Novel enmein-type diterpenoid hybrids coupled with nitrogen mustards: Synthesis of promising candidates for anticancer therapeutics

Article abstract of DOI:10.1016/j.ejmech.2018.01.069

Natural derived enmein-type diterpenoids exert cytotoxicity against a wide range of human cancer cells. Yet their medicinal applications are hindered by insufficient potency for chemotherapy. Hence, a series of novel enmein-type diterpenoid hybrids coupled with nitrogen mustards were designed and synthesized to increase antitumor efficacy while reducing systemic toxicity. Most conjugates exhibited stronger antiproliferative activities than parent diterpenoids and nitrogen mustards, especially for multidrug-resistant tumor cell line Bel-7402/5-FU. Among them, compound E2 showed the most potent inhibitory activities in human leukemia HL-60 cells, human prostate cancer PC-3 cells, human liver cancer Bel-7402 cells and drug-resistant human liver cancer Bel-7402/5-FU cells with IC₅₀ values of 7.83 μM, 3.97 μM, 0.77 μM and 2.07 μM, respectively. Additionally, high selectivity with selectivity index over 130 was also observed from cytotoxic evaluation between L-02 human normal liver cells and Bel-7402 malignant liver cells. Further studies on mechanism of action indicated that E2 induced both apoptosis and G1 phase cell cycle arrest in Bel-7402 hepatoma cells. Moreover, the dysfunction in mitochondrial pathway was also involved in E2 initiated apoptotic activation, which entailed the loss of mitochondrial membrane potential followed by upregulating the bax/bcl-2 ratio and increasing the expression of cytochrome c, p53, caspase-3 and -9. Overall, E2 has the potential to emerge as a promising drug candidate for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2018.01.069

European Journal of Medicinal Chemistry 146 (2018) 588e598
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Novel enmein-type diterpenoid hybrids coupled with nitrogen
mustards: Synthesis of promising candidates for anticancer
therapeutics
Xiang Gao a, Jia Li a, Mingying Wang a, Shengtao Xu b, Weiwei Liu c, Linghe Zang d,
,
, **
Zhanlin Li a, Huiming Hua a *, Jinyi Xu b, Dahong Li a
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
b Department of Medicinal Chemistry and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009,
PR China
c Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
d School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural derived enmein-type diterpenoids exert cytotoxicity against a wide range of human cancer cells.
Yet their medicinal applications are hindered by insufficient potency for chemotherapy. Hence, a series of
novel enmein-type diterpenoid hybrids coupled with nitrogen mustards were designed and synthesized
to increase antitumor efficacy while reducing systemic toxicity. Most conjugates exhibited stronger
antiproliferative activities than parent diterpenoids and nitrogen mustards, especially for multidrug-
resistant tumor cell line Bel-7402/5-FU. Among them, compound E2 showed the most potent inhibi-
tory activities in human leukemia HL-60 cells, human prostate cancer PC-3 cells, human liver cancer Bel-
Received 25 November 2017
Received in revised form
18 January 2018
Accepted 19 January 2018
Keywords:
7402 cells and drug-resistant human liver cancer Bel-7402/5-FU cells with IC50 values of 7.83
mM,
6,7-Seco-ent-kaurane diterpenoid
Nitrogen mustard
Hybrid
Antiproliferative activity
Selectivity
3.97 M, 0.77 M and 2.07 M, respectively. Additionally, high selectivity with selectivity index over 130
m
m
m
was also observed from cytotoxic evaluation between L-02 human normal liver cells and Bel-7402
malignant liver cells. Further studies on mechanism of action indicated that E2 induced both
apoptosis and G1 phase cell cycle arrest in Bel-7402 hepatoma cells. Moreover, the dysfunction in
mitochondrial pathway was also involved in E2 initiated apoptotic activation, which entailed the loss of
mitochondrial membrane potential followed by upregulating the bax/bcl-2 ratio and increasing the
expression of cytochrome c, p53, caspase-3 and -9. Overall, E2 has the potential to emerge as a promising
drug candidate for cancer therapy.
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
chemotherapy [3]. Therefore, consistent explorations in search of
efficacious and safe antitumor candidates for potential clinical ap-
Cancer claimed the lives of 8.8 million around the world in 2015
alone [1]. A heavy toll surpassed only by heart disease [2]. Con-
fronted with this severe health problem, researchers understand-
ably exert their endeavor on pharmacological improvement that
will facilitate the treatment for tumor. Through continuous efforts
made by scientists worldwide, cancer has become increasingly
survivable over recent decades, not least for rapid advances of
plications remain the focus of medicinal chemistry study.
Anticancer drugs stand profound benefits from extensive
structural resources of natural origin [4]. Vast amount of unique
scaffolds accompanied with diverse bioactivities offer a myriad of
opportunities for the design of new drugs. The ent-kaurane diter-
penoids (Fig. 1), including oridonin (1), are renowned for its various
bioactive properties stretching from antifungal, antibacterial, anti-
inflammatory, to antitumor [5e7]. Since its first isolation and
elucidation from genus Isodon (Rabdosia) in 1976 [8], oridonin is
most researched natural product of this genus. Its extensive efficacy
against various malignant tumors having been studied and
* Corresponding author.
** Corresponding author.
E-mail addresses: huimhua@163.com (H. Hua), lidahong0203@163.com (D. Li).
https://doi.org/10.1016/j.ejmech.2018.01.069
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
589
biologically active natural structures, new anticancer drug candi-
dates can be achieved with mollified toxicity and enhanced safety.
