Synthesis and biological evaluation of pentacyclic triterpenoid derivatives as potential novel antibacterial agents

Article abstract of DOI:10.1016/j.bioorg.2021.104692

A series of ursolic acid (UA), oleanolic acid (OA) and 18β-glycyrrhetinic acid (GA) derivatives were synthesized by introducing a range of substituted aromatic side-chains at the C-2 position after the hydroxyl group at C-3 position was oxidized. Their antibacterial activities were evaluated in vitro against a panel of four Staphylococcus spp. The results revealed that the introduction of aromatic side-chains at the C-2 position of GA led to the discovery of potent triterpenoid derivatives for inhibition of both drug sensitive and resistant S. aureus, while the other two series derivatives of UA and OA showed no significant antibacterial activity even at high concentrations. In particular, GA derivative 33 showed good potency against all four Staphylococcus spp. (MIC = 1.25–5 μmol/L) with acceptable pharmacokinetics properties and low cytotoxicity in vitro. Molecular docking was also performed using S. aureus DNA gyrase to rationalize the observed antibacterial activity. This series of GA derivatives has strong potential for the development of a new type of triterpenoid antibacterial agent.

Full text of DOI:10.1016/j.bioorg.2021.104692

Bioorganic Chemistry 109 (2021) 104692
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of pentacyclic triterpenoid derivatives
as potential novel antibacterial agents
,
Panpan Wua b, Borong Tua, Jinfeng Lianga, Shengzhu Guoa, Nana Caoa, Silin Chena,
Zhujun Luoa, Jiahao Lia, Wende Zhenga, Xiaowen Tanga, Dongli Lia, Xuetao Xua,
,b,*
Wenfeng Liua, Xi Zhenga, Zhaojun Shenga, Adam P. Robertsc, Kun Zhanga
,
,d,*
Weiqian David Honga
a School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, PR China
b Department of Pharmaceutical Engineering, Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, PR China
c Centre for Drugs and Diagnostics, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom
d Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of ursolic acid (UA), oleanolic acid (OA) and 18β-glycyrrhetinic acid (GA) derivatives were synthesized
by introducing a range of substituted aromatic side-chains at the C-2 position after the hydroxyl group at C-3
position was oxidized. Their antibacterial activities were evaluated in vitro against a panel of four Staphylococcus
spp. The results revealed that the introduction of aromatic side-chains at the C-2 position of GA led to the dis-
covery of potent triterpenoid derivatives for inhibition of both drug sensitive and resistant S. aureus, while the
other two series derivatives of UA and OA showed no significant antibacterial activity even at high concentra-
tions. In particular, GA derivative 33 showed good potency against all four Staphylococcus spp. (MIC = 1.25–5
Pentacyclic triterpenes
18β-glycyrrhetinic acid
Natural product derivatives
Gram-positive bacteria
Antibacterial
μ
mol/L) with acceptable pharmacokinetics properties and low cytotoxicity in vitro. Molecular docking was also
performed using S. aureus DNA gyrase to rationalize the observed antibacterial activity. This series of GA de-
rivatives has strong potential for the development of a new type of triterpenoid antibacterial agent.
1. Introduction
Potent pharmacological activities of these triterpenes have been
demonstrated including their ability to inhibit the growth of various
Many of the medical achievements of the last century could be lost
through the spread of antimicrobial resistance [1–3]. Previously curable
infectious diseases may become untreatable and spread throughout the
world, which has already started to happen [4,5]. In particular, anti-
biotic resistant Staphylococcus aureus remains a serious clinical problem.
Normal treatments become less effective as resistance develops [6,7].
Herein, the development of new antibiotics is an urgent issue, meaning
that the development of new classes of antibiotics to circumvent existing
antimicrobial resistance is constantly needed [8–11].
bacterial pathogens [14,15], against some infectious viruses [13,16,17],
induce cancer cell differentiation and apoptosis [18,19], and prevent
herbivore infections in the host [20,21]. Some pentacyclic triterpenoids
have already emerged as new series of chemotherapeutics and some of
them are currently in clinical trials [22,23]. Moreover, with these pro-
nounced pharmacological activities, medicinal chemists were attracted
by the safety characteristics of pentacyclic triterpenoids while compared
with other clinically available chemotherapeutic agents that often suffer
serious side effects [24,25]. However, the antibacterial activity of pen-
tacyclic triterpenoids is relatively weak [26]. In a recent report by
Huang and co-workers, tri-hydroxyl groups were introduced in ring A
while an ester moiety was formed at C-20 of the oleanane-type
The natural products of ursolic acid (UA), oleanolic acid (OA) and
18β-glycyrrhetinic acid (GA) (Fig. 1), are biologically active pentacyclic
triterpenoids which are secondary metabolites of various plants [12,13].
Abbreviations: UA, ursolic acid; OA, oleanolic acid; GA, 18β-glycyrrhetinic acid; MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration;
IZ, inhibition zone; SAR, structure-activity relationship; DMPK, drug metabolism and pharmacokinetics.
* Corresponding authors at: School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, PR China.
E-mail addresses: xizheng@pharmacy.rutgers.edu (X. Zheng), Adam.Roberts@lstmed.ac.uk (A.P. Roberts), kzhang@gdut.edu.cn (K. Zhang), davidhwq@liverpool.
ac.uk (W.D. Hong).
https://doi.org/10.1016/j.bioorg.2021.104692
Received 25 November 2020; Received in revised form 21 January 2021; Accepted 22 January 2021
Available online 3 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
compounds 1–9 (Fig. 2). To our knowledge, their activity against bac-
teria were not evaluated or reported yet, so these derivatives of UA were
assessed for their in vitro antibacterial activities in this study initially.
Three series of novel UA, OA and GA derivatives were prepared with
modifications at C-2 and C-3 positions of selected pentacyclic triterpe-
noids in two high yielding steps as detailed in Scheme 1[34]. Jones re-
agent was used to oxidize the three pentacyclic triterpenoids to give the
ketone intermediates 10, 11, and 12. Three series target derivatives
were then produced by Claisen Schmidt condensation at C-2 position of
the ketone intermediates of UA, OA and GA, in which derivatives 13–34
were obtained from parent compound GA, derivatives 35 and 36 were
obtained from UA and derivatives 37 and 38 were obtained from OA.
They were also evaluated for the in vitro antibacterial activities in this
study as showed in Table 1 and Table 2.
2.2. Antibacterial activity
Fig. 1. The chemical structure of GA, UA and OA.
The antibacterial activity of all the pentacyclic triterpenoids de-
rivatives were assayed against four Gram-positive bacteria. All bacterial
strains were cultured in Muller Hinton agar at 37 ◦C overnight.
triterpene GA to enhance their antimicrobial property [14]. Previous
structure–activity relationship (SAR) studies of GA have suggested that
the carboxylic acid group at C-20 and ring A are involved in various
biological activities [27–29].
The antimicrobial activity of the pentacyclic triterpenoid derivatives
against three sensitive strains of Gram-positive bacteria were firstly
assessed by a Kirby–Bauer assay and summarized in Table 1. The dosage
of each examined derivative was 80 nmol in this assay. The sizes of the
inhibition zone (IZ) diameter showed that the GA derivatives (13–34)
were more potent than the parent compound of GA, the oxidized in-
termediates (GA-O, UA-O and OA-O) and all others derivatives of UA
(1–9, 35 and 36) and OA (37 and 38), in which the IZ diameter was in
the range from 6.89 ± 0.78 to 15.93 ± 0.12 mm of three examined
Gram-positive strains. However, all the tested derivatives exhibited no
obviously inhibitory activity against the two Gram-negative bacteria;
Salmonella typhimurium (CMCC 50115) and Escherichia coli (CMCC
44102) (data not shown). The difference of antibacterial activities
among the series of GA derivatives (13–34) during this agar disk
diffusion assay were not fully demonstrated, so a microtiter plate dilu-
tion method was conducted to determine the minimal inhibitory
We have focused on the modifications at the C-2 and C-3 positions of
UA, OA and GA and report a series of GA derivatives displaying in vitro
antibacterial activity against both antibiotic sensitive and resistant
Staphylococcus spp. which are significantly higher than that of the parent
compound and provide a basis for onward development of triterpenoids
as antibacterial agents.
2. Results and discussion
2.1. Derivative design
Previously, multiple series of pentacyclic triterpenoids derivatives
were obtained by modification at positions of C-3 and C-28 in our group
to evaluate their potential for α-glucosidase inhibition [30–33], such as
Fig. 2. Structures of ursolic acid derivative 1–9. The fragments in blue are the introduced groups; the numbers within the structures are the crucial modification sites.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
Scheme 1. Synthesis of three series of pentacyclic triterpenoids derivatives at positions of C-2 and C-3. Reagents and conditions: (a) Jones reagent, acetone, 0 ◦C to
rt, 2 h, 91–96%; (b) R5-CHO, KOH, EtOH, rt, 3 h, 66–93%.
concentration (MIC) and the minimal bactericidal concentration (MBC)
in 96-well plates. After incubation for 24 h, the plates were evaluated for
the presence or absence of bacterial growth. Each sample concentration
was repeated four times and Gatifloxacin was employed as positive
control in the assay. The final concentration of DMSO in the 96-well
plate had no effect on bacterial growth.
GA derivatives of 21, 32 and 33 against two strains of drug sensitive
Staphylococcus aureus (ATCC 6538 and ATCC 29213) and Staphylococcus
epidermidis (ATCC 12228). As presented in Fig. 3, all tested strains of
bacteria were effectively inhibited at the MICs of derivatives 21, 32 and
33 with a slight growth close to the end of the kinetic study. The bac-
terial growth was totally inhibited at higher concentrations of 2 × MIC
and 4 × MIC until the end of the assay, which were also in accordance
with the biological assays determining MBCs. While the bacterial strains
were incubated with 0.5 × MIC of 21, 32 and 33, the number of bacteria
initially decreased at a rapid rate, then gradually increased, and growth
inhibition was maintained for 6–8 h. Furthermore, similar growth in-
hibition patterns were observed for all three Staphylococcus spp. tested.
The MIC and MBC results of the derivatives determined by the micro-
dilution method were presented in Table 2. The results suggested that
GA derivatives 13–34 (MIC = 1.25–100 μmol/L, MBC = 2.5–100 μmol/
L) had inhibitory activity against all four Staphylococcus spp., which was
in accordance with the agar disk diffusion study results (Table 1). These
assays of GA derivatives displayed considerable effect on inhibition of
Methicillin-resistant Staphylococcus aureus (MRSA) with MIC range from
5 to 100 μmol/L (Table 2). The results also confirmed that there is no
improvement to the inhibition of the tested bacteria by introducing
exocyclic , β-unsaturated ketone group at the similar (C-2) position of
2.3. Molecular docking
α
OA and UA (35–38). Since GA derivatives are structurally different from
OA and UA derivatives in terms of their natural product cores, it suggests
that the difference in antibacterial activity of three series derivatives
might be related to the structural differences in ring C and ring E, such as
the position of the carboxylic acid and/or the carbonyl group. Amongst
the GA derivatives, different sizes of the aromatic side-chains are well
tolerated at the C-2 position e.g. phenyl ring (13, MIC = 6.25–12.5
In order to rationalize the observed antibacterial activity and to
investigate the interactions of the newly prepared compounds in the
DNA gyrase catalytic site, compounds with significant antibacterial ac-
tivity and the target protein from S. aureus DNA gyrase (PDB code: 5cdq)
were selected for molecular docking with the SYBYL-X 2.0 program. The
binding model of compound 33 and gyrase-DNA is depicted in Fig. 4,
which revealed that the compound is well filled in the binding pocket
[39]. As show in Fig. 4A, in this binding mode, the molecular structure of
the compound exhibits a large bend and the carboxyl amide group in
compound 33 is in close proximity (3.17 Å) with amino acid residue
ASP512 and has the potentially of hydrogen bonding interaction. It can
be seen from the molecular surface of the compound and protein that the
high electrostatic potential position of the compound structure was
located in the corresponding high electrostatic potential region of the
protein and vice versa, which is propitious to form more stable ligand-
protein complexes. The 2D hydrophobic interaction diagram (Fig. 4B)
showed that 33 accommodated in the hydrophobic sub-pocket of the
active site surrounded by the hydrophobic site chains of the amino acids
Gly82, Gly459, Ser438, Gly436, Gly584, Glu435, Asp508, Asp510,
Pro80, Val511, Hls81, Arg33, which enhanced the bonding force be-
tween the compound and the protein. Noticeably, the vast majority of
the hydrophobic forces were concentrated in the site substituted with
aromatic rings.
μ
mol/L) vs. quinolone ring (34, MIC = 5
μmol/L) vs. biaryl rings (20 &
23, MIC = 6.25–12.5 mol/L). A number of mono- or di-substituted
μ
phenyl, or other heterocyclic aryl. side-chains also promoted reason-
able activities against the tested Gram-positive bacteria. In general, for
this series of GA derivatives, the potency differences between the sen-
sitive strain and resistant strains of S. aureus are small (≤2 fold) and the
differences between corresponding MIC values and MBC values are also
quite small (≤2 fold). Overall, this series of GA derivatives showed
consistent activity against all four tested Staphylococcus spp., which
were significantly higher than both the parent compound (GA) and the
ketone intermediate (10). In particular, compound 33 demonstrated the
highest activity (MIC = 1.25 μmol/L) against all four Staphylococcus spp.
within this series.
The time killing kinetic studies were performed over a period of 20 h’
assay at 37 ◦C according to previously reported study with a slight
modification [35–38]. Fig. 3 displayed the time-kill curves of selected
3

