points were taken on an XT-4 micro melting
1
point apparatus and uncorrected. H NMR spectra were recorded with a Bruker AV-300
or ACF 500 spectrometer in the indicated solvents (TMS as internal standard): the values
of the chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in
Hz. High-resolution mass spectra were recorded using an Agilent QTOF 6520.
5.1.1 20(S)-Protopanaxadiol (PPD)
PPD were prepared from total ginsenosides by alkaline hydrolysis according to the
literature [23]. mp. 197–199ꢀ; ESI-MS m/z 461.5 [M + H]+; 1H NMR (CDCl3, 300 MHz)
δ 5.16 (t, J = 7.8 Hz, 1H), 3.58 (m, 1H), 3.20 (m, 1H), 1.70 (s, 3H), 1.63 (s, 3H),1.18 (s,
13
3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s, 6H), 0.78 (s, 3H); C NMR (CDCl3, 75 MHz) δ
131.7, 125.0, 77.8, 74.2, 70.8, 55.8, 53.4, 51.5, 50.0, 47.7, 39.7, 38.9, 38.9, 37.0, 34.7,
34.5, 31.7, 31.2, 28.0, 27.3, 26.9, 26.4, 25.7, 22.3, 18.2, 17.7, 16.8, 16.1, 15.7, 15.3. HR-
MS (ESI) m/z: calculated for C30H52NaO3[M + Na]+: 483.3809, found: 483.3818.
5.1.2 (20S,24S)-Epoxy-dammarane-3β,12β,25-triol (3) and (20S,24R)-Epoxy-
dammarane-3β,12β,25-triol (4)
To a solution of PPD (500 mg, 1.08 mmol) and DMAP (20 mg, 0.16 mmol) in dry
pyridine (4 mL), acetic anhydride (0.42 mL, 4.43 mmol) was slowly added, and the
mixture stirred for 24 h at room temperature.The reaction mixture was then diluted with
ethyl acetate, washed with 10% HCl, water and brine successively, dried over anhydrous
sodium sulfate, concentrated and the residue was purified by column chromatography,
eluting with petroleum ether :ethyl acetate 10:1 mixture. 3β, 12β-diacetoxy-20 (S)-
protopanaxadiol (1) was obtained as a white solid (515mg, 86%).
A solution of 1 (208mg, 0.38mmol) in dichloromethane (10 ml) was cooled to -3°C.
Then a solution of m-CPBA (185mg, 75%, 0.76mmol) in dichloromethane (10 ml) was
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added slowly and stirred for 2 h. The organic solution was washed with saturated. aq.
NaHCO3, saturated. aq. Na2S2O3, water and brine successively, and dried over Na2SO4.
The dichloromethane was evaporated in vacuo and the residue was purified over silica gel
(8:1 petroleum ether-ethyl acetate) to get (20S, 24)-epoxy-3β, 12β-diacetoxy-dammarane-
25-ol (2) as a white solid (148mg, 69%).
Potassium hydroxide (255 mg, 4.55 mmol) was added to a solution of 2 (405 mg, 0.72
mmol) in methanol (10 mL). The resulting mixture was refluxed for 2 h. The solvent was
removed under reduced pressure, ethyl acetate was added,then washed with water and
bine, dried over anhydrous sodium sulfate, concentrated in vacuo and the residue was
purified over silica gel (2:1-1:1 petroleum ether-ethyl acetate), and then crystallized from
ethyl acetate yielded (20S, 24S)-Epoxy-dammarane-3β, 12β, 25-triol (3) as white pellet-
like crystal (166 mg, 48%) and from acetone yielded (20S, 24R)-Epoxy-dammarane-3β,
12β, 25-triol (4) as white lump-like crystal (155 mg, 45%).
(20S,24S)-Epoxy-dammarane-3β,12β,25-triol (3). mp. 224–225ꢀ; ESI-MS m/z 477.3
[M + H]+; 1H NMR (CDCl3, 300 MHz) δ 3.87 (dd, J = 10.2Hz, 5.1 Hz, 1H), 3.52 (td, J =
10.2Hz, 4.8 Hz, 1H), 3.19 (dd, J = 10.8Hz, 5.4 Hz, 1H), 2.25 (td, J = 10.5Hz, 4.2 Hz, 1H),
1.27 (s, 3H), 1.23 (s, 3H), 1.10 (s, 3H), 1.01 (s, 3H), 0.97(s, 3H) , 0.91 (s, 3H), 0.88 (s,
13
3H), 0.78 (s, 3H); C NMR (CDCl3, 75 MHz) δ 87.3, 87.0, 78.7, 70.4, 69.9, 55.8, 52.0,
50.1, 48.8, 48.7, 39.6, 38.8 (overlapping signal), 37.0, 34.6, 32.1, 31.5, 31.5, 28.7, 28.4,
27.9, 27.8, 27.3, 24.9, 24.1, 18.2, 17.6, 16.1, 15.3, 15.2. HR-MS (ESI) m/z: calculated for
C30H53O4[M + H]+: 477.3938, found: 477.3946.
(20S,24R)-Epoxy-dammarane-3β,12β,25-triol (4). mp. 167–169ꢀ; ESI-MS m/z 477.3
1
[M + H]+; H NMR (CDCl3, 300 MHz) δ 3.84 (dd, J = 8.4Hz, 6.6 Hz, 1H), 3.52 (td, J =
10.5Hz, 4.8 Hz, 1H), 3.19 (dd, J = 10.8Hz, 5.1 Hz, 1H), 2.19 (td, J = 10.8Hz, 3.6 Hz, 1H),
1.28 (s, 3H), 1.27 (s, 3H), 1.10 (s, 3H), 0.99 (s, 3H), 0.97(s, 3H) , 0.90 (s, 3H), 0.86 (s,
13
3H), 0.78 (s, 3H); C NMR (CDCl3, 75 MHz) δ 86.4, 85.3, 78.7, 70.9, 70.0, 55.9, 51.9,
50.4, 49.3, 47.9, 39.7, 38.9, 38.8, 37.1, 34.7, 32.5, 31.2, 31.1, 28.5, 27.9, 27.8, 27.5, 27.39,
26.0, 24.9, 18.2, 18.1, 16.2, 15.3, 15.2. HR-MS (ESI) m/z: calculated for C30H53O4 [M +
H]+: 477.3938, found: 477.3934.
5.1.3 (20S,24S)-Epoxy-25-hydroxy-dammarane-3,12-dione (5)
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To a solution of 3 (100 mg, 0.21 mmol) in dry dichloromethane (6 mL) was added
pyridinium chlorochromate (131 mg, 0.60 mmol), the mixture was stirred at room
temperature for 8 h, then filtrated, concentrated in vacuo and the residue was purified
over silica gel (4:1 petroleum ether-ethyl acetate) to give the product as a white solid (75
mg, 75%). mp. 169–171ꢀ; ESI-MS m/z 473.4 [M + H]+; 1H NMR (CDCl3, 300 MHz) δ
3.69-3.73 (m, 1H), 3.01 (d, J = 9.8Hz, 1H), 2.49-2.58 (m, 1H), 2.45-2.47 (m, 1H), 2.24-
2.29 (m, 1H), 2.20-2.30 (m, 2H), 1.40 (s, 3H), 1.25 (s, 6H),1.20 (s, 3H), 1.10 (s, 3H), 1.04
(s, 3H), 0.88 (s, 3H), 0.87 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 216.6, 216.6, 87.6, 85.3,
70.2, 57.3, 56.9, 55.1, 53.7, 47.3, 40.2, 39.8, 39.1, 37.3, 36.5, 33.8, 33.6, 31.9, 29.3, 27.7,
26.7, 26.6, 26.2, 24.8, 23.9, 21.0, 19.7, 16.5, 15.7, 15.2. HR-MS (ESI) m/z: calculated for
C30H48NaO4 [M + Na]+: 495.3445, found: 495.3453.
5.1.4 (20S,24S)-Epoxy-12β,25-dihydroxy-dammarane-3-one (6)
To a solution of 3 (86 mg, 0.18mmol) in dry dichloromethane (8 mL) was added
pyridinium chlorochromate (87 mg, 0.40 mmol), the mixture was stirred at room
temperature for 8 h,then filtered, concentrated in vacuo, and the residue was purified over
silica gel (1:1 petroleum ether-ethyl acetate) to give the product as a white solid (55 mg,
65%). mp. 168–170ꢀ; ESI-MS m/z 475.3 [M + H]+; 1H NMR (CDCl3, 500 MHz) δ 3.89
(dd, J = 10.5Hz, 5.0 Hz, 1H), 3.54 (td, J = 10.0Hz, 4.5Hz, 1H), 2.49-2.54 (m, 1H), 2.45
(ddd, J = 11.0Hz, 8.0Hz, 3.5Hz, 1H), 2.27 (td, J = 10.5Hz, 4.5Hz, 1H), 1.28 (s, 3H), 1.23
(s, 3H), 1.11 (s, 3H), 1.08 (s, 3H), 1.053 (s, 3H), 1.046 (s, 3H), 0.96 (s, 3H), 0.93 (s, 3H);
13C NMR (CDCl3, 125 MHz) δ 217.5, 87.3, 87.0, 70.2, 69.9, 55.2, 52.1, 49.4, 48.8, 48.8,
47.2, 39.6, 39.5, 36.7, 33.9, 33.9, 32.1, 32.0, 31.5, 28.7, 28.4, 27.8, 26.6, 24.9, 24.2, 20.8,
19.6, 17.6, 15.9, 15.1. HR-MS (ESI) m/z: calculated for C30H51O4 [M + H]+: 475.3782,
found: 475.3780.
5.1.5 (20S,24R)-Epoxy-25-hydroxy-dammarane-3,12-dione (7)
To a solution of 4 (160 mg, 0.33 mmol) in dry dichloromethane (10 mL) was added
pyridiniumchlorochromate (217 mg, 1.00 mmol), the mixture was stirred at room
temperature for 8 h, then filtered, concentrated in vacuo and the residue was purified over
silica gel (3:1 petroleum ether-ethyl acetate) to give the product as a white solid (119 mg,
76%). mp. 157–159ꢀ; ESI-MS m/z 473.4 [M + H]+; 1H NMR (CDCl3, 300 MHz) δ 3.69
10