With well-established synthetic route and extensive clinical use
[37e39], nitrogen mustard compounds 6, 12 and 16 were naturally
selected to be our targets of research. Thus, it is imperative that
necessary modification is performed to obtain desired anti-
resistant cytotoxic efficacy with concurrently heightened selec-
tivity and safety.
Aforementioned predicament inspired us to conjugate two
enmein-type diterpenoid cores with different alkylating pharma-
cophores through linkers, a well-established structure modification
strategy for chemotherapy [35e38]. In this article, we outlined the
synthetic approach to introduce three nitrogen mustards (butyric
acid methyl merphalan ester 6, chlorambucil 12, and benzoic acid
mustard 16) to 14- or 6,14-hydroxyl groups of enmein-type de-
rivatives. Resulting hybrids then were evaluated against human
tumor HL-60, PC-3, Bel-7402, and Bel-7402/5-FU cell lines and
normal liver L-02 cell line for antiproliferative activities. Addition-
ally, further mechanisms regarding cell cycle progression, induction
of apoptosis, dysfunction of mitochondrial membrane potential and
expression of apoptosis-related proteins were also investigated.
Overall, a novel hybrid molecule E2 with excellent selectivity and
potent cytotoxic activity against multidrug resistant cell line was
developed as a promising lead for cancer therapeutics.
Fig. 1. Chemical structures of natural ent-kaurane diterpenoids oridonin (1), enmein-
type derivatives (2 and 3).
reported throughout coming decades [9e15], which includes
cytotoxicity effects for human breast cancer [16], gastric cancer
[17], gallbladder cancer [18], cervical carcinoma [19], hepatocellular
carcinoma [20] and leukemia [21]. Another particular epitome,
enmein-type 6,7-seco-ent-kaurane diterpenoid derivative (2) from
oridonin has been under intensive study stem from its complex ring
skeleton and broad cytotoxic activities against a wide range of
malignant tumors [22e30]. Moreover, the accessibility provided by
one-step conversion of commercially available oridonin to target
enmein-type diterpenoid structure is immensely constructive [30].
However, enmein-type diterpenoid derivatives are still shunned by
cancer therapy mainly due to the insufficient efficacy, despite
having a number of pharmacological advantages like safe, unique,
and extensive antiproliferative potency in possession [30,31]. Upon
well designed synthesis, natural product derivatives obtained with
significantly enhanced anticancer potency would be immensely
helpful to the exploration for new therapeutic agents. Hence,
enmein-type diterpenoid holds great potential as an apt structural
core for further modification.
As the very first chemical drug reach clinical application, ni-
trogen mustards (Fig. 2) break the dawn for chemotherapy after
second world war [32]. Thereafter, strong killing effects against a
broad spectrum of malignant tumors prompted nitrogen mustards
to gain a prevalent use in the field of cancer therapy [33]. However,
its non-specific DNA alkylating effects require dangerously high
dosage to achieve practical plasma concentration, which in turn
drive up systemic toxicity risking irreversible side-effects and
develop drug-resistance limiting therapeutic use [34]. Improve-
ments were made to increase cure rate and rate of long term sur-
vival for application of mustard drug, which include joint treatment
of nitrogen mustard with radiotherapy and/or other cytotoxic
agents. Yet the underlying problems of toxicity and selectivity still
loom [33]. However, there were already a lot of published studies in
which anticancer drugs were linked up with natural product
derived core via covalent ester bond [35,36], with which notable
improvement including specific anti-resistant efficacy, enhanced
selectivity and increased overall cytotoxicity against malignant
tumors were observed. It is our constant interest that through
introducing pharmacophores of strong antitumor efficacy to
2. Result and discussion
2.1. Chemistry
First, three nitrogen mustard intermediates were synthesized as
showed in Scheme 1 according to literature procedures [37e39].
The reflux of melphalan 4 in anhydrous methanol with the thionyl
chloride which was added dropwise afforded the correspondent
ester 5. The target derivative 6 was obtained through the esterifi-
cation of the primary amine of 5 in which succinic anhydride was
added dropwise to an acetic acid solution of compound 5 [37]. Aryl
substitution of acetanilide 7 with succinic anhydride in a solvent of
anhydrous AlCl3 and CS2 led to compound 8. The subsequent 4-(4-
aminophenyl) butyric acid 9 was acquired through standard Wolff-
Kishner reductive reaction in which target compound 8 was mixed
with potassium hydroxide, hydrazine hydrate then diethylene
glycol. Treatment of butyric acid 9 with dropwise added thionyl
chloride in anhydrous ethanol under 0 ꢀC gave the ethyl group
protected ester 10. Then the intermediate 10 were mixed with 25%
acetic acid then reacted with dropwise added ethylene oxide to
provide bis-(2-hydroxyethyl)-amino substituted 11. The final
chloration and deprotection was succeeded through two-step
process with reflux of compound 11 with phosphorus oxychloride
then reacted with hydrochloric acid sequentially to yield chlor-
ambucil 12 [38]. Condensation of 4-aminobenzoic acid 13 with
anhydrous ethanol afforded 14 with protected ester side chain.
Treatment of compound 14 with ethylene oxide added dropwise in
Fig. 2. Chemical structures of butyric acid methyl merphalan ester (6), chlorambucil (12), and the benzoic acid mustard (16).