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
Table 1
Biological evaluation of series pentacyclic triterpenoids derivatives expressed as the inhibition zone (mm).a
Compound code
R5
Bacterium and Inhibition Zone (mm)
Dosage: 80 nmol
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus epidermidis
(ATCC 6538)
(ATCC 29213)
(ATCC 12228)
GA
–
–
–
–
6.78 ± 0.22
6.76 ± 0.33
6.29 ± 0.43
<6b
6.85 ± 0.20
6.55 ± 0.27
6.81 ± 0.22
<6
7.46 ± 0.18
7.24 ± 0.19
7.08 ± 0.25
<6
UA
OA
1~12
13
9.85 ± 0.22
9.66 ± 0.18
9.09 ± 0.18
14
15
8.25 ± 0.22
8.96 ± 0.36
8.39 ± 0.88
9.00 ± 0.57
7.92 ± 0.22
8.56 ± 0.08
16
9.31 ± 0.23
9.58 ± 0.39
8.99 ± 0.75
17
18
10.01 ± 0.09
7.34 ± 0.05
10.08 ± 0.27
6.91 ± 0.41
11.21 ± 0.07
6.89 ± 0.78
19
9.86 ± 1.24
10.74 ± 0.38
9.09 ± 0.73
20
21
22
9.55 ± 0.08
10.03 ± 0.17
10.01 ± 0.06
9.68 ± 0.55
10.39 ± 0.36
10.23 ± 0.21
9.88 ± 0.03
9.90 ± 0.77
10.59 ± 0.17
23
24
10.19 ± 0.25
8.55 ± 0.29
9.69 ± 0.53
8.47 ± 0.30
9.89 ± 0.56
9.09 ± 0.23
25
26
27
9.37 ± 0.38
8.99 ± 1.01
7.01 ± 0.18
9.38 ± 0.26
9.55 ± 0.19
8.43 ± 0.81
9.68 ± 0.41
9.39 ± 0.70
6.99 ± 0.93
28
29
10.25 ± 0.99
7.25 ± 0.28
10.68 ± 1.14
7.18 ± 0.37
11.20 ± 0.22
7.20 ± 0.33
30
11.21 ± 1.33
13.23 ± 0.88
12.39 ± 0.11
31
32
7.66 ± 0.28
8.90 ± 0.09
7.54 ± 0.50
8.75 ± 0.17
7.89 ± 0.03
9.11 ± 0.20
33
34
14.83 ± 0.55
7.91 ± 0.28
15.60 ± 0.46
8.11 ± 0.23
15.93 ± 0.12
8.37 ± 0.88
35
36
37
38
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
Gatifloxacinc
a
–
19.12 ± 0.73
17.13 ± 0.64
18.67 ± 0.25
Results are expressed as the diameter of inhibition zone (mm), values represent the means of three independent replicates ± SD.
< 6, no measureable inhibition zone.
b
c
The dosage of gatifloxacin used in the inhibition zone assay was 1 nmol.
2.4. Pharmacokinetics and cytotoxicity
protein, but 33 was noticeably more soluble than 34 in aqueous me-
dium. For metabolic stability, although their turnover rates by rat he-
patocytes were both reasonably low, 33 showed much lower clearance
than 34 by human microsome in vitro. In addition, from the in vitro
toxicity assessment, both 33 and 34 showed low cytotoxicity (CC50 > 64
Two GA derivatives, 33 and 34 were assessed for their DMPK
properties (Table 3). In terms of physiochemical properties, both com-
pounds have similar lipophilicity and were highly bound to plasma
4

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
Table 2
Biological evaluation of pentacyclic triterpenoids derivatives expressed in MICa and MBCb (
μ
mol/L).
Compound code
R5
MICs and MBCs of Selected Bacterium (μmol/L)
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus epidermidis
Methicillin-resistant Staphylococcus aureus
(ATCC 6538)
(ATCC 29213)
(ATCC 12228)
(MRSA)
MICa
MBCb
MIC
MBC
MIC
MBC
MIC
MBC
GA
–
–
–
–
200
NTc
200
NT
NT
NT
NT
12.5
200
NT
NT
NT
NT
25
＞200
＞200
＞200
＞200
50
NT
NT
NT
NT
100
UA
＞200
＞200
＞200
6.25
NT
＞200
＞200
＞200
12.5
＞200
＞200
＞200
12.5
OA
NT
1~12
13
NT
12.5
14
15
12.5
12.5
12.2
25
6.25
6.25
12.5
12.5
25
25
25
100
50
100
50
12.5
16
12.5
12.5
6.25
12.5
12.5
12.5
50
100
17
18
12.5
12.5
25
25
12.5
12.5
25
25
12.5
25
25
50
50
100
100
100
19
6.25
12.5
6.25
12.5
6.25
12.5
50
50
20
21
22
6.25
6.25
25
12.5
12.5
50
3.125
3.125
25
12.5
6.25
50
12.5
3.125
25
25
50
50
50
50
6.25
50
50
100
23
24
6.25
12.5
12.5
25
6.25
12.5
12.5
25
12.5
12.5
12.5
12.5
25
50
50
100
25
26
27
6.25
12.5
12.5
12.5
25
3.125
12.5
6.25
6.25
25
6.25
25
12.5
50
12.5
50
25
50
50
12.5
12.5
12.5
12.5
25
28
29
6.25
6.25
12.5
12.5
6.25
6.25
12.5
12.5
6.25
12.5
12.5
25
25
25
25
50
30
12.5
25
12.5
25
12.5
25
50
50
31
32
12.5
12.5
6.25
6.25
12.5
12.5
6.25
25
50
25
50
25
3.125
1.5625
3.125
12.5
33
34
1.25
5
2.5
5
1.25
5
2.5
10
1.25
5
2.5
5
5
5
5
10
35
36
37
38
＞200
＞200
＞200
＞200
0.2
NT
NT
NT
NT
0.2
＞200
＞200
＞200
＞200
0.2
NT
NT
NT
NT
0.2
＞200
＞200
＞200
＞200
0.2
NT
NT
NT
NT
0.2
＞200
＞200
＞200
＞200
NT
NT
NT
NT
NT
NT
Gatifloxacin
a
–
MIC (µmol/mL), minimum inhibitory concentration, i.e., the lowest concentration of the compound that completely inhibits the growth of bacteria.
MBC (µmol/mL), minimum bactericidal concentration, i.e., the lowest concentration of the compound that completely kills the bacteria.
NT, not tested.
b
c
5

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
Fig. 3. The time killing kinetic studies of GA derivatives 21, 32 and 33 against three Staphylococcus spp. Including two strains of Staphylococcus aureus (ATCC 6538
and ATCC 29213) and Staphylococcus epidermidis (ATCC 12228), exposed to four different concentrations of derivative 21 (Fig. 3A, B, C), 32 (Fig. 3D, E, F) and 33
(Fig. 3G, H, I) according to their respective MICs (n = 4).
Fig. 4. The poses of compound 33 docked in the cleavage site of S. aureus DNA gyrase with surface of electrostatic potential (A) and hydrophobic interaction (B).
μ
M) against BV2 microglial cells that demonstrated sufficient safety
3. Conclusion
margin (>50 folds) in comparison to the antibacterial activity in vitro.
The in vitro DMPK and safety profiles of both compounds indicated they
were suitable for further optimization as early leads for either an oral or
IV administrative antibiotic series.
In summary, a number of derivatives of pentacyclic triterpenoids:
UA, OA and GA, were synthesized and tested for their antibacterial ac-
tivity. Amongst this group of natural product derivatives, those modified
from GA showed significantly higher potency than both their parent and
other analogues derived from UA or OA cores. The modification in this
6