ACCEPTED MANUSCRIPT
(dd, J = 10.0 Hz, 6.5 Hz, 1H), 2.93 (d, J = 9.5 Hz, 1H), 2.56 (td, J = 10.5 Hz, 4.5 Hz, 1H),
2.48-2.50 (m, 1H), 2.42 (ddd, J = 11.5 Hz, 7.5 Hz, 4.0 Hz, 1H), 2.20-2.30 (m, 2H), 1.26
(s, 3H), 1.24 (s, 3H),1.21 (s, 3H), 1.11 (s, 3H), 1.10 (s, 3H), 1.06 (s, 3H), 1.03 (s, 3H),
0.78 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 216.7, 216.7, 85.0, 83.5, 71.1, 57.1, 55.7, 55.1,
53.5, 47.3, 40.3, 39.8, 39.1, 37.2, 35.0, 33.7, 33.5, 32.0, 29.6, 27.6, 26.6, 26.5, 25.6, 24.9,
24.4, 20.9, 19.7, 16.5, 15.7, 15.2. HR-MS (ESI) m/z: calculated for C30H49O4 [M + H]+:
473.3625, found: 473.3639.
5.1.6 (20S,24R)-Epoxy-12β,25-dihydroxy-dammarane-3-one (8)
To a solution of 4 (100mg, 0.21mmol) in dry dichloromethane (8 ml) was added
pyridinium chlorochromate (87mg, 0.40mmol), the mixture was stirred at room
temperature for 3 h. Then filtrated, concentrated in vacuo and the residue was purified
over silica gel (5:1 petroleum ether-ethyl acetate) to give the product as a white solid (65
mg, 66%). mp. 162–164ꢀ; ESI-MS m/z 475.3 [M + H]+; 1H NMR (CDCl3, 500 MHz) δ
3.85 (dd, J = 8.6 Hz, 6.0 Hz, 1H), 3.53 (td, J = 10.5 Hz, 4.5 Hz, 1H), 2.48-2.54 (m, 1H),
2.42 (ddd, J = 11.5 Hz, 7.5 Hz, 4.0 Hz, 1H), 2.21 (td, J = 11.5 Hz, 3.5 Hz, 1H), 1.28 (s,
6H), 1.10 (s, 3H), 1.07 (s, 3H), 1.04 (s, 3H), 1.02 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H); 13C
NMR (CDCl3, 125 MHz) δ 217.4, 86.4, 85.3, 70.7, 70.0, 55.3, 51.9, 49.7, 49.4, 47.8, 47.2,
39.7, 39.5, 36.7, 34.0, 33.9, 32.5, 31.6, 31.1, 28.5, 27.8, 27.4, 26.6, 26.0, 24.9, 20.8, 19.6,
17.9, 15.9, 15.1. HR-MS (ESI) m/z: calculated for C30H51O4 [M + H]+: 475.3782, found:
475.3790.
5.1.7 (20S,24S)-Epoxy-12β-O-acetyl-dammarane-3β,25-diol (14)
To a solution of 3 (500 mg, 1.05 mmol) and DMAP (20 mg, 0.16 mmol) in dry
pyridine (4 ml), acetic anhydride (0.30 ml, 3.15 mmol) was slowly added. The reaction
mixture was stirred at room temperature for 5 h, then extracted with ethyl acetate, and the
organic layer was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, concentrated, and purified by column chromatography
(petroleum ether: ethyl acetate = 8:1) to yield a white solid (500mg, 85%): (20S, 24S)-
Epoxy-3β, 12β-diacetoxy-dammarane -25-ol (10).
To a solution of 10 (100 mg, 0.18 mmol) in methanol (10 ml), Potassium hydroxide
(21 mg, 0.36 mmol) was added. The reaction mixture was stirred at room temperature for
6 h. The methanol was evaporated in vacuo, ethyl acetate was added. The organic layer
11