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X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
Scheme 1. Synthesis of nitrogen mustards derivatives. Reagents and conditions: (a) SOCl2, MeOH, reflux, 12 h; (b) succinic anhydrides, DMAP, DCM, rt, 18 h; (c) AlCl3 anhydrous,
succinic anhydride, rt, 8 h; (d) KOH, hydrazine monohydrate, diethylene glycol, reflux, 8h; (e) SOCl2, ethanol anhydrous, rt, 4 h; (f) acetic acid, ethylene oxide, rt, 24 h; (g) POCl3,
0.5 h, then 10% HCl, 12 h; (h) ethanol, H2SO4, reflux, 5 h; (i) ethylene oxide, H2O, HCOOH, rt, 24 h; (j) POCl3, 0.5 h, then 10% HCl, 12 h.
acetic acid aqueous solution gave bis-(2-hydroxyethyl)-amino
substituted ethyl benzoate 15. Reaction of intermediate 15 with
thionyl chloride in benzene for reflux, and then acidation with
concentrated chloric acid for reflux provided benzoic acid mustard,
the target compound 16 [39]. Second, the synthesis of enmein-type
derivatives is outlined in Scheme 2 [40]. Treatment of commercially
acquired oridonin 1 with sodium periodate in water gave ester 2 for
over 90% yield. Oxidation of 2 with Jones reagent at 0 ꢀC afforded
ketone 3 in a similar high yield. Final step, a serial of enmein-
coupled nitrogen mustard conjugates (E1eE6) were obtained, as
exhibited in Scheme 2, through the combination of compounds 2 or
exploration.
Among the mustard molecules (6, 12 and 16), melphalan de-
rivative (6) inhibited three malignant cell lines (HL-60, PC-3 and
Bel-7402) with IC50 values of 12.36 M, 57.27 M and 46.45 M,
respectively. Chlorambucil (12) exhibited poor inhibitory effects to
both malignant and normal liver cells, while benzoic acid mustard
m
m
m
(16) exhibited a large IC50 value of over 80 mM to PC-3 prostate
cancer cells. Almost all conjugated hybrids (E1eE6) exerted supe-
rior antiproliferative effects to ent-kaurane diterpenoid cores (1e3)
against four malignant tumor cell lines. However, a more mixed
picture emerged when comparing antiproliferative activities of
target hybrids with that of nitrogen mustards. The IC50 values
against drug-resistant Bel-7402/5-FU cells were significantly
3
and compounds 6, 12 or 16 under the condition of 4-
dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDCI) in
dichloromethane (DCM).
reduced from above 100 mM of three mustards to below 16 mM for
all the target conjugates. But melphalan coupled compounds E3
and E6 demonstrated that sizeable increase in antiproliferative
activities against Bel-7402 hepatoma cells accompanied with steep
2.2. Biological evaluation
decline against PC-3 prostate cancer cells. Moreover, above 100
of half inhibition rate of melphalan derivatives against normal
human liver cells were still above 100 M throughout hybridizing.
As to benzoic acid mustard hybrids (E2 and E5), dismal effects
against PC-3 cells (IC50 > 80 M) were substantially reversed by
conjugating with two diterpenoid cores (3.97 M of E2 and 7.03
of E5). Additionally, notable selectivity improvement of hybrid
compounds was noticed through selectivity index (SI) elevation
from 2.6 (1), 1.6 (3), and 0.8 (2) of diterpenoids to 16.3 (E4), 33.6
(E1), 39.3 (E5), and above 130 (E2) for tested conjugates. Among
hybrid derivatives, E2 of enmein-type diterpenoid (2) coupled with
benzoic acid mustard (16) demonstrated the most effective anti-
proliferative activities against HL-60, PC-3, Bel-7402 and Bel-7402/
mM
2.2.1. Cytotoxic activity and structure-activity relationships
analysis
m
The antiproliferative evaluation of ent-kaurane diterpenoids
(1e3), nitrogen mustards (6, 12 and 16), and corresponding hybrids
(E1eE6) were carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) cell viability and cytotoxicity
assay. The resulting IC50 values against four human cancer cell lines
(HL-60, PC-3, Bel-7402 and Bel-7402/5-FU), and one normal cell
line (L-02) were summarized in Table 1 with 5-FU as positive
control. Besides promising antiproliferative activities against tumor
cells, good selectivity between normal and tumor human liver cells
was also observed, which is a key character in antitumor drug
m
m
mM

X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
591
Scheme 2. Synthesis of enmein-type derivatives. Reagents and conditions: (a) sodium periodate, H2O, rt, 6 h; (b) Jones reagent, DCM, rt, 0.5 h; (c) EDCI, DMAP, DCM, rt, 8e12 h.
5-FU cells with IC50 values of 7.83
mM, 3.97
mM, 0.77
mM and
2.2.2. Cell cycle analysis
2.07 M, respectively, and displayed the lowest toxicity through SI
m
Uninterrupted cell cycle progression is vital to the biochemical
processes surrounding cell division and replication. Thus, cell cycle
arrest at certain phase of mitosis leads to suppression of cell pro-
liferation and in the event of tumor growth [41,42]. As potent DNA
alkylating agents, nitrogen mustards arrest preparative G1 phase of
of over 130 between normal and malignant human liver cells,
which rendered it the most desirable molecule for intense mech-
anism study.

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X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
Table 1
Antiproliferative activities of enmein derivatives, nitrogen mustards and their hybrids against four cancer and one normal cell lines.
