Molecules 29 04957

molecules
Article
Thiazolidinedione-Conjugated Lupeol Derivatives as Potent
Anticancer Agents Through a Mitochondria-Mediated
Apoptotic Pathway
Siqi Deng 1 , Yinxu Zhao 1 , Xiaoshan Guo 1 , Xian Hong 2 , Gang Li 2 , Yuchun Wang 1 , Qingyi Li 1 , Ming Bu 1, *
and Ming Wang 1, *
1 College of Pharmacy, Qiqihar Medical University, Qiqihar 161006, China; 17735745701@163.com (S.D.);
zyx99813@163.com (Y.Z.); xiaoshan990519@163.com (X.G.); wyc58811@163.com (Y.W.);
qingyili2022@163.com (Q.L.)
2 Research Institute of Medicine & Pharmacy, Qiqihar Medical University, Qiqihar 161006, China;
hongxian24@163.com (X.H.); lgyjy@qmu.edu.cn (G.L.)
* Correspondence: buming@qmu.edu.cn (M.B.); wangming_qy@163.com (M.W.)
Abstract: To improve the potential of lupeol against cancer cells, a privileged structure, thiazolidine-
dione, was introduced into its C-3 hydroxy group with ester, piperazine-carbamate, or ethylenedi-
amine as a linker, and three series of thiazolidinedione-conjugated compounds (6a–i, 9a–i, and 12a–i)
were prepared. The target compounds were evaluated for their cytotoxic activities against human
lung cancer A549, human breast cancer MCF-7, human hepatocarcinoma HepG2, and human hepatic
LO2 cell lines, and the results revealed that most of the compounds displayed improved potency
over lupeol. Compound 12i exhibited significant activity against the HepG2 cell line, with an IC50
value of 4.40 µM, which is 9.9-fold more potent than lupeol (IC50 = 43.62 µM). Mechanistic studies
suggested that 12i could induce HepG2 cell apoptosis, as evidenced by AO/EB staining and annexin
V-FITC/propidium iodide dual staining assays. Western blot analysis suggested that compound
12i can upregulate Bax expression, downregulate Bcl-2 expression, and activate the mitochondria-
mediated apoptotic pathway. Collectively, compound 12i is worthy of further investigation to support
Citation: Deng, S.; Zhao, Y.; Guo, X.; the discovery of effective agents against cancer.
Hong, X.; Li, G.; Wang, Y.; Li, Q.; Bu,
M.; Wang, M. Thiazolidinedione- Keywords: triterpenoid; lupeol; thiazolidinedione; hybrids; antitumor; apoptotic
Conjugated Lupeol Derivatives as
Potent Anticancer Agents Through a
Mitochondria-Mediated Apoptotic
Pathway. Molecules 2024, 29, 4957.
1. Introduction
https://doi.org/10.3390/
molecules29204957
Cancer is the leading cause of death and has a serious impact on human health
worldwide [1,2]. Chemotherapy is a traditional method for treating cancer [3]. However,
Academic Editor: Gabriella Marucci despite the effectiveness of chemotherapeutic drugs, they can also cause adverse reactions
Received: 30 August 2024 in normal cells and are associated with multidrug resistance (MDR) [4]. Therefore, it is
Revised: 13 October 2024 urgent to develop more effective drugs with low toxicity. Natural products have a long
Accepted: 18 October 2024 history of medicinal use and are one of the main sources of new antitumor drugs [5,6]. At
Published: 20 October 2024 present, many plant-based antitumor drugs are in clinical use, such as camptothecin [7],
paclitaxel [8], and podophyllotoxin [9].
Lupeol (1) (Figure 1) is a triterpenoid widely found in fruits and vegetables, such
as grapes, white cabbage, green pepper, strawberry, and others [10,11]. It has numerous
Copyright: © 2024 by the authors.
biological activities, including those against arthritis, inflammation, diabetes, cancer, renal
Licensee MDPI, Basel, Switzerland.
toxicity, heart diseases, and hepatic toxicity, among others [12–17]. Of these, its antitumor
This article is an open access article
activity has received the most attention, and many reports in the literature have discussed
distributed under the terms and
the antitumor effects of lupeol. T.R. Min et al. reported that lupeol can induce cancer
conditions of the Creative Commons
cell apoptosis by suppressing EGFR/STAT3 activity [18]. Bhattacharyya et al. reported
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
that lupeol showed antitumor activity by inhibiting angiogenesis in a mouse model of
4.0/).
melanoma [19]. Zhang X. et al. revealed that lupeol can induce autophagy by inhibiting
Molecules 2024, 29, 4957. https://doi.org/10.3390/molecules29204957 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, x FOR PEER REVIEW 2 of 26
Molecules 2024, 29, 4957 2 of 25
[19]. Zhang X. et al. revealed that lupeol can induce autophagy by inhibiting the Akt-
mTOR
the pathwaypathway
Akt-mTOR and activating an autophagy-inhibited
and activating an autophagy-inhibitedepithelial–mesenchymal transi-
epithelial–mesenchymal
tion (EMT) [20]. Homa Fatma et al. reported that lupeol can inhibit cancer
transition (EMT) [20]. Homa Fatma et al. reported that lupeol can inhibit cancer cell cell proliferation
by regulatingby
proliferation the PI3K/AkT/mTOR
regulating and RAS/BRAF/MEK/ERK
the PI3K/AkT/mTOR pathways, inducing
and RAS/BRAF/MEK/ERK apop-
pathways,
tosis in cancer
inducing apoptosiscellsin [21].
cancerNigam et al.Nigam
cells [21]. showed that
et al. topical
showed lupeol
that applications
topical (200
lupeol applica-
µg/mouse)
tions can prevent
(200 µg/mouse) can7,12-dimethylbenz(a)anthracene
prevent 7,12-dimethylbenz(a)anthracene(DMBA)-induced DNA damage
(DMBA)-induced DNA
(DNA strand
damage (DNAbreaks)
strand in murine
breaks) in skin [22].skin [22].
murine
Figure 1.
Figure 1. Structures
Structures of
of lupeol
lupeol and
and the
the design
design of
of TZD-conjugated
TZD-conjugatedlupeol
lupeolderivatives.
derivatives.
Because
Because of
of lupeol’s
lupeol’s moderate
moderate antitumor
antitumor activity
activity and
and low
low water
water solubility,
solubility, medicinal
medicinal
chemists
chemists have carried out structural modification studies based on it, resulting in
have carried out structural modification studies based on it, resulting in many
many
structurally novel lupeol derivatives. For example, lupeol-3-succinate induced autophagy
structurally novel lupeol derivatives. For example, lupeol-3-succinate induced autophagy
in tumor cells, and lupeol carbamate–quaternary ammonium salt derivatives have an
in tumor cells, and lupeol carbamate–quaternary ammonium salt derivatives have an anti-
anti-proliferative activity that is tens of times higher than their parent compounds in
proliferative activity that is tens of times higher than their parent compounds in hepato-
hepatocellular carcinoma [23,24].
cellular carcinoma [23,24].
Thiazolidinedione (TZD) is a five-membered thiazolidine ring with carbonyl groups
Thiazolidinedione (TZD) is a five-membered thiazolidine ring with carbonyl groups
at the two and four positions. It has a high dipole moment and can form hydrogen bonds
at the two and four positions. It has a high dipole moment and can form hydrogen bonds
with target proteins, which could be favorable in the binding of biomolecules. TZD was
with target proteins, which could be favorable in the binding of biomolecules. TZD was
introduced in the late 1990s as a potential antidiabetic agent for treating type II diabetes [25].
introduced in the late 1990s as a potential antidiabetic agent for treating type Ⅱ diabetes
In addition, TZD has many biological activities, including antibacterial, anticancer, anti-
[25]. In addition, TZD has many biological activities, including antibacterial, anticancer,
arthritic, anti-inflammatory, antioxidant, and so on [26]. TZD regulates apoptosis and
anti-arthritic, anti-inflammatory, antioxidant, and so on [26]. TZD regulates apoptosis and
proliferation in various cancer cell lines [27,28]. Steven D. Knight et al. have reported that
proliferation in various cancer cell lines [27,28]. Steven D. Knight et al. have reported that
the TZD derivative GSK1059615 can exert anticancer effects by inhibiting PI3K-α [29]. Liu
theal.TZD
et derivative
reported a TZD GSK1059615 can anticancer
with potential exert anticancer effects
activity by inhibiting
through PI3K-α [29].and
the Raf/MEK/ERK Liu
PI3K/Akt signaling pathways [30]. Several anticancer drugs containing TZD moieties are
et al. reported a TZD with potential anticancer activity through the Raf/MEK/ERK and
Molecules 2024, 29, 4957 PI3K/Akt
et al. reportedsignaling
a TZD pathways [30]. Several
with potential anticancer
anticancer drugs
activity containing
through TZD moieties
the Raf/MEK/ERK are
3and
of 25
currently
PI3K/Akt undergoing
signaling pathwaysclinical trials, including
[30]. Several chemical
anticancer Ⅰ (S-49076
drugs hydrochloride)
containing TZD moieties for are
the
treatment of solid tumors
currently undergoing and trials,
clinical chemical Ⅱ (inolitazone
including chemical Ⅰ (S-49076 hydrochloride)
hydrochloride) for the treatment for the of
colorectal
treatment cancer
of solid (Figure
tumors 1).
and Therefore,
chemical Ⅱ
TZD moieties
(inolitazone can be considered
hydrochloride)
currently undergoing clinical trials, including chemical I (S-49076 hydrochloride) for the for privileged
the treatment struc-
of
tures in thecancer
colorectal
treatment ofdesign of antitumor
solid(Figure
tumors 1).and agents TZD
Therefore,
chemical [31,32].
moieties canhydrochloride)
II (inolitazone be considered for privileged struc-
the treatment
tures Wein decided
the design to incorporate
of antitumor the TZD
agents moiety
[31,32]. into the C-3
of colorectal cancer (Figure 1). Therefore, TZD moieties can be considered privileged position of lupeol with ester,
piperazine–carbamate,
We decided
structures or
ofethylenediamine
to incorporate
in the design the TZD
antitumor as
moiety
agents a linker.
into theInC-3
[31,32]. thisposition
way, weofdesigned
lupeol with the ester,
TZD-
conjugated
We decided compounds
piperazine–carbamate, or6a–i, 9a–i,
the and
TZD12a–i
ethylenediamine
to incorporate as atolinker.
moiety improve
into In C-3
the theposition
this potential
way, we of of lupeol
designed
lupeol theagainst
with TZD-
ester,
different
conjugated cancer
piperazine–carbamate, cells (Figure
compounds or6a–i, 1). Theand
9a–i,
ethylenediaminetarget compounds
12a–ias to improve
a linker. Inwerethe synthesized,
this potential
way, and their
of lupeol
we designed anti-
against
the TZD-
different
tumor cancer
activities cells
against (Figure
the 1).
human The target
non-small compounds
cell lung were
cancer
conjugated compounds 6a–i, 9a–i, and 12a–i to improve the potential of lupeol against synthesized,
cells (A549), and their
human anti-
breast
tumor cells
cancer
different activities
cancer against
(MCF-7),
cells the human
human
(Figure 1). Thenon-small
hepatocellular cell lung cancer
target carcinomas
compounds cells cells (A549),
were(HepG2), human
and human
synthesized, and their breast
hepatic
anti-
cancer
cells
tumor cells were
(LO2) (MCF-7),
activities against human
evaluated. hepatocellular
In
the human addition, thecarcinomas
non-small cell lung cells
mechanism of (HepG2),
cancer action
cellsof and human
compound
(A549), human hepatic
12i was
breast
cells (LO2)
further
cancer were evaluated.
investigated.
cells (MCF-7), human In addition, thecarcinomas
hepatocellular mechanismcells of action
(HepG2), of compound
and human12i was
hepatic
further
cells investigated.
(LO2) were evaluated. In addition, the mechanism of action of compound 12i was
2. Results
further and Discussion
investigated.
2. Results
2.1. Synthesis andof Discussion
Lupeol Derivatives
2. Results
2.1. Synthesis and Discussion
2.1.1. Synthesis Lupeol
of Derivatives5a–i
of Intermediates
2.1. Synthesis of Lupeol Derivatives
2.1.1. Synthesis of Intermediates 5a–i via the Knoevenagel condensation reaction of 2,4-
2.1.1.Intermediates 3a–i were obtained
Synthesis of Intermediates 5a–i
Intermediates(2)
thiazolidinedione 3a–i were
with obtained
different via the in
aldehydes Knoevenagel
the presence condensation
of piperidinereaction of 2,4-
as the catalyst
Intermediates 3a–i were obtained via the Knoevenagel condensation reaction of
thiazolidinedione
[33]. Intermediates(2) with
3a–i different
were aldehydes
alkylated in thebromoacetate,
with ethyl presence of piperidine
yielding as the catalyst
intermediates
2,4-thiazolidinedione (2) with different aldehydes in the presence of piperidine as the
[33]. Subsequent
4a–i. Intermediates 3a–i wereunder
hydrolysis alkylated
acidicwith ethyl bromoacetate,
conditions yielded 5a–i.yielding intermediates
This synthetic route is
catalyst [33]. Intermediates 3a–i were alkylated with ethyl bromoacetate, yielding interme-
4a–i.
detailedSubsequent
in Scheme hydrolysis
1. under acidic conditions yielded 5a–i. This synthetic route is
diates 4a–i. Subsequent hydrolysis under acidic conditions yielded 5a–i. This synthetic
detailed in Scheme 1.
route is detailed in Scheme 1.
Scheme 1. Synthesis of intermediates 5a–i. (i) Different aldehydes, piperidine, EtOH, reflux, 8–10 h,
Scheme
76%–88%; Synthesis
Scheme 1. (ii) of intermediates
ethyl bromoacetate, K2CO 5a–i.
5a–i.
3, KI,(i) Different
(i) Different
DMF, aldehydes,
aldehydes,
50 °C, piperidine,
piperidine,
5–7 h, 67%–82%; EtOH,
EtOH,
(iii) reflux,
reflux,
AcOH, 8–10
HCl,8–10 h,h,
reflux,
76%–88%;
76–88%; (ii)
(ii) ethyl
ethyl bromoacetate,
bromoacetate, KK CO
CO , KI,
KI, DMF,
DMF, 50
50 ◦ C,5–7
°C, 5–7h,
h,67%–82%;
67–82%; (iii) AcOH,
AcOH, HCl,
HCl, reflux,
reflux,
5–8 h, 58%–72%. 22 3
3
5–8 h,
5–8 h, 58–72%.
58%–72%.
2.1.2. Synthesis of Target Compounds 6a–i
2.1.2.
2.1.2. Synthesis
Synthesis of
of Target
Target Compounds
Compounds 6a–i 6a–i
The esterification of lupeol (1) with intermediates 5a–i in the presence of 1-ethyl-3-
The
The esterification
esterification of
of lupeol
lupeol(1)
(1)with intermediates
with(EDCI)
intermediates 5a–iininthe
5a–i thepresence
presenceofof1-ethyl-3-(3-
1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide
dimethylaminopropyl)carbodiimide (EDCI) and
and 4-dimethylaminopyridine
4-dimethylaminopyridine (DMAP)
(DMAP)
led to
(3-dimethylaminopropyl)carbodiimide
led to target compounds 6a–i. The (EDCI)
synthetic and
route is 4-dimethylaminopyridine
detailed in Scheme 2. (DMAP)
target compounds 6a–i. The synthetic route is detailed in Scheme
led to target compounds 6a–i. The synthetic route is detailed in Scheme 2. 2.
(ⅰ) R
(ⅰ) R
O
O O
S O
S N
HO N O
HO Lup (1) O O 6a-i
Lup (1) O 6a-i
Scheme 2. Synthesis of target compounds 6a–i. (i) 5a–i, EDCI, DMAP, CH22Cl22,, rt,
DMAP, CH rt, 8–10
8–10 h,
h, 35%–
35–70%.
Scheme 2. Synthesis of target compounds 6a–i. (i) 5a–i, EDCI, DMAP, CH2Cl2, rt, 8–10 h, 35%–
70%.
2.1.3.
70%. Synthesis of Target Compounds 9a–i
2.1.3.ASynthesis
substitution reaction
of Target of the C-39a–i
Compounds hydroxyl lupeol group and 4-nitrophenyl chloro-
2.1.3. Synthesis
formate of TargetofCompounds
in the presence 9a–i compound 7; compound 7 was then reacted
pyridine produced
with piperazine to introduce a carbamate at the C-3 position to produce compound 8.
Compound 8 reacted with compounds 5a–i via amidation to obtain compounds 9a–i. The
synthetic route is detailed in Scheme 3.
A substitution reaction of the C-3 hydroxyl lupeol group and 4-nitrophenyl chlo-
A substitution
roformate reaction
in the presence of of the C-3produced
pyridine hydroxylcompound
lupeol group and 4-nitrophenyl
7; compound chlo-
7 was then re-
roformate in the presence of pyridine produced compound 7; compound 7 was
acted with piperazine to introduce a carbamate at the C-3 position to produce compound then re-
acted with piperazine
8. Compound 8 reactedto with
introduce a carbamate
compounds at the
5a–i via C-3 position
amidation to produce
to obtain compound
compounds 9a–i.
Molecules 2024, 29, 4957 4 of 25
8.
TheCompound 8 reacted
synthetic route with compounds
is detailed in Scheme 3.5a–i via amidation to obtain compounds 9a–i.
The synthetic route is detailed in Scheme 3.
Scheme 3. Synthesis of target products 9a–i. (i) 4-nitrophenyl chloroformate, pyridine, CH2Cl2, 0-rt,
Scheme
1–2 h, 80%; Synthesis
3. Synthesis ofoftarget
(ii) piperazine, target products
Etproducts
3N, CH2Cl
9a–i.4–5
9a–i.
2, rt, (i)
(i)h,4-nitrophenyl
4-nitrophenyl chloroformate,
chloroformate,
75%; (iii) 5a–i, pyridine,
pyridine,
EDCI, DMAP, CH2ClCH CH
Cl26–8
2, 2rt,
Clh,
,20-rt,2,
1–2
0-rt, h,
1–2
38%–62%. 80%; (ii) piperazine,
h, 80%; Et3N,Et
(ii) piperazine, CH 2Cl
3 N, CH2, rt, 4–5
2 Cl h, 4–5
2 , rt, 75%;h,(iii)
75%; 5a–i,
(iii)EDCI, DMAP,
5a–i, EDCI, CH2ClCH
DMAP, 2, rt, 6–8
2 Cl h,
2 , rt,
38%–62%.
6–8 h, 38–62%.
2.1.4. Synthesis of Target Compounds 12a–i
2.1.4. Synthesis of
2.1.4. Synthesis of Target
Target Compounds
Compounds 12a–i12a–i
A substitution reaction occurred between compound 7 and N-Boc-ethylenediamine
A substitution
A presence
substitution reaction
reactionoccurred
occurred betweencompound
between compound 7 and
7 andN-Boc-ethylenediamine
N-Boc-ethylenediamine in
in the of Et 3N, producing compound 10. Then, the N-Boc protection was re-
the
in presence
the presence of Et N, producing compound
of3 Et3N, producing compound 10. Then, the
10. Then, N-Boc protection
the compound was
N-Boc protection removed
wasthen
re-
moved using a trifluoroacetic acid (TFA) treatment to obtain 11, which
using
moved a using
trifluoroacetic
a acid (TFA)
trifluoroacetic acidtreatment
(TFA) to obtain
treatment tocompound
obtain 11, which
compound then
11, reacted
which then
reacted with compounds 5a–i in the presence of EDCI, N-hydroxybenzotrizole (HOBT),
with
reactedcompounds 5a–i in the
with compounds 5a–ipresence
in (DIPEA)of EDCI,ofN-hydroxybenzotrizole
the presence EDCI, (HOBT),(HOBT),
N-hydroxybenzotrizole and N,
and N, N-diisopropylethylamine
N-diisopropylethylamine (DIPEA) to to obtain
obtain compounds
compounds 12a–i.
12a–i. The The synthetic
synthetic route
route is
and N, N-diisopropylethylamine
is detailed in Scheme 4. (DIPEA) to obtain compounds 12a–i. The synthetic route
detailed in Scheme
is detailed in Scheme 4.4.
Scheme
Scheme 4. 4. Synthesis
Synthesis of
of target
target products 12a–i.(i)
products12a–i. (i)N-Boc-ethylenediamine,
N-Boc-ethylenediamine,Et Et33N,
N, CH
CH22Cl22, rt, 10–12 h,
Scheme 4. Synthesis
79%; (ii) CF of target
Cl22, ,00products 12a–i. (i)70%;
◦ C-rt,overnight, N-Boc-ethylenediamine, EtDIPEA,
3N, CH2Cl 2, 2rt, 10–12 h,
79%; 33COOH,
COOH, CH
CH 22Cl °C-rt, overnight, 70%;(iii)
(iii)5a–i,
5a–i,EDCI,
EDCI,HOBT,
HOBT, DIPEA,CHCH Cl22Cl
, 0-rt, 8–
2 , 0-rt,
79%;
10 h, (ii) CF
32%–58%.
8–10 h, 32–58%.3COOH, CH2Cl2, 0 °C-rt, overnight, 70%; (iii) 5a–i, EDCI, HOBT, DIPEA, CH2Cl2, 0-rt, 8–
10 h, 32%–58%.
2.2.
2.2. Biological
Biological Evaluation
Evaluation
2.2. Biological
2.2.1. Evaluation
2.2.1. In Vitro Antiproliferative
In Vitro Antiproliferative Activity
Activity and
and Structure–Activity
Structure–Activity Relationship
Relationship Studies
Studies
2.2.1.The
In Vitro Antiproliferative
antitumor Activity and6a–i,
activities of compounds Structure–Activity Relationship
9a–i, and 12a–i toward humanStudies
lung cancer
The antitumor activities of compounds 6a–i, 9a–i, and 12a–i toward human lung can-
A549,Thehuman breast cancer MCF-7, human hepatocarcinoma HepG2, and human hepatic
cer A549,antitumor activities
human breast of compounds
cancer 6a–i, 9a–i,
MCF-7, human and 12a–i toward
hepatocarcinoma human
HepG2, lung
and can-
human
LO2 cells were
cer A549, determined
human using the
breast cancer (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
MCF-7, human hepatocarcinoma HepG2, and human
bromide (MTT) assay. Lupeol and cisplatin were used as the positive controls. The IC50
results are shown in Table 1.
Table 1 shows that all of the synthesized lupeol-3-thiazolidinedione derivatives (6a–i,
9a–i, and 12a–i) showed significantly higher antitumor activities than the parent lupeol
against all three cancer cell lines. Compounds 9a–i contained a piperazine–carbamate
linker, and compounds 12a–i contained an ethylenediamine linker, which showed good
Molecules 2024, 29, 4957 5 of 25
antitumor activity. Compound 12i showed the most significant antitumor activity against
the three cancer cell lines, with the IC50 values ranging from 4.40 to 10.06 µM. The antitumor
activity against HepG2 cells was 9.91-fold higher than that of lupeol. In the A549 cell line,
compounds 6c, 6e, 6f, 6i, 9d, 9e, 9f, 9g, 12a, 12f, and 12i showed the most significant
antitumor activities, with IC50 values ranging from 9.24 to 11.71 µM. In the MCF-7 cell
lines, compounds 6h, 6i, 9f, 12d, 12f, and 12i showed significant antitumor activity, with
the IC50 values ranging from 8.70 to 11.34 µM. In the HepG2 cell line, compounds 6d, 6i,
9d, 9f, 9i, 12c, 12f, and 12i showed the most significant antitumor activity, with IC50 values
ranging from 4.40 to 10.86 µM. In addition, the IC50 values of all compounds tested were
greater than 28 µM, thus demonstrating that these compounds exhibited relatively weak
cytotoxicity against the human hepatic LO2 cells and were selective to a certain extent for
cancer cells.
Table 1. IC50 values of lupeol derivatives 6a–i, 9a–i, and 12a–i tested against a panel of cell lines for
48 h. SI (selectivity index) = IC50 values on L02 cells/HepG2 cells.
IC50 (µM) *
Compounds SI
A549 MCF-7 HepG2 LO2
6a 17.30 ± 1.58 23.99 ± 1.87 22.72 ± 1.59 92.56 ± 1.03 4.07
6b 14.16 ± 1.72 13.78 ± 0.75 16.58 ± 1.38 70.48 ± 1.51 4.25
6c 10.37 ± 0.82 22.12 ± 1.42 18.20 ± 1.27 76.44 ± 1.22 4.2
6d 14.19 ± 1.43 15.41 ± 1.22 10.86 ± 0.78 39.25 ± 1.07 3.61
6e 9.43 ± 0.74 12.52 ± 0.74 15.48 ± 0.86 70.82 ± 0.93 4.57
6f 10.34 ± 0.86 16.10 ± 0.89 19.22 ± 1.57 85.34 ± 1.12 4.44
6g 14.94 ± 1.21 12.79 ± 0.22 15.34 ± 1.32 60.57 ± 1.84 3.95
6h 14.03 ± 1.39 11.34 ± 0.85 12.82 ± 0.97 58.36 ± 0.78 4.55
6i 11.35 ± 0.91 8.70 ± 0.12 9.48 ± 0.71 45.53 ± 1.33 4.80
9a 18.71 ± 1.25 19.07 ± 1.78 20.46 ± 1.62 79.14 ± 1.25 3.87
9b 15.47 ± 0.64 14.04 ± 0.87 18.11 ± 1.73 80.49 ± 1.79 4.44
9c 12.74 ± 0.68 16.27 ± 0.76 15.71 ± 1.52 65.11 ± 0.93 4.14
9d 11.71 ± 0.75 18.12 ± 1.93 9.52 ± 0.61 42.17 ± 1.28 4.43
9e 10.37 ± 0.38 18.60 ± 1.27 16.52 ± 1.26 63.31 ± 1.14 3.83
9f 9.24 ± 0.48 9.19 ± 0.46 10.81 ± 0.45 56.08 ± 0.77 5.19
9g 9.91 ± 0.42 12.04 ± 0.75 14.98 ± 0.74 62.77 ± 0.62 4.19
9h 16.45 ± 1.69 13.83 ± 0.58 15.33 ± 0.98 68.93 ± 1.19 4.50
9i 13.78 ± 0.84 20.13 ± 1.59 6.91 ± 0.31 35.40 ± 0.72 5.12
12a 10.53 ± 0.56 16.83 ± 1.29 16.51 ± 0.54 71.03 ± 0.57 4.30
12b 17.05 ± 1.28 19.11 ± 1.63 17.67 ± 0.79 76.87 ± 0.69 4.35
12c 13.39 ± 0.79 14.29 ± 0.78 10.74 ± 0.12 40.68 ± 0.31 3.79
12d 16.37 ± 0.43 9.12 ± 0.24 15.75 ± 0.68 59.91 ± 1.03 3.80
12e 13.77 ± 0.76 14.38 ± 0.72 12.93 ± 0.52 57.67 ± 1.14 4.46
12f 9.68 ± 0.52 10.02 ± 0.22 9.43 ± 0.78 35.53 ± 0.34 3.77
12g 15.70 ± 1.42 16.44 ± 0.87 14.29 ± 0.56 63.22 ± 1.31 4.42
12h 13.40 ± 1.08 17.32 ± 1.28 18.62 ± 1.49 77.51 ± 1.27 4.14
12i 10.06 ± 0.62 8.74 ± 0.34 4.40 ± 0.02 29.23 ± 0.53 6.69
Lupeol 35.86 ± 1.18 62.03 ± 1.79 43.62 ± 1.37 - -
Cisplatin 4.96 ± 0.48 5.79 ± 0.49 4.65 ± 0.54 - -
* The data are expressed as means ± standard deviation (SD) from three independent experiments.
Based on the preliminary conformational relationships, we found that the introduction