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
out using eluent (petroleum ether/ethyl acetate, 3 : 1, containing 0.5%
formic acid). Derivatives 13–34 could be prepared by Claisen Schmidt
condensation of intermediate 10 with corresponding aldehydes in the
presence of ethanolic potassium hydroxide in good yield at room tem-
perature. All the results were detailed below.
Table 3
DMPK and cytotoxicity data for selected GA derivatives 33 and 34.
33
34
LogDa
3.40
533
3.40
75
Solubility at pH 7.4b
Human PPB (% Free)c
0.28
21.6
<3.00
>64
0.05
26.2
54.40
>64
d
Rat Heps. Clint
e
Human Mics. Clint
f
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-
CC50
a
Octanol/water partitioning, pH 7.4, measured value.
b
c
d
e
f
Aqueous solubility in pH 7.4 PBS buffer (
μM).
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-
Human plasma protein binding (%free).
L⋅minꢀ 1 1 × 106 cellsꢀ 1).
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (GA-O, 10, C30H44O4). Yield: 96%; white
solid; mp: 291–292 ◦C; 1H NMR (400 MHz, CDCl3) δ 5.75 (s, 1H), 3.03 –
2.88 (m, 1H), 2.71 – 2.55 (m, 1H), 2.45 (s, 1H), 2.42 – 2.31 (m, 1H), 2.22
(d, J = 10.7 Hz, 1H), 2.11 – 1.98 (m, 2H), 1.94 (d, J = 13.5 Hz, 1H), 1.91
– 1.79 (m, 1H), 1.77 – 1.50 (m, 4H), 1.50 – 1.35 (m, 7H), 1.35 – 1.30 (m,
1H), 1.30 – 1.20 (m, 8H), 1.18 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.05 –
0.99 (m, 1H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 217.3, 199.8,
181.2, 169.9, 128.6, 61.2, 55.7, 48.4, 47.9, 45.5, 43.9, 43.5, 41.2, 39.9,
37.9, 36.9, 34.4, 32.3, 32.1, 31.1, 28.7, 28.6, 26.7, 26.6, 23.5, 21.6,
Rat hepatocytes intrinsic clearance (
μ
Human microsome intrinsic clearance (
μ
L⋅minꢀ 1⋅mgꢀ 1).
The concentration of the compound that reduced mammalian cell viability to
50% (μM), cycloheximide as positive control (CC50 = 0.25 ± 0.03).
work was mainly focused on the C-2 position of the pentacyclic tri-
terpenoid scaffolds. With a wide range of side-chains substituted at the
C-2 position of the GA scaffold, it indicated that this position can tolerate
different sized and types of aromatic rings as substitutions, maintaining
reasonable antibacterial activities. This finding lays a solid foundation
for future optimization, and the SAR at this position, and indeed other
positions of the GA scaffold, is being investigated in more detail in
ongoing studies. Using molecular docking, we demonstrated the selected
lead compound 33 can fit in well within the binding site of the S. aureus
DNA gyrase, although further computational and experimental studies
are still required to investigate this preliminary observation. Pre-
liminary assessments of DMPK and safety properties suggested that the
two selected lead compounds are well positioned for further optimiza-
tion and development. Other key aspects of the next stage optimization/
development are to broaden the antibacterial spectrum of the GA de-
rivatives against Gram-negative bacteria and to further understand the
mechanism of action and resistance potential of this novel series of semi-
synthetic compounds.
19.0,
18.7,
15.8.
HRMS
(ESI):
C30H45O4
(469.3312)
[M+H]+=469.3314.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-
((Z)-benzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (13, C37H48O4). According to the general
procedure, derivative 13 was prepared by Claisen Schmidt condensation
of intermediate 10 with benzaldehyde in the presence of ethanolic po-
tassium hydroxide at room temperature. Purification of product by flash
column chromatography was carried out using eluent (petroleum ether/
ethyl acetate, 6 : 1, containing 0.5% formic acid). Yield: 88%; white
solid; mp: 262–263 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 7.5 Hz,
2H), 7.49 (s, 1H), 7.41 (t, J = 7.4 Hz, 2H), 7.35 – 7.30 (m, 1H), 5.83 (s,
1H), 4.28 (d, J = 16.7 Hz, 1H), 2.58 (s, 1H), 2.29 (t, J = 16.5 Hz, 2H),
2.14 – 1.96 (m, 3H), 1.90 (t, J = 12.0 Hz, 1H), 1.81 – 1.64 (m, 2H), 1.63 –
1.49 (m, 3H), 1.49 – 1.41 (m, 5H), 1.40 – 1.29 (m, 2H), 1.25 (d, J = 9.5
Hz, 6H), 1.21 (s, 3H), 1.20 – 1.14 (m, 6H), 1.13 – 1.03 (m, 2H), 0.89 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 207.8, 199.5, 181.9, 170.0, 137.2,
135.9, 134.0, 130.5, 128.6, 128.5, 128.4, 59.4, 53.3, 48.3, 45.4, 45.0,
44.6, 43.8, 43.38, 41.0, 37.7, 36.2, 31.9, 31.5, 30.8, 29.7, 28.6, 28.4,
26.5, 26.4, 23.2, 22.5, 19.6, 18.0, 15.4. HRMS (ESI): C37H49O4
(557.3625) [M+H]+=557.3632.
4. Experimental section
4.1. Chemistry materials and methods
All reagents were purchased from Adamas Reagent Ltd. (Shanghai
China) in analytical reagent grade and were used directly without
further purification. Flash chromatography was carried out using silica
gel (200–300 mesh) which was supplied by Inno-chem Co., Ltd. (Beijing
China). Analytical TLC was performed on pre-coated silica gel F254
plates (0.25 mm; E. Merck), and the products were visualized under UV
(254 nm) or by treated with an ethanolic solution of p-anisaldehyde
spray followed by heating. All derivatives of GA, UA and OA were
characterized by 1H NMR, 13C NMR and HRMS. The antimicrobial ac-
tivity was assayed by using a Multi-model Plate Reader (Infinite 200).
The purities of all tested compounds were confirmed by analytical HPLC
with a dual pump Shimadzu LC 20A system equipped with a C18 column
(250 mm × 4.6 mm, 5 µM YMC). Analytical method conditions: flow
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-
rate = 0.5 mL/min, injection volume = 10 μL, isocratic elution system =
80% solvent A (70% water, 20% acetonitrile, 5% glacial acetic acid, 5%
tetrahydrofuran) and 20% solvent B (acetonitrile) at room temperature
and run time = 15 min. The purities of all compounds are over 95% and
Rt are between 7.6~9.2 min.
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-(thiophen-2-ylmethy-
lene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13, 14b-icosahy-
dropicene-2-carboxylic acid (14, C35H46O4S). According to the general
procedure, derivative 14 was prepared by Claisen Schmidt condensation
of intermediate 10 with formylthiophene in the presence of ethanolic
potassium hydroxide at room temperature. Purification of product by
flash column chromatography was carried out using eluent (petroleum
ether/ethyl acetate, 4 : 1, containing 0.5% formic acid). Yield: 85%;
white solid; mp: 294–295 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.73 (s, 1H),
7.47 (d, J = 5.0 Hz, 1H), 7.33 (d, J = 3.4 Hz, 1H), 7.10 (dd, J = 4.9, 3.9
Hz, 1H), 5.86 (s, 1H), 4.37 – 4.25 (m, 1H), 2.62 (s, 1H), 2.24 (t, J = 14.6
Hz, 2H), 2.13 – 1.96 (m, 3H), 1.89 (td, J = 13.4, 4.1 Hz, 1H), 1.77 – 1.62
4.1.1. General procedure for the synthesis of GA derivatives (13~34)
GA derivatives 13–34 were obtained according to Scheme 1. GA was
◦
dissolved in acetone at 0 C; Jones reagent was added to the reaction
mixture drop-wisely until the solution colour was stable in light brown,
which implied that the Jones reagent was in slight excess to oxidize the
C-3 hydroxyl group into ketone to produce the intermediate 10. Puri-
fication of compound 10 by flash column chromatography was carried
7