Compound
IC50
(m
M)a
HL-60
PC-3
Bel-7402
Bel-7402/5-FU
L-02
SIb
1
2
3
6
12
16
E1
E2
E3
E4
E5
E6
5-FU
14.30 0.43
28.07 2.08
20.61 1.34
12.36 0.65
11.63 0.42
6.02 0.34
6.35 0.27
7.83 0.42
17.58 0.42
9.94 0.35
5.57 0.22
18.47 0.36
2.37 0.20
10.97 0.87
30.55 1.60
22.56 1.32
57.27 2.40
40.35 1.78
>80
7.32 0.38
3.97 0.50
>100
7.36 0.47
32.09 1.38
15.87 1.03
46.45 2.17
>100
23.94 1.34
2.08 0.14
0.77 0.08
5.73 0.30
3.06 0.20
1.60 0.21
6.50 0.28
18.49 1.20
10.51 0.46
44.27 1.36
19.57 1.12
>100
>100
>100
8.49 0.15
2.07 0.13
7.93 0.47
15.58 0.42
2.90 0.15
12.76 0.34
>80
18.94 0.75
27.06 1.25
25.46 1.20
68.57 2.08
>100
32.86 2.39
69.88 3.57
>100
2.6
0.8
1.6
1.5
NCd
1.4
33.6
>130
NCd
16.3
39.3
NCd
>100
5.50 0.43
7.03 0.35
>100
49.76 2.14
62.84 2.79
>100
23.86 1.05
NTc
a
IC50: half inhibitory concentrations measured by the MTT assay. The values are expressed as averages standard deviations of three independent experiments.
b
c
SI: selectivity index. It was calculated as: SI ¼ IC50(L-O2)/IC50(Bel-7402)
.
NT: not tested.
d
NC: not calculated.
cell-division cycle by alkylation of DNA molecules. To examine the
antiproliferative mechanism of E2 on cell cycle of Bel-7402 cells,
flow cytometry was applied to measure DNA content of cell nuclei.
Bel-7402 cells were treated with indicated concentrations of E2
increased relatively to correspondent E2 concentrations added,
from 63.55% of untreated control to 66.71%, 69.71% and 72.09%,
respectively. Hence, E2 arrested Bel-7402 cell cycle at G1 phase.
(0.4 mM, 0.8 mM and 1.2 mM) for 48 h, then stained with propidium
iodide (PI) for subsequent cytometry analysis. As showed in Fig. 3,
the percentage shares of G1 phase in cell cycle progression were
2.2.3. The morphological analysis by Hoechst 33258 staining
Cell cycle arrest could proceed to apoptosis, and eventually cell
death. This occurs when a cell senses that the DNA damage
Fig. 3. Influence of the cell cycle arrest of G1 phase by E2 in Bel-7402 cells: Bel-7402 cells were incubated with the indicated concentrations of E2 for 24 h and the cells were stained
with PI. Cellular DNA content, for cell cycle distribution analysis, was measured using a flow cytometer. The diagrams showed the distribution of the cells according to their DNA
content. The inserts gave the percentages in different cell cycle phases.

X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
593
prevents it to function properly. And the aberrant morphological
changes in chromatin is a strong indication of apoptotic process
[43]. Thus, Hoechst 33258 staining were carried out under fluo-
rescent microscope on E2 treated Bel-7402 cells to determine
whether apoptosis was triggered during inhibition of cell growth.
The stain results were compiled in Fig. 4, in which control treated
with vehicle solvent demonstrated that hazy light-blue in nuclei
overlapped among indistinct cell borders, while clear shape of in-
[45]. In an effort to uncover the participation of mitochondria
pathway in E2 induced apoptosis of Bel-7402 cells, the changes of
mitochondrial membrane potentials were assessed. Bel-7402 cells
were treated with different concentrations of E2 (0.4
mM, 0.8 mM
and 1.2 M) and vehicle solvent for control and put into 48 h in-
m
cubation, then staining with the lipophilic cationic fluorescent
mitochondrial probe 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzi-
midazol-carbocyanine iodide (JC-1). After that, flow cytometry
analysis was established to measure the number of cells with
collapsed mitochondrial membrane potential. The results were
summarized in Fig. 6, which showed that the share of cells with
discernable apoptotic morphology increased in a dose-dependent
manner from 7.20% of control, to 14.38%, 20.93% and 40.00% for
different concentrations of E2 applied, respectively. It was clear that
E2 could induce apoptosis in Bel-7402 cells through mitochondria
pathway.
dividual cell with sheen blue color after treatment of E2 (0.4
mM,
0.8 M and 1.2 M) revealed chromatin condensation and nuclear
m
m
disintegration, sign of apoptotic process. The stain results certified
apoptosis induction was involved in E2 treated Bel-7402 cells.
2.2.4. Cell apoptosis assay
Apoptosis is a natural process of self-destruction by degradative
enzymes in cells that genetically programmed to have a limited
lifespan or damaged by toxic chemical agents. However, tumor cells
can divide relentlessly without programmed cell death, which
mainly due to imbalance between proliferation and apoptosis
during carcinogenesis. Thus, chemical induced apoptosis in tumor
cells is key to cancer therapy [44]. Therefore, to further confirm E2
induced apoptosis in Bel-7402 cells, an annexin V-FITC/PI binding
assay was performed. Bel-7402 cells were treated with E2 at indi-
2.2.6. Effects on apoptosis-related proteins
Apoptosis through intrinsic pathway is a programmed process
of cell death centered by mitochondrion and mediated by a group
of apoptosis related proteins. Among them, bcl-2 family proteins
function as an indispensable regulator of the cell apoptosis by
altering the ratio of bcl-2/bax following an apoptotic stimulus [46].