of thiazolidinedione, as well as these three types of linkers (ester, piperazine-carbamate,
and ethylenediamine), significantly enhances the antitumor activity of the compounds.
This discovery underscores the potential of these specific molecular modifications in the
development of more effective cancer therapies. We also found that the effect of the
substituents on the antitumor activity of compounds 6i, 9i, and 12i was more significant
than that of the compounds 6a–h, 9a–h, and 12a–h. This indicates that the antitumor
activity of the aliphatic substituent is superior to that of the aromatic substituents. The
antitumor activities of compounds 6c–f, 9c–f, and 12c–f, containing electron donor groups
such as -CH3 , -C(CH3 )3 , and -OCH3 , among others, are generally superior to those of
Molecules 2024, 29, 4957 6 of 25
compounds 6a–b, 9a–b, and 12a–b, containing electron-withdrawing groups such as -F

and -Cl.
Compound 12i showed the strongest antitumor activity. Thus, we further investigated
its effects and mechanism regarding in vitro anti-hepatocellular carcinoma activity.
2.2.2. Compound 12i Induced Apoptosis in HepG2 Cells

To further evaluate compound 12i-induced HepG2 cell apoptosis, HepG2 cells were
treated with this compound (at 0, 2, 4, and 8 µM), followed by staining with Acridine
Orange/Ethidium bromide (AO/EB). The morphological variations in the cells were ob-
served using fluorescence microscopy, as shown in Figure 2A, and the nuclei of the cells
in the control group were intact and stained green by AO. With the increasing concentra-
tion of compound 12i, the cell membrane ruptured; the nuclei crumpled; and the green
fluorescence gradually weakened and turned orange–red. Subsequently, we used Annexin
V-FITC/PI double staining to characterize the effect of compound 12i based on the total
apoptosis rate of HepG2 cells. The apoptosis rate was detected via flow cytometry, as
shown in Figure 2B,C. The total apoptosis rate of the HepG2 cells increased with the in-
creasing concentrations of compound 12i compared with the blank control. It significantly
increased from 5.75% in the blank control to 11.46%, 22.40%, and 60.50%. Thus, compound
12i induces HepG2 apoptosis.
Figure 2. Compound
Figure 2. Compound12i induces HepG2HepG2
12i induces cell apoptosis. (A) HepG2
cell apoptosis. (A)cells were cells
HepG2 treated withtreated
were 12i (2, 4,with 12i (2, 4,
and 8 µM) or 0.1% DMSO (blank control) and then stained with AO/EB. Normal cells were stained
and 8 µM) or 0.1% DMSO (blank control) and then stained with AO/EB. Normal cells were stained
green with AO staining solution with intact nuclei, apoptotic cells were stained red with EB staining
solution. (B) Detection of apoptosis in HepG2 cells treated with various concentrations (0, 2, 4, and
8 µM) of 12i for 48 h using an Annexin V-FITC/PI double-staining assay. (C) Percentage of cell apop-
tosis for different concentrations of 12i (* p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the
blank control group). Error bars are representative of three independent experiments.
Molecules 2024, 29, 4957 7 of 25
green with AO staining solution with intact nuclei, apoptotic cells were stained red with EB staining
solution. (B) Detection of apoptosis in HepG2 cells treated with various concentrations (0, 2, 4, and
8 µM) of 12i for 48 h using an Annexin V-FITC/PI double-staining assay. (C) Percentage of cell
apoptosis for different concentrations of 12i (* p < 0.05, ** p < 0.01, and *** p < 0.001 compared with
the blank control group). Error bars are representative of three independent experiments.
2.2.3. Effects of 12i on Reactive Oxygen Species (ROS) Generation

ROS are a key element in cancer therapy [34]. To evaluate whether compound 12i
can affect ROS production in HepG2 cells, we used 20 ,70 -dichlorofluorescein diacetate
(DCFH-DA) staining to determine the ROS levels. After 48 h, morphological variations
in the cells were observed using fluorescence microscopy, as shown in Figure 3A. The
green fluorescence of the 12i-treated experiments was more obvious than that of the blank
control, and the green fluorescence of 12i (8 µM) was the most obvious. According to
the above data, compound 12i induces ROS production dose-dependently in HepG2 cells.
Figure
Flow3B–D. The above
cytometry datatoindicates
was used that compound
detect changes in HepG212i cellsignificantly
ROS content,increased
and the the pro-are
results
duction
shownofinROS, which
Figure may
3B–D. Thealso be responsible
above forthat
data indicates its induction
compoundof12i apoptosis.
significantly increased
the production of ROS, which may also be responsible for its induction of apoptosis.
Figure
Figure 3. Compound
3. Compound 12i induces
12i induces ROSROS generation
generation in HepG2
in HepG2 cells.
cells. (A) (A) HepG2
HepG2 cells
cells were were with
treated treated
12iwith and(2,
(2, 4,12i 4, and
8 µM) and80.1% and 0.1%
µM)DMSO (blankDMSO (blank
control) control)
and then andwith
stained thenDCFH-DA.
stained with DCFH-DA.
(B,C) Deter-
mination of ROS content
(B,C) Determination ofin HepG2
ROS cells.
content in (D) ROScells.
HepG2 content
(D)for
ROSdifferent
content12i
forconcentrations (* p < 0.05,
different 12i concentrations
** (*
p <p <
0.01, and *** p < 0.001 compared with the blank control group). Error bars represent
0.05, ** p < 0.01, and *** p < 0.001 compared with the blank control group). Error bars represent three
independent experiments.
three independent experiments.
2.2.4.
2.2.4. Effects
Effects of of onon
12i12i Mitochondria
Mitochondria Membrane
Membrane Potential
Potential (MMP)
(MMP)
MMP MMP depolarization
depolarization is one
is one of of
thethe landmark
landmark events
events of of apoptosis
apoptosis [35].
[35]. ToTo investigate
investigate
thethe impact
impact of of onon
12i12i MMP
MMP depolarization
depolarization in in HepG2
HepG2 cells,
cells, wewe used
used 5,50 ,6,6,6,60 -tetrachloro-
5,5′,6,6,6,6′-tetrachloro-
1,10 ,3,30 -tetraethylioiodine
1,1′,3,3′-tetraethylioiodine carbocyan
carbocyan (JC-1)
(JC-1) staining.
staining. When
When thethe mitochondrial
mitochondrial membrane
membrane
potential is high, JC-1 aggregates in the substrate to form a polymer (J-aggregates),
potential is high, JC-1 aggregates in the substrate to form a polymer (J-aggregates), which which
produces red fluorescence. When the mitochondrial membrane
produces red fluorescence. When the mitochondrial membrane potential is low, JC-1 can-potential is low, JC-1
cannot accumulate in the mitochondrial matrix and exists as a monomer
not accumulate in the mitochondrial matrix and exists as a monomer to produce green to produce green
fluorescence. As shown in Figure 4A, the compound 12i-treated experimental groups
showed increased green fluorescence and decreased red fluorescence, with increasing
concentrations compared with the blank control. Thus, compound 12i could significantly
reduce Δψm. The flow cytometry results are shown in Figure 4B,C. The compound 12i
Molecules 2024, 29, 4957 8 of 25
fluorescence. As shown in Figure 4A, the compound 12i-treated experimental groups