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
(m, 1H), 1.60 – 1.48 (m, 4H), 1.48 – 1.40 (m, 5H), 1.28 – 1.23 (m, 8H),
1.23 – 1.19 (m, 6H), 1.17 (s, 3H), 1.13 (s, 2H), 0.88 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 206.8, 199.6, 181.1, 170.0, 139.7, 132.7, 130.8,
130.5, 130.1, 128.9, 127.6, 59.7, 53.2, 48.5, 45.2, 44.0, 43.6, 41.3, 37.9,
36.2, 32.1, 31.7, 31.1, 30.1, 29.9, 28.8, 28.6, 26.8, 26.6, 23.4, 22.7,
icosahydropicene-2-carboxylic acid (17, C37H47NO6). According to the
general procedure, derivative 17 was prepared by Claisen Schmidt
condensation of intermediate 10 with 4-nitrobenzaldehyde in the pres-
ence of ethanolic potassium hydroxide at room temperature. Purifica-
tion of product by flash column chromatography was carried out using
eluent (petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic
acid). Yield: 89%; white solid; mp: 230–231 ◦C; 1H NMR (400 MHz,
CDCl3) δ 8.26 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.53 – 7.43
(m, 1H), 5.74 (s, 1H), 4.44 (s, 3H), 4.25 – 4.16 (m, 1H), 3.38 – 3.24 (m,
1H), 2.62 (s, 1H), 2.41 – 2.18 (m, 2H), 2.18 – 2.04 (m, 1H), 2.04 – 1.84
(m, 3H), 1.84 – 1.72 (m, 1H), 1.70 – 1.51 (m, 5H), 1.48 – 1.38 (m, 5H),
1.29 (d, J = 16.3 Hz, 3H), 1.24 – 1.17 (m, 6H), 1.17 – 1.14 (m, 3H), 1.12
– 1.05 (m, 2H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.9, 200.0,
178.9, 171.8, 146.9, 142.15, 137.52, 134.19, 130.5, 129.5, 128.8,
127.8, 123.3, 59.0, 53.2, 45.5, 44.8, 43.9, 43.4, 43.3, 41.0, 37.4, 36.1,
31.6, 31.5, 31.1, 30.7, 28.9, 28.2, 27.9, 26.2, 26.0, 22.7, 22.1, 19.2,
17.6, 14.9. HRMS (ESI): C37H48NO6 (602.3476) [M+H]+=602.3478.
19.9,
18.2,
16.0.
HRMS
(ESI):
C35H47O4S
(563.3190)
[M+H]+=563.3187.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
3-methoxybenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (15, C38H50O5). According to the general
procedure, derivative 15 was prepared by Claisen Schmidt condensation
of intermediate 10 with 3-methoxybenzaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic acid).
Yield: 83%; white solid; mp: 183–184 ◦C; 1H NMR (400 MHz, CDCl3) δ
8.68 (d, J = 4.3 Hz, 1H), 7.69 (td, J = 7.7, 1.4 Hz, 1H), 7.45 (d, J = 7.9
Hz, 1H), 7.42 (s, 1H), 7.14 (dt, J = 42.3, 21.1 Hz, 1H), 5.79 (s, 1H), 4.38
(d, J = 18.1 Hz, 1H), 2.59 (s, 1H), 2.46 (d, J = 18.2 Hz, 1H), 2.24 (d, J =
10.8 Hz, 2H), 2.11 – 1.93 (m, 3H), 1.86 (td, J = 13.4, 3.7 Hz, 2H), 1.78 –
1.59 (m, 3H), 1.59 – 1.46 (m, 4H), 1.46 – 1.39 (m, 5H), 1.27 – 1.19 (m,
8H), 1.19 – 1.13 (m, 8H), 1.11 – 0.97 (m, 2H), 0.83 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 207.83, 199.32, 181.64, 169.81, 159.42, 137.16,
137.10, 134.33, 129.41, 128.62, 122.78, 115.70, 114.48, 59.38, 55.35,
53.32, 48.27, 45.46, 45.06, 44.48, 43.82, 43.35, 40.99, 37.71, 36.26,
31.91, 31.53, 30.89, 29.65, 28.61, 28.45, 26.56, 26.40, 23.27, 22.58,
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-
((Z)-3-chlorobenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,
9,10,11,12,12a,12b,13,14b-icosahydropi-
cene-2-carboxylic acid (18, C37H47ClO4). According to the general pro-
cedure, derivative 18 was prepared by Claisen Schmidt condensation of
intermediate 10 with 3-chlorobenzaldehyde in the presence of ethanolic
potassium hydroxide at room temperature. Purification of product by
flash column chromatography was carried out using eluent (petroleum
ether/ethyl acetate, 6 : 1, containing 0.5% formic acid). Yield: 81%;
white solid; mp: 235–236 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.43 (s, 1H),
7.37 (s, 2H), 7.32 (t, J = 7.7 Hz, 1H), 7.29 – 7.25 (m, 2H), 5.80 (s, 1H),
4.68 (s, 1H), 4.20 (d, J = 16.7 Hz, 1H), 2.54 (s, 1H), 2.23 (d, J = 15.3 Hz,
2H), 2.11 – 1.94 (m, 3H), 1.94 – 1.81 (m, 1H), 1.80 – 1.60 (m, 1H), 1.60
– 1.47 (m, 3H), 1.46 – 1.32 (m, 6H), 1.25 (d, J = 9.2 Hz, 3H), 1.20 (s,
19.61, 18.05, 15.52. HRMS (ESI):
C38H51O5 (587.3731)
[M+H]+=587.3734.
3H), 1.18 (s, 3H), 1.16 (s, 6H), 1.07 (d, J = 16.0 Hz, 2H), 0.86 (s, 3H).13
C
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-
NMR (100 MHz, CDCl3) δ 207.6, 199.4, 181.8, 170.0, 137.9, 135.7,
135.6, 134.5, 130.5, 129.8, 128.8, 128.5, 128.2, 59.5, 53.7, 48.5, 45.7,
45.2, 44.4, 44.0, 43.6, 41.2, 37.9, 36.5, 32.1, 31.7, 31.1, 29.7, 28.8,
28.6, 26.8, 26.6, 23.4, 22.7, 19.7, 18.2, 15.6. HRMS (ESI): C37H48ClO4
(591.3236) [M+H]+=591.3235.
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-(pyridin-2-ylmethylene)-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (16, C36H47NO4). According to the general
procedure, derivative 16 was prepared by Claisen Schmidt condensation
of intermediate 10 with 2-pyridinecarboxaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 5 : 1, containing 0.5% formic acid).
Yield: 79%; white solid; mp: 202–203 ◦C; 1H NMR (400 MHz, CDCl3) δ
7.43 (s, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 7.7 Hz, 1H), 7.03 (s,
1H), 6.86 (dd, J = 8.2, 2.3 Hz, 1H), 5.79 (s, 1H), 4.27 (d, J = 17.0 Hz,
1H), 3.83 (s, 3H), 2.54 (s, 1H), 2.34 – 2.16 (m, 2H), 2.10 – 1.94 (m, 3H),
1.87 (td, J = 13.4, 3.8 Hz, 2H), 1.79 – 1.60 (m, 2H), 1.52 (dd, J = 19.4,
11.4 Hz, 4H), 1.45 – 1.38 (m, 5H), 1.25 (d, J = 6.8 Hz, 9H), 1.21 (s, 3H),
1.19 – 1.13 (m, 2H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.9,
199.4, 182.0, 169.9, 159.6, 137.4, 137.3, 134.5, 129.5, 128.8, 115.8,
114.7, 59.6, 55.5, 53.6, 48.4, 45.6, 45.2, 44.0, 43.5, 41.2, 37.9, 36.5,
32.1, 31.7, 31.1, 29.8, 28.7, 28.6, 26.7, 26.6, 23.4, 22.7, 19.8, 18.2,
15.6. HRMS (ESI): C36H48NO4 (558.3578) [M+H]+=558.3585.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-((Z)-4-((trifluoromethyl)
thio)benzylidene)-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene
ꢀ 2-carboxylic acid (19, C38H47F3O4S). According to the general pro-
cedure, derivative 19 was prepared by Claisen Schmidt condensation of
intermediate 10 with 4-(trifluoromethylthio)benzaldehyde in the pres-
ence of ethanolic potassium hydroxide at room temperature. Purifica-
tion of product by flash column chromatography was carried out using
eluent (petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic
acid). Yield: 82%; white solid; mp: 258–259 ◦C; 1H NMR (400 MHz,
CDCl3) δ 7.66 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.42 (s, 1H),
5.83 (s, 1H), 4.24 (d, J = 17.0 Hz, 1H), 2.56 (s, 1H), 2.33 – 2.18 (m, 2H),
2.13 – 1.94 (m, 3H), 1.88 (td, J = 13.3, 3.6 Hz, 1H), 1.79 – 1.69 (m, 1H),
1.65 (t, J = 13.6 Hz, 1H), 1.60 – 1.48 (m, 2H), 1.48 – 1.33 (m, 6H), 1.33 –
1.25 (m, 2H), 1.24 (s, 3H), 1.21 (s, 3H), 1.19 (s, 3H), 1.16 (d, J = 2.4 Hz,
6H), 1.07 (d, J = 14.0 Hz, 2H), 0.87 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 207.6, 199.7, 181.8, 170.6, 138.5, 136.3, 136.2, 135.4, 131.3, 131.2,
128.7, 128.1, 124.4, 59.5, 53.6, 48.5, 45.7, 45.2, 44.6, 44.0, 43.6, 41.2,
37.9, 36.5, 32.1, 31.7, 31.1, 30.3, 29.7, 28.8, 28.6, 26.7, 26.6, 23.4,
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-
2,4a,6a,6b,9,9,12a-heptamethyl-11-((Z)-4-nitrobenzylidene)-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
8