Bcl-2 protein exerts inhibitory effects on pro-apoptotic proteins
through physical interaction and builds up drug resistance in tumor
cells while bax protein facilitates permeabilization of mitochon-
drial outer membrane and the release of pro-apoptotic proteins,
including cytochrome c, from the inter-membrane place into the
outer cytoplasm [47e51]. Thereafter, apoptotic protease activating
factor-1 (Apaf-1) binds cytochrome c and dATP to form a caspase-
activating polymeric platform known as the apoptosome, which
in turn binds and cleaves procaspase-9 protein, releasing its
mature, activated form, caspase-9. Once released, proapoptotic
protein caspase-9 then cleave and activate caspase-3 directly
[52,53]. And as an essential member of “executioner” caspases,
caspase-3 cleaves and activates a cascade of key substrates, to usher
in the final degradation phase of cell apoptosis [42,54]. P53 exerts
crucial mediate effect over different cellular responses to DNA
damage, which mainly include G1 phase cell cycle arrest and
apoptosis. Hence, induction of p53 protein by DNA alkylating
agents such as nitrogen mustards substantially strengthen
apoptotic process in tumor cells [55]. To explore the mediative
mechanism of aforementioned apoptosis related proteins behind
E2 induced apoptosis in Bel-7402 cells, correspondent Western blot
analysis was performed, and subsequent results were summarized
in Fig. 7. After 48 h treatment of E2, upregulation of pro-apoptotic
bax, caspase-3, -9, cytochrome c, and p53 proteins expression,
and suppression of anti-apoptotic bcl-2 protein expression were
detected in a concentration-dependent fashion. Thus, it was
confirmed that apoptosis induction in E2 treated cells followed
mitochondria mechanism mediated by apoptotic related proteins.
cated concentrations (0.4 mM, 0.8 mM and 1.2 mM) then the per-
centages of apoptotic cells were measured by flow cytometry. As
shown in Fig. 5, 48 h after the treatment of E2 gave substantial rise
to the apoptotic ratios from 2.33% of vehicle control to 18.87%
(0.4 mM), 35.53% (0.8 mM) and 60.92% (1.2 mM) for different con-
centrations applied in a dose-dependent manner. Aforementioned
results manifested the conspicuous apoptotic effects that induced
by E2 in Bel-7402 cells.
2.2.5. Mitochondria membrane potential analysis
Notable signs of apoptosis manifest itself through palpable
changes in membrane integrity undergone by mitochondria. Both
the inner and the outer mitochondrial membranes were involved in
these changes, which sequentially lead to the dissipation of the
inner transmembrane potential, promote mitochondrial perme-
ability transition and induce the release of pro-apoptotic proteins
3. Conclusion
A series of novel enmein hybrids coupled with nitrogen mus-
tards were designed and synthesized for anticancer therapeutics.
Antiproliferative evaluation showed that efficacy enhancement
accompanied with improved selectivity for tested hybrids. The
most effective one, conjugate E2 exhibited cytotoxicity against HL-
60, PC-3, Bel-7402 and drug-resistant Bel-7402/5-FU cell lines with
IC50 values of 7.83 mM, 3.97 mM, 0.77 mM and 2.07 mM, respectively.
Additionally, the SI between normal and malignant liver cells sur-
passed 130. Further mechanism study of E2 demonstrated that
strong apoptotic effects exerted were attributed to G1 phase cell
cycle arrest, collapse of mitochondrial membrane potential and
Fig. 4. Hoechst staining of E2 treated Bel-7402 cells. Cells were treated with the
indicated concentrations of E2 or vehicle control for 48 h, stained with Hoechst 33258
and examined with a fluorescent microscope.

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X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
Fig. 5. Apoptosis in Bel-7402 cells by treatment with E2: Bel-7402 cells were incubated with different concentrations of E2 for 24 h and the cells were stained with annexin V-FITC
and PI, followed by flow cytometry analysis.
mediation between pro- and anti-apoptotic proteins through
mitochondrial pathway. All considered, E2 represents a promising
drug candidate for anticancer therapeutics that merit further
investigation.
48 h to give compound 2. The mixture was extracted by DCM
(3 ꢂ 50 mL). The organic layer was combined, washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. Com-
pound 2 (500 mg, 1.4 mmol) was dissolved in 50 mL acetone, then
Jones reagent (1700 m
L, 4.3 mmol) was added dropwise at 0 ꢀC to
give compound 3 after 30 min. Isopropanol was added to quench
the reaction then poured into 20 mL water, for extraction with DCM
(3 ꢂ 20 mL). The organic layer was combined, washed with brine,
dried over anhydrous Na2SO4 and concentrated in vacuo. Enmein-
type cores (36 mg, 0.1 mmol) were dissolved in 5 mL DCM and
conjugated with the corresponding nitrogen mustards (0.2 mmol),
using EDCI (0.4 mmol) and DMAP (0.02 mmol). The mixture was
stirred for 8e12 h under room temperature, then poured into 10 mL
10% HCl and 10 mL water, for extraction with DCM (3 ꢂ 10 mL). The
organic layer was combined, washed with brine, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The target products were
obtained through purification by flash column chromatography
(MeOH/DCM 1:200, v/v).