showed increased green fluorescence and decreased red fluorescence, with increasing
concentrations compared with the blank control. Thus, compound 12i could significantly
reduce ∆ψm. The flow cytometry results are shown in Figure 4B,C. The compound 12i
concentration was positively correlated with the monomer rate of JC-1; the monomer rate of
JC-1
Molecules 2024, 29, x FOR PEER REVIEW
increased from 1.95% in the blank control group to 35.8% in the highest concentration
9 of 26
group (8 µM). Altogether, these data suggest that compound 12i decreased the membrane
potential of HepG2 cells.
Figure 4.
Figure Compound 12i
4. Compound induces MMP
12i induces MMP in in HepG2
HepG2 cells.
cells. (A)
(A)HepG2
HepG2cells
cellswere
weretreated
treatedwith
with12i (2,4,
12i(2, 4,
and 8 µM) or 0.1% DMSO (blank control) and then stained with JC-1. (B) Determination
and 8 µM) or 0.1% DMSO (blank control) and then stained with JC-1. (B) Determination of MMP of MMP
depolarization rate
depolarization rate in
inHepG2
HepG2cells.
cells.(C)
(C)Percentage
Percentageofof
decreasing
decreasing MMPMMPforfor
different
different concentrations
12i 12i concentra-
(* p < (*
tions 0.05, p < 0.01,
p <**0.05, ** p <and
0.01, p < 0.001
***and *** p <compared
0.001 compared
with thewith
blankthe blankgroup).
control controlError
group).
barsError bars
represent
represent three independent
three independent experiments.experiments.
2.2.5. Effects
2.2.5. Effects of
of 12i on the
12i on the Mitochondrial
Mitochondrial Apoptosis
Apoptosis Pathway
Pathway
Compound 12i
Compound 12i induces
inducesHepG2
HepG2cell
cellapoptosis.
apoptosis. ToTo
further demonstrate
further thisthis
demonstrate mechanism,
mecha-
we used a Western blot to detect the expression levels of the relevant apoptotic
nism, we used a Western blot to detect the expression levels of the relevant apoptotic proteins.
pro-
The GAPDH
teins. The GAPDHexpression level was
expression levelused
wasas an internal
used controlcontrol
as an internal group.group.
Figure Figure
5A,B shows
5A,B
that compound
shows 12i can12i
that compound downregulate the anti-apoptotic
can downregulate proteinprotein
the anti-apoptotic Bcl-2 and upregulate
Bcl-2 the
and upregu-
pro-apoptotic protein Bax in HepG2 cells. Compound 12i also activated
late the pro-apoptotic protein Bax in HepG2 cells. Compound 12i also activated the ex- the expression
pression levels of both cleaved caspase-7 and cleaved caspase-9 dose dependently. These
data suggest that compound 12i induces HepG2 cell apoptosis, possibly via the mitochon-
drial apoptotic pathway.
Molecules 2024, 29, 4957 9 of 25
levels
Molecules 2024, 29, x FOR PEER REVIEW of both cleaved caspase-7 and cleaved caspase-9 dose dependently. These 10 ofdata
26
suggest that compound 12i induces HepG2 cell apoptosis, possibly via the mitochondrial
apoptotic pathway.
Figure 5. Compound 12i inhibited the mitochondrial apoptosis pathway. (A) Western blot analysis
of the expression levels of Bax, Bcl-2, caspase-7, cleaved-caspase-7, caspase-9, and cleaved-caspase-
9Figure
proteins
Figure in HepG2 cells
Compound
5.5.Compound treated
inhibited
12iinhibited
12i with
the 12i
the (2, 4, and 8 µM)
mitochondrial
mitochondrial and 0.1%
apoptosis
apoptosis DMSO (A)
pathway.
pathway. (the blank
(A)Western control
Western group).
blotanalysis
blot analysis
(B) Statistical
ofofthe
theexpressionanalysis
expressionlevels (* p < 0.05,
levelsofofBax, **
Bax,Bcl-2,p < 0.01,
Bcl-2,caspase-7,and *** p < 0.001 compared
caspase-7,cleaved-caspase-7, with
cleaved-caspase-7,caspase-9,the blank
caspase-9, and control group).
and cleaved-caspase-
cleaved-caspase-9
9Error barsin
proteins
proteins inrepresent threetreated
HepG2 cells independent
with
with 12i12iexperiments.
(2,
(2,4,4,and
and88µM)µM)andand0.1%
0.1%DMSO
DMSO(the (theblank
blankcontrol
controlgroup).
group).
(B)
(B)Statistical
Statisticalanalysis
analysis(*(*pp<<0.05,
0.05,****p p< 0.01, and
< 0.01, and ******
p <p 0.001 compared
< 0.001 comparedwith
with thethe
blank
blankcontrol
controlgroup).
group).
2.2.6.bars
Error Effects of 12ithree
represent on the PI3K/AKT/mTOR
independent experiments. Pathway
Error bars represent three independent experiments.
The literature reports suggest that lupeol can exert antitumor effects through the
2.2.6.Effects
2.2.6. Effectsofof12i
phosphoinositide onthe
12i3-kinase
on thePI3K/AKT/mTOR
PI3K/AKT/mTOR Pathway
Pathway
(PI3K)/protein kinase B (AKT)/mammalian target of the ra-
pamycin The literature reports
(mTOR) pathway,suggest
The literature suggest that lupeol
and thiazolidinedione can
that lupeol canhasexert antitumor
also
exert effects
been shown
antitumor through
to
effects induce the
through phos-
cancer
the
phoinositide
cell apoptosis3-kinase
phosphoinositide via 3-kinase(PI3K)/protein
the PI3K/AKT/mTOR kinase
(PI3K)/protein B (AKT)/mammalian
pathway.
kinase BTherefore, targettarget
we predict
(AKT)/mammalian of
thatthecompound
rapamycin
of the ra-
(mTOR)
12i
pamycin pathway,
may also exhibit
(mTOR) and
pathway, thiazolidinedione
antitumor andactivity has also
through
thiazolidinedione thebeen shown
PI3K/AKT/mTOR
has also beento induce cancer
pathway.
shown to induce cell apopto-
Therefore,
cancer
sis apoptosis
cell via the
Western PI3K/AKT/mTOR
blotting
via was conducted
the PI3K/AKT/mTOR pathway. Therefore,
to determine
pathway. we predict we
the Therefore,
expression that compound
of the that 12i
key proteins
predict may
Akt,also
compound P-
exhibit
Akt, antitumor
PI3K, P-PI3K, activity
mTOR, through
and the
P-mTOR. PI3K/AKT/mTOR
As shown in pathway.
Figure 6A,B,
12i may also exhibit antitumor activity through the PI3K/AKT/mTOR pathway. Therefore, Therefore,
compound Western
12i was
blotting
able
Western was conducted
to reduce
blotting the
was to determine
phosphorylation
conducted the expression
levels
to determineof P-Akt, of the key
P-PI3K,
the expression of proteins
and P-mTOR
the Akt, inP-Akt,
key proteins PI3K,
a dose-de-
Akt, P-
P-PI3K,
pendent
Akt, mTOR,
manner.
PI3K, and
P-PI3K,The P-mTOR.
resultsand
mTOR, As shown
indicate
P-mTOR. in Figure
that compound
As shown in6A,B, compound
12iFigure
can inhibit 12i
6A,B,the was able
PI3K/AKT/mTOR
compound to reduce
12i was
the to
phosphorylation
pathway.
able levels of P-Akt,
reduce the phosphorylation P-PI3K,
levels and P-mTOR
of P-Akt, P-PI3K, in anda dose-dependent
P-mTOR in a dose-de- manner.
The results
pendent indicate
manner. Thethat compound
results thatcan
indicate 12i inhibit the
compound 12iPI3K/AKT/mTOR pathway.
can inhibit the PI3K/AKT/mTOR
pathway.
Figure
Figure6.6. Compound
Compound12i 12i inhibits
inhibits the
the PI3K/Akt/mTOR
PI3K/Akt/mTORpathway. pathway.(A) (A)Western
Westernblot blotanalysis
analysisofofthe
the
expression levels of Akt, P-Akt, PI3K, P-PI3K, mTOR, and P-mTOR in HepG2
expression levels of Akt, P-Akt, PI3K, P-PI3K, mTOR, and P-mTOR in HepG2 cells after exposure cells after exposureto
tothe
theblank
Figure blank controland
6. Compound
control and
12idifferent
inhibits
different concentrations (2,(2,4,4,and
the PI3K/Akt/mTOR
concentrations and 8 8µM)
µM)
pathway. ofof 12i
(A)
12i for
4848h.h.(B)
Western
for (B)The
blot The percentage
analysis of theof
percentage
of cell apoptosis
expression levels for different concentrations of 12i (*: p < 0.05, **: p < 0.01, ***: p < 0.001, compared to
cell apoptosis forofdifferent
Akt, P-Akt, PI3K, P-PI3K,
concentrations mTOR,
of 12i (*: p <and P-mTOR
0.05, in HepG2
**: p < 0.01, ***: p < cells after
0.001, exposure
compared to
the
totheblank
theblank control
blankcontrol group)
controlgroup) Error
and different bars are representatives
concentrations (2, 4, and of three independent experiments.
Error bars are representatives of 8three
µM)independent
of 12i for 48 experiments.
h. (B) The percentage
of cell apoptosis for different concentrations of 12i (*: p < 0.05, **: p < 0.01, ***: p < 0.001, compared to
2.2.7.
the Compound
blank 12i IsError
control group) Predicted
bars aretorepresentatives
Bind to PI3K of three independent experiments.
2.2.7. Compound 12i Is Predicted to Bind to PI3K

Molecules2024,
Molecules 29,x4957
2024,29, FOR PEER REVIEW 1110ofof26
25
2.2.7.InCompound 12i Is Predicted

order to investigate to Bind
the binding to PI3K
mode of compound 12i to PI3K, molecular dock-
In order
ing studies to investigate
based the binding
on the crystal structure mode of compound
of PI3K (PDB: 4OYS) 12i to PI3K,
were molecularwhich
performed, dock-
ing studies
revealed thatbased on the crystal
12i interacts structurereactive
with different of PI3Ksites
(PDB: in4OYS) werevariable
the 4OYS performed, which
pocket, as
revealed
shown that 12i7. interacts
in Figure Amino acid with different
residues reactive
TRY-670 andsites in the
ILE-685 4OYS
form variablebonds
hydrogen pocket,
withas
shown in Figure 7. Amino acid residues TRY-670 and ILE-685 form
the carbonyl groups at positions C34 and C36 in compound 12i. Perhaps this hydrogen hydrogen bonds with
the carbonyl
bonding groupspushes
interaction at positions C34 and
the bonds andC36 in compound
substituents intoPerhaps
deep 12i. this lumen
the narrow hydrogenof
4OYS. Perhaps it can more efficiently promote the binding of the carbonyl grouplumen
bonding interaction pushes the bonds and substituents deep into the narrow of
at posi-
4OYS. Perhaps it can more efficiently promote the binding of the carbonyl
tion C31 to the active center lysine LYS-636 in the narrow grooves of 4OYS, which may group at position
C31 to
affect thegrowth
cell active and
center lysine LYS-636
reproduction inregulation
or the the narrowofgrooves of 4OYS,
mitochondrial which may
function. affect
In partic-
cell growth and reproduction or the regulation of mitochondrial function.
ular, the binding of the protein–ligand complex could reach −9.07 kcal/mol, which is a In particular,
the binding
good indicationof the
thatprotein–ligand
compound 12icomplex
has goodcould −9.07 kcal/mol,
reach activity
inhibitory which is a good
against PI3K.
indication that compound 12i has good inhibitory activity against PI3K.
Figure 7. There was an eutectic structure of compound 12i with PI3K (PDB: 4OYS). (A) Modeled
Figure 7. Thereclose-up
and enlarged was an of
eutectic structure
the surface of compound
mosaic 12i with
of PI3K tetramer PI3K
and (PDB: 4OYS).
12i binding. (A) Modeled
(B) Close-up of 12i
and enlarged close-up of the surface mosaic of PI3K tetramer and 12i binding. (B) Close-up
interactions in the PI3K allosteric binding pocket. Here, 12i was rendered as a rod and coloredof 12i
interactions in the PI3K allosteric binding pocket. Here, 12i was rendered as a rod and colored
according to atom type. Cyan denoted carbon, blue denoted nitrogen, and red denoted oxygen. Key ac-
cording to atom type. Cyan denoted carbon, blue denoted nitrogen, and red
residual atoms in PI3K that interacted with the compound were denoted in cyan. denoted oxygen. Key
residual atoms in PI3K that interacted with the compound were denoted in cyan.
3. Materials and Methods
3.3.1.
Materials and Methods
Chemistry
3.1.1.
3.1. Reagents and Instruments
Chemistry
3.1.1. Thiazolidinedione was purchased from Anhui Zesheng Technology Co., Ltd. (Shang-
Reagents and Instruments
hai, Thiazolidinedione
China), and the otherwasreagents
purchased (analytical
from Anhuigrade) were purchased
Zesheng Technologyfrom Tianjin
Co., Ltd. Fuyu
(Shang-
Fine Chemical Co., Ltd. (Tianjin, China). The progress of all chemical reactions
hai, China), and the other reagents (analytical grade) were purchased from Tianjin Fuyu was care-
fully monitored via thin-layer chromatography (TLC) using silica gel GF254
Fine Chemical Co., Ltd. (Tianjin, China). The progress of all chemical reactions was care- (Qingdao
Ocean
fully Chemicalvia
monitored Co.,thin-layer
Ltd., Qingdao, China) as the
chromatography stationary
(TLC) using phase. Column
silica gel GF254chromatog-
(Qingdao
raphy purified the intermediates and
Ocean Chemical Co., Ltd., Qingdao, China) as target derivatives (300–400-mesh
the stationary silica gel,
phase. Column chromatog- Yantai
1 13
Yinlong Silica Gel Co., Ltd., Yantai, China). H NMR and C NMR analyses of the target
raphy purified the intermediates and target derivatives (300–400-mesh silica gel, Yantai
compounds were conducted using a Bruker NMR instrument (Bruker Avance DRX400).
Yinlong Silica Gel Co., Ltd., Yantai, China). 1H NMR and 13C NMR analyses of the target
The target compounds were also characterized using a Bruker high-resolution mass spec-
compounds were conducted using a Bruker NMR instrument (Bruker Avance DRX400).
trometer (Bruker Esquire 6000). The Supplementary Material includes HRMS, 1H NMR,
The target compounds were also characterized using a Bruker high-resolution mass spec-
and 13C NMR spectral data of all derivatives.
trometer (Bruker Esquire 6000). The Supplementary Material includes HRMS, 1H NMR,
and 13C
3.1.2. NMR spectral
Synthesis data of all 3a–i
of Intermediates derivatives.
Compound 2 (TZD, 500 mg, 4.26 mmol) was refluxed with different aldehydes
3.1.2. Synthesis of Intermediates 3a–i
(1.00 equiv) and piperidines (1.00 equiv) in EtOH (10 mL), stirring for 8–10 h, and moni-
toredCompound 2 (TZD,
via TLC until 500 mg, was
the reaction 4.26 mmol) wasThe
complete. refluxed
solventwith
wasdifferent
pouredaldehydes
into water,(1.00
and
equiv)
the pH of the system was adjusted to 7 with HCl (37%). The organic phase wasmonitored
and piperidines (1.00 equiv) in EtOH (10 mL), stirring for 8–10 h, and separated,
via
andTLC
the until the reaction
aqueous wasextracted
phase was complete.with
The ethyl
solvent was poured
acetate, washed into water,
with and the
saturated pH
salted
of the system
water, was anhydrous
dried with adjusted toNa
7 with HCl (37%). The organic phase was separated, and the
2 SO4 , and concentrated under reduced pressure. The crude
aqueous
productsphase
(3a–i)was
wereextracted
directlywith ethyl
carried acetate,
out in the washed
next stepwith saturated
of the salted water, dried
reaction.
with anhydrous Na2SO4, and concentrated under reduced pressure. The crude products
(3a–i) were directly carried out in the next step of the reaction.

Molecules 2024, 29, 4957 11 of 25

Compounds 3a–i (500 mg, 1.69–2.93 mmol), K2 CO3 (1.20 equiv), KI (1.00 equiv), and
ethyl bromoacetate (1.00 equiv) were added to a round bottom flask, containing 8 mL of
dimethylformamide (DMF). TLC monitored the reaction until there were no ingredients
left. The sample was extracted with dichloromethane (CH2 Cl2 ) and washed with saturated
salted water; the organic phases were then separated and combined. The organic phase
was dried with anhydrous Na2 SO4 and filtered. The solvent was removed under reduced
pressure, and the residue was purified via silica gel column chromatography (PE/EA = 20:1,
v/v) to obtain 4a–i.

A mixture of compounds 4a–i (400 mg, 1.04–1.56 mmol), HCl (37%, 2 mL), and
AcOH (3 mL) was refluxed for 5–8 h, using TLC to monitor the reaction. After the reac-
tion, the solution was extracted with ethyl acetate, washed with saturated salted water,
dried over Na2 SO4 , and concentrated under reduced pressure to yield the corresponding
intermediates 5a–i.
3.1.5. Synthesis of Compounds 6a–i