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
22.7, 19.7, 18.2, 15.6. HRMS (ESI): C38H48F3O4S (657.3220)
temperature. Purification of product by flash column chromatography
was carried out using eluent (petroleum ether/ethyl acetate, 4 : 1,
containing 0.5% formic acid). Yield: 76%; white solid; mp: 232–233 ◦C;
1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.5 Hz, 1H), 7.54 (s, 1H), 6.69
(d, J = 8.4 Hz, 1H), 5.77 (s, 1H), 4.03 (dd, J = 16.3, 7.6 Hz, 1H), 2.50 (d,
J = 4.4 Hz, 1H), 2.29 – 2.19 (m, 1H), 2.14 – 1.98 (m, 3H), 1.98 – 1.79 (m,
2H), 1.78 – 1.68 (m, 1H), 1.68 – 1.59 (m, 1H), 1.59 – 1.54 (m, 2H), 1.54
– 1.47 (m, 2H), 1.46 – 1.32 (m, 9H), 1.23 (s, 3H), 1.21 – 1.16 (m, 9H),
1.07 (d, J = 13.6 Hz, 2H), 0.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ
206.7, 199.8, 181.7, 171.1, 150.5, 149.4, 140.7, 138.1, 130.6, 129.7,
128.3, 122.9, 59.2, 53.8, 48.3, 46.1, 45.1, 43.8, 43.5, 43.4, 41.0, 37.7,
36.7, 31.9, 31.6, 30.9, 29.1, 28.6, 28.4, 26.5, 26.4, 23.2, 22.8, 19.4,
18.2, 15.51.
[M+H]+=657.3222.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-((Z)-4-(pyridin-2-yl)ben-
zylidene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosa-
hydropicene-2-carboxylic acid (20, C42H51NO4). According to the general
procedure, derivative 20 was prepared by Claisen Schmidt condensation
of intermediate 10 with 4-(2-Pyridinyl)benzaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic acid).
Yield: 89%; white solid; mp: 239–240 ◦C; 1H NMR (400 MHz, CDCl3) δ
8.68 (d, J = 4.3 Hz, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.81 – 7.66 (m, 2H),
7.60 (d, J = 8.3 Hz, 2H), 7.55 – 7.46 (m, 1H), 7.21 (t, J = 4.9 Hz, 1H),
5.79 (d, J = 9.9 Hz, 1H), 4.36 – 4.25 (m, 1H), 2.56 (d, J = 14.7 Hz, 1H),
2.38 – 2.18 (m, 2H), 2.11 – 1.95 (m, 3H), 1.93 – 1.81 (m, 1H), 1.80 –
1.49 (m, 7H), 1.45 – 1.40 (m, 5H), 1.26 (s, 3H), 1.23 (s, 3H), 1.20 – 1.15
(m, 9H), 1.06 (d, J = 13.1 Hz, 2H), 0.86 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 207.7, 199.3, 181.2, 169.9, 156.7, 149.5, 138.9, 137.0, 136.6,
136.5, 134.7, 130.9, 129.1, 128.7, 127.0, 126.9, 125.4, 122.3, 120.8,
59.5, 53.4, 48.3, 45.5, 45.1, 44.8, 43.8, 43.4, 41.1, 37.7, 36.3, 31.9,
31.6, 29.7, 29.7, 28.6, 28.4, 26.6, 26.5, 22.6, 19.6, 18.1, 15.5. HRMS
(ESI): C42H52NO4 (634.3891) [M+H]+=634.3903.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
4-(1H-1,2,4-triazol-1-yl)benzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-
10,13-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-ico-
sahydropicene-2-carboxylic acid (23, C39H49N3O4). According to the
general procedure, derivative 23 was prepared by Claisen Schmidt
condensation of intermediate 10 with 4-(1H-1,2,4-triazol-1-yl) benzal-
dehyde in the presence of ethanolic potassium hydroxide at room tem-
perature. Purification of product by flash column chromatography was
carried out using eluent (petroleum ether/ethyl acetate, 4 : 1, containing
1
0.5% formic acid). Yield: 75%; white solid; mp: 204–205 ◦C; H NMR
(400 MHz, CDCl3) δ 8.66 (s, 1H), 8.15 (s, 1H), 7.73 (d, J = 8.6 Hz, 2H),
7.64 (d, J = 8.6 Hz, 2H), 7.49 (s, 1H), 5.82 (s, 1H), 4.30 (d, J = 16.8 Hz,
1H), 2.57 (s, 1H), 2.35 – 2.19 (m, 2H), 2.12 – 1.95 (m, 3H), 1.94 – 1.82
(m, 1H), 1.81 – 1.70 (m, 1H), 1.65 (t, J = 13.5 Hz, 1H), 1.61 – 1.49 (m,
4H), 1.49 – 1.40 (m, 3H), 1.40 – 1.30 (m, 2H), 1.27 – 1.21 (m, 7H), 1.21
– 1.15 (m, 9H), 1.07 (d, J = 15.1 Hz, 2H), 0.87 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 207.4, 199.7, 180.7, 170.7, 152.3, 140.8, 136.4, 136.1,
135.5, 135.3, 131.8, 128.6, 128.6, 123.0, 119.9, 59.5, 53.6, 48.5, 45.7,
45.1, 44.5, 43.8, 43.5, 41.3, 37.8, 36.5, 32.0, 31.7, 31.1, 29.7, 28.7,
28.6, 26.7, 26.6, 23.4, 22.8, 19.7, 18.2, 15.6. HRMS (ESI): C39H50N3O4
(624.3796) [M+H]+=624.3793.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-((6-(trifluoromethyl)pyr-
idin-3-yl)methylene)-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (21, C37H46F3NO4). According to the general
procedure, derivative 21 was prepared by Claisen Schmidt condensation
of intermediate 10 with 2-trifluoromethyl-pyridine-5-carbaldehyde in
the presence of ethanolic potassium hydroxide at room temperature.
Purification of product by flash column chromatography was carried out
using eluent (petroleum ether/ethyl acetate, 4 : 1, containing 0.5%
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
1
◦
formic acid). Yield: 84%; white solid; mp: 174–175 C; H NMR (500
MHz, Chloroform-d) δ 8.88 – 8.66 (m, 1H), 8.01 (dd, J = 8.2, 1.6 Hz,
1H), 7.71 (d, J = 8.2 Hz, 1H), 7.43 (s, 1H), 5.80 (s, 1H), 4.21 (d, J = 17.9
Hz, 1H), 2.54 (s, 1H), 2.30 – 2.21 (m, 2H), 2.08 – 1.94 (m, 3H), 1.86 (td,
J = 13.5, 4.1 Hz, 1H), 1.79 – 1.69 (m, 1H), 1.63 (t, J = 13.6 Hz, 1H), 1.59
– 1.47 (m, 4H), 1.45 – 1.39 (m, 5H), 1.36 – 1.31 (m, 1H), 1.30 – 1.20 (m,
8H), 1.19 – 1.13 (m, 8H), 1.10 – 1.03 (m, 1H), 0.86 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 206.9, 199.6, 181.6, 170.9, 151.7, 147.1, 138.7,
137.6, 134.8, 131.5, 128.6, 123.0, 120.3, 59.4, 53.8, 48.5, 45.9, 45.2,
44.5, 43.9, 43.6, 41.2, 37.8, 36.6, 32.1, 31.7, 31.1, 29.6, 28.8, 28.5,
26.7, 26.6, 23.4, 22.8, 19.7, 18.2, 15.6. HRMS (ESI): C37H47F3NO4
(626.3452) [M+H]+=626.3455.
3-fluoro-4-methoxybenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-
dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (24, C38H49FO5). According to the general
procedure, derivative 24 was prepared by Claisen Schmidt condensation
of intermediate 10 with 3-fluoro-4-methoxybenzaldehyde in the pres-
ence of ethanolic potassium hydroxide at room temperature. Purifica-
tion of product by flash column chromatography was carried out using
eluent (petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic
acid). Yield: 85%; white solid; mp: 188–189 ◦C; 1H NMR (400 MHz,
CDCl3) δ 7.37 (s, 1H), 7.28 – 7.22 (m, 2H), 6.98 (t, J = 8.8 Hz, 1H), 5.82
(s, 1H), 4.21 (d, J = 16.8 Hz, 1H), 3.89 (s, 3H), 2.57 (s, 1H), 2.25 (d, J =
15.8 Hz, 2H), 2.13 – 1.96 (m, 3H), 1.93 – 1.81 (m, 1H), 1.79 – 1.61 (m,
2H), 1.60 – 1.48 (m, 4H), 1.48 – 1.34 (m, 6H), 1.31 – 1.22 (m, 3H), 1.20
(d, J = 8.4 Hz, 6H), 1.15 (s, 6H), 1.07 (d, J = 13.7 Hz, 2H), 0.87 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 207.6, 199.6, 182.3, 170.1, 152.0, 148.1,
136.03, 133.12, 129.3, 128.8, 127.3, 118.2, 113.2, 59.5, 56.3, 53.4,
48.4, 45.4, 45.2, 44.7, 43.9, 43.5, 41.1, 37.8, 36.3, 32.0, 31.6, 31.0,
29.9, 28.7, 28.5, 26.7, 26.6, 23.3, 22.7, 19.7, 18.2, 15.5. HRMS (ESI):
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-11-
((2,6-dichloropyridin-3-yl)methylene)-2,4a,6a,6b,9,9,12a-heptamethyl-
10,13-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-ico-
sahydropicene-2-carboxylic acid (22, C36H45Cl2NO4). According to the
general procedure, derivative 22 was prepared by Claisen Schmidt
condensation of intermediate 10 with 2,6-dichloropyridine-3-carbalde-
hyde in the presence of ethanolic potassium hydroxide at room
C
38H50FO5 (605.3637) [M+H]+=605.3642.
9