4. Experimental
4.1. Chemistry
Chemicals and solvents were purchased from commercial
sources. Further purification by standard methods were employed
when necessary. Melting points were determined on an XT-4 micro
melting point apparatus and uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker ARX-400 NMR spectrometer in
the indicated solvents (TMS as internal standard): the values of the
chemical shifts were expressed in
d values (ppm) and the coupling
constants (J) in Hz. Mass spectra were obtained on Agilent 1100 Ion
trap mass spectrometer. HR-MS were carried out on Agilent Q-TOF
B.05.01 (B5125.2).
4.1.1.1. Compound E1.
4.1.1. General procedures to synthesize enmein-type diterpenoids
and E1ꢁE6
(3R,3aR,6aS,8aR,11S,13aS,13bS,14R)-3-hydroxy-4,4-dimethyl-10-
methylene-8,9-dioxododecahydro-1H,8H-8a,11-
Oridonin (1.08 g, 3.0 mmol) was dissolved in 50 mL water then
mixed with sodium periodate (2.57 g, 12.0 mmol) and stirred for
methanocyclohepta[c]furo[3,4-e]chromen-14-yl
4-(4-(bis(2-
chloroethyl)amino)phenyl)butanoate. Brown oil, yield: 18.9%. 1H

X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
595
Fig. 6. Effect of E2 on the mitochondrial membrane potentials of Bel-7402 cells: Bel-7402 cells were incubated with the indicated concentrations of E2 for 48 h prior to staining with
JC-1.
NMR (CDCl3, 400 M Hz),
d
(ppm): 7.04, 6.62 (each 2H, d, J ¼ 8.4 Hz,
198.49, 166.56, 165.56, 150.10, 147.94, 132.35 (ꢂ2), 120.13, 117.64,
110.91 (ꢂ2), 101.77, 77.22, 76.84, 76.09, 74.61, 73.97, 60.13, 53.84,
53.32, 50.17, 48.53, 41.00, 40.08, 37.10, 32.90, 31.03, 29.64, 23.29,
23.05, 19.79; HRMS (ESI) m/z calcd for C31H37Cl2NO7 [MþNa]þ
628.1947, found 628.1968.
Ar-H), 6.20 (1H, s, 14-CH), 5.72 (1H, s, 6-CH), 5.55 (1H, s, 17-CH2),
5.33 (1H, s, 17-CH2), 4.57 (1H, dd, J ¼ 11.5, 5.8 Hz, 1-CH), 4.05, 3.93
(each 1H, d, J ¼ 9.4 Hz, 20-CH2), 3.69, 3.61 (each 4H, t, J ¼ 6.0 Hz,
130,140-CH2, 150,160-CH2), 3.12 (1H, d, J ¼ 9.3 Hz, 13-CH), 2.52 (2H, m,
40-CH2), 2.27 (2H, m, 20-CH2), 1.84 (2H, m, 30-CH2), 1.02 (3H, s, 18-
CH3), 0.95 (3H, s, 19-CH3). 13C NMR (CDCl3, 100 M Hz),
d (ppm):
4.1.1.3. Compound E3.
(3S,3aR,6aS,8aR,11S,13aS,13bS,14R)-4,4-dimethyl-10-methylene-
8,9-dioxododecahydro-1H,8H-8a,11-methanocyclohepta[c]furo
197.55, 171.81, 166.42, 147.62, 144.10, 129.79, 129.68, 120.56, 112.79,
112.66, 101.40, 75.65, 75.07, 73.36, 53.93, 53.80, 53.25, 49.59, 47.89,
40.52, 40.40, 40.29, 36.97, 33.97, 33.93, 33.84, 32.87, 31.39, 29.99,
26.40, 26.21, 23.12, 22.93, 19.83; HRMS (ESI) m/z calcd for
[3,4-e]chromene-3,14-diyl
bis(4-(((S)-3-(4-(bis(2-chloroethyl)
amino)phenyl)-1-methoxy-1-oxopropan-2-yl)amino)-4-
C
34H43Cl2NO7 [MþH]þ 648.2458, found 648.2489.