ACCEPTED MANUSCRIPT
was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and
purified over silica gel (petroleum ether: ethyl acetate=4:1) to get (20S, 24S)-Epoxy-12β-
O-acetyl-dammarane-3β, 25-diol (14) as a white solid (60 mg, 65%). mp. 200–203ꢀ;
ESI-MS m/z 519.4 [M + H]+; 1H NMR (CDCl3, 300 MHz) δ 4.83 (td, J=10.6 Hz, 5.4 Hz,
1H), 3.63 (t, J=8.6 Hz, 6.3Hz, 1H), 3.20 (dd, J=10.9 Hz, 4.1Hz, 1H), 2.13-2.28 (m, 1H),
2.01 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.09 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.93 (s,
13
3H), 0.85 (s, 3H), 0.78 (s, 3H); C NMR (CDCl3, 75 MHz) δ 170.8, 85.7, 84.7, 78.9,
75.8, 70.7, 56.0, 52.6, 50.4, 49.9, 46.6, 40.0, 39.8, 39.1, 39.0, 37.3, 34.7, 31.2, 28.6, 28.2,
27.8, 27.4, 27.1, 25.9, 24.2, 23.0, 22.1, 18.4, 17.6, 16.2, 15.7, 15.6. HR-MS (ESI) m/z:
calculated for C32H54NaO5 [M + Na]+: 541.3863, found: 541.3863.
5.1.8 (20S,24S)-Epoxy-3β,25-dihydroxy-dammarane-12-one (15)
To a solution of 3 (100 mg, 0.21 mmol) and DMAP (10 mg, 0.08 mmol) in dry
pyridine (4 ml) was slowly added acetic anhydride (0.03 ml, 0.32 mmol), the mixture was
stirred for 12 h at room temperature, then diluted with ethyl acetate, washed with 10%
HCl, water and brine successively, dried over anhydrous sodium sulfate, concentrated
and the residue was purified by column chromatography, eluting with petroleum ether :
ethyl acetate 3:1 mixture. (20S, 24S)-Epoxy-3β-acetoxy-dammarane-12β, 25-diol (9) was
obtained as a white solid (94 mg, 86%).
To a solution of 9 (120 mg, 0.23 mmol) in dry dichloromethane (5 mL) was added
pyridiniumchlorochromate (120 mg, 0.54 mmol), the mixture was stirred at room
temperature for 10 h, then filtrated, concentrated in vacuo and the residue was purified
over silica gel (4:1 petroleum ether-ethyl acetate) to give (20S, 24S)-Epoxy-3β-acetoxy-
25-hydroxy-dammarane-12-one (11) as a white solid (85 mg, 72%).
To a solution of 11 (85 mg, 0.16 mmol) in methanol (5 mL) was added potassium
hydroxide (40 mg, 0.71 mmol), the mixture was refluxed for 5 h. The solvent was
removed in vacuo and ethyl acetate was added, then washed with water and brine, dried
over anhydrous Na2SO4. The residue was purified by column chromatography over silica
gel (2:1 petroleum ether-ethyl acetate) to afford (20S, 24S)-Epoxy-3β, 25-dihydroxy-
dammarane-12-one (15) as white powder (68 mg, 87%). mp. 162–164ꢀ; ESI-MS m/z
475.4 [M + H]+; 1H NMR (CDCl3, 500 MHz) δ 3.71 (dd, J = 10.5 Hz, 6.0 Hz, 1H), 3.20
12