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
2H), 7.03 – 6.93 (m, 1H), 6.88 (d, J = 15.4 Hz, 1H), 5.85 (s, 1H), 2.57 (s,
1H), 2.34 – 2.22 (m, 4H), 2.13 (d, J = 17.4 Hz, 1H), 2.08 – 1.94 (m, 3H),
1.94 – 1.83 (m, 1H), 1.79 – 1.61 (m, 2H), 1.59 – 1.48 (m, 3H), 1.48 –
1.40 (m, 3H), 1.39 – 1.33 (m, 1H), 1.30 – 1.23 (m, 6H), 1.20 (s, 3H), 1.17
(d, J = 6.0 Hz, 6H), 1.13 (s, 3H), 1.07 (d, J = 13.5 Hz, 2H), 0.88 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 206.6, 199.9, 181.5, 170.2, 140.9, 139.1,
137.9, 134.2, 132.4, 129.6, 128.9, 127.4, 123.0, 59.5, 53.7, 48.5, 45.4,
45.2, 44.0, 43.6, 43.0, 41.2, 37.9, 36.4, 32.1, 31.8, 31.1, 29.8, 28.8,
28.6, 26.7, 26.6, 23.4, 22.8, 21.5, 19.8, 18.3, 15.5. HRMS (ESI):
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-
2,4a,6a,6b,9,9,12a-heptamethyl-11-((Z)-4-methylbenzylidene)-10,13-
dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (25, C38H50O4). According to the general
procedure, derivative 25 was prepared by Claisen Schmidt condensation
of intermediate 10 with p-tolualdehyde in the presence of ethanolic
potassium hydroxide at room temperature. Purification of product by
flash column chromatography was carried out using eluent (petroleum
ether/ethyl acetate, 6 : 1, containing 0.5% formic acid). Yield: 79%;
white solid; mp: 292–293 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.45 (s, 1H),
7.40 (d, J = 8.1 Hz, 2H), 7.18 (t, J = 7.4 Hz, 2H), 5.81 (s, 1H), 2.56 (s,
1H), 2.34 (s, 3H), 2.31 – 2.18 (m, 2H), 2.11 – 1.95 (m, 3H), 1.87 (td, J =
13.4, 3.9 Hz, 1H), 1.78 – 1.60 (m, 2H), 1.55 (s, 4H), 1.47 – 1.39 (m, 5H),
1.40 – 1.29 (m, 2H), 1.29 – 1.22 (m, 3H), 1.21 (s, 3H), 1.19 (s, 3H), 1.15
(d, J = 2.6 Hz, 6H), 1.07 (d, J = 13.7 Hz, 2H), 0.87 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 207.9, 199.6, 181.4, 169.9, 138.8, 137.5, 133.3,
133.3, 130.7, 129.4, 128.8, 59.6, 53.5, 48.5, 45.5, 45.2, 44.9, 44.0, 43.5,
41.2, 37.9, 36.4, 32.1, 31.7, 31.1, 29.9, 28.8, 28.6, 26.7, 26.6, 23.4,
22.7, 21.5, 19.8, 18.2, 15.6. HRMS (ESI): C38H51O4 (571.3782)
[M+H]+=571.3784.
C
40H53O4 (597.3938) [M+H]+=597.3939.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-
2,4a,6a,6b,9,9,12a-heptamethyl-11-((5-methylpyrazin-2-yl)methylene)-
10,13-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-ico-
sahydropicene-2-carboxylic acid (28, C36H48N2O4). According to the
general procedure, derivative 28 was prepared by Claisen Schmidt
condensation of intermediate 10 with 2-pyrazinecarboxaldehyde,5-
methyl- in the presence of ethanolic potassium hydroxide at room
temperature. Purification of product by flash column chromatography
was carried out using eluent (petroleum ether/ethyl acetate, 4 : 1,
containing 0.5% formic acid). Yield: 88%; white solid; mp: 213–214 ◦C;
1H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 7.1 Hz, 2H), 7.40 (s, 1H), 5.80
(s, 1H), 4.48 (d, J = 18.4 Hz, 1H), 2.60 (s, 1H), 2.56 (s, 3H), 2.50 (d, J =
19.7 Hz, 1H), 2.29 – 2.20 (m, 1H), 2.13 – 1.95 (m, 3H), 1.87 (td, J =
13.3, 3.6 Hz, 1H), 1.80 – 1.61 (m, 2H), 1.59 – 1.50 (m, 3H), 1.50 – 1.31
(m, 5H), 1.28 – 1.21 (m, 8H), 1.20 – 1.11 (m, 9H), 1.07 (d, J = 13.4 Hz,
2H), 0.87 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.6, 199.4, 181.6,
169.8, 152.0, 148.5, 146.2, 144.3, 139.9, 130.8, 128.8, 59.3, 53.6, 48.5,
45.6, 45.3, 45.2, 44.0, 43.6, 41.2, 37.9, 36.1, 32.1, 31.7, 31.1, 29.9,
28.8, 28.6, 26.7, 26.6, 23.4, 22.6, 21.5, 19.8, 18.2, 15.6. HRMS (ESI):
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-((Z)-4-(trifluoromethyl)
benzylidene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-ico-
sahydropicene-2-carboxylic acid (26, C38H47F3O4). According to the
general procedure, derivative 26 was prepared by Claisen Schmidt
condensation of intermediate 26 with 4-trifluoromethylbenzaldehyde in
the presence of ethanolic potassium hydroxide at room temperature.
Purification of product by flash column chromatography was carried out
using eluent (petroleum ether/ethyl acetate, 6 : 1, containing 0.5%
C
36H49N2O4 (573.3687) [M+H]+=573.3692.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
1
◦
formic acid). Yield: 87%; white solid; mp: 280–281 C; H NMR (400
MHz, CDCl3) δ 7.64 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.46 (s,
1H), 5.81 (s, 1H), 4.24 (d, J = 16.8 Hz, 1H), 2.55 (s, 1H), 2.25 (d, J =
16.3 Hz, 2H), 2.12 – 1.93 (m, 3H), 1.93 – 1.81 (m, 1H), 1.79 – 1.69 (m,
1H), 1.65 (t, J = 13.5 Hz, 1H), 1.60 – 1.47 (m, 4H), 1.47 – 1.38 (m, 3H),
1.38 – 1.30 (m, 1H), 1.24 (dd, J = 16.6, 7.9 Hz, 8H), 1.19 (s, 3H), 1.17
(d, J = 1.9 Hz, 6H), 1.07 (d, J = 13.3 Hz, 2H), 0.87 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 207.3, 199.5, 180.6, 170.3, 139.4, 139.4, 136.2,
135.3, 130.3, 128.6, 125.4, 59.3, 53.6, 48.4, 45.7, 45.1, 44.3, 43.7, 43.4,
41.1, 37.7, 36.4, 31.9, 31.6, 30.9, 29.5, 28.6, 28.4, 26.6, 26.4, 23.2,
4-bromo-2-fluorobenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-
dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (29, C37H46BrFO4). According to the general
procedure, derivative 29 was prepared by Claisen Schmidt condensation
of intermediate 10 with 2-bromo-4-fluorobenzaldehyde in the presence
of ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic acid).
Yield: 68%; white solid; mp: 282–283 ◦C; 1H NMR (400 MHz, DMSO‑d6)
δ 12.12 (s, 1H), 7.69 – 7.59 (m, 1H), 7.52 – 7.39 (m, 2H), 7.36 (s, 1H),
5.45 (s, 1H), 3.80 (d, J = 16.9 Hz, 1H), 2.64 (s, 1H), 2.45 (d, J = 18.7 Hz,
1H), 2.17 – 2.03 (m, 2H), 1.86 – 1.66 (m, 5H), 1.66 – 1.48 (m, 3H), 1.44
– 1.34 (m, 6H), 1.31 – 1.17 (m, 5H), 1.12 – 1.07 (m, 9H), 0.98 (s, 3H),
0.89 – 0.82 (m, 1H), 0.77 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ
206.0, 199.0, 178.1, 171.0, 162.0, 156.2, 137.4, 132.2, 128.0, 127.8,
127.3, 123.0, 119.7, 58.6, 52.6, 48.7, 45.5, 45.0, 43.7, 43.5, 43.2, 41.2,
22.6, 19.6, 18.1, 15.4. HRMS (ESI):
C38H48F3O4 (625.3499)
[M+H]+=625.3501.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-((E)-3-(p-tolyl)allyli-
dene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (27, C40H52O4). According to the general
procedure, derivative 27 was prepared by Claisen Schmidt condensation
of intermediate 10 with trans-4-methylcinnamaldehyde in the presence
of ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic acid).
Yield: 83%; white solid; mp: 294–296 ◦C; 1H NMR (400 MHz, CDCl3) δ
7.38 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 7.0 Hz, 2H), 7.11 (d, J = 7.9 Hz,
38.0, 36.2, 35.6, 32.1, 29.5, 28.9, 28.3, 26.6, 26.3, 23.2, 23.0, 19.4,
79
18.2, 16.8, 15.4. HRMS (ESI): C
H
BrFNaO4 (675.2456) [M +
37 46
Na]+=675.2460, C37
H
BrFNaO4 (677.3442) [M + Na]+=677.2448.
81
46
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
3-chloro-4-fluorobenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-
10

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (30, C37H46ClFO4). According to the general
procedure, derivative 30 was prepared by Claisen Schmidt condensation
of intermediate 10 with 3-chloro-4-fluorobenzaldehyde in the presence
of ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic acid).
Yield: 66%; mp: 133–134 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.42 (dd, J =
7.1, 2.0 Hz, 1H), 7.40 – 7.33 (m, 1H), 7.03 (t, J = 8.7 Hz, 1H), 6.45 (s,
1H), 5.78 (s, 1H), 3.73 (d, J = 14.8 Hz, 1H), 2.58 – 2.49 (m, 1H), 2.29 –
2.20 (m, 2H), 2.12 – 1.81 (m, 5H), 1.79 – 1.60 (m, 3H), 1.60 – 1.54 (m,
2H), 1.46 – 1.39 (m, 6H), 1.29 (s, 3H), 1.28 – 1.23 (m, 6H), 1.20 – 1.18
(m, 3H), 1.16 – 1.13 (m, 3H), 1.07 (s, 2H), 0.87 (s, 3H). 13C NMR (126
MHz, CDCl3) δ 207.3, 199.5, 182.1, 170.3, 157.8, 135.0, 134.8, 133.2,
133.0, 130.0, 128.6, 121.1, 116.6, 59.3, 53.4, 48.31, 45.6, 45.1, 44.1,
43.8, 43.4, 41.0, 37.7, 36.3, 31.9, 31.5, 30.9, 29.6, 28.6, 28.4, 26.6,
26.4, 23.2, 22.6, 19.6, 18.1, 15.4. HRMS (ESI): C37H47ClFO4 (631.2960)
[M + Na]+=631.2933.
129.9, 128.7, 125.8, 120.0, 59.4, 54.2, 53.8, 48.4, 45.8, 45.2, 44.1, 43.9,
43.5, 41.1, 37.8, 36.6, 32.0, 31.7, 31.0, 29.6, 28.7, 28.5, 26.7, 26.6,
23.3, 22.8, 19.7, 18.2, 15.5. HRMS (ESI): C37H49FNO5 (606.3589)
[M+H]+=606.3590.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
4-acetamidobenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (33, C39H51NO5). According to the general
procedure, derivative 31 was prepared by Claisen Schmidt condensation
of intermediate 10 with 4-Acetamidobenzaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 2 : 3, containing 0.5% formic acid).
Yield 82.3%; yellow solid; mp: 214–216 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 12.21 (s, 1H), 10.13 (s, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.45
(d, J = 8.2 Hz, 2H), 7.30 (s, 1H), 5.47 (s, 1H), 3.93 (d, J = 17.1 Hz, 1H),
2.67 (s, 1H), 2.48 (s, 4H), 2.10 (d, J = 13.0 Hz, 2H), 1.87 – 1.61 (m, 6H),
1.59 – 1.45 (m, 3H), 1.41 (d, J = 21.5 Hz, 5H), 1.35 (d, J = 13.4 Hz, 2H),
1.29 – 1.12 (m, 4H), 1.11 – 1.06 (m, 8H), 1.04 (s, 2H), 0.96 (s, 3H), 0.76
(s, 2H). 13C NMR (126 MHz, DMSO‑d6) δ 206.2, 199.1, 178.1, 170.9,
169.0, 140.3, 136.3, 132.8, 131.7, 130.4, 128.0, 119.1, 58.6, 52.4, 48.6,
45.0, 44.9, 43.8, 43.6, 43.5, 41.2, 38.0, 36.0, 32.0, 31.3, 30.8, 29.9,
29.0, 28.2, 26.6, 26.3, 24.6, 23.2, 22.9, 19.6, 18.1, 15.5. HRMS (ESI):
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR)-11-((Z)-
2,4-dimethoxybenzylidene)-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (31, C39H52O6). According to the general
procedure, derivative 31 was prepared by Claisen Schmidt condensation
of intermediate 10 with 2,4-dimethoxybenzaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 6 : 1, containing 0.5% formic acid).
Yield: 85%; mp: 209–210 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H),
7.41 (d, J = 8.6 Hz, 1H), 6.51 (dd, J = 8.6, 2.3 Hz, 1H), 6.44 (d, J = 2.3
Hz, 1H), 5.79 (s, 1H), 4.17 (d, J = 16.6 Hz, 1H), 3.82 (s, 3H), 3.80 (s,
3H), 2.54 (s, 1H), 2.27 – 2.14 (m, 2H), 2.12 – 1.92 (m, 3H), 1.87 (td, J =
13.4, 3.9 Hz, 1H), 1.78 – 1.59 (m, 2H), 1.59 – 1.46 (m, 4H), 1.46 – 1.38
(m, 5H), 1.38 – 1.30 (m, 1H), 1.29 – 1.21 (m, 3H), 1.21 – 1.11 (m, 12H),
1.06 (d, J = 13.8 Hz, 2H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
207.4, 199.6, 181.0, 169.8, 161.6, 160.2, 132.7, 131.6, 131.2, 128.9,
118.2, 104.4, 98.6, 59.7, 55.7, 55.5, 53.6, 48.5, 45.5, 45.2, 44.7, 43.9,
43.5, 41.2, 37.9, 36.6, 32.1, 31.8, 31.1, 29.9, 28.7, 28.6, 26.7, 26.6,
23.4, 22.9, 19.8, 18.3, 15.6. HRMS (ESI): C39H53O6 (617.3837)
[M+H]+=617.3842.
C
39H51NNaO5 (636.3659) [M + Na]+=636.3649.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-(quinolin-8-ylmethy-
lene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (34, C40H49NO4). According to the general
procedure, derivative 31 was prepared by Claisen Schmidt condensation
of intermediate 10 with 8-quinolinecarboxaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 2 : 1, containing 0.5% formic acid).
Yield 89%; white solid; mp: 210–211 ◦C; 1H NMR (500 MHz, CDCl3) δ
8.99 (dd, J = 4.2, 1.8 Hz, 1H), 8.65 (s, 1H), 8.16 (dd, J = 8.3, 1.7 Hz,
1H), 7.87 (d, J = 7.2 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 7.7
Hz, 1H), 7.45 (dd, J = 8.2, 4.2 Hz, 1H), 5.77 (s, 1H), 4.29 (d, J = 17.1 Hz,
1H), 2.55 (s, 1H), 2.32 – 2.25 (m, 1H), 2.22 (dd, J = 13.2, 3.5 Hz, 1H),
2.09 – 2.00 (m, 2H), 1.98 – 1.92 (m, 1H), 1.87 (td, J = 13.6, 4.1 Hz, 1H),
1.75 (t, J = 10.5 Hz, 1H), 1.66 (d, J = 13.6 Hz, 1H), 1.58 (td, J = 16.3,
14.0, 6.3 Hz, 3H), 1.51 (d, J = 13.3 Hz, 2H), 1.43 (d, J = 14.9 Hz, 6H),
1.27 (s, 3H), 1.26 (d, J = 1.8 Hz, 6H), 1.24 (s, 6H), 1.19 (s, 2H), 0.87 (s,
3H). 13C NMR (126 MHz, CDCl3) δ 207.2, 199.6, 181.8, 170.1, 149.8,
147.1, 136.4, 134.8, 134.8, 134.3, 130.2, 128.5, 128.5, 128.3, 126.2,
121.3, 59.3, 53.7, 48.2, 45.7, 45.1, 44.1, 43.8, 43.4, 40.9, 37.7, 36.6,
31.9, 31.6, 30.9, 29.5, 28.6, 28.5, 26.5, 26.4, 23.3, 22.9, 19.5, 18.1,
15.5. HRMS (ESI): C40H49NNaO4 (630.3554) [M + Na]+=630.3556.
(2S,4aS,6aS,6bR,8aR,12aS,12bR,14bR,Z)-11-
((5-fluoro-2-methoxypyridin-3-yl)methylene)-2,4a,6a,6b,9,9,12a-hepta-
methyl-10,13-dioxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahy-
dropicene-2-carboxylic acid (32, C37H48FNO5). According to the general
procedure, derivative 32 was prepared by Claisen Schmidt condensation
of intermediate 10 with 5-fluoro-2-methoxynicotinaldehyde in the
presence of ethanolic potassium hydroxide at room temperature. Puri-
fication of product by flash column chromatography was carried out
using eluent (petroleum ether/ethyl acetate, 5 : 1, containing 0.5%
4.1.2. General procedure for the synthesis of UA derivatives 35, 36
UA derivatives 35, 36 were obtained according to Scheme 1. UA was
1
◦
formic acid). Yield: 72%; white solid; mp: 194–195 C; H NMR (400
MHz, CDCl3) δ 7.95 (d, J = 2.9 Hz, 1H), 7.59 – 7.52 (m, 1H), 7.50 (dd, J
= 8.6, 2.8 Hz, 1H), 5.79 (s, 1H), 4.17 – 4.04 (m, 1H), 3.96 (s, 2H), 2.53
(s, 1H), 2.35 – 2.21 (m, 1H), 2.16 (dd, J = 16.6, 1.5 Hz, 1H), 2.12 – 1.93
(m, 3H), 1.87 (td, J = 13.3, 3.7 Hz, 1H), 1.78 – 1.60 (m, 2H), 1.60 – 1.47
(m, 4H), 1.47 – 1.40 (m, 5H), 1.40 – 1.26 (m, 3H), 1.27 – 1.22 (m, 4H),
1.21 – 1.12 (m, 10H), 1.12 – 1.03 (m, 2H), 0.87 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 206.9, 199.4, 182.0, 170.2, 158.6, 154.9, 136.5, 133.0,
◦
dissolved in acetone at 0 C; Jones reagent was added to the reaction
system drop-wisely until the solution colour was stable in light brown,
implied that the Jones reagent was in slight excess to oxidize the C-3
hydroxyl group into ketone to provide intermediate 11 without further
purification. Derivatives 35, 36 were prepared by Claisen Schmidt
condensation of intermediate 11 with corresponding aldehydes in the
presence of ethanolic potassium hydroxide in good yield at room
11