oxobutanoate). Brown oil, yield: 14.1%. 1H NMR (CDCl3, 400 M Hz),
d
(ppm): 6.98 (4H, dd, J ¼ 8.7, 2.5 Hz, Ar-H), 6.63 (4H, dd, J ¼ 8.7,
4.1.1.2. Compound E2.
(3R,3aR,6aS,8aR,11S,13aS,13bS,14R)-3-hydroxy-4,4-dimethyl-10-
methylene-8,9-dioxododecahydro-1H,8H-8a,11-
3.3 Hz, Ar-H), 6.22 (1H, s, 6-CH), 6.15 (1H, s, 14-CH), 6.02 (1H, d,
J ¼ 7.6 Hz, NH) 5.96 (1H, d, J ¼ 7.6 Hz, NH), 5.73 (1H, s, 17-CH2), 5.60
(1H, s, 17-CH2), 4.76 (2H, dd, J ¼ 7.8, 5.6 Hz, 50-CH, 500-CH), 4.56 (1H,
dd, J ¼ 10.8, 5.3 Hz, 1-CH), 4.11, 3.96 (each 1H, d, J ¼ 9.4 Hz, 20-CH2),
3.72 (3H, s, 1800-CH3), 3.74 (3H, s, 180-CH3), 3.62, 3.71 (each 8H, t,
J ¼ 6.4 Hz, 130,140-CH2, 150,160-CH2, 1300,1400-CH2, 1500,1600-CH2), 3.19
(1H, d, J ¼ 10.0 Hz,13-CH), 3.01 (4H, m, 60-CH2, 600-CH2), 2.62 (2H, m,
200-CH2), 2.53 (2H, m, 300-CH2), 2.53 (2H, m, 20-CH2), 2.39 (2H, m, 30-
CH2), 1.06 (3H, s, 19-CH3), 1.01 (3H, s, 19-CH3). 13C NMR (CDCl3,
methanocyclohepta[c]furo[3,4-e]chromen-14-yl
4-(bis(2-
chloroethyl)amino)benzoate. White power, yield: 11.7%. 1H NMR
(CDCl3, 400 M Hz),
d
(ppm): 7.87, 6.62 (each 2H, d, JA ¼ JB ¼ 8.8 Hz,
Ar-H), 6.24 (1H, s, 14-CH), 5.85 (1H, s, 6-CH), 5.56 (1H, s, 17-CH2),
5.36 (1H, s, 17-CH2), 4.57 (1H, dd, J ¼ 11.5, 5.8 Hz, 1-CH), 4.01, 4.11
(each 1H, d, J ¼ 9.4 Hz, 20-CH2), 3.64, 3.79 (each 4H, t, J ¼ 7.0 Hz,
80,90-CH2, 100,110-CH2), 3.32 (1H, d, J ¼ 9.4 Hz, 13-CH), 1.05 (3H, s, 18-
100 M Hz),
d (ppm): 197.76, 172.16, 172.00, 171.88, 171.27, 170.79,
CH3), 0.98 (3H, s, 19-CH3). 13C NMR (CDCl3, 100 M Hz),
d (ppm):
170.41, 166.98, 147.51, 145.10, 144.96, 130.64, 130.56, 124.98, 120.95,

596
X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
J ¼ 6.0 Hz, 130,140-CH2, 150,160-CH2), 3.15 (1H, d, J ¼ 9.4 Hz, 13-CH),
2.50 (2H, m, 40-CH2), 2.30 (2H, m, 20-CH2), 1.82 (2H, m, 30-CH2), 1.21
(s, 3H, 18-CH3), 1.06 (s, 3H, 19-CH3). 13C NMR (CDCl3, 100 M Hz),
d
(ppm): 197.17, 175.28, 173.29, 165.97, 147.01, 143.97, 130.99, 129.75
(ꢂ2), 121.44, 112.58 (ꢂ2), 74.41, 73.13, 71.36, 59.46, 53.81, 50.79,
47.50, 45.95, 40.38, 40.31 (ꢂ2), 36.38, 33.82, 33.32, 33.05, 32.26,
29.53, 26.47, 26.20, 23.63, 23.06, 19.16; HRMS (ESI) m/z calcd for
C
34H41Cl2NO7 [MþH]þ 646.2351, found 646.2333.
4.1.1.5. Compound E5.
(3aR,6aS,8aR,11S,13aS,13bS,14R)-4,4-dimethyl-10-methylene-3,8,9-
trioxododecahydro-1H,8H-8a,11-methanocyclohepta[c]furo[3,4-e]
chromen-14-yl 4-(bis(2-chloroethyl)amino)benzoate. White po-
wer, yield: 20.2%. 1H (CDCl3, 400 M Hz)
d 7.83, 6.61 (each 2H, d,
J ¼ 8.92 Hz, Ar-H), 6.27 (1H, s, 14-CH), 5.83 (1H, s, 17-CH2), 5.61 (1H,
s,17-CH2), 4.62 (1H, dd, J ¼ 11.6, 5.7 Hz,1-CH), 4.37, 4.09 (each 1H, d,
J ¼ 10.2 Hz, 20-CH2), 3.78, 3.62 (each 4H, t, J ¼ 7.0 Hz, 80,90-CH2,
100,110-CH2), 3.33 (1H,d, J ¼ 9.5 Hz, 13-CH), 1.22 (3H, s, 18-CH3), 1.07
(3H, s, 19-CH3). 13C NMR (CDCl3, 100 M Hz),
d (ppm): 197.53, 175.28,
165.59, 165.52, 150.24, 147.17, 132.34 (ꢂ2), 121.22, 117.28, 110.93
(ꢂ2), 77.20, 76.74, 74.46, 73.27, 71.43, 59.43, 53.29, 50.80, 47.73,
45.97, 40.62, 40.03, 36.44, 33.06, 32.27, 29.41, 23.64, 23.13, 19.20;
HRMS (ESI) m/z calcd for C31H35Cl2NO7 [MþNa]þ 626.1793, found
626.1851.