A mixture of lupeol (1, 50 mg, 0.12 mmol), intermediates 5a–i (0.18mmol), EDCI
(34.42 mg, 0.18 mmol), and DMAP (14.66 mg, 0.12 mmol) in CH2 Cl2 (6 mL) were stirred
for 8–12 h. Throughout this period, TLC analysis monitored the process until the starting
material completely disappeared. The solvent was then removed under reduced pressure,
resulting in crude product formation. Further purification involved separation via silica
gel column chromatography using CH2 Cl2 as a developing agent. This process produced
target products 6a–i.
Lupeol-3-[(1-Ethyl)-2]-5-(4-Fluorobenzylidene)-2,4-Thiazolidinedione (6a)
White solid, yield: 65%; mp: 176.8–178.5 ◦ C.1 H NMR (600 MHz, chloroform-d) δ 7.90
(s, 1H, CH2 =CH2 ), 7.52 (dd, J = 8.5, 5.2 Hz, 2H, Ar-H), 7.19 (t, J = 8.4 Hz, 2H, Ar-H), 4.68
(d, J = 2.4 Hz, 1H, CH2 =CH2 ), 4.56 (s, 1H, CH2 =CH2 ), 4.54 (d, J = 4.7 Hz, 1H, CH), 4.47 (s,
2H, CH2 ), 2.37 (td, J = 11.0, 5.8 Hz, 1H), 1.94–1.88 (m, 1H), 1.68 (s, 3H, CH3 ), 1.67–1.61 (m,
6H), 1.48 (ddd, J = 13.0, 8.2, 3.1 Hz, 2H), 1.38 (q, J = 8.3, 7.9 Hz, 6H), 1.34 (d, J = 2.7 Hz, 1H),
1.29 (d, J = 2.5 Hz, 1H), 1.26 (d, J = 7.9 Hz, 2H), 1.23–1.09 (m, 3H), 1.06 (dd, J = 12.9, 4.7 Hz,
1H), 1.02 (s, 3H, CH3 ), 1.00–0.95 (m, 2H), 0.93 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.83 (s, 3H,
CH3 ), 0.78 (d, J = 4.5 Hz, 6H, CH3 ×2). 13 C NMR (150 MHz, chloroform-d) δ 167.29 (C33),
165.99 (C35), 165.68 (C31), 164.77 (C20), 163.08 (C40), 151.09 (C38), 133.39 (C37), 132.56
(C42), 132.50 (C36), 120.85 (C39), 116.85 (C34), 116.70 (C41), 109.52 (C29), 55.43 (C3), 50.41
(C5), 48.39 (C9), 48.12 (C18), 43.12 (C19), 42.96 (C17), 42.56 (C14), 40.95 (C32), 40.12 (C8),
38.38 (C22), 38.14 (C13), 37.97 (C1), 37.16 (C4), 35.68 (C10), 34.27 (C16), 29.94 (C7), 29.84
(C21), 28.04 (C15), 27.54 (C12), 25.17 (C2), 23.69 (C23), 21.07 (C24), 19.40 (C11), 18.26 (C30),
18.13 (C28), 16.52 (C6), 16.24 (C25), 16.09 (C26), 14.65 (C27). HRMS (ESI) m/z: calcd for
C42 H56 NO4 FNaS [M+Na]+ 712.3812, found 712.3804.
Lupeol-3-[(1-Ethyl)-2]-5-(4-Chlorobenzylidene)-2,4-Thiazolidinedione (6b)
Yellow solid, yield: 70%; mp: 168.6–170.4 ◦ C. 1 H NMR (600 MHz, chloroform-d) δ
7.88 (s, 1H, CH2 =CH2 ), 7.46 (s, 4H, Ar-H), 4.68 (d, J = 2.4 Hz, 1H, CH2 =CH2 ), 4.56 (d,
J = 3.1 Hz, 1H, CH2 =CH2 ), 4.54 (d, J = 4.8 Hz, 1H, CH), 4.47 (d, J = 1.7 Hz, 2H, CH2 ), 2.37
(dt, J = 11.1, 5.5 Hz, 1H), 1.91 (ddd, J = 13.0, 6.9, 2.7 Hz, 1H), 1.68 (s, 3H, CH3 ), 1.67–1.61
(m, 6H), 1.48 (td, J = 8.2, 3.9 Hz, 2H), 1.40–1.36 (m, 6H), 1.34–1.32 (m, 1H), 1.31–1.29 (m,
1H), 1.26 (d, J = 7.6 Hz, 2H), 1.24–1.14 (m, 3H), 1.06 (dd, J = 12.9, 4.7 Hz, 1H), 1.02 (s, 3H,
CH3 ), 1.00–0.95 (m, 2H), 0.93 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.83 (s, 3H, CH3 ), 0.78 (d,
J = 3.8 Hz, 6H, CH3 ×2). 13 C NMR (150 MHz, chloroform-d) δ 167.13 (C33), 165.94 (C35),
165.59 (C31), 151.09 (C20), 137.02 (C40), 133.18 (C38, C42), 131.68 (C37), 131.52 (C36), 129.76
(C39, C41), 121.80 (C34), 109.52 (C29), 83.57 (C3), 55.43 (C5), 50.41 (C9), 48.39 (C18), 48.12
Molecules 2024, 29, 4957 12 of 25
(C19), 43.12 (C17), 42.96 (C14), 42.58 (C32), 40.95 (C8), 40.12 (C22), 38.38 (C13), 38.14 (C1),
37.97 (C4), 37.16 (C10), 35.68 (C16), 34.27 (C7), 29.94 (C21), 28.05 (C15), 27.54 (C12), 25.17
(C2), 23.70 (C23), 21.07 (C24), 19.40 (C11), 18.26 (C30), 18.13 (C28), 16.52 (C6), 16.24 (C25),
16.09 (C26), 15.06 (C27). HRMS (ESI) m/z: calcd for C42 H56 NO4 NaSCl [M+Na]+ 728.3516,
found 728.3510.
Lupeol-3-[(1-Ethyl)-2]-5-(4-Methylbenzylidene)-2,4-Thiazolidinedione (6c)
Light-yellow solid, yield: 62%; mp: 182.7–184.5 ◦ C. 1 H NMR (600 MHz, chloroform-d)
δ 7.91 (s, 1H, CH2 =CH2 ), 7.42 (d, J = 7.7 Hz, 2H, Ar-H), 7.29 (d, J = 7.8 Hz, 2H, Ar-H), 4.68 (s,
1H, CH2 =CH2 ), 4.56 (s, 1H, CH2 =CH2 ), 4.54 (d, J = 4.5 Hz, 1H, CH), 4.47 (s, 2H, CH2 ), 2.41
(s, 3H, CH3 ), 2.36 (dd, J = 11.0, 5.8 Hz, 1H), 1.94–1.87 (m, 1H), 1.68 (s, 3H, CH3 ), 1.67–1.59
(m, 6H), 1.47 (dd, J = 15.3, 6.0 Hz, 2H), 1.45–1.36 (m, 6H), 1.36 (d, J = 4.5 Hz, 1H), 1.34 (s, 1H),
1.32–1.24 (m, 2H), 1.24–1.08 (m, 3H), 1.06 (dd, J = 12.9, 4.4 Hz, 1H), 1.01 (s, 3H, CH3 ), 0.97
(d, J = 12.0 Hz, 2H), 0.93 (s, 3H, CH3 ), 0.84 (d, J = 15.6 Hz, 6H, CH3 ), 0.78 (s, 6H, CH3 ×2).
13 C NMR (150 MHz, chloroform-d) δ 167.70 (C33), 166.07 (C35), 165.87 (C31), 151.08 (C20),
141.69 (C40), 134.82 (C37), 130.54 (C38, C42), 130.48 (C36), 130.17 (C39, C41), 119.89 (C34),
109.95 (C29), 83.46 (C3), 55.43 (C5), 50.40 (C9), 48.38 (C18), 48.12 (C19), 43.11 (C17), 42.95
(C14), 42.49 (C32), 40.94 (C8), 40.11 (C22), 38.81 (C13), 38.13 (C1), 37.95 (C4), 37.15 (C10),
36.04 (C16), 34.27 (C7), 29.93 (C21), 28.03 (C15), 27.54 (C12), 25.17 (C2), 23.68 (C23), 21.75
(C43), 21.06 (C24), 19.40 (C11), 18.25 (C30), 18.12 (C28), 16.50 (C6), 16.22 (C25), 16.08 (C26),
14.64 (C27). HRMS (ESI) m/z: calcd for C43 H59 NO4 NaS [M+Na]+ 708.4062, found 708.4055.
Lupeol-3-[(1-Ethyl)-2]-5-(4-Tert-Butylphenyl)-2,4-Thiazolidinedione (6d)
White solid, yield: 45%; mp: 188.6–190.5 ◦ C. 1 H NMR (600 MHz, chloroform-d) δ 7.92
(s, 1H, CH2 =CH2 ), 7.51 (d, J = 8.3 Hz, 2H, Ar-H), 7.47 (d, J = 8.3 Hz, 2H, Ar-H), 4.68 (d,
J = 2.5 Hz, 1H, CH2 =CH2 ), 4.56 (d, J = 2.7 Hz, 1H, CH2 =CH2 ), 4.55–4.51 (m, 1H, CH), 4.47
(d, J = 2.9 Hz, 2H, CH2 ), 2.37 (td, J = 11.0, 5.8 Hz, 1H), 1.94–1.88 (m, 1H), 1.68 (s, 3H, CH3 ),
1.67–1.60 (m, 6H), 1.49–1.45 (m, 2H), 1.38 (d, J = 4.2 Hz, 6H), 1.35 (s, 9H, CH3 ×3), 1.32–1.29
(m, 1H), 1.29–1.22 (m, 2H), 1.22–1.09 (m, 3H), 1.09–1.04 (m, 1H), 1.01 (s, 3H, CH3 ), 0.97 (d,
J = 11.1 Hz, 2H), 0.93 (s, 3H, CH3 ), 0.85 (s, 3H, CH3 ), 0.83 (s, 3H, CH3 ), 0.78 (s, 6H, CH3 ×2).
13 C NMR (150 MHz, chloroform-d) δ 167.74 (C33), 166.08 (C35), 165.89 (C31), 154.72 (C20),
151.08 (C40), 134.69 (C37), 130.46 (C38, C42), 130.05 (C36), 126.46 (C39, C41), 120.00 (C34),
109.92 (C29), 83.47 (C3), 55.43 (C5), 50.40 (C9), 48.39 (C18), 48.13 (C19), 43.11 (C17), 42.96
(C14), 42.51 (C32), 40.95 (C8), 40.12 (C22), 38.38 (C13), 38.14 (C1), 37.95 (C4), 37.15 (C10),
35.68 (C16), 35.23 (C43), 34.27 (C7), 31.20 (C44, C45, C46), 29.94 (C21), 28.04 (C15), 27.54
(C12), 25.17 (C2), 23.68 (C23), 21.07 (C24), 19.40 (C11), 18.26 (C30), 18.13 (C28), 16.50 (C6),
16.23 (C25), 16.08 (C26), 14.65 (C27). HRMS (ESI) m/z: calcd for C46 H65 NO4 NaS [M+Na]+
750.4532, found 750.4532.
Lupeol-3-[(1-Ethyl)-2]-5-(2-Methoxybenzylidene)-2,4-Thiazolidinedione (6e)
(s, 1H, CH2 =CH2 ), 7.45 (dd, J = 7.8, 1.6 Hz, 1H, Ar-H), 7.43–7.39 (m, 1H, Ar-H), 7.05 (t,
J = 7.6 Hz, 1H, Ar-H), 6.95 (d, J = 8.3 Hz, 1H, Ar-H), 4.68 (d, J = 2.4 Hz, 1H, CH2 =CH2 ),
4.56 (d, J = 2.4 Hz, 1H, CH2 =CH2 ), 4.54 (t, J = 5.7 Hz, 1H, CH), 4.47 (d, J = 3.1 Hz, 2H,
CH2 ), 3.90 (s, 3H, OCH3 ), 2.37 (td, J = 11.1, 5.8 Hz, 1H), 1.91 (ddd, J = 13.1, 7.1, 2.9 Hz,
1H), 1.68 (s, 3H, CH3 ), 1.67–1.58 (m, 6H), 1.51–1.46 (m, 2H), 1.39 (q, J = 4.9, 3.9 Hz, 6H),
1.36 (d, J = 3.9 Hz, 1H), 1.34–1.32 (m, 1H), 1.32–1.25 (m, 2H), 1.25–1.08 (m, 3H), 1.06 (dd,
J = 12.8, 4.7 Hz, 1H), 1.02 (s, 3H, CH3 ), 1.01–0.95 (m, 2H), 0.93 (s, 3H, CH3 ), 0.86 (s, 3H,
CH3 ), 0.83 (s, 3H, CH3 ), 0.78 (d, J = 5.8 Hz, 6H, CH3 ×2). 13 C NMR (150 MHz, chloroform-d)
δ 168.13 (C33), 166.17 (C35), 165.88 (C31), 158.71 (C20), 151.09 (C40), 132.60 (C37), 130.50
(C36), 129.59 (C39), 122.41 (C41), 121.03 (C38, C42), 111.32 (C34), 109.92 (C29), 83.41 (C3),
55.66 (C43), 55.44 (C5), 50.40 (C9), 48.39 (C18), 48.13 (C19), 43.11 (C17), 42.96 (C14), 42.43
(C32), 40.95 (C8), 40.12 (C22), 38.39 (C13), 38.14 (C1), 37.96 (C4), 37.16 (C10), 35.68 (C16),
34.28 (C7), 29.94 (C21), 28.05 (C15), 27.54 (C12), 25.18 (C2), 23.69 (C23), 21.07 (C24), 19.40
Molecules 2024, 29, 4957 13 of 25
(C11), 18.26 (C30), 18.13 (C28), 16.51 (C6), 16.24 (C25), 16.08 (C26), 14.65 (C27). HRMS (ESI)
m/z: calcd for C43 H59 NO5 NaS [M+Na]+ 724.4012, found 724.4009.
Lupeol-3-[(1-Ethyl)-2]-5-(2,3,4-Trimethoxybenzylidene)-2,4-Thiazolidinedione (6f)
δ 8.19 (s, 1H, CH2 =CH2 ), 7.21 (d, J = 8.7 Hz, 1H, Ar-H), 6.78 (d, J = 8.8 Hz, 1H, Ar-H), 4.68
(d, J = 2.5 Hz, 1H, CH2 =CH2 ), 4.57–4.56 (m, 1H, CH2 =CH2 ), 4.54 (d, J = 4.5 Hz, 1H, CH),
4.47 (d, J = 3.9 Hz, 2H, CH2 ), 3.94 (d, J = 9.8 Hz, 6H, OCH3 ×2), 3.89 (s, 3H, OCH3 ), 2.37
(td, J = 11.0, 5.8 Hz, 1H), 1.94–1.87 (m, 1H), 1.68 (s, 3H, CH3 ), 1.67–1.61 (m, 6H), 1.50–1.46
(m, 2H), 1.45–1.37 (m, 6H), 1.36 (d, J = 4.0 Hz, 1H), 1.34 (d, J = 2.3 Hz, 1H), 1.25 (s, 2H),
1.23–1.11 (m, 3H), 1.06 (dd, J = 12.9, 4.5 Hz, 1H), 1.02 (s, 3H, CH3 ), 1.00–0.95 (m, 2H), 0.93 (s,
3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.83 (s, 3H, CH3 ), 0.78 (d, J = 4.4 Hz, 6H, CH3 ×2). 13 C NMR
(150 MHz, chloroform-d) δ 168.16 (C33), 166.22 (C35), 165.92 (C31), 156.51 (C20), 154.10
(C40), 151.11 (C37), 142.52 (C38), 130.08 (C36), 124.83 (C42), 120.44 (C39), 119.69 (C41),
109.52 (C34), 107.73 (C29), 83.42 (C3), 62.04 (C43), 61.12 (C44), 56.31 (C45), 55.45 (C5), 50.41
(C9), 48.82 (C18), 48.14 (C19), 43.12 (C17), 42.96 (C14), 42.43 (C32), 40.96 (C8), 40.13 (C22),
38.40 (C13), 38.15 (C1), 37.97 (C4), 37.16 (C10), 35.69 (C16), 34.28 (C7), 29.95 (C21), 29.84
(C11), 28.05 (C15), 27.55 (C12), 25.18 (C2), 23.69 (C23), 21.07 (C24), 18.27 (C30), 18.13 (C28),
16.51 (C6), 16.24 (C25), 16.09 (C26), 14.66 (C27). HRMS (ESI) m/z: calcd for C45 H63 NO7 NaS
[M+Na]+ 784.4223, found 784.4225.
Lupeol-3-[(1-Ethyl)-2]-5-(2-Furanylmethylene)-2,4-Thiazolidinedione (6g)
Light-brown solid, yield: 48%; mp: 185.4–187.2 ◦ C. 1 H NMR (600 MHz, chloroform-
d) δ 7.68 (d, J = 1.7 Hz, 1H, CH2 =CH2 ), 7.67 (s, 1H, CH2 =CH2 ), 6.81 (d, J = 3.5 Hz, 1H,
CH2 =CH2 ), 6.59 (dd, J = 3.6, 1.8 Hz, 1H, CH2 =CH2 ), 4.68 (d, J = 2.4 Hz, 1H, CH2 =CH2 ), 4.56
(dd, J = 2.5, 1.4 Hz, 1H, CH2 =CH2 ), 4.55–4.52 (m, 1H, CH), 4.45 (d, J = 3.1 Hz, 2H, CH2 ), 2.37
(dt, J = 11.1, 5.5 Hz, 1H), 1.91 (ddd, J = 11.2, 7.0, 2.7 Hz, 1H), 1.68 (s, 3H, CH3 ), 1.66–1.59 (m,
6H), 1.50–1.46 (m, 2H), 1.42–1.37 (m, 6H), 1.36 (d, J = 4.3 Hz, 1H), 1.34 (d, J = 2.8 Hz, 1H),
1.29 (ddd, J = 21.6, 11.7, 7.4 Hz, 2H), 1.24–1.08 (m, 3H), 1.06 (dd, J = 12.8, 4.7 Hz, 1H), 1.01 (s,
3H, CH3 ), 1.00–0.95 (m, 2H), 0.93 (s, 3H, CH3 ), 0.85 (s, 3H, CH3 ), 0.83 (s, 3H, CH3 ), 0.78 (s,
6H, CH3 ×2). 13 C NMR (150 MHz, chloroform-d) δ 168.47 (C35), 166.11 (C33), 165.57 (C31),
151.09 (C20), 149.81 (C37), 146.78 (C40), 120.31 (C34), 118.85 (C38), 118.31 (C36), 113.38
(C39), 109.52 (C29), 83.41 (C3), 55.43 (C5), 50.40 (C9), 48.39 (C18), 48.13 (C19), 43.12 (C17),
42.96 (C14), 42.41 (C32), 40.95 (C8), 40.12 (C22), 38.38 (C13), 38.14 (C1), 37.95 (C4), 37.15
(C10), 35.68 (C16), 34.27 (C7), 29.94 (C21), 28.03 (C15), 27.54 (C12), 25.17 (C2), 23.68 (C23),
21.06 (C24), 19.40 (C11), 18.25 (C30), 18.13 (C28), 16.52 (C6), 16.23 (C25), 16.08 (C26), 14.65
(C27). HRMS (ESI) m/z: calcd for C40 H55 NO5 NaS [M+Na]+ 684.3699, found 684.3698.
Lupeol-3-[(1-Ethyl)-2]-5-(2-Thienylmethylene)-2,4-Thiazolidinedione (6h)
Yellow solid, yield: 64%; mp: 177.6–179.5 ◦ C. 1 H NMR (600 MHz, chloroform-d) δ 8.10
(s, 1H, CH2 =CH2 ), 7.68 (d, J = 5.0 Hz, 1H, CH2 =CH2 ), 7.42 (d, J = 3.8 Hz, 1H, CH2 =CH2 ),
7.20 (t, J = 4.4 Hz, 1H, CH2 =CH2 ), 4.68 (d, J = 2.4 Hz, 1H, CH2 =CH2 ), 4.56 (s, 1H, CH2 =CH2 ),
4.54 (d, J = 4.7 Hz, 1H, CH), 4.46 (d, J = 2.3 Hz, 2H, CH2 ), 2.37 (q, J = 5.3 Hz, 1H), 1.93–1.88
(m, 1H), 1.68 (s, 3H, CH3 ), 1.65 (d, J = 10.4 Hz, 6H), 1.49–1.46 (m, 2H), 1.40–1.36 (m, 6H), 1.36
(s, 1H), 1.34 (d, J = 2.8 Hz, 1H), 1.31–1.25 (m, 2H), 1.24–1.16 (m, 3H), 1.08–1.05 (m, 1H), 1.01
(s, 3H, CH3 ), 0.98–0.95 (m, 2H), 0.93 (s, 3H, CH3 ), 0.85 (s, 3H, CH3 ), 0.83 (s, 3H, CH3 ), 0.78 (s,
6H, CH3 ×2). 13 C NMR (150 MHz, chloroform-d) δ 167.04 (C35), 166.04 (C33), 165.52 (C31),
151.73 (C20), 137.66 (C37), 133.81 (C40), 132.40 (C34), 128.83 (C38), 127.21 (C36), 119.47
(C39), 109.91 (C29), 83.49 (C3), 55.43 (C5), 50.40 (C9), 48.39 (C18), 48.13 (C19), 43.11 (C17),
42.96 (C14), 42.62 (C32), 40.95 (C8), 40.12 (C22), 38.39 (C13), 38.14 (C1), 37.96 (C4), 37.15
(C10), 35.68 (C16), 34.27 (C7), 29.94 (C21), 28.04 (C15), 27.54 (C12), 25.17 (C2), 23.68 (C23),
21.45 (C24), 19.85 (C11), 18.26 (C30), 18.13 (C28), 16.51 (C6), 16.24 (C25), 16.08 (C26), 14.65
(C27). HRMS (ESI) m/z: calcd for C40 H55 NO4 NaS2 [M+Na]+ 700.3470, found 700.3479.
Molecules 2024, 29, 4957 14 of 25
Lupeol-3-[(1-Ethyl)-2]-5-(2-Methylpropylidene)-2,4-Thiazolidinedione (6i)
White solid, yield: 45%; mp: 176.5–178.4 ◦ C. 1 H NMR (600 MHz, chloroform-d)
δ 6.97 (d, J = 9.7 Hz, 1H, CH2 =CH2 ), 4.68 (d, J = 2.5 Hz, 1H, CH2 =CH2 ), 4.56 (s, 1H,
CH2 =CH2 ), 4.52 (dd, J = 11.5, 4.7 Hz, 1H, CH), 4.40 (d, J = 4.1 Hz, 2H, CH2 ), 2.46–2.41
(m, 1H), 2.38 (dt, J = 11.1, 5.4 Hz, 1H), 1.94–1.88 (m, 1H), 1.68 (s, 3H, CH3 ), 1.67–1.60 (m,
6H), 1.58 (d, J = 18.1 Hz, 1H), 1.50–1.46 (m, 2H), 1.41–1.37 (m, 6H, CH3 ×2), 1.36 (s, 1H),
1.34 (d, J = 2.2 Hz, 1H), 1.25 (s, 2H), 1.24–1.17 (m, 3H), 1.14 (dd, J = 6.6, 1.7 Hz, 6H), 1.06
(dd, J = 12.8, 4.6 Hz, 1H), 1.02 (s, 3H, CH3 ), 1.01–0.96 (m, 2H), 0.93 (s, 3H, CH3 ), 0.84 (d,
J = 2.7 Hz, 6H, CH3 ×2), 0.78 (s, 6H, CH3 ×2). 13 C NMR (150 MHz, chloroform-d) δ 168.36
(C35), 166.10 (C31), 165.45 (C33), 152.03 (C20), 145.50 (C36), 123.54 (C34), 109.52 (C29),
83.46 (C3), 55.44 (C5), 50.41 (C9), 48.39 (C18), 48.13 (C19), 43.12 (C17), 42.97 (C14), 42.31
(C32), 40.95 (C8), 40.12 (C22), 38.38 (C13), 38.14 (C1), 37.93 (C4), 37.16 (C10), 35.69 (C16),
34.28 (C7), 32.09 (C37), 29.94 (C21), 29.84 (C15), 28.03 (C38), 27.55 (C12), 25.18 (C2), 23.67
(C23), 21.36 (C24), 21.07 (C39), 19.41 (C11), 18.27 (C30), 18.13 (C28), 16.46 (C6), 16.24 (C25),
16.09 (C26), 14.66 (C27). HRMS (ESI) m/z: calcd for C39 H59 NO4 NaS [M+Na]+ 660.4062,
found 660.4068.
3.1.6. Synthesis of Lupeol-3-(4-Nitrobenzoate) (7)