ACCEPTED MANUSCRIPT
(dd, J = 11.0 Hz, 4.5 Hz, 1H), 2.96 (d, J = 9.5 Hz, 1H), 2.53 (td, J = 10.5 Hz, 4.5 Hz, 1H),
2.20-2.22 (m, 2H), 1.20 (s, 3H), 1.19(s, 3H), 1.09 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.94
(s, 3H), 0.80 (s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 211.0, 87.5, 85.2, 78.5,
70.2, 57.2, 55.9, 55.7, 54.3, 40.3, 39.6, 38.8, 38.5, 37.6, 36.5, 34.2, 31.9, 29.6, 27.9, 27.6,
27.1, 26.5, 26.1, 24.7, 23.9, 18.3, 16.5, 16.0, 15.5, 15.2. HR-MS (ESI) m/z: calculated for
C30H51O4 [M + H]+: 475.3782, found: 475.3791.
5.1.9 (20S,24S)-Epoxy-12β-O-(2-carboxybenzoyl)-dammarane-3β,25-diol (16)
To a solution of 3 (200 mg, 0.42 mmol) and DMAP (20 mg, 0.16 mmol) in dry
pyridine (4 ml), acetic anhydride (0.06 ml, 0.63 mmol) was slowly added. The reaction
mixture was stirred at room temperature for 5 h, then extracted with ethyl acetate, and the
organic layer was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, concentrated, and purified by column chromatography
(petroleum ether: ethyl acetate = 4:1) to yield a white solid (178mg, 82%): (20S, 24S)-
Epoxy-3β-O-acetyl-dammarane-12β, 25-diol (9).
To a solution of 9 (178 mg, 0.34 mmol) and DMAP (12 mg, 0.10 mmol) in dry
pyridine (4 ml), phthalic anhydride (164mg, 1.11 mmol) was added. The reaction mixture
was stirred at 115oC for 20 h, then extracted with ethyl acetate, and the organic layer was
washed with 10% HCl, water and brine successively, dried over anhydrous sodium
sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl
acetate = 1:1) to yield the product as a white solid (124mg, 55%): (20S, 24S)-Epoxy-3β-
O-acetyl-12β-O-(2-carboxy benzoyl)-dammarane-25-ol (12).
To a solution of 12 (100 mg, 0.15 mmol) in methanol (10 ml), Potassium hydroxide
(17 mg, 0.30 mmol) was added. The reaction mixture was stirred at room temperature for
6 h. The methanol was evaporated in vacuo, ethyl acetate was added. The organic layer
was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and
purified over silica gel (dichloromethane : methanol=80:1) to get (20S, 24R)-Epoxy-12β-
O-(2-carboxy benzoyl)-dammarane-3β, 25-diol (16) as a white solid (56 mg, 60%). mp.
1
192–195 ; ESI-MS m/z 625.4 [M + H]+; H NMR (CDCl3, 300 MHz) δ 7.71-7.75 (m,
2H), 7.44-7.52 (m, 2H), 5.11 (td, J=10.5 Hz, 5.5 Hz, 1H), 3.55 (dd, J=7.0 Hz, 6.2 Hz,
1H), 3.18 (dd, J=11.0 Hz, 4.9 Hz, 1H), 2.04-2.18 (m, 2H), 1.27 (s, 3H),1.11 (s, 3H), 1.05
(s, 3H),1.01 (s, 3H), 0.98 (s, 3H),0.97 (s, 3H),0.86 (s, 3H),0.75 (s, 3H); 13C NMR (CDCl3,
13