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
temperature.
(4aS,6aS,6bR,12aR)-2,2,6a,6b,9,9,12a-hepta-
(1S,2R,4R,6aS,6bR,12aR)-1,2,6a,6b,9,9,12a-
methyl-10-oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octade-
cahydropicene-4a(2H)-carboxylic acid (OA-O, 12, C30H46O3). Yield: 91%;
white solid; mp: 209–210 ◦C; 1H NMR (400 MHz, CDCl3) δ 5.31 (t, J =
3.3 Hz, 1H), 2.84 (dd, J = 13.8, 4.1 Hz, 1H), 2.63 – 2.47 (m, 1H), 2.42 –
2.32 (m, 1H), 2.07 – 1.80 (m, 4H), 1.80 – 1.68 (m, 2H), 1.68 – 1.55 (m,
4H), 1.52 – 1.45 (m, 3H), 1.43 (s, 1H), 1.41 – 1.17 (m, 6H), 1.15 (s, 3H),
1.09 (s, 3H), 1.04 (d, J = 6.0 Hz, 5H), 0.99 – 0.85 (m, 7H), 0.81 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 217.7, 183.2, 143.8, 122.6, 55.6, 47.6,
47.1, 46.8, 46.0, 42.0, 41.3, 39.5, 39.3, 37.0, 34.3, 34.0, 33.2, 32.6,
32.4, 30.8, 27.9, 26.7, 26.0, 23.7, 23.7, 23.2, 21.6, 19.8, 17.2, 15.2.
HRMS (ESI): C30H46NaO3 (477.3339) [M + Na]+=477.3342.
heptamethyl-10-oxo-11-((Z)-4-(trifluoromethyl)benzylidene)-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydropi-
cene-4-carboxylic acid (35, C38H49F3O3). According to the general pro-
cedure, derivative 35 was prepared by Claisen Schmidt condensation of
intermediate 11 with 4-trifluoromethylbenzaldehyde in the presence of
ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 8 : 1, containing 0.5% formic acid).
Yield: 91%; white solid; mp: 171–172 ◦C; 1H NMR (400 MHz, CDCl3) δ
7.67 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.5 Hz, 3H), 5.27 (t, J = 3.2 Hz,
1H), 2.98 (d, J = 16.3 Hz, 1H), 2.34 – 2.14 (m, 2H), 2.09 – 1.97 (m, 1H),
1.94 (dd, J = 8.6, 3.2 Hz, 2H), 1.86 (td, J = 13.7, 4.0 Hz, 1H), 1.78 – 1.61
(m, 4H), 1.59 – 1.46 (m, 3H), 1.46 – 1.18 (m, 6H), 1.14 (d, J = 7.9 Hz,
9H), 1.08 – 0.99 (m, 1H), 0.96 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 5.7 Hz,
3H), 0.87 (s, 3H), 0.81 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.6,
183.7, 139.6, 139.6, 138.3, 136.0, 135.8, 130.4, 125.6, 125.50, 125.46,
53.4, 52.8, 48.2, 45.5, 45.4, 44.1, 42.4, 39.6, 39.3, 39.0, 36.8, 36.6,
32.2, 30.8, 29.7, 28.1, 24.2, 23.7, 23.6, 22.9, 21.3, 20.4, 17.2, 16.9,
15.6. ESI-MS m/z 609.4 [Mꢀ H]ꢀ . HRMS (ESI): C38H49F3NaO3
(633.3526) [M + Na]+=633.3525.
(4aS,6aS,6bR,12aR)-2,2,6a,6b,9,9,12a-hepta-
methyl-10-oxo-11-((Z)-4-(trifluoromethyl)benzylidene)-
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylic acid (37, C38H49F3O3). According to the general
procedure, derivative 37 was prepared by Claisen Schmidt condensation
of intermediate 12 with 4-trifluoromethylbenzaldehyde in the presence
of ethanolic potassium hydroxide at room temperature. Purification of
product by flash column chromatography was carried out using eluent
(petroleum ether/ethyl acetate, 3 : 1, containing 0.5% formic acid).
Yield: 90%; white solid; mp: 268–269 ◦C; 1H NMR (400 MHz, CDCl3) δ
7.64 (d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.5 Hz, 3H), 5.33 (t, J = 3.1 Hz,
1H), 2.95 (d, J = 16.4 Hz, 1H), 2.86 (dd, J = 13.5, 4.0 Hz, 1H), 2.28 (d, J
= 16.3 Hz, 1H), 2.06 – 1.88 (m, 3H), 1.85 – 1.55 (m, 7H), 1.55 – 1.31 (m,
7H), 1.20 (s, 4H), 1.17 (s, 3H), 1.15 (s, 3H), 1.00 – 0.89 (m, 6H), 0.86 (s,
3H), 0.82 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.7, 182.9, 143.9,
139.6, 136.0, 135.7, 130.4, 125.5, 125.5, 125.4, 122.4, 53.3, 46.8, 46.1,
45.6, 45.5, 44.2, 42.2, 41.4, 39.4, 36.5, 34.0, 33.2, 32.5, 32.0, 30.8,
29.9, 29.8, 27.8, 25.9, 23.8, 23.7, 23.2, 22.8, 20.5, 16.8, 15.4. HRMS
(ESI): C38H49F3NaO3 (633.3526) [M + Na]+=633.3522.
(1S,2R,4R,6aS,6bR,12aR)-1,2,6a,6b,9,9,12a-
heptamethyl-11-((Z)-4-methylbenzylidene)-10-oxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,
12b,13,14b-icosahydropi-
cene-4-carboxylic acid (36, C38H52O3). According to the general pro-
cedure, derivative 36 was prepared by Claisen Schmidt condensation of
intermediate 12 with 4-methylbenzaldehyde in the presence of etha-
nolic potassium hydroxide at room temperature. Purification of product
by flash column chromatography was carried out using eluent (petro-
leum ether/ethyl acetate, 8 : 1, containing 0.5% formic acid). Yield:
87%; white solid: mp: 142–143 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.53 (s,
1H), 7.35 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 5.28 (s, 1H), 3.03
(d, J = 16.2 Hz, 1H), 2.38 (s, 3H), 2.31 – 2.24 (m, 1H), 2.22 (d, J = 11.4
Hz, 1H), 2.09 – 1.93 (m, 3H), 1.86 (td, J = 13.4, 3.8 Hz, 1H), 1.78 – 1.61
(m, 3H), 1.59 – 1.45 (m, 4H), 1.45 – 1.33 (m, 4H), 1.26 (s, 3H), 1.14 (d,
J = 3.3 Hz, 9H), 0.96 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.4 Hz, 3H), 0.87
(s, 3H), 0.81 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.9, 183.8, 138.8,
138.2, 137.8, 133.3, 133.1, 130.6, 129.4, 125.8, 53.3, 52.9, 48.2, 45.5,
45.3, 44.3, 42.4, 39.6, 39.3, 39.0, 36.9, 36.4, 32.3, 30.8, 29.8, 28.2,
24.3, 23.8, 23.6, 22.8, 21.5, 21.3, 20.5, 17.2, 16.9, 15.6. ESI-MS m/z
(4aS,6aS,6bR,12aR)-2,2,6a,6b,9,9,12a-hepta-
methyl-11-((Z)-4-methylbenzylidene)-10-oxo-
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-
4a(2H)-carboxylic acid (38, C38H52O3). According to the general pro-
cedure, derivative 38 was prepared by Claisen Schmidt condensation of
intermediate 12 with 4-methylbenzaldehyde in the presence of etha-
nolic potassium hydroxide at room temperature. Purification of product
by flash column chromatography was carried out using eluent (petro-
leum ether/ethyl acetate, 3 : 1, containing 0.5% formic acid). Yield:
93%; white solid; mp: 154–155 ◦C; 1H NMR (400 MHz, CDCl3) δ 7.52 (s,
1H), 7.33 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 5.33 (t, J = 3.1
Hz, 1H), 3.00 (d, J = 16.3 Hz, 1H), 2.85 (dd, J = 13.6, 3.8 Hz, 1H), 2.38
(s, 3H), 2.28 (d, J = 15.9 Hz, 1H), 2.07 – 1.88 (m, 3H), 1.83 – 1.69 (m,
3H), 1.69 – 1.54 (m, 3H), 1.54 – 1.44 (m, 3H), 1.44 – 1.30 (m, 3H), 1.28
– 1.16 (m, 6H), 1.14 (d, J = 8.7 Hz, 6H), 0.92 (d, J = 7.2 Hz, 6H), 0.85 (s,
3H), 0.81 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.9, 183.9, 143.9,
138.8, 137.7, 133.3, 133.0, 130.6, 129.3, 122.6, 53.2, 46.8, 46.1, 45.6,
45.3, 44.4, 42.1, 41.3, 39.4, 36.4, 34.0, 33.2, 32.5, 32.0, 30.8, 29.9,
27.9, 25.9, 23.8, 23.7, 23.2, 22.8, 21.5, 20.5, 16.8, 15.4. HRMS (ESI):
555.4 [Mꢀ H]ꢀ . HRMS (ESI): C38H52NaO3 (579.3809) [M
Na]+=579.3812.
+
4.1.3. General procedure for the synthesis of UA derivatives 37, 38
OA derivatives 37, 38 were obtained according to Scheme 1. OA was
◦
dissolved in acetone at 0 C; Jones reagent was added to the reaction
system drop-wisely until the solution colour was stable in light brown,
implied that the Jones reagent was in slight excess to oxidize the C-3
hydroxyl group into ketone to provide the intermediate 12. Purification
of compound 12 by flash column chromatography was carried out using
eluent (petroleum ether/ethyl acetate, 1 : 1, containing 0.5% formic
acid). Derivatives 37, 38 were prepared by Claisen Schmidt condensa-
tion of intermediate 16 with corresponding aldehydes in the presence of
ethanolic potassium hydroxide in good yield at room temperature.
C
38H52NaO3 (579.3809) [M + Na]+=579.3803.
12