Lupeol (1, 500 mg, 1.17 mmol) and 4-nitrophenyl chloroformate (471 mg, 2.34 mmol)
were reacted in CH2 Cl2 (10 mL) under a N2 environment, after which pyridine (277 µL,
3.51 mmol) was added at 0 ◦ C. The mixture was stirred for 30 min at 0 ◦ C. Then, the
reaction was continued at room temperature for 30–90 min. The reaction’s progress was
monitored using TLC until there was no starting material left. The solvent was washed
with distilled water twice and then saturated in a NaCl solution, dried over Na2 SO4 ,
and concentrated under reduced pressure. Crude product 7 was purified via column
chromatography (CH2 Cl2 ).
Lupeol-3-(4-Nitrobenzoate) (7)
(d, J = 9.2 Hz, 2H, Ar-H), 7.38 (d, J = 9.1 Hz, 2H, Ar-H), 4.69 (d, J = 2.4 Hz, 1H, CH2 =CH2 ),
4.57 (dd, J = 2.6, 1.4 Hz, 1H, CH2 =CH2 ), 4.44 (dd, J = 11.4, 5.1 Hz, 1H), 2.38 (td, J = 11.0,
5.8 Hz, 1H), 1.92 (ddt, J = 13.8, 10.4, 5.2 Hz, 1H), 1.88–1.70 (m, 4H), 1.68 (s, 3H, CH3 ),
1.68–1.62 (m, 2H), 1.54–1.47 (m, 2H), 1.46–1.38 (m, 6H), 1.36 (s, 1H), 1.34 (d, J = 3.0 Hz, 1H),
1.30 (d, J = 2.8 Hz, 1H), 1.29–1.11 (m, 3H), 1.08 (dd, J = 12.7, 4.7 Hz, 1H), 1.04 (s, 3H, CH3 ),
1.02–1.00 (m, 1H), 0.99 (s, 3H, CH3 ), 0.95 (s, 3H, CH3 ), 0.89 (d, J = 8.2 Hz, 6H, CH3 ×2), 0.82
(dd, J = 9.1, 2.8 Hz, 1H), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 155.92
(C32), 152.57 (C31), 151.08 (C22), 145.37 (C35), 125.39 (C29), 121.99 (C33, C37), 109.53 (C34,
C36), 87.78 (C3), 55.48 (C5), 50.46 (C9), 48.40 (C18), 48.12 (C19), 43.12 (C17), 42.98 (C14),
40.97 (C20), 40.12 (C8), 38.43 (C13), 38.30 (C1), 38.14 (C4), 37.20 (C10), 35.68 (C16), 34.28
(C7), 29.95 (C21), 28.09 (C15), 27.56 (C12), 25.18 (C2), 23.68 (C23), 21.10 (C24), 19.42 (C11),
18.27 (C30), 18.13 (C28), 16.52 (C6), 16.31 (C25), 16.11 (C26), 14.66 (C27).
3.1.7. Synthesis of Lupeol-3-Piperazinecarboxylate (8)

A solution of compound 7 (430 mg, 0.73 mmol), piperazine (125 mg, 1.46 mmol),
and Et3 N (202 µL, 1.46 mmol) was reacted at room temperature for 4–5 h. The reaction’s
progress was monitored using TLC until no starting material remained. The sample was
then concentrated under reduced pressure to remove the excess solvent. The residue was
purified using silica gel column chromatography (CH2 Cl2 /MeOH = 40:1, v/v) to produce
intermediate 8.
Lupeol-3-Piperazinecarboxylate (8)
White solid, yield: 75%; mp: 190.7–192.5 ◦ C. 1 H NMR (600 MHz, chloroform-d) δ
4.68 (d, J = 2.4 Hz, 1H, CH2 =CH2 ), 4.57 (t, J = 2.0 Hz, 1H, CH2 =CH2 ), 4.36 (dd, J = 11.8,
4.5 Hz, 1H), 3.46 (t, J = 5.2 Hz, 4H, CH2 ×2), 2.84 (s, 4H, CH2 ×2), 2.38 (td, J = 11.0, 5.8 Hz,
Molecules 2024, 29, 4957 15 of 25
1H), 2.20–2.15 (m, 1H), 1.91 (ddt, J = 13.7, 10.5, 5.2 Hz, 1H), 1.71 (dd, J = 8.6, 4.5 Hz, 1H),
1.69–1.68 (m, 3H, CH3 ), 1.67–1.61 (m, 3H), 1.59 (dd, J = 12.6, 3.2 Hz, 1H), 1.52–1.46 (m, 2H),
1.43–1.33 (m, 8H), 1.33–1.27 (m, 2H), 1.25 (d, J = 5.4 Hz, 1H), 1.23–1.16 (m, 2H), 1.07 (dd,
J = 12.9, 4.6 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.00 (dt, J = 13.4, 3.4 Hz, 2H), 0.94 (s, 3H, CH3 ),
0.88 (s, 3H, CH3 ), 0.84 (d, J = 12.4 Hz, 6H, CH3 ×2), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz,
chloroform-d) δ 155.72 (C31), 151.12 (C22), 109.50 (C29), 82.12 (C3), 55.48 (C5), 50.42 (C9),
48.40 (C18), 48.13 (C19), 45.94 (C32, C35), 43.12 (C17), 42.95 (C14), 40.96 (C26), 40.12 (C33,
C34), 38.47 (C8), 38.26 (C22), 38.17 (C13), 37.18 (C4), 35.70 (C1), 34.32 (C10), 29.94 (C16),
28.19 (C7), 27.56 (C21), 25.21 (C15), 24.30 (C12), 21.07 (C2), 19.40 (C23, C24), 18.34 (C11),
18.13 (C30), 16.96 (C28), 16.27 (C6), 16.10 (C25), 14.67 (C27).

A solution of compound 8 (50 mg, 0.09 mmol), intermediates 5a–i (0.14 mmol), EDCI
(26 mg, 0.14 mmol), and DMAP (6.10 mg, 0.05 mmol) in CH2 Cl2 (6 mL) was stirred
for 6–8 h. The reaction’s progress was monitored using TLC until no starting material
remained. The solvent was then removed under reduced pressure to provide the crude
product. Further purification involved separation via silica gel column chromatography
(CH2 Cl2 /MeOH = 160:1, v/v) to produce compounds 9a–i.
Lupeol-3-[(1-Piperazinyl)-4]-5-(4-Fluorobenzylidene)-2,4-Thiazolidinedione (9a)
(s, 1H, CH2 =CH2 ), 7.52 (dd, J = 8.7, 5.3 Hz, 2H, Ar-H), 7.18 (t, J = 8.5 Hz, 2H, Ar-H), 4.69
(d, J = 2.5 Hz, 1H, CH2 =CH2 ), 4.58–4.56 (m, 1H, CH2 =CH2 ), 4.55 (s, 2H, CH2 ), 4.40 (dd,
J = 11.6, 4.4 Hz, 1H, CH), 3.56 (d, J = 62.4 Hz, 8H, CH2 ×4), 2.38 (td, J = 11.1, 5.8 Hz, 1H),
1.91 (ddd, J = 13.6, 10.9, 8.7 Hz, 1H), 1.69 (s, 3H, CH3 ), 1.68–1.53 (m, 6H), 1.52–1.47 (m,
2H), 1.46–1.38 (m, 6H), 1.37 (s, 1H), 1.35 (d, J = 5.7 Hz, 1H), 1.34–1.29 (m, 2H), 1.27–1.11
(m, 3H), 1.08 (dd, J = 12.9, 4.6 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.03–0.98 (m, 2H), 0.95 (s, 3H,
CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s, 3H, CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR
(C31), 151.12 (C20), 133.39 (C41), 132.49 (C46), 132.43 (C43), 129.62 (C42), 121.13 (C39),
116.82 (C44), 116.67 (C46), 110.16 (C29), 82.85 (C3), 55.47 (C5), 50.44 (C9), 48.40 (C18), 48.14
(C19), 44.81 (C32, C33), 43.12 (C17), 42.96 (C14), 42.51 (C37), 42.27 (C34, C35), 40.97 (C8),
40.13 (C22), 38.45 (C13), 38.27 (C4), 38.16 (C1), 37.19 (C10), 35.69 (C16), 34.31 (C7), 29.95
(C21), 28.25 (C15), 27.56 (C12), 25.21 (C2), 24.27 (C23), 21.08 (C24), 19.41 (C11), 18.34 (C30),
C47 H64 N3 O5 NaSF [M+Na]+ 824.4448, found 824.4444.
Lupeol-3-[(1-Piperazinyl)-4]-5-(4-Chlorobenzylidene)-2,4-Thiazolidinedione (9b)
δ 7.87 (s, 1H, CH2 =CH2 ), 7.45 (s, 4H, Ar-H), 4.69 (d, J = 2.5 Hz, 1H, CH2 =CH2 ), 4.58–4.56 (m,
1H, CH2 =CH2 ), 4.55 (s, 2H, CH2 ), 4.40 (dd, J = 11.7, 4.4 Hz, 1H, CH), 3.56 (d, J = 60.2 Hz,
8H, CH2 ×4), 2.38 (td, J = 11.0, 5.8 Hz, 1H), 1.95–1.88 (m, 1H), 1.69 (s, 3H, CH3 ), 1.63
(ddd, J = 20.1, 12.4, 3.8 Hz, 6H), 1.52–1.47 (m, 2H), 1.44–1.38 (m, 6H), 1.37 (s, 1H), 1.35 (d,
J = 5.9 Hz, 1H), 1.34–1.27 (m, 2H), 1.27–1.11 (m, 3H), 1.08 (dd, J = 12.9, 4.6 Hz, 1H), 1.03 (s,
3H, CH3 ), 1.02–0.98 (m, 2H), 0.95 (s, 3H, CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s,
3H, CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 167.62 (C36), 165.97 (C38),
163.26 (C40), 155.44 (C45), 151.12 (C31), 136.92 (C20), 133.17 (C41), 131.76 (C42), 131.47 (C43,
C47), 129.73 (C44, C46), 122.06 (C39), 109.50 (C29), 82.86 (C3), 55.46 (C5), 50.44 (C9), 48.40
(C18), 48.13 (C19), 44.80 (C32, C33), 43.12 (C17), 42.96 (C14), 42.53 (C37), 42.27 (C34, C35),
40.96 (C8), 40.12 (C22), 38.45 (C13), 38.26 (C4), 38.16 (C1), 37.19 (C10), 35.69 (C16), 34.30
(C7), 29.94 (C21), 28.25 (C15), 27.56 (C12), 25.20 (C2), 24.26 (C23), 21.08 (C24), 19.41 (C11),
18.34 (C30), 18.13 (C28), 16.99 (C6), 16.28 (C25), 16.10 (C26), 14.66 (C27). HRMS (ESI) m/z:
calcd for C47 H64 N3 O5 NaSCl [M+Na]+ 840.4153, found 840.4153.
Molecules 2024, 29, 4957 16 of 25
Lupeol-3-[(1-Piperazinyl)-2]-5-(4-Methylbenzylidene)-2,4-Thiazolidinedione (9c)
J = 2.5 Hz, 1H, CH2 =CH2 ), 4.57 (t, J = 2.0 Hz, 1H, CH2 =CH2 ), 4.55 (s, 2H, CH2 ), 4.40 (dd,
J = 11.7, 4.4 Hz, 1H, CH), 3.63–3.48 (m, 8H, CH2 ×4), 2.41 (s, 3H, CH3 ), 2.37 (dt, J = 11.2,
5.6 Hz, 1H), 1.95–1.88 (m, 1H), 1.69 (d, J = 6.8 Hz, 3H, CH3 ), 1.68–1.59 (m, 6H), 1.52–1.46 (m,
2H), 1.46–1.37 (m, 6H), 1.37 (s, 1H), 1.35 (d, J = 5.7 Hz, 1H), 1.34–1.28 (m, 2H), 1.28–1.10 (m,
3H), 1.08 (dd, J = 12.9, 4.6 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.02–0.97 (m, 2H), 0.95 (s, 3H, CH3 ),
0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s, 3H, CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz,
chloroform-d) δ 168.20 (C36), 166.25 (C38), 163.36 (C40), 155.50 (C45), 151.11 (C31), 141.58
(C20), 134.83 (C41), 130.56 (C42), 130.48 (C43, C47), 130.15 (C44, C46), 120.16 (C39), 109.50
(C29), 82.82 (C3), 55.47 (C5), 50.43 (C9), 48.40 (C18), 48.13 (C19), 44.79 (C32, C33), 43.12
(C17), 42.96 (C14), 42.41 (C37), 42.24 (C34, C35), 40.96 (C8), 40.12 (C22), 38.45 (C13), 38.26
(C4), 38.16 (C1), 37.57 (C10), 35.69 (C16), 34.30 (C7), 29.94 (C21), 28.24 (C15), 27.56 (C12),
25.20 (C2), 24.26 (C23), 21.74 (C48), 21.08 (C24), 19.41 (C11), 18.33 (C30), 18.12 (C28), 16.99
(C6), 16.27 (C25), 16.09 (C26), 14.66 (C27). HRMS (ESI) m/z: calcd for C48 H67 N3 O5 NaS
[M+Na]+ 820.4699, found 820.4700.
Lupeol-3-[(1-Piperazinyl)-4]-5-(4-Tert-Butylphenyl)-2,4-Thiazolidinedione (9d)
7.91 (s, 1H, CH2 =CH2 ), 7.50 (d, J = 8.2 Hz, 2H, Ar-H), 7.46 (d, J = 8.5 Hz, 2H, Ar-H), 4.69
(d, J = 2.6 Hz, 1H, CH2 =CH2 ), 4.57 (t, J = 2.0 Hz, 1H, CH2 =CH2 ), 4.55 (s, 2H, CH2 ), 4.40
(dd, J = 11.7, 4.4 Hz, 1H, CH), 3.56 (d, J = 64.3 Hz, 8H, CH2 ×4), 2.38 (td, J = 11.0, 5.8 Hz,
1H), 1.95–1.88 (m, 1H), 1.69 (s, 3H, CH3 ), 1.68–1.57 (m, 6H), 1.52–1.47 (m, 2H), 1.45–1.38
(m, 6H), 1.37–1.36 (m, 1H), 1.34 (s, 9H, CH3 ×3), 1.32–1.28 (m, 2H), 1.28–1.11 (m, 3H), 1.08
(dd, J = 13.0, 4.5 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.02–0.99 (m, 2H), 0.95 (s, 3H, CH3 ), 0.89 (s,
3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s, 3H, CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz,
(C20), 134.70 (C41), 130.50 (C42), 130.39 (C43, C47), 126.42 (C44, C46), 120.27 (C39), 109.51
(C29), 82.82 (C3), 55.47 (C5), 50.43 (C9), 48.40 (C18), 48.13 (C19), 44.80 (C32, C33), 43.12
(C17), 42.96 (C14), 42.41 (C37), 42.24 (C34, C35), 40.96 (C8), 40.12 (C22), 38.45 (C13), 38.26
(C4), 38.16 (C1), 37.19 (C10), 35.69 (C16), 35.21 (C48), 34.31 (C7), 31.20 (C49, C50, C51), 29.95
(C21), 28.25 (C15), 27.56 (C12), 25.20 (C2), 24.26 (C23), 21.08 (C24), 19.41 (C11), 18.34 (C30),
C51 H73 N3 O5 NaS [M+Na]+ 862.5169, found 862.5168.
Lupeol-3-[(1-Piperazinyl)-4]-5-(2-Methoxybenzylidene)-2,4-Thiazolidinedione (9e)
δ 8.29 (s, 1H, CH2 =CH2 ), 7.45 (dd, J = 7.8, 1.5 Hz, 1H, Ar-H), 7.43–7.39 (m, 1H, Ar-H), 7.04
(t, J = 7.5 Hz, 1H, Ar-H), 6.94 (d, J = 8.3 Hz, 1H, Ar-H), 4.69 (d, J = 2.6 Hz, 1H, CH2 =CH2 ),
4.57 (t, J = 2.1 Hz, 1H, CH2 =CH2 ), 4.55 (s, 2H, CH2 ), 4.40 (dd, J = 11.7, 4.5 Hz, 1H, CH), 3.89
(s, 3H, OCH3 ), 3.56 (d, J = 61.1 Hz, 8H, CH2 ×4), 2.38 (td, J = 11.0, 5.8 Hz, 1H), 1.95–1.88 (m,
1H), 1.69 (s, 3H, CH3 ), 1.68–1.58 (m, 6H), 1.52–1.46 (m, 2H), 1.46–1.38 (m, 6H), 1.37–1.36
(m, 1H), 1.35 (d, J = 5.6 Hz, 1H), 1.32–1.29 (m, 2H), 1.27–1.21 (m, 3H), 1.08 (dd, J = 12.9,
4.5 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.02–0.98 (m, 2H), 0.95 (s, 3H, CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s,
3H, CH3 ), 0.84 (s, 3H, CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 168.61
(C36), 166.22 (C38), 163.49 (C40), 158.65 (C45), 151.11 (C31), 132.50 (C20), 130.58 (C42),
129.57 (C41), 122.49 (C43), 121.74 (C44), 121.01 (C47), 111.27 (C46), 110.35 (C39), 109.50
(C29), 82.81 (C3), 55.63 (C5), 55.47 (C9), 48.40 (C18), 48.13 (C19), 44.80 (C32, C33), 43.12
(C17), 42.96 (C14), 42.31 (C37), 42.22 (C34, C35), 40.96 (C8), 40.12 (C22), 38.45 (C13), 38.26
(C4), 38.16 (C1), 37.18 (C10), 35.69 (C16), 34.30 (C7), 29.94 (C21), 28.24 (C15), 27.56 (C12),
25.20 (C2), 24.26 (C23), 22.78 (C48), 21.08 (C24), 19.41 (C11), 18.33 (C30), 18.12 (C28), 16.99
[M+Na]+ 836.4648, found 836.4649.
Molecules 2024, 29, 4957 17 of 25
Lupeol-3-[(1-Piperazinyl)-4]-5-(2,3,4-Trimethoxybenzylidene)-2,4-Thiazolidinedione (9f)
J = 2.6 Hz, 1H, CH2 =CH2 ), 4.57 (t, J = 2.0 Hz, 1H, CH2 =CH2 ), 4.55 (s, 2H, CH2 ), 4.40 (dd,
J = 11.6, 4.4 Hz, 1H, CH), 3.93 (d, J = 8.2 Hz, 6H, OCH3 ×2), 3.88 (s, 3H, OCH3 ), 3.56 (d,
J = 61.1 Hz, 8H, CH2 ×4), 2.38 (td, J = 11.1, 5.8 Hz, 1H), 1.95–1.88 (m, 1H), 1.69 (s, 3H, CH3 ),
1.68–1.54 (m, 6H), 1.52–1.47 (m, 2H), 1.46–1.38 (m, 6H), 1.37 (s, 1H), 1.35 (d, J = 5.5 Hz, 1H),
1.31 (dd, J = 12.7, 2.7 Hz, 2H), 1.27–1.10 (m, 3H), 1.08 (dd, J = 12.9, 4.5 Hz, 1H), 1.03 (s, 3H,
CH3 ), 1.03–0.98 (m, 2H), 0.95 (s, 3H, CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s, 3H,
CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 168.60 (C36), 166.27 (C38),
163.65 (C40), 156.43 (C45), 155.54 (C31), 154.06 (C20), 151.12 (C41), 142.94 (C42), 130.04
(C43), 124.78 (C44), 120.50 (C47), 119.55 (C46), 109.50 (C39), 107.72 (C29), 82.82 (C3), 62.05
(C48), 61.09 (C49), 56.28 (C50), 55.47 (C5), 50.43 (C9), 48.40 (C18), 48.13 (C19), 44.81 (C32,
C33), 43.12 (C17), 42.96 (C14), 42.31 (C37), 42.23 (C34, C35), 40.96 (C8), 40.12 (C22), 38.45
(C13), 38.26 (C4), 38.16 (C1), 37.19 (C10), 35.69 (C16), 34.30 (C7), 29.94 (C21), 28.24 (C15),
27.56 (C12), 25.20 (C2), 24.26 (C23), 21.08 (C24), 19.40 (C11), 18.33 (C30), 18.12 (C28), 16.99
[M+Na]+ 896.4860, found 896.4853.
Lupeol-3-[(1-Piperazinyl)-4]-5-(2-Furanylmethylene)-2,4-Thiazolidinedione (9g)
Light-brown solid, yield: 57%; mp: 189.3–191.2 ◦ C. 1 H NMR (600 MHz, chloroform-
d) δ 7.68 (d, J = 1.8 Hz, 1H, CH2 =CH2 ), 7.66 (s, 1H, CH2 =CH2 ), 6.80 (d, J = 3.5 Hz, 1H,
CH2 =CH2 ), 6.58 (dd, J = 3.6, 1.8 Hz, 1H, CH2 =CH2 ), 4.69 (d, J = 2.5 Hz, 1H, CH2 =CH2 ), 4.57
(d, J = 2.6 Hz, 1H, CH2 =CH2 ), 4.53 (s, 2H, CH2 ), 4.39 (dd, J = 11.7, 4.4 Hz, 1H, CH), 3.55 (d,
J = 63.0 Hz, 8H, CH2 ×4), 2.38 (td, J = 11.1, 5.8 Hz, 1H), 1.95–1.88 (m, 1H), 1.69 (s, 3H, CH3 ),
1.68–1.58 (m, 6H), 1.52–1.46 (m, 2H), 1.46–1.37 (m, 6H), 1.37 (s, 1H), 1.35 (d, J = 5.7 Hz, 1H),
1.33–1.27 (m, 2H), 1.27–1.11 (m, 3H), 1.08 (dd, J = 12.9, 4.6 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.01
(dd, J = 13.7, 3.7 Hz, 2H), 0.95 (s, 3H, CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s, 3H,
CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 168.93 (C36), 165.96 (C38),
163.40 (C40), 155.60 (C45), 151.11 (C31), 149.85 (C20), 146.73 (C41), 120.82 (C43), 119.06
(C42), 118.19 (C39), 113.33 (C44), 109.50 (C29), 82.81 (C3), 55.47 (C5), 50.43 (C9), 48.40 (C18),
48.13 (C19), 44.78 (C32, C33), 43.12 (C17), 42.96 (C14), 42.30 (C37), 42.21 (C34, C35), 40.96
(C8), 40.12 (C22), 38.44 (C13), 38.25 (C4), 38.15 (C1), 37.18 (C10), 35.68 (C16), 34.30 (C7),
29.94 (C21), 28.24 (C15), 27.56 (C12), 25.20 (C2), 24.25 (C23), 21.07 (C24), 19.40 (C11), 18.33
(C30), 18.12 (C28), 16.99 (C6), 16.27 (C25), 16.09 (C26), 14.66 (C27). HRMS (ESI) m/z: calcd
for C45 H63 N3 O6 NaS [M+Na]+ 796.4335, found 796.4327.
Lupeol-3-[(1-Piperazinyl)-4]-5-(2-Thienylmethylene)-2,4-Thiazolidinedione (9h)
Light-yellow solid, yield: 53%; mp: 191.7–193.4 ◦ C. 1 H NMR (600 MHz, chloroform-
d) δ 8.09 (s, 1H, CH2 =CH2 ), 7.67 (d, J = 5.0 Hz, 1H, CH2 =CH2 ), 7.41 (d, J = 3.7 Hz, 1H,
CH2 =CH2 ), 7.20–7.18 (m, 1H, CH2 =CH2 ), 4.69 (d, J = 2.5 Hz, 1H, CH2 =CH2 ), 4.57 (t,
J = 2.0 Hz, 1H, CH2 =CH2 ), 4.54 (s, 2H, CH2 ), 4.40 (dd, J = 11.7, 4.4 Hz, 1H, CH), 3.55 (d,
J = 61.5 Hz, 8H, CH2 ×4), 2.38 (td, J = 11.0, 5.8 Hz, 1H), 1.91 (ddd, J = 13.1, 7.1, 2.9 Hz, 1H),
1.69 (s, 3H, CH3 ), 1.68–1.59 (m, 6H), 1.52–1.47 (m, 2H), 1.43–1.38 (m, 6H), 1.37 (s, 1H), 1.35
(d, J = 5.7 Hz, 1H), 1.33–1.26 (m, 2H), 1.26–1.17 (m, 3H), 1.08 (dd, J = 12.9, 4.6 Hz, 1H), 1.03
(s, 3H, CH3 ), 1.00 (d, J = 3.6 Hz, 2H), 0.95 (s, 3H, CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ),
0.84 (s, 3H, CH3 ), 0.79 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 167.52 (C36),
165.91 (C38), 163.51 (C40), 155.56 (C45), 151.11 (C31), 137.71 (C20), 133.69 (C41), 132.30
(C43), 128.79 (C42), 127.20 (C39), 119.28 (C44), 109.50 (C29), 82.83 (C3), 55.47 (C5), 50.43
(C9), 48.40 (C18), 48.13 (C19), 44.79 (C32, C33), 43.12 (C17), 42.96 (C14), 42.55 (C37), 42.24
(C34, C35), 40.96 (C8), 40.12 (C22), 38.45 (C13), 38.26 (C4), 38.16 (C1), 37.18 (C10), 35.69
(C16), 34.30 (C7), 29.94 (C21), 28.24 (C15), 27.56 (C12), 25.20 (C2), 24.26 (C23), 21.08 (C24),
19.41 (C11), 18.33 (C30), 18.12 (C28), 16.99 (C6), 16.27 (C25), 16.09 (C26), 14.66 (C27). HRMS
(ESI) m/z: calcd for C45 H63 N3 O5 NaS2 [M+Na]+ 812.4107, found 812.4107.
Molecules 2024, 29, 4957 18 of 25
Lupeol-3-[(1-Piperazinyl)-4]-5-(2-Methylpropylidene)-2,4-Thiazolidinedione (9i)
(d, J = 9.7 Hz, 1H, CH2 =CH2 ), 4.69 (d, J = 2.6 Hz, 1H, CH2 =CH2 ), 4.57 (t, J = 2.0 Hz, 1H,
CH2 =CH2 ), 4.48 (s, 2H, CH2 ), 4.39 (dd, J = 11.7, 4.5 Hz, 1H, CH), 3.54 (d, J = 65.5 Hz, 8H,
CH2 ×4), 2.47–2.42 (m, 1H), 2.38 (td, J = 11.0, 5.8 Hz, 1H), 1.91 (ddd, J = 13.2, 6.9, 2.7 Hz, 1H),
1.68 (s, 3H, CH3 ), 1.68–1.53 (m, 6H), 1.52–1.46 (m, 2H), 1.43–1.37 (m, 6H), 1.37 (d, J = 2.7 Hz,
1H), 1.35 (d, J = 5.7 Hz, 1H), 1.33–1.29 (m, 2H), 1.22 (ddd, J = 18.2, 14.3, 10.7 Hz, 3H), 1.14
(d, J = 6.7 Hz, 6H, CH3 ×2), 1.08 (dd, J = 12.9, 4.5 Hz, 1H), 1.03 (s, 3H, CH3 ), 1.02–0.98 (m,
2H), 0.95 (s, 3H, CH3 ), 0.89 (s, 3H, CH3 ), 0.86 (s, 3H, CH3 ), 0.84 (s, 3H, CH3 ), 0.79 (s, 3H,
CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 167.98 (C36), 165.19 (C38), 163.64 (C40), 155.55
(C31), 151.12 (C20), 145.43 (C41), 122.96 (C39), 109.50 (C29), 82.83 (C3), 55.47 (C5), 50.44
(C9), 48.40 (C18), 48.14 (C19), 44.77 (C32, C33), 43.48 (C17), 43.12 (C14), 42.96 (C37), 42.19
(C34, C35), 40.96 (C8), 40.13 (C22), 38.45 (C13), 38.26 (C4), 38.16 (C1), 37.19 (C10), 35.69
(C16), 34.31 (C7), 32.09 (C42), 29.95 (C21), 28.24 (C15), 27.56 (C12), 25.21 (C2), 24.26 (C23),
21.37 (C43, C44), 21.08 (C24), 19.41 (C11), 18.34 (C30), 18.13 (C28), 16.99 (C6), 16.27 (C25),
16.10 (C26), 14.66 (C27). HRMS (ESI) m/z: calcd for C44 H67 N3 O5 NaS [M+Na]+ 772.4699,
found 772.4700.
3.1.9. Synthesis of Lupeol-3-[(1-Ethylenediaminyl)-4]-Tert-Butoxycarbonyl (10)