ACCEPTED MANUSCRIPT
75 MHz) δ172.8, 167.7, 131.6, 130.2, 129.5, 129.2, 86.0, 85.0, 79.0, 76.6, 71.1, 56.1,
52.9, 50.2, 50.2, 46.9, 39.8, 39.2, 39.1, 39.0, 37.3, 34.8, 31.4, 28.2, 27.6, 27.4, 27.3, 27.2,
26.4, 24.0, 23.7, 18.4, 18.0, 16.3, 15.8, 15.6. HR-MS (ESI) m/z: calculated for
C38H56NaO7 [M + Na]+: 647.3918, found: 647.3922.
5.1.11 (20S,24S)-Epoxy-12β-O-cinnamoyl-dammarane-3β,25-diol (17)
To a solution of 3 (200 mg, 0.42 mmol) and DMAP (20 mg, 0.16 mmol) in dry pyridine
(4 ml), acetic anhydride (0.06 ml, 0.63 mmol) was slowly added. After stirring at room
temperature for 5 h. The reaction mixture was extracted with ethyl acetate, and the
organic layer was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, concentrated, and purified by column chromatography
(petroleum ether: ethyl acetate = 2:1) to yield a white solid (163mg, 75%): (20S, 24S)-
Epoxy-3β-O-acetyl-dammarane-12β, 25-diol (9).
To a solution of 9 (163 mg, 0.31 mmol) and DMAP (20 mg, 0.16 mmol) in dry
pyridine (4 mL), cinnamoyl chloride (154 mg, 0.93 mmol) was added. The reaction
mixture was stirred at 120oC for 25 h, then diluted with ethyl acetate, and the organic
layer was washed with 10% HCl, water and brine successively, dried over anhydrous
sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:
ethyl acetate = 4:1) to yield the product as a white solid (104mg, 52%): (20S, 24S)-
Epoxy-3β-O-acety1-12β-O-benzene acryloyl-dammarane-25-ol (13).
To a solution of 13 (104 mg, 0.16 mmol) in methanol (10 mL), Potassium hydroxide
(18 mg, 0.32 mmol) was added. The reaction mixture was stirred at room temperature for
6 h. The methanol was evaporated in vacuo, ethyl acetate was added. The organic layer
was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and
purified over silica gel (petroleum ether: ethyl acetate = 2:1) to get (20S, 24S)-Epoxy-
12β-O-benzene acryloyl-dammarane-3β, 25-diol (17) as a white solid (63 mg, 66%). mp.
188–192ꢀ; ESI-MS m/z 607.4 [M + H]+; 1H NMR (CDCl3, 300 MHz) δ 7.68 (d, J=16.0
Hz, 1H), 7.52-7.55 (m, 2H), 7.41 (t, J=3.4 Hz, 3.1Hz, 3H), 6.38 (d, J=16.0 Hz, 1H), 5.02
(td, J=10.5 Hz, 5.5 Hz, 1H), 3.63 (dd, J=7.0 Hz, 6.2 Hz, 1H), 3.23 (dd, J=11.0 Hz, 4.9 Hz,
1H), 2.11-2.29 (m, 2H), 1.26 (s, 3H), 1.18 (s, 3H), 1.08 (s, 3H), 1.04 (s, 3H), 1.00(s, 3H),
0.99 (s, 3H), 0.87 (s, 3H), 0.79 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 166.7, 145.1, 134.6,
130.6, 129.1, 128.3, 118.9, 85.7, 84.7, 79.0, 75.8, 70.7, 56.0, 52.6, 50.6, 50.0, 46.8, 40.2,
14

ACCEPTED MANUSCRIPT
39.8, 39.1, 39.0, 37.4, 34.7, 31.1, 28.7, 28.2, 27.8, 27.5, 27.2, 25.9, 24.2, 23.1, 18.5, 17.7,
16.2, 15.8, 15.6. HR-MS (ESI) m/z: calculated for C39H58NaO5 [M + Na]+: 629.4190,
found: 629.4192.
5.1.12 (20S,24R)-Epoxy-12β-O-acetyl-dammarane-3β,25-diol (22)
To a solution of 4 (500 mg, 1.05 mmol) and DMAP (20 mg, 0.16 mmol) in dry
pyridine (4 mL), acetic anhydride (0.30 mL, 3.15 mmol) was slowly added. The reaction
mixture was stirred at room temperature for 5 h, then extracted with ethyl acetate, and the
organic layer was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, concentrated, and purified by column chromatography
(petroleum ether: ethyl acetate = 8:1) to yield a white solid (500mg, 85%): (20S, 24R)-
Epoxy-3β, 12β-diacetoxy-dammarane-25-ol (19).
To a solution of 19 (100 mg, 0.18 mmol) in methanol (10 mL), Potassium hydroxide
(21 mg, 0.36 mmol) was added. The reaction mixture was stirred at room temperature for
6 h. The methanol was evaporated in vacuo, ethyl acetate was added. The organic layer
was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and
purified over silica gel (petroleum ether: ethyl acetate=4:1) to get (20S, 24R)-Epoxy-12β-
O-acetyl-dammarane-3β, 25-diol (22) as a white solid (60 mg, 65%). mp. 205–208ꢀ;
ESI-MS m/z 519.4 [M + H]+; 1H NMR (CDCl3, 300 MHz) δ 4.83 (td, J=10.6 Hz, 5.3 Hz,
1H), 3.65 (t, J=7.1 Hz, 6.7 Hz, 1H), 3.19 (dd, J=11.2 Hz, 4.2 Hz, 1H), 2.14-2.33 (m, 1H),
2.01 (s, 3H), 1.28 (s, 3H), 1.26 (s, 3H), 1.10 (s, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.94 (s,
13
3H), 0.85 (s, 3H), 0.77 (s, 3H); C NMR (CDCl3, 75 MHz) δ 170.8, 85.9, 83.5, 78.8,
75.8, 71.2, 56.2, 56.0, 52.4, 50.7, 50.0, 46.5, 39.8, 39.1, 39.0, 37.3, 34.7, 31.4, 28.6, 28.2,
27.7, 27.4, 27.0, 26.3, 24.4, 22.4, 22.0, 18.4, 17.8, 16.2, 15.7, 15.6. HR-MS (ESI) m/z:
calculated for C32H54NaO5 [M + Na]+: 541.3863, found: 541.3865.
5.1.13 (20S,24R)-Epoxy-3β,25-dihydroxy-dammarane-12-one (23)
To a solution of 4 (100 mg, 0.21 mmol) and DMAP (10 mg, 0.08 mmol) in dry
pyridine (4 ml) was slowly added acetic anhydride (0.03 ml, 0.32 mmol), the mixture was
stirred for 12 h at room temperature,then diluted with ethyl acetate, washed with 10%
HCl, water and brine successively, dried over anhydrous sodium sulfate, concentrated
and the residue was purified by column chromatography,eluting with petroleum
15