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
4.2. Methods for biological assessments
bound in the original crystal structure. The top pose and protein were
loaded into work area and the MOLCAD (Molecular Computer Aided
Design) program was employed to visualize the binding mode between
the protein and the ligand. MOLCAD calculates and exhibits the surfaces
of channels and cavities. And the protein–ligand complexes were moved
to LigPlus program to determine the hydrophobic interaction.
4.2.1. Microorganisms and Culture media
The bacterial strains of Staphylococcus aureus (ATCC 6538), Staphy-
lococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228),
Methicillin-resistant Staphylococcus aureus (MRSA), Salmonella typhimu-
rium (CMCC 50115) and Escherichia coli (CMCC 44102) were obtained
from Guangdong Culture Collection Center (Guangdong, People’s Re-
public of China). All the six strains were cultured in Mueller-Hinton Agar
(MHA) and Mueller-Hinton broth (MHB).
4.2.6. Pharmacokinetic properties assays
The DMPK results showed in the Table 3 were assessed through a
high through-put platform kindly provided by AstraZeneca U.K. The
methods of the five assays, including LogD7.4, aqueous solubility, plasma
protein binding, microsome and hepatocyte clearance measurements
have been reported previously [46,47].
4.2.2. Agar disk diffusion method
The antimicrobial activities were determined according to the stan-
dard agar disk diffusion method with a slight modification [38,40–42].
A 0.5 McFarland (1 × 107 to 1 × 108 CFU/mL) concentration of the
bacterial suspension was uniformly inoculated onto MHA solidified in
120 mm petri dishes. Once the dishes were prepared, 6 mm-diameter
4.2.7. Cytotoxicity test
BV2 microglial cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal
bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (Gibco, CA,
USA), and incubated at 37 ◦C under humidified atmosphere containing
5% CO2.
discs of filter paper containing 5 μL of the triterpenoids derivatives,
which had been diluted ten times with dimethyl sulfoxide (DMSO), were
pressed gently against the surface of the agar. Discs containing gati-
floxacin was used as positive control, while DMSO was used as the
negative control. The dishes were incubated in a constant temperature
incubator at 37 ◦C for 24 h. The inhibition zone (IZ) diameter was
measured by a vernier caliper. All experiments were performed in
triplicate.
The cytotoxicity of GA derivatives (33 and 34) was tested on BV2
cells by MTT assay. Briefly, BV2 cells were seeded in 96-well plates at a
density of 5 × 103 cells/well. After incubation overnight, the medium
was replaced with fresh medium containing various concentrations of
33 and 34 (0, 1, 2, 4, 8, 16, 32, 64 μM). After incubating for another 24
h, the cells were washed with PBS, and then incubated with fresh me-
dium containing MTT (0.5 mg/mL) for 4 h. Subsequently, 200 L of
4.2.3. Broth microdilution method
μ
The minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC) were determined by a microdilution
method in 96-well plates according to Clinical and Laboratory Standards
Institute (CLSI), with a slight modification [37,38,43]. A dilution series
of the triterpenoids derivatives were obtained with DMSO as the solvent
DMSO was added to each well, and the optical density was recorded at
550 nm by a Multiskan GO microplate reader (Thermo Fisher Scientific,
MA, USA). The cell viability was calculated from: cell viability = (OD
sample/OD control) × 100%, where the sample represents the cells
treated with 33 and 34 solution and the control means non-treated cells.
by two-fold serial dilution. Each well received 5
centration of the triterpenoids derivative and 195
μL of a specific con-
μL of MHB inoculated
Author contributions
with the test microorganism (1.5 × 105 CFU/mL); the final concentra-
tion of the examined derivative was reached. Gatifloxacin was used as
positive control and DMSO was used as negative control. The micro-
plates were incubated in a bacteriological oven for 24 h at 37 ◦C, and the
antibacterial results of the tested derivatives were monitored by
measuring the absorbance at 600 nm using a Multimodel Plate Reader
(Infinite 200). The lowest concentration without visible growth was
defined as the MIC.
Performing the experimental work and drafting the manuscript:
(PPW, BRT, NNC, SLC, XTX, WDH). Performing the bioactivity test:
(PPW, WDZ, JHL, SZG, WFL). Performing the experimental statistical
analysis (PPW, ZJS, XWT). The director as well as the designer of the
manuscript: (WDH, APR, HM, XZ). The project coordinator: (WDH, DLL,
KZ).
The minimum bactericidal concentrations (MBCs) were determined
Declaration of Competing Interest
based on the MIC results [38,44,45]: serial sub-cultivation of a 5 μL
aliquot near the MIC in microtiter plates containing 195
μ
L of Mueller
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
◦
Hinton broth per well; incubation for 24 h at 37 C. The lowest con-
centration of antimicrobial agent that killed at least 99.9% of the
starting inoculum was defined as the MBC endpoint, which was deter-
mined by measuring the absorbance at 600 nm using a Multimodel Plate
Reader (Infinite 200). All experiments were conducted in triplicate.
Acknowledgments
This study was supported by National Natural Science Foundation of
China (No. 81803390). Natural Science Foundation of Guangdong
Province (No. 008187040035), Special Fund Project of Science and
Technology Innovation Strategy of Guangdong Province 2018 and 2020
[No. Jiangke(2018)352 and Jiangke(2020)182], the project of Jiang-
men city social welfare innovation platform construction (No.
2016350100170008351, 2018090103460022105). The authors are also
grateful to the foundation of Department of Education of Guangdong
Province (No. 2020KZDZX1202, 2018KTSCX236, 2017KSYS010 and
2016KCXTD005) and the Youth Foundation of Wuyi University (No.
2017td01). The authors want to thank AstraZeneca U.K. for providing
the in vitro DMPK assessments for this work.
4.2.4. Killing kinetic studies
The killing kinetic assay on the Gram-positive strains [35–38],
including Staphylococcus aureus (ATCC 6538), Staphylococcus aureus
(ATCC 29213) and Staphylococcus epidermidis (ATCC 12228), was per-
formed against three selected derivatives 21, 32 and 33 in 96-well plates
and four different concentrations (0.5 × MIC, 1 × MIC, 2 × MIC, 4 ×
MIC) of each derivative were assayed. The microplates were incubated
for 20 h at 37 ◦C, and the growth of bacteria was monitored by
measuring the absorbance at 600 nm using a Multimodel Plate Reader
(Infinite 200) every 2 h.
4.2.5. Molecular docking
Molecular docking was carried out using the Surflex-Dock GeomX
module of SYBYL-X 2.0. Briefly, potential ligand binding sites (Proto-
Mol) were defined for the protein–ligand complex based on the ligand
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
13

P. Wu et al.
Bioorganic Chemistry 109 (2021) 104692
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