Compound 7 (500 mg, 0.85 mmol) and N-Boc-ethylenediamine (408 µL, 2.55 mmol)
were reacted in the presence of Et3 N (354 µL, 2.55 mmol) for 1–2 h. The reaction was
monitored via TLC until the raw material was completely reacted. Crude compound 10
was purified using silica gel chromatography (CH2 Cl2 /MeOH = 62:1, v/v); this produced
the target product, namely 10.
Lupeol-3-[(1-Ethylenediaminyl)-4]-Tert-Butoxycarbonyl (10)
(d, J = 5.8 Hz, 1H, NH), 4.92 (s, 1H, NH), 4.68 (d, J = 2.5 Hz, 1H, CH), 4.57 (s, 1H, CH2 =CH2 ),
4.33 (dd, J = 12.1, 4.2 Hz, 1H, CH2 =CH2 ), 3.27 (d, J = 15.7 Hz, 4H, CH2 ×2), 2.38 (td, J = 11.1,
5.8 Hz, 1H), 1.91 (ddd, J = 13.2, 7.2, 3.0 Hz, 1H), 1.68 (s, 3H, CH3 ), 1.65 (d, J = 3.3 Hz, 2H),
1.52–1.45 (m, 5H), 1.44 (d, J = 3.7 Hz, 9H), 1.40–1.35 (m, 7H), 1.31–1.24 (m, 5H), 1.23–1.17
(m, 3H), 1.07 (dd, J = 12.9, 4.6 Hz, 1H), 1.03 (s, 3H, CH3 ), 0.98 (d, J = 4.1 Hz, 1H), 0.94 (s,
3H, CH3 ), 0.87 (s, 3H, CH3 ), 0.84 (s, 3H, CH3 ), 0.79 (s, 6H, CH3 ×2). 13 C NMR (150 MHz,
chloroform-d) δ 151.09 (C34), 109.48 (C31), 81.59 (C22), 79.59 (C29), 55.52 (C3), 50.42 (C35),
48.38 (C5), 48.11 (C9), 43.10 (C18), 42.93 (C19), 41.35 (C17), 40.94 (C14), 40.88 (C8), 40.10
(C32), 38.47 (C21), 38.14 (C33), 37.15 (C13), 35.67 (C4), 34.31 (C1), 31.70 (C10), 29.92 (C16),
29.81 (C7), 28.49 (C38, C37, C36), 28.05 (C20), 27.53 (C15), 25.19 (C12), 24.20 (C2), 22.77
(C23), 21.04 (C11), 19.39 (C24), 18.29 (C30), 18.11 (C28), 16.61 (C6), 16.27 (C25), 16.08 (C26),
14.63 (C27).
3.1.10. Synthesis of Intermediate 11

Excess CF3 COOH was added dropwise to a stirred solution of intermediate 10 in
CH2 Cl2 (8 mL) under nitrogen at 0 ◦ C and monitored using TLC. After the reaction, the
solvent was removed under reduced pressure to produce light-yellow oil 11, which was
used in the next step without further purification.