ACCEPTED MANUSCRIPT
ether :ethyl acetate 10:1 mixture. (20S, 24R)-Epoxy-3β-acetoxy-dammarane-12β, 25-diol
(18) was obtained as a white solid (94 mg, 86%).
To a solution of 18 (120 mg, 0.23 mmol) in dry dichloromethane (5 mL) was added
pyridinium chlorochromate (120 mg, 0.54 mmol), the mixture was stirred at room
temperature for 10 h, then filtered, concentrated in vacuo and the residue was purified
over silica gel (10:1 petroleum ether-ethyl acetate) to give (20S, 24R)-Epoxy-3β-acetoxy
-25-hydroxy-dammarane-12-one (20) as a white solid (85 mg, 72%).
To a solution of 20 (85 mg, 0.16 mmol) in methanol (5 mL) was added potassium
hydroxide (40 mg, 0.71 mmol), and the mixture was refluxed for 5 h. The solvent was
removed in vacuo and ethyl acetate was added, and then washed with water and brine,
dried over anhydrous Na2SO4. The residue was purified by column chromatography over
silica gel (2:1 petroleum ether-ethyl acetate) to afford (20S, 24R)-Epoxy-3β, 25-
dihydroxy-dammarane-12-one (23) as white powder (68 mg, 87%). mp. 193–195ꢀ; ESI-
MS m/z 475.4 [M + H]+; 1H NMR (CDCl3, 500 MHz) δ 3.69 (dd, J = 8.5 Hz, 6.0 Hz, 1H),
3.19 (dd, J = 11.5 Hz, 5.0 Hz, 1H), 2.90 (d, J = 10.0 Hz, 1H), 2.57 (td, J = 10.5 Hz, 4.5
Hz, 1H), 2.19-2.21 (m, 2H), 1.207(s, 3H), 1.198 (s, 3H), 1.11 (s, 3H), 1.10 (s, 3H), 0.99
13
(s, 3H), 0.93 (s, 3H), 0.80 (s, 3H), 0.76 (s, 3H); C NMR (CDCl3, 125 MHz) δ 211.4,
85.1, 83.5, 78.5, 71.1, 57.0, 55.7, 55.7, 54.2, 40.3, 39.7, 38.8, 38.5, 37.6, 35.1, 34.2, 32.0,
29.6, 27.9, 27.5, 27.1, 26.5, 25.5, 24.9, 24.3, 18.3, 16.6, 16.0, 15.5, 15.2. HR-MS (ESI)
m/z: calculated for C30H51O4 [M + H]+: 475.3782, found: 475.3791.
5.1.14 (20S,24R)-Epoxy-12β-O-(2-carboxy benzoyl)-dammarane-3β,25-diol (24)
To a solution of 4 (200 mg, 0.42 mmol) and DMAP (20 mg, 0.16 mmol) in dry
pyridine (5 mL), acetic anhydride (0.06 mL, 0.63 mmol) was slowly added. The reaction
mixture was stirred at room temperature for 5 h, then extracted with ethyl acetate, and the
organic layer was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, concentrated, and purified by column chromatography
(petroleum ether: ethyl acetate = 4:1) to yield a white solid (178 mg, 82%): (20S, 24R)-
Epoxy-3β-O-acetyl-dammarane-12β, 25-diol (18).
To a solution of 18 (178 mg, 0.34 mmol) and DMAP (12 mg, 0.10 mmol) in dry
pyridine (4 mL), phthalic anhydride (164 mg, 1.11 mmol) was added. The reaction
mixture was stirred at 120oC for 25 h, then extracted with ethyl acetate, and the organic
16