EDCI (0.80 equiv), HOBT (0.10 equiv), and DIPEA (1.50 equiv) were added to a stirred
solution of intermediate 5a–i (70 mg, 0.20–0.31 mmol) in CH2 Cl2 (8 mL) at 0 ◦ C. After 0.5 h,
intermediate 11 (0.50 equiv) was added, and the reaction mixture was stirred for 8–12 h at
room temperature. The mixtures were concentrated. The residue was purified using silica
gel chromatography (CH2 Cl2 /MeOH = 80:1, v/v). This process produced target products
in the form of compounds 12a–i.
Molecules 2024, 29, 4957 19 of 25
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(4-Fluorobenzylidene)-2,4-Thiazolidinedione (12a)
7.88 (s, 1H, CH2 =CH2 ), 7.52 (dd, J = 8.8, 5.1 Hz, 2H, Ar-H), 7.17 (t, J = 8.3 Hz, 2H, Ar-H),
7.13 (d, J = 5.3 Hz, 1H, NH), 5.26 (d, J = 6.9 Hz, 1H, CH2 =CH2 ), 5.19 (t, J = 6.1 Hz, 1H,
CH2 =CH2 ), 4.39 (s, 2H, CH2 ), 4.33–4.26 (m, 1H, CH), 3.48–3.25 (m, 4H, CH2 ×2, 1H, NH),
1.78–1.70 (m, 3H), 1.70–1.64 (m, 3H, CH3 ), 1.64 (s, 4H), 1.58 (d, J = 8.5 Hz, 1H), 1.56–1.53
(m, 1H), 1.49–1.46 (m, 2H), 1.40 (d, J = 3.6 Hz, 1H), 1.35 (t, J = 5.1 Hz, 2H), 1.32 (dd, J = 13.4,
4.3 Hz, 2H), 1.25 (d, J = 6.4 Hz, 3H), 1.23–1.17 (m, 3H), 1.06 (s, 1H), 1.03 (s, 1H), 1.00 (d,
J = 6.9 Hz, 2H), 0.94 (s, 3H, CH3 ), 0.86 (t, J = 11.5 Hz, 9H, CH3 ×3), 0.79 (s, 3H, CH3 ), 0.73 (s,
3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 167.58 (C34), 165.90 (C36), 165.71 (C38),
164.69 (C43), 163.00 (C31), 158.49 (C22), 139.97 (C39), 133.22 (C45), 132.47 (C41), 132.41
(C40), 130.23 (C37), 119.01 (C42), 116.80 (C44), 116.66 (C29), 82.29 (C3), 55.57 (C5), 50.44
(C9), 48.80 (C18), 43.99 (C19), 42.43 (C17), 42.28 (C14), 41.16 (C35), 39.29 (C8), 38.54 (C32),
38.13 (C21), 37.07 (C33), 36.79 (C13), 36.42 (C4), 34.49 (C1), 34.23 (C10), 29.82 (C16), 28.13
(C7), 27.71 (C20), 27.14 (C15), 24.15 (C12), 22.65 (C2), 21.75 (C23), 21.72 (C11), 18.26 (C24),
calcd for C45 H62 N3 O5 NaSF [M+Na]+ 798.4292, found 798.4301.
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(4-Chlorobenzylidene)-2,4-Thiazolidinedione (12b)
(s, 1H, CH2 =CH2 ), 7.45 (s, 4H, Ar-H), 6.96 (s, 1H), 5.04 (s, 1H, NH), 4.70 (s, 1H, CH2 =CH2 ),
4.58 (s, 1H, CH2 =CH2 ), 4.38 (s, 2H, CH2 ), 4.31–4.23 (m, 1H, CH), 3.37 (d, J = 45.4 Hz, 4H,
CH2 ×2), 2.38 (td, J = 11.0, 5.8 Hz, 1H), 1.93 (q, J = 11.1, 10.7 Hz, 1H), 1.70 (s, 3H, CH3 ),
1.69–1.58 (m, 6H), 1.50–1.46 (m, 2H), 1.38 (d, J = 10.0 Hz, 6H), 1.32 (s, 1H), 1.31–1.23 (m, 3H),
1.23–1.15 (m, 3H), 1.07 (d, J = 11.8 Hz, 1H), 1.02 (s, 3H), 0.99 (s, 1H), 0.94 (s, 3H, CH3 ), 0.84 (d,
J = 19.4 Hz, 6H, CH3 ×2), 0.78 (d, J = 5.5 Hz, 6H, CH3 ×2), 0.72 (d, J = 9.0 Hz, 1H). 13 C NMR
(C31), 136.95 (C22), 133.01 (C39), 131.80 (C37), 131.44 (C41, C45), 129.74 (C42, C44), 122.19
(C44), 109.53 (C29), 82.43 (C3), 55.62 (C5), 50.49 (C9), 48.43 (C18), 48.13 (C19), 44.11 (C17),
43.13 (C14), 42.94 (C35), 42.23 (C8), 40.96 (C32), 40.31 (C21), 40.12 (C33), 38.52 (C13), 38.15
(C4), 37.16 (C1), 35.69 (C10), 34.29 (C16), 29.96 (C7), 29.84 (C20), 28.15 (C15), 27.56 (C12),
25.25 (C2), 24.16 (C23), 21.06 (C11), 19.47 (C24), 18.30 (C30), 18.13 (C28), 16.66 (C6), 16.26
(C25), 16.09 (C26), 14.71 (C27). HRMS (ESI) m/z: calcd for C45 H62 N3 O5 NaSCl [M+Na]+
814.3996, found 814.4000.
Lupeol-3-[(1-Ethylenediaminyl)-2]-5-(4-Methylbenzylidene)-2,4-Thiazolidinedione (12c)
Light-brown solid, yield: 45%; mp: 192.4–194.2 ◦ C. 1 H NMR (600 MHz, chloroform-d)
δ 7.88 (s, 1H, CH2 =CH2 ), 7.40 (d, J = 7.8 Hz, 2H, Ar-H), 7.27 (d, J = 8.5 Hz, 2H, Ar-H), 7.16
(d, J = 5.0 Hz, 1H, NH), 5.25 (dd, J = 13.4, 5.6 Hz, 1H, CH2 =CH2 ), 5.20 (d, J = 6.1 Hz, 1H,
CH2 =CH2 ), 4.39 (s, 2H, CH2 ), 4.30 (td, J = 9.6, 8.7, 4.7 Hz, 1H, CH), 3.43–3.27 (m, 4H, CH2 ×2,
1H, NH), 2.40 (d, J = 3.0 Hz, 3H, CH3 ), 2.02 (h, J = 6.4 Hz, 1H), 1.76 (ddd, J = 18.5, 11.5, 4.6
Hz, 2H), 1.69 (s, 1H), 1.64 (s, 3H, CH3 ), 1.57 (q, J = 8.8, 8.3 Hz, 2H), 1.48 (dt, J = 13.4, 8.8 Hz,
3H), 1.41 (t, J = 7.4 Hz, 3H), 1.35 (s, 1H), 1.31 (td, J = 7.9, 3.4 Hz, 3H), 1.25 (d, J = 4.6 Hz, 3H),
1.14 (s, 1H), 1.03 (d, J = 4.4 Hz, 3H), 1.00 (d, J = 5.3 Hz, 3H), 0.94 (s, 3H, CH3 ), 0.89–0.83 (m,
9H, CH3 ×3), 0.78 (d, J = 3.5 Hz, 3H, CH3 ), 0.73 (d, J = 6.4 Hz, 3H, CH3 ). 13 C NMR (150
MHz, chloroform-d) δ 168.02 (C34), 166.04 (C36), 141.61 (C38), 139.95 (C43), 134.76 (C31),
134.40 (C22), 133.36 (C39), 130.46 (C41, C45), 130.14 (C42, C44), 126.21 (C37), 119.02 (C40),
115.78 (C29), 82.33 (C3), 55.55 (C5), 50.40 (C9), 48.81 (C18), 44.73 (C19), 43.94 (C17), 42.42
(C14), 42.27 (C35), 41.16 (C8), 40.31 (C32), 39.29 (C21), 38.52 (C33), 38.12 (C13), 37.06 (C4),
36.79 (C1), 36.42 (C10), 34.49 (C16), 34.22 (C7), 33.44 (C46), 32.47 (C20), 29.82 (C15), 28.11
(C12), 27.72 (C2), 27.14 (C23), 24.13 (C11), 22.68 (C24), 21.75 (C30), 18.26 (C28), 17.81 (C6),
16.42 (C25), 16.12 (C26), 14.87 (C27). HRMS (ESI) m/z: calcd for C46 H65 N3 O5 NaS [M+Na]+
794.4543, found 794.4537.
Molecules 2024, 29, 4957 20 of 25
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(4-Tert-Butylphenyl)-2,4-Thiazolidinedione (12d)
(s, 1H, CH2 =CH2 ), 7.50–7.43 (m, 4H, Ar-H), 7.23–7.15 (m, 1H, NH), 5.26 (d, J = 7.0 Hz, 2H,
CH2 =CH2 ), 4.40 (s, 2H, CH2 ), 4.33 (p, J = 6.2, 5.1 Hz, 1H, CH), 3.47–3.26 (m, 4H, CH2 ×2, 1H,
NH), 2.02 (tt, J = 12.8, 6.5 Hz, 1H), 1.75 (s, 1H), 1.71–1.66 (m, 3H), 1.63 (s, 3H, CH3 ), 1.57–1.54
(m, 1H), 1.49 (ddd, J = 15.0, 10.6, 6.1 Hz, 3H), 1.44–1.37 (m, 5H), 1.33 (s, 9H, CH3 ×3), 1.29
(d, J = 4.1 Hz, 1H), 1.25 (s, 2H), 1.23–1.18 (m, 2H), 1.15 (s, 1H), 1.04 (d, J = 4.8 Hz, 2H), 0.99
(dd, J = 10.9, 4.4 Hz, 4H), 0.94 (d, J = 6.8 Hz, 3H), 0.89–0.83 (m, 9H, CH3 ×3), 0.79 (s, 3H,
CH3 ), 0.72 (d, J = 20.0 Hz, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 168.05 (C34),
166.16 (C36), 163.17 (C38), 158.40 (C43), 154.65 (C31), 139.93 (C22), 134.68 (C39), 130.39 (C41,
C45), 126.39 (C42, C44), 120.13 (C40), 119.00 (C20), 115.76 (C29), 82.28 (C3), 55.55 (C37),
50.41 (C5), 48.78 (C9), 44.73 (C18), 43.88 (C17), 42.42 (C14), 41.15 (C35), 40.31 (C8), 39.29
(C32), 38.13 (C46), 37.07 (C33), 36.78 (C13), 36.41 (C4), 35.16 (C1), 34.47 (C10), 34.23 (C19),
33.42 (C7), 32.46 (C16), 31.16 (C47, C48, C49), 29.80 (C21), 28.08 (C15), 27.72 (C12), 27.12
(C2), 24.19 (C23), 22.66 (C11), 21.74 (C24), 18.25 (C30), 17.80 (C28),16.65 (C6), 16.42 (C25),
16.12 (C26), 14.87 (C27). HRMS (ESI) m/z: calcd for C49 H71 N3 O5 NaS [M+Na]+ 836.5012,
found 836.5015.
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(2-Methoxybenzylidene)-2,4-Thiazolidinedione (12e)
δ 8.28 (s, 1H, CH2 =CH2 ), 7.45 (d, J = 7.7 Hz, 1H, Ar-H), 7.41 (t, J = 7.9 Hz, 1H, Ar-H), 7.04 (t,
J = 7.5 Hz, 1H, Ar-H), 6.99 (s, 1H, NH), 6.94 (d, J = 8.3 Hz, 1H, Ar-H), 5.26 (d, J = 7.0 Hz, 1H,
CH2 =CH2 ), 5.13 (d, J = 6.4 Hz, 1H, CH2 =CH2 ), 4.38 (s, 2H, CH2 ), 4.33–4.28 (m, 1H, CH),
3.90 (s, 3H, OCH3 ), 3.44–3.27 (m, 4H, CH2 ×2, 1H, NH), 1.76–1.71 (m, 3H), 1.70–1.65 (m,
3H), 1.64 (s, 3H, CH3 ), 1.58 (dd, J = 14.3, 8.2 Hz, 3H), 1.51–1.44 (m, 3H), 1.40–1.30 (m, 5H),
1.23–1.19 (m, 3H), 1.02 (d, J = 5.5 Hz, 3H), 1.00 (d, J = 6.1 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H,
CH3 ), 0.90–0.83 (m, 9H, CH3 ×3), 0.78 (s, 3H, CH3 ), 0.73 (s, 3H, CH3 ). 13 C NMR (150 MHz,
(C22), 132.56 (C39), 130.53 (C45), 129.57 (C41), 122.48 (C40), 121.35 (C37), 121.00 (C42),
119.03 (C44), 111.28 (C29), 82.27 (C3), 55.64 (C5), 55.54 (C46), 50.39 (C9), 48.81 (C18), 43.95
(C17), 42.44 (C14), 42.29 (C35), 41.83 (C8), 40.37 (C21), 39.30 (C20), 38.49 (C13), 38.12 (C1),
37.06 (C4), 36.81 (C10), 36.43 (C19), 34.50 (C16), 34.24 (C7), 28.12 (C15), 27.74 (C12), 27.14
(C32), 24.13 (C2), 22.69 (C23), 21.76 (C24), 21.71 (C11), 18.27 (C30), 17.83 (C28), 16.66 (C6),
16.42 (C25), 16.14 (C26), 14.92 (C27). HRMS (ESI) m/z: calcd for C46 H65 N3 O6 NaS [M+Na]+
810.4492, found 810.4501.
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(2,3,4-Trimethoxybenzylidene)-2,4-
Thiazolidinedione (12f)
(s, 1H, CH2 =CH2 ), 7.20 (d, J = 8.7 Hz, 1H, Ar-H), 6.84 (s, 1H, NH), 6.77 (d, J = 8.7 Hz, 1H,
Ar-H), 5.26 (d, J = 6.8 Hz, 1H, CH2 =CH2 ), 5.10 (s, 1H, CH2 =CH2 ), 4.38 (s, 2H, CH2 ), 4.34 (d,
J = 7.3 Hz, 1H, CH), 3.96–3.91 (m, 6H, OCH3 ), 3.88 (s, 3H, OCH3 ), 3.58–3.14 (m, 4H, CH2 ×2,
1H, NH), 1.77 (dd, J = 13.5, 4.8 Hz, 1H), 1.73 (d, J = 2.7 Hz, 1H), 1.69 (d, J = 13.1 Hz, 2H),
1.63 (s, 3H, CH3 ), 1.60 (s, 1H), 1.56 (d, J = 6.8 Hz, 1H), 1.50 (s, 2H), 1.43–1.34 (m, 4H), 1.30
(td, J = 13.4, 4.0 Hz, 3H), 1.25 (s, 3H), 1.21 (d, J = 15.7 Hz, 2H), 1.06 (d, J = 5.6 Hz, 1H), 1.03
(s, 3H), 1.00 (dd, J = 12.1, 6.5 Hz, 5H), 0.94 (s, 3H, CH3 ), 0.87 (d, J = 16.3 Hz, 6H, CH3 ×2),
0.79 (s, 3H, CH3 ), 0.73 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 168.40 (C34),
166.17 (C36), 165.91 (C38), 158.34 (C43), 156.46 (C31), 154.05 (C22), 142.48 (C39), 139.98
(C45), 130.08 (C41), 124.79 (C40), 120.47 (C37), 119.48 (C42), 119.02 (C44), 107.68 (C29), 82.22
(C3), 62.03 (C46), 61.08 (C47), 56.28 (C48), 55.57 (C5), 50.42 (C9), 48.81 (C18), 43.92 (C19),
42.44 (C17), 42.28 (C14), 41.19 (C35), 40.37 (C8), 39.31 (C32), 38.56 (C21), 38.16 (C33), 37.09
(C13), 36.80 (C4), 36.43 (C1), 34.50 (C10), 34.25 (C16), 32.48 (C7), 29.82 (C20), 28.12 (C15),
27.73 (C12), 27.15 (C2), 24.17 (C23), 22.66 (C11), 21.76 (C24), 18.28 (C30), 17.83 (C28), 16.67
Molecules 2024, 29, 4957 21 of 25
[M+Na]+ 870.4703, found 870.4707.
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(2-Furanylmethylene)-2,4-Thiazolidinedione (12g)
Light yellow solid, yield: 42%; mp: 187.8–189.2 ◦ C. 1 H NMR (600 MHz, chloroform-d)
δ 8.07 (d, J = 8.8 Hz, 1H, CH2 =CH2 ), 7.67 (s, 1H, NH), 7.26–7.13 (m, 1H, CH2 =CH2 ), 6.88
(d, J = 8.7 Hz, 1H, CH2 =CH2 ), 6.80 (d, J = 3.7 Hz, 1H, CH2 =CH2 ), 6.58 (d, J = 3.5 Hz, 1H,
CH2 =CH2 ), 5.27–5.24 (m, 1H, CH2 =CH2 ), 4.39 (s, 2H, CH2 ), 4.30 (tt, J = 10.9, 5.2 Hz, 1H,
CH), 3.49–3.18 (m, 4H, CH2 ×2, 1H, NH), 2.09–1.91 (m, 1H), 1.69 (d, J = 12.5 Hz, 3H), 1.63
(s, 3H, CH3 ), 1.61–1.53 (m, 3H), 1.52–1.47 (m, 2H), 1.42 (dd, J = 9.3, 5.2 Hz, 2H), 1.37 (d,
J = 9.6 Hz, 2H), 1.31 (d, J = 8.5 Hz, 1H), 1.25 (s, 4H), 1.16 (s, 1H), 1.04 (d, J = 5.0 Hz, 3H), 0.99
(dd, J = 10.6, 3.9 Hz, 4H), 0.95 (d, J = 4.5 Hz, 3H, CH3 ), 0.89–0.83 (m, 9H, CH3 ×3), 0.78 (s,
3H, CH3 ), 0.72 (d, J = 18.2 Hz, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ 168.82 (C34),
166.43 (C36), 165.82 (C38), 163.09 (C40), 149.75 (C31), 146.79 (C22), 140.90 (C39), 126.19
(C43), 120.34 (C37), 118.37 (C41), 115.79 (C42), 113.41 (C29), 82.44 (C3), 55.53 (C5), 50.70
(C9), 50.40 (C18), 48.80 (C19), 44.76 (C17), 43.82 (C14), 42.44 (C35), 42.27 (C8), 41.78 (C32),
41.17 (C21), 40.32 (C33), 39.29 (C13), 38.48 (C4), 38.11 (C1), 37.25 (C10), 37.07 (C15), 36.78
(C16), 36.41 (C7), 34.48 (C20), 32.47 (C12), 29.81 (C2), 28.11 (C23), 24.14 (C11), 22.67 (C6),
calcd for C43 H61 N3 O6 NaS [M+Na]+ 770.4179, found 770.4187.
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(2-Thienylmethylene)-2,4-Thiazolidinedione (12h)
Light yellow solid, yield: 47%; mp: 193.5–195.3 ◦ C. 1 H NMR (600 MHz, chloroform-
d) δ 8.09 (s, 1H, CH2 =CH2 ), 7.66 (d, J = 5.0 Hz, 1H, CH2 =CH2 ), 7.42 (d, J = 3.9 Hz, 1H,
CH2 =CH2 ), 7.19 (q, J = 4.5 Hz, 1H, CH2 =CH2 ), 7.15–7.07 (m, 1H, NH), 5.27 (d, J = 6.9 Hz,
1H, CH2 =CH2 ), 5.13 (t, J = 6.3 Hz, 1H, CH2 =CH2 ), 4.38 (s, 2H, CH2 ), 4.29 (dd, J = 11.7,
4.5 Hz, 1H, CH), 3.45–3.26 (m, 4H, CH2 ×2, 1H, NH), 1.78–1.71 (m, 3H, CH3 ), 1.68 (d,
J = 12.1 Hz, 2H), 1.64 (s, 3H, CH3 ), 1.61 (s, 1H), 1.56 (dd, J = 16.4, 7.2 Hz, 2H), 1.49 (q,
J = 7.9 Hz, 3H), 1.41 (d, J = 4.7 Hz, 1H), 1.36 (d, J = 9.6 Hz, 2H), 1.33–1.30 (m, 2H), 1.26
(d, J = 9.8 Hz, 5H), 1.04 (s, 2H), 1.01–0.99 (m, 3H), 0.96 (s, 3H, CH3 ), 0.86 (d, J = 12.9 Hz,
9H, CH3 ×3), 0.78 (s, 3H, CH3 ), 0.74 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ
167.32 (C36), 165.78 (C34), 158.55 (C38), 139.97 (C40), 137.71 (C31), 133.75 (C22), 132.26
(C39), 129.90 (C43), 128.80 (C37), 127.08 (C41), 119.33 (C42), 119.03 (C29), 82.28 (C3), 55.53
(C5), 50.43 (C9), 48.82 (C18), 44.15 (C19), 42.46 (C17), 42.28 (C14), 41.95 (C35), 41.18 (C8),
40.34 (C32), 39.31 (C21), 38.52 (C33), 38.11 (C13), 37.08 (C4), 36.80 (C1), 36.43 (C10), 34.50
(C15), 34.25 (C16), 29.82 (C7), 28.11 (C20), 27.77 (C12), 27.15 (C2), 24.15 (C23), 22.70 (C11),
21.76 (C6), 18.26 (C24), 17.83 (C30), 16.67 (C28), 16.43 (C25), 16.09 (C26), 14.97 (C27). HRMS
(ESI) m/z: calcd for C43 H61 N3 O5 NaS2 [M+Na]+ 786.3950, found 786.3953.
Lupeol-3-[(1-Ethylenediaminyl)-4]-5-(2-Methylpropylidene)-2,4-Thiazolidinedione (12i)
7.03–7.00 (m, 1H, NH), 6.96 (d, J = 9.7 Hz, 1H, CH2 =CH2 ), 5.31–5.25 (m, 1H, CH2 =CH2 ),
5.22 (t, J = 6.2 Hz, 1H, CH2 =CH2 ), 4.34 (s, 1H, CH), 4.32 (s, 2H, CH2 ), 3.43–3.26 (m, 4H,
CH2 ×2, 1H, NH), 1.77–1.73 (m, 2H), 1.71–1.66 (m, 3H), 1.63 (s, 3H, CH3 ), 1.55 (dd, J = 16.0,
7.2 Hz, 3H, CH3 ), 1.40–1.37 (m, 3H, CH3 ), 1.31 (s, 1H), 1.30–1.30 (m, 2H), 1.29 (s, 2H), 1.27
(s, 6H), 1.05 (s, 3H), 0.99 (s, 2H), 0.95 (s, 2H), 0.89 (s, 3H, CH3 ), 0.88 (s, 6H, CH3 ×2), 0.87
(s, 3H, CH3 ), 0.80 (s, 3H, CH3 ), 0.74 (s, 3H, CH3 ). 13 C NMR (150 MHz, chloroform-d) δ
167.76 (C34), 165.89 (C36), 165.05 (C38), 158.30 (C31), 145.30 (C22), 139.95 (C39), 123.00
(C37), 119.01 (C29), 82.13 (C3), 55.59 (C5), 50.47 (C9), 48.80 (C18), 43.68 (C19), 42.44 (C17),
42.28 (C1), 41.49 (C10), 41.19 (C8), 40.38 (C32), 39.31 (C21), 38.61 (C33), 38.16 (C13), 37.10
(C4), 36.80 (C1), 36.43 (C10), 34.49 (C15), 34.27 (C16), 32.06 (C40), 29.82 (C7), 28.10 (C20),
27.72 (C12), 27.14 (C2), 24.18 (C23), 22.65 (C11), 21.73 (C6), 21.39 (C41, C42), 18.28 (C24),
C42 H65 N3 O5 NaS [M+Na]+ 746.4543, found 746.4545.
Molecules 2024, 29, 4957 22 of 25
3.2. Cell Culture

The human cancer cell lines HepG2, A549, and MCF-7 were purchased from the cell
bank of the Chinese Academy of Sciences (Shanghai, China). The human hepatic cell line
LO2 was obtained from iCell Bioscience Inc. (Shanghai, China). These cell lines were
cultured in Dulbecco’s Modified Eagle Medium containing 10% (v/v) fetal bovine serum
(FBS, Wisent) and 1% (v/v) Penicillin–Streptomycin Solution mixture (Beyotime, Shanghai,
China) at 37 ◦ C under 5% CO2 .
3.3. In Vitro Antiproliferative Assay