ACCEPTED MANUSCRIPT
layer was washed with 10% HCl, water and brine successively, dried over anhydrous
sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:
ethyl acetate = 1:1) to yield the product as a white solid (124 mg, 55%): (20S, 24R)-
Epoxy-3β-O-acetyl-12β-O-(2-carboxy benzoyl)-dammarane-25-ol (21).
To a solution of 21 (100 mg, 0.15 mmol) in methanol (10 mL), Potassium hydroxide
(17 mg, 0.30 mmol) was added. The reaction mixture was stirred at room temperature for
6 h. The methanol was evaporated in vacuo, ethyl acetate was added. The organic layer
was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and
purified over silica gel (dichloromethane: methanol=80:1) to get (20S, 24R)-Epoxy-12β-
O-(2-carboxy benzoyl)-dammarane-3β, 25-diol (24) as a white solid (56 mg, 60%). mp.
1
196–198ꢀ; ESI-MS m/z 625.4 [M + H]+; H NMR (CDCl3, 300 MHz) δ 7.84-7.86 (m,
1H), 7.68-7.70 (m, 1H), 7.56-7.59 (m, 2H), 5.12 (td, J=10.5 Hz, 5.0 Hz, 1H), 3.56 (t,
J=7.5 Hz, 6.5 Hz, 1H), 3.26 (dd, J=10.5 Hz, 4.5 Hz, 1H), 2.11-2.19 (m, 2H), 1.28 (s, 3H),
1.09 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.03(s, 3H) , 1.00 (s, 3H), 0.87 (s, 3H), 0.76 (s,
3H); 13C NMR (CDCl3, 75 MHz) δ 170.4, 167.4, 133.1, 131.4, 131,2, 130.8, 129.5, 128.9,
85.9, 83.1, 78.8, 76.7, 71.3, 55.8, 52.4, 50.2, 50.0, 46.5, 39.6, 38.8, 38.7, 38.6, 37.1, 34.5,
31.5, 27.9, 27.7, 27.1, 27.0, 26.5, 26.2, 23.9, 21.9, 18.2, 17.8, 16.0, 15.5, 15.3. HR-MS
(ESI) m/z: calculated for C38H56NaO7 [M + Na]+: 647.3918, found: 647.3916.
5.2 Pharmacology
Antibacterial activity assay
The minimum inhibitory concentration (MIC) of the synthesized compounds was
determined against several bacterial strains using a standard LB medium dilution
technique as described below. The compounds were dissolved to 50 mM in DMSO and
transferred into 100 µl of Luria-Bertani medium as a serial dilution from 128 ꢀg/ml to 2
ꢀg/ml in 96-well NUNC Microwell™ plates. Bacteria were grown at 37°C in 5 ml LB
with shaking until the optical density reached 0.6-0.7, and 5 µl of the culture was added
to each well. The plate was incubated in a FLUOstar Optima plate reader (BMG Labtech)
at 37°C with 600 rpm shaking. The OD600 of the culturewas taken every 10 min using LB
17

ACCEPTED MANUSCRIPT
as the blank. The samples were tested in triplicate and the growth pattern of each sample
was compared to cells exposed to equal amounts of DMSO.
Synergistic antibacterial activity assay
The synergistic antibacterial activity of the synthesized compounds was determined
against S. aureus strain USA300 and B. subtilis strain 168 together with kanamycin or
chloramphenicol. Kanamycin was dissolved in MilliQ water to 50 mM and
chloramphenicol was dissolved in DMSO. Using a standard LB medium dilution
technique, kanamycin or chloramphenicol was transferred into 100 µl of Luria-Bertani
medium as a serial dilution from 1 ꢀg/ml to 0.0020 ꢀg/ml or from 4 ꢀg/ml to 0.0078
ꢀg/ml, respectively (minimum bactericidal or bacteriostatic concentrations) in 96-well
NUNC Microwell™ plates. Bacteria were grown at 37°C in 5 ml LB with shaking until
the optical density reached 0.6-0.7, 5 µl of the culture and the compounds to be tested at
half of their minimum inhibitory concentrations were added to each well. The plate was
incubated in a FLUOstar Optima plate reader (BMG Labtech) at 37°C with 600 rpm
shaking. The OD600 of the culture was taken every 10 min using LB as the blank. The
samples were tested in triplicate and the growth pattern of each sample was compared to
cells exposed to equal amounts of DMSO. The FICI was calculated using fractional
inhibitory concentration (FIC) according to the formulae from the literature [19] (Table
3).
MTT cytotoxicity assay
The MTT assay was employed in cytotoxicity assay in vitro, which was performed in
96-well plates. Human cervical (HeLa) and human epithelial kidney (HEK-293) cells at
the log phase of their growth cycle (5×104 cell/mL) were added to each well (100
µL/well), then treated in three replicates at various concentrations of the samples (0.39-
100 µg/mL), and incubated for 24 h at 37
in a humidified atmosphere of 5% CO2.
After 72 h, 20 µL of MTT solution (5 mg/mL) per well was added to each cultured
medium, which was incubated for another 4 h. Then DMSO was added to each well (150
µL/well). After 10 min at room temperature, the OD of each well was measured on a
Microplate Reader (BIO-RAD Instruments Inc NO.550) at the wavelength of 490 nm. In
these experiments, the negative reference agent was 0.1% DMSO, and 5-fluorouracil was
used as the positive reference with the concentration of 10 µg/mL.
18

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Acknowledgements
The authors are grateful to National Natural Science Foundation of China
(No.81001358), Promotive research fund for excellent young and middle-aged scientists
of Shandong Province (No. BS2010YY073), Shandong Provincial Natural Science
Foundation, China (No. ZR2012HM036), Open Project Program of State Key Laboratory
of Natural Medicines, China Pharmaceutical University (No.201201), Taishan Scholar
Project to Fenghua Fu and Australian NHMRC grant APP1008014 for financial support.
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Figure captions
Table 1. In vitro antibacterial activity of ocotillol-type derivatives. (MIC: µg/mL).
Table 2. Antibacterial activity of compounds PPD, 3, 5, 16 and 24 against MRSA.
(MIC: µg/mL).
Table 3. Synergistic effect of different antibiotics with compounds PPD, 3 and 16
against S. aureus USA 300 and B. subtilis 168.
Table 4. Cytotoxic activity of compounds 3, 5 and 16 against HEK-293 and HeLa
cells.
Figure 1. Natural triterpenoid products.
Figure 2. The synergistic effects of compounds PPD, 3 and 16 with two conventional
antibiotics against CA- MRSA USA300 and B. subtilis 168
Figure 3. The preliminary SARs of ocotillol-type triterpenoid derivatives.
Scheme 1. Synthesis of triols 3 and 4.
Scheme 2. Synthesis of oxidation derivatives (5-8, 15, 23) of 3 and 4.
Scheme 3. Synthesis of ester derivatives (14, 16, 17, 22, 24).

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Table 1.
In vitro antibacterial activity of ocotillol-type derivatives. (MIC: µg/mL)
S. aures
B. sub
E. coli
P. aer
A. baum
Strain
16
16
8
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
64
HBA
PPD
3
4
5
6
7
8
14
15
16
17
22
23
24
KANa
32
8
8
>128
>128
>128
>128
>128
>128
64
>128
128
64
128
64
16
>128
>128
>128
>128
64
>128
128
128
>128
64
32
128
128
>128
>128
16
64
>128
128
>128
>128
32
4
>128
>128
64
>128
64
>128
>128
128
16
128
128
128
>128
64
>128
>128
1
>128
>128
8
>128
>128
1
1
0.25
a KAN: kanamycin

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Table 2.
Antibacterial activity of compounds PPD, 3, 5, 16 and 24 against MRSAa.
(MIC: µg/mL)
compound
MRSA USA 300
16
32
8
HBA
PPD
3
16
32
64
1
5
16
24
KANb
a MRSA: methicillin-resistant S. aureus; b KAN: kanamycin

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Table 3䟏
Synergistic effect of different antibiotics with compounds PPD, 3 and 16 against S.
aureus USA 300 and B. subtilis 168
MIC (µg/mL)
MBC (µg/mL)
FICI (FIC index) d
Compound
S. aureus
B.sub
S. aureus
B.sub
S. aureus
B.sub
USA300
168
USA300
168
USA300
168
KANa
CHLb
1
4
0.25
2
4
N/Ac
2
1
N/A
-
-
-
-
PPD+KAN
3+KAN
0.25
0.125
0.0078
4
0.0625
0.2
0.25
0.26
0.14
0.008
1.13
1.5
0.25
1.03
<0.008
0.53
1.25
<0.004
1
1
16+KAN
PPD+CHL
3+CHL
<0.0020
1
2
<0.0020
N/A
N/A
N/A
N/A
4
2
N/A
16+CHL
0.016
<0.0078
<0.0078
0.005
a KAN: kanamycin; b CHL: chloramphenicol; c N/A: not applicable.
d
FICI: according to the literature [19]: FIC of drug A (FIC A) = MIC of drug A in
combination/MIC of drug A alone; FIC of drug B (FIC B) = MIC of drug B in combination/MIC
of drug B alone; hence FICI = FIC A + FIC B. “Synergy” was defined when FICI was less than or
equal to 0.5; while “additive” in which the FICI was greater than 0.5 and less than or equal to 1.0;
whereas “indifferent” when the FICI was greater than 1.0 and less than or equal to 2.0; and
“antagonistic” in cases which the FICI was greater than 2.0.

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Table 4.
Cytotoxic activity of compounds 3, 5 and 16 against HeLa and HEK-293 cells
IC50a (µg/mL)
SIb (HeLa / HEK-293) SI (HeLa / HEK-293)
S.aureus RN4220 CA-MRSA USA300
Compound
HeLa
HEK-293
47.64±3.46 154.26±10.53
46.98±4.12 170.19±12.34
54.39±5.70 175.32±16.09
6.0
2.9
13.6
-
19.3
10.6
43.8
-
6.0
2.9
1.7
-
19.3
10.6
5.5
-
3
5
16
5-FUc
0.80±0.13
-
a IC50 is the concentrations required to inhibit 50% of cell growth and the results are expressed as
the mean ± S.D. of three independent experiments.
b SI: Selectivity index (IC50 / MIC).
c 5-FU: 5-fluorouracil.

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Figure 1. Natural triterpenoid products

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Figure 2. The synergistic effects of compounds PPD, 3 and 16 with two conventional
antibiotics against CA- MRSA USA300 and B. subtilis 168.

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Figure 3. The preliminary SARs of ocotillol-type triterpenoid derivatives.