An MTT assay evaluated the cytotoxicity of lupeol derivatives toward the A549, MCF-7,
HepG2, and LO2 cell lines. The cells were inoculated into 96-well plates (3 × 103 cells/well)
and cultured for 24 h at 37 ◦ C in 5% CO2 . After that, a gradient concentration of the test drug
was added to continue the incubation for 48 h. Subsequently, the MTT solution continued to
incubate with the cells for 4 h. Afterward, the upper waste layer was discarded, and DMSO
was added (Kermel, Tianjin, China). After the formazan completely dissolved, the absorbance
of each well at 490 nm was measured with a microplate reader. The mean values were derived
from three independent experiments.
3.4. Apoptosis Analysis Using Flow Cytometry

HepG2 cells were inoculated into 6-well plates for 24 h, attached via cell adhesion, and
then treated with 12i (0, 2, 4, and 8 µM) for 48 h. The cells were harvested and treated with
Annexin V-FITC/PI staining solution, and the data were gathered using a flow cytometer
(FACS Calibur, BD, Milpitas, CA, USA).
3.5. Analysis of Apoptosis Using Confocal Microscopy

HepG2 cells were seeded in 24-well laser confocal plates for 24 h. Then, gradient
concentrations (0, 2, 4, and 8 µM) of 12i were added and co-incubated for 48 h. The
supernatant was then discarded, the cells were washed using PBS, and 5 µL each of AO and
EB dyes was added at 25 ◦ C for 20 min. Then, the dyes were removed, and PBS was added.
Finally, the samples were collected and imaged using confocal microscopy (LSM710, Zeiss,
Oberkochen, Germany).
3.6. Mitochondrial Membrane Potential Analysis Using Flow Cytometry

HepG2 cells were inoculated into 6-well plates for 24 h, attached via cell adhesion,
and then treated with 12i (0, 2, 4, and 8 µM) for 48 h. The cells were cleaned with PBS
and treated with JC-1 solution at 25 ◦ C for 20 min, and the data were gathered using a
flow cytometer.
3.7. Mitochondrial Membrane Potential Analysis Using Confocal Microscopy

and then treated with 12i (0, 2, 4, and 8 µM) for 48 h. The cells were cleaned with PBS and
treated with JC-1 solution at 25 ◦ C for 20 min, and the samples were collected and imaged
using confocal microscopy.
3.8. ROS Content Detection Using Flow Cytometry

and treated with 12i (0, 2, 4, and 8 µM) for 48 h. After the cells were washed with PBS, the
supernatant was removed and incubated with 500 µL of 10 µm/L DCFH-DA solution at
25 ◦ C for 20 min, and the data were gathered using a flow cytometer.
3.9. Measurement of ROS Using Confocal Microscopy

and treated with 12i (0, 2, 4, and 8 µM) for 48 h. After the cells were washed with PBS, the
Molecules 2024, 29, 4957 23 of 25
supernatant was removed and incubated with 500 µL of 10 µm/L DCFH-DA solution at
25 ◦ C for 20 min, and the samples were collected and imaged using confocal microscopy.
3.10. Western Blot Analysis

HepG2 cells were seeded in 6-well plates at a density of 1 × 106 overnight and treated
with 12i (2, 4, and 8 µM) for 48 h. They were then washed twice with cooled PBS to
remove the medium and lysed using a lysis buffer containing RIPA (Beyotime, Shanghai,
China). The protein levels were quantified using a bicinchoninic acid protein assay kit
(Beyotime, Shanghai, China). The proteins were separated via SDS-PAGE electrophoresis
and transferred to PVDF membranes (Beyotime, Shanghai, China). After blocking with 5%
skim milk for 2 h, the membranes were incubated with the primary antibody overnight at
4 ◦ C, followed by 2 h with the specific secondary antibody. The antibodies were sourced
commercially, including Bax (5023T, Cell Signaling Technology, Danvers, MA, USA), Bcl2
(3498T, Cell Signaling Technology, Danvers, MA, USA), Caspase-7 (12827T, Cell Signaling
Technology, Danvers, MA, USA), Caspase-9 (9508T, Cell Signaling Technology, Danvers,
MA, USA), Cleaved-Caspase-7 (8438T, Cell Signaling Technology, Danvers, MA, USA),
Cleaved-Caspase-9 (7237T, Cell Signaling Technology, Danvers, MA, USA), GAPDH (2118T,
Cell Signaling Technology, Danvers, MA, USA), PI3K (4249T, Cell Signaling Technology,
Danvers, MA, USA), p-PI3K (4228T, Cell Signaling Technology, Danvers, MA, USA), AKT
(4691T, Cell Signaling Technology, Danvers, MA, USA), p-AKT (4060T, Cell Signaling
Technology, Danvers, MA, USA), mTOR (2972T, Cell Signaling Technology, Danvers, MA,
USA). Immunoreactive bands were visualized using BeyoECL Plus (Beyotime, Shanghai,
China) and scanned in a ChemiDoc MP Imaging System (Bio-Rad, Shanghai, China).
4. Conclusions
In conclusion, we successfully designed and synthesized a series of novel lupeol-3-
thiazolidinedione derivatives by altering the linker between lupeol and thiazolidinedione.
An MTT assay was conducted to evaluate the in vitro antiproliferative activity of all com-
pounds against three cancer cell lines and a normal cell line. We successfully selected
compound 12i, which exhibited strong antitumor effects in HepG2 cells, with an IC50 of
4.40 µM that was 9.9-fold higher than that of the parent lupeol. According to the results of
the in vitro antitumor activity test, the IC50 value of compound 12i is marginally higher
than that of the marketed drug cisplatin. In addition, compound 12i was weakly cytotoxic
to human hepatic cell LO2 and, to some extent, selective for cancer cells. Mechanistic
studies demonstrated that compound 12i promotes ROS production, decreases mitochon-
drial membrane potential, and induces HepG2 cell apoptosis through the mitochondria
pathway. Thus, compound 12i provides a lead structure for the development of novel
anticancer drugs.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules29204957/s1. Supplementary File: HRMS, 1 H NMR,
and 13 C NMR spectral data of all derivatives.
Author Contributions: Conceptualization, M.B. and M.W.; methodology, M.B., M.W. and X.H.;
chemistry experiments, S.D. and X.G.; biological experiments, Y.Z.; data curation, S.D. and Y.Z.;
formal analysis, Y.W., G.L. and Q.L.; writing-original draft preparation, S.D.; writing-review and
editing, M.B. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Fundamental Research Funds for the Education Depart-
ment of Heilongjiang Province (No. 2020-KYYWF-0024).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article and Supplementary Material, further inquiries can be directed to the corresponding authors.
Molecules 2024, 29, 4957 24 of 25
Conflicts of Interest: The authors reported no potential conflicts of interest.
References
1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA. Cancer. J. Clin. 2021, 71, 209–249. [CrossRef]
[PubMed]
2. Siegel, R.L.; Miller, K.D.; Wagle, N.S.; Jemal, A. Cancer statistics, 2023. CA. Cancer. J. Clin. 2023, 73, 17–48. [CrossRef] [PubMed]
3. Fujita, S.; Kotake, K. Chemotherapy. Nihon. Rinsho. 2014, 72, 102–107. [PubMed]
4. Liu, S.; Khan, A.R.; Yang, X.; Dong, B.; Ji, J.; Zhai, G. The reversal of chemotherapy-induced multidrug resistance by nanomedicine
for cancer therapy. J. Control. Release. 2021, 335, 1–20. [CrossRef]
5. Mir, R.H.; Mir, P.A.; Uppal, J.; Chawla, A.; Patel, M.; Bardakci, F.; Adnan, M.; Mohi-Ud-Din, R. Evolution of Natural Product
Scaffolds as Potential Proteasome Inhibitors in Developing Cancer Therapeutics. Metabolites 2023, 13, 509. [CrossRef]
6. Naeem, A.; Hu, P.; Yang, M.; Zhang, J.; Liu, Y.; Zhu, W.; Zheng, Q. Natural Products as Anticancer Agents: Current Status and
Future Perspectives. Molecules 2022, 27, 8367. [CrossRef]
7. Wang, X.; Zhuang, Y.; Wang, Y.; Jiang, M.; Yao, L. The recent developments of camptothecin and its derivatives as potential
anti-tumor agents. Eur. J. Med. Chem. 2023, 260, 115710. [CrossRef]
8. Song, Z.; Lu, Q.; Tao, A.; Wu, T. Synthesis and Anti-cancer Activity of Paclitaxel-Coumarin Conjugate. Curr. Org. Synth. 2021,
18, 587–591. [CrossRef]
9. Xiao, J.; Gao, M.; Sun, Z.; Diao, Q.; Wang, P.; Gao, F. Recent advances of podophyllotoxin/epipodophyllotoxin hybrids in
anticancer activity, mode of action, and structure-activity relationship: An update (2010–2020). Eur. J. Med. Chem. 2020, 208,
112830. [CrossRef]
10. Imam, S.; Azhar, I.; Hasan, M.M.; Ali, M.S.; Ahmed, S.W. Two triterpenes lupanone and lupeol isolated and identified from
Tamarindus indica linn. Pak. J. Pharm. Sci. 2007, 20, 125–127.
11. You, Y.J.; Nam, N.H.; Kim, Y.; Bae, K.H.; Ahn, B.Z. Antiangiogenic activity of lupeol from Bombax ceiba. Phytother. Res. 2003,
17, 341–344. [CrossRef] [PubMed]
12. Fernández, M.A.; de las Heras, B.; García, M.D.; Sáenz, M.T.; Villar, A. New insights into the mechanism of action of the
anti-inflammatory triterpene lupeol. J. Pharm. Pharmacol. 2001, 53, 1533–1539. [CrossRef] [PubMed]
13. Molnár, J.; Gyémánt, N.; Tanaka, M.; Hohmann, J.; Bergmann-Leitner, E.; Molnár, P.; Deli, J.; Didiziapetris, R.; Ferreira, M.J.
Inhibition of multidrug resistance of cancer cells by natural diterpenes, triterpenes and carotenoids. Curr. Pharm. Des. 2006,
12, 287–311. [CrossRef] [PubMed]
14. Zhang, L.; Zhang, Y.; Zhang, L.; Yang, X.; Lv, Z. Lupeol, a dietary triterpene, inhibited growth, and induced apoptosis through
down-regulation of DR3 in SMMC7721 cells. Cancer. Invest. 2009, 27, 163–170. [CrossRef]
15. Liu, K.; Zhang, X.; Xie, L.; Deng, M.; Chen, H.; Song, J.; Long, J.; Li, X.; Luo, J. Lupeol and its derivatives as anticancer and
anti-inflammatory agents: Molecular mechanisms and therapeutic efficacy. Pharmacol. Res. 2021, 164, 105373. [CrossRef]
16. Najid, A.; Simon, A.; Cook, J.; Chable-Rabinovitch, H.; Delage, C.; Chulia, A.J.; Rigaud, M. Characterization of ursolic acid as a
lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells. FEBS. Lett. 1992,
299, 213–217. [CrossRef]
17. Nagumo, A.; Takanashi, K.; Hojo, H.; Suzuki, Y. Cytotoxicity of bacteriohopane-32-ol against mouse leukemia L1210 and P388
cells in vitro. Toxicol. Lett. 1991, 58, 309–313. [CrossRef]
18. Min, T.R.; Park, H.J.; Ha, K.T.; Chi, G.Y.; Choi, Y.H.; Park, S.H. Suppression of EGFR/STAT3 activity by lupeol contributes to the
induction of the apoptosis of human non-small cell lung cancer cells. Int. J. Oncol. 2019, 55, 320–330. [CrossRef]
19. Bhattacharyya, S.; Mitra, D.; Ray, S.; Biswas, N.; Banerjee, S.; Majumder, B.; Mustafi, S.M.; Murmu, N. Reversing effect of Lupeol
on vasculogenic mimicry in murine melanoma progression. Microvasc. Res. 2019, 121, 52–62. [CrossRef]
20. Zhang, X.; Gao, Z.; Chen, K.; Zhuo, Q.; Chen, M.; Wang, J.; Lai, X.; Wang, L. Lupeol inhibits the proliferation and migration
of MDA-MB-231 breast cancer cells via a novel crosstalk mechanism between autophagy and the EMT. Food. Funct. 2022,
13, 4967–4976. [CrossRef]
21. Fatma, H.; Jameel, M.; Siddiqui, A.J.; Kuddus, M.; Buali, N.S.; Bahrini, I.; Siddique, H.R. Chemotherapeutic potential of lupeol
against cancer in pre-clinical model: A systematic review and meta-analysis. Phytomedicine 2024, 132, 155777. [CrossRef]
[PubMed]
22. Nigam, N.; Prasad, S.; Shukla, Y. Preventive effects of lupeol on DMBA induced DNA alkylation damage in mouse skin. Food.
Chem. Toxicol. 2007, 45, 2331–2335. [CrossRef] [PubMed]
23. Bu, M.; Wang, L.; Luo, R.; Xu, T.; Lin, Y.; Ge, P.; Liu, J. Lupinol-3β-succinate induce apoptosis of HepG2 cells by regulating the
mitochondrial apoptosis pathway. Drugs Clin. Drugs Clin. 2024, 39, 549–556.
24. Tian, S.; Zhao, Y.; Deng, S.; Hou, L.; Song, J.; Wang, M.; Bu, M. Lupeol-3-carbamate Derivatives: Synthesis and Biological
Evaluation as Potential Antitumor Agents. Molecules 2024, 29, 3990. [CrossRef]
25. Ortiz, A.; Sansinenea, E. Synthetic thiazolidinediones: Potential antidiabetic compounds. Curr. Org. Chem. 2011, 15, 108–127.
[CrossRef]
26. Kaur Manjal, S.; Kaur, R.; Bhatia, R.; Kumar, K.; Singh, V.; Shankar, R.; Kaur, R.; Rawal, R.K. Synthetic and medicinal perspective
of thiazolidinones: A review. Bioorg. Chem. 2017, 75, 406–423. [CrossRef]
Molecules 2024, 29, 4957 25 of 25
27. Mansure, J.J.; Nassim, R.; Kassouf, W. Peroxisome proliferator-activated receptor gamma in bladder cancer: A promising
therapeutic target. Cancer. Biol. Ther. 2009, 8, 6–15. [CrossRef]
28. Fu, X.; Mao, Q.; Zhang, B.; Lv, J.; Ping, K.; Zhang, P.; Lin, F.; Zhao, J.; Feng, Y.; Yang, J.; et al. Thiazolidinedione-Based Structure
Modification of Celastrol Provides Thiazolidinedione-Conjugated Derivatives as Potent Agents against Non-Small-Cell Lung
Cancer Cells through a Mitochondria-Mediated Apoptotic Pathway. J. Nat. Prod. 2022, 85, 1147–1156. [CrossRef]
29. Knight, S.D.; Adams, N.D.; Burgess, J.L.; Chaudhar, A.M.; Darcy, M.G.; Donatelli, C.A.; Luengo, J.I.; Newlander, K.A.; Parrish,
C.A.; Ridgers, L.H.; et al. Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin.
ACS. Med. Chem. Lett. 2010, 1, 39–43. [CrossRef]
30. Liu, K.; Rao, W.; Parikh, H.; Li, Q.; Guo, T.L.; Grant, S.; Kellogg, G.E.; Zhang, S. 3,5-Disubstituted-thiazolidine-2,4-dione analogs
as anticancer agents: Design, synthesis and biological characterization. Eur. J. Med. Chem. 2012, 47, 125–137. [CrossRef]
31. Sun, J.; Chen, Y.H.; Liu, H.Y.; Hondo, E.; Zhou, Y.; Wu, Y.F. Thiazolidinedione: A Privileged Scaffold for the Development of
Anticancer Agents. Curr. Top. Med. Chem. 2021, 21, 2529–2545. [CrossRef] [PubMed]
32. Sethi, N.S.; Prasad, D.N.; Singh, R.K. An Insight into the Synthesis and SAR of 2,4-Thiazolidinediones (2,4-TZD) as Multifunctional
Scaffold: A Review. Mini. Rev. Med. Chem. 2020, 20, 308–330. [CrossRef] [PubMed]
33. Wu, S.; Zhang, Y.; He, X.; Che, X.; Wang, S.; Liu, Y.; Jiang, Y.; Liu, N.; Dong, G.; Yao, J.; et al. From antidiabetic to antifungal:
Discovery of highly potent triazole-thiazolidinedione hybrids as novel antifungal agents. Chem. Med. Chem. 2014, 9, 2639–2646.
[CrossRef] [PubMed]
34. Sahoo, B.M.; Banik, B.K.; Borah, P.; Jain, A. Reactive Oxygen Species (ROS): Key Components in Cancer Therapies. Anticancer.
Agents. Med. Chem. 2022, 22, 215–222. [CrossRef]
35. Jin, X.Y.; Chen, H.; Li, D.D.; Li, A.L.; Wang, W.Y.; Gu, W. Design, synthesis, and anticancer evaluation of novel quinoline
derivatives of ursolic acid with hydrazide, oxadiazole, and thiadiazole moieties as potent MEK inhibitors. J. Enzyme. Inhib. Med.
Chem. 2019, 34, 955–972. [CrossRef]
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Molecules 2024, 29, 4957. https://doi.org/10.3390/molecules29204957 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 4957 2 of 25

Based on the preliminary conformational relationships, we found that the introduction

compounds 6a–b, 9a–b, and 12a–b, containing electron-withdrawing groups such as -F

2.2.2. Compound 12i Induced Apoptosis in HepG2 Cells

2.2.3. Effects of 12i on Reactive Oxygen Species (ROS) Generation

fluorescence. As shown in Figure 4A, the compound 12i-treated experimental groups

2.2.7. Compound 12i Is Predicted to Bind to PI3K

2.2.7.InCompound 12i Is Predicted

3.1.3. Synthesis of Intermediates 4a–i

3.1.3. Synthesis of Intermediates 4a–i

3.1.4. Synthesis of Intermediates 5a–i

3.1.5. Synthesis of Compounds 6a–i

3.1.6. Synthesis of Lupeol-3-(4-Nitrobenzoate) (7)

3.1.7. Synthesis of Lupeol-3-Piperazinecarboxylate (8)

3.1.8. Synthesis of Compounds 9a–i

3.1.9. Synthesis of Lupeol-3-[(1-Ethylenediaminyl)-4]-Tert-Butoxycarbonyl (10)

3.1.10. Synthesis of Intermediate 11

3.1.11. Synthesis of Compounds 12a–i

3.2. Cell Culture

3.3. In Vitro Antiproliferative Assay

3.4. Apoptosis Analysis Using Flow Cytometry

3.5. Analysis of Apoptosis Using Confocal Microscopy

3.6. Mitochondrial Membrane Potential Analysis Using Flow Cytometry

3.7. Mitochondrial Membrane Potential Analysis Using Confocal Microscopy

3.8. ROS Content Detection Using Flow Cytometry

3.9. Measurement of ROS Using Confocal Microscopy

3.10. Western Blot Analysis

Conflicts of Interest: The authors reported no potential conflicts of interest.

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