4.1.1.6. Compound E6.
(3aR,6aS,8aR,11S,13aS,13bS,14R)-4,4-dimethyl-10-methylene-3,8,9-
trioxododecahydro-1H,8H-8a,11-methanocyclohepta[c]furo[3,4-e]
chromen-14-yl 4-(((S)-3-(4-(bis(2-chloroethyl)amino)phenyl)-1-
methoxy-1-oxopropan-2-yl)amino)-4-oxobutanoate. Brown oil,
yield: 35.5%. 1H NMR (CDCl3, 400 M Hz),
d (ppm): 6.97, 6.63 (each
2H, d, J ¼ 8.6 Hz, Ar-H), 6.25 (1H, d, J ¼ 1.2 Hz, 14-CH), 6.02 (1H, d,
J ¼ 7.6 Hz, 6-OH), 5.70 (1H, s, 17-CH2), 5.64 (1H, s, 17-CH2), 4.74 (1H,
dt, J ¼ 7.8, 5.7 Hz, 50-CH), 4.58 (1H, dd, J ¼ 11.5, 5.7 Hz, 1-CH), 4.34,
4.03 (each 1H, d, J ¼ 10.2 Hz, 20-CH2), 3.73 (3H, s, 180-CH3), 3.63,
3.71 (each 4H, t, J ¼ 6.2 Hz, 130,140-CH2, 150,160-CH2), 3.22 (1H, d,
J ¼ 8.6 Hz, 13-CH), 2.63 (2H, m, 2' -CH2), 2.51 (2H, m, 3' -CH2), 2.38
(1H, m, 6' -CH2), 2.08 (1H, m, 6' -CH2), 1.21 (3H, s, 18-CH3), 1.06 (3H,
s, 19-CH3). 13C NMR (CDCl3, 100 M Hz),
d (ppm): 197.17, 175.21,
172.28, 171.98, 170.63, 166.32, 146.93, 145.03, 130.53 (ꢂ2), 124.84,
121.65, 112.32 (ꢂ2), 77.24, 74.47, 73.53, 71.32, 59.45, 53.60, 53.42,
52.27, 50.77, 47.43, 45.95, 40.38 (ꢂ2), 40.16, 36.62, 36.37, 33.04,
32.25, 30.43, 29.51, 29.44, 23.62, 23.02, 19.17; HRMS (ESI) m/z calcd
for C38H46Cl2N2O10 [MþNa]þ 783.2460, found 783.2422.
4.2. MTT assay
Fig. 7. Western blot of apoptosis-related proteins in Bel-7402 cells after exposure to
different concentrations (0, 0.4 mM, 0.8 mM and 1.2 mM) of E2 for 48 h.
Antiproliferative activity was examined by the MTT assay
following the method described previously [56]. Flat-bottomed 96-
well plates were applied to undertake the assay. All tested cells
were added to each well, then treated with different concentrations
of the target compounds in triplicate, and incubated for 72 h. After
120.53, 112.37, 112.28, 101.94, 101.74, 77.23, 76.12, 75.74, 75.16,
74.12, 73.89, 60.17, 59.88, 53.81, 53.65, 53.57, 53.48, 53.27, 52.34,
52.26, 49.83, 49.46, 48.46, 48.00, 40.54, 40.37, 37.03, 36.91, 36.60,
32.84, 31.38, 30.55, 30.18, 30.02, 29.74, 29.56, 29.31, 23.18, 23.08,
22.95, 19.69; HRMS (ESI) m/z calcd for C56H70Cl4N4O14 [MþH]þ
1163.3643, found 1163.3792.
that, MTT solution (20 mL, 5 mg/mL) and DMSO (150 mL/well) was
added to each well sequentially. 10 min of incubation at room
temperature was followed. The absorbance (OD) value was
measured under 490 nm wavelength on a microplate reader (BIO-
RAD Instruments Inc. 550, Hercules, CA, USA). 5-FU was used as the
positive reference in this assay. Half inhibition rate (IC50) of four
tumor and one normal cell growth was collected for antitumor
evaluation.
4.1.1.4. Compound E4.
(3aR,6aS,8aR,11S,13aS,13bS,14R)-4,4-dimethyl-10-methylene-3,8,9-
trioxododecahydro-1H,8H-8a,11-methanocyclohepta[c]furo[3,4-e]
chromen-14-yl 4-(4-(bis(2-chloroethyl)amino)phenyl)butanoate.
Yellow oil, yield: 25.6%. 1H NMR (CDCl3, 400 M Hz),
d
(ppm): 7.06,
4.3. Cell cycle analysis
6.64 (each 2H, d, J ¼ 8.6 Hz, Ar-H), 6.25 (1H, s, 14-CH), 5.70 (1H, s,
17-CH2), 5.62 (1H, s, 17-CH2), 4,59 (1H, dd, J ¼ 11.5, 5.7 Hz, 1-CH),
4.35, 4.03 (each 1H, d, J ¼ 10.2 Hz, 20-CH2), 3.69, 3.62 (each 4H, t,
The cell cycle of Bel-7402 cell line treated with E2 was evaluated
by flow cytometry. Bel-7402 cells were seeded in 6-well plates and

X. Gao et al. / European Journal of Medicinal Chemistry 146 (2018) 588e598
597
incubated at 37 ꢀC for 24 h. Then, E2 dissolved in DMSO was added
in indicated concentrations for incubation in triplicate manner.
Blank solvent of DMSO was added to control cells. After 48 h in-
cubation, all cells were centrifuged and fixed in 70% ethanol at 4 ꢀC
Science Foundation of Liaoning Province (20170540858), General
Scientific Research Projects of Department of Education in Liaoning
Province (2017LQN05), and Career Development Support Plan for
Young and Middle-aged Teachers in Shenyang Pharmaceutical
University.
overnight and suspended again in PBS mixed with 100
mL RNase A
and 400 L PI. Cell cycle distribution of DNA content was assessed
m
on a flow cytometer (FACS Calibur Becton-Dickinson, Franklin Lake,
NJ, USA).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.01.069.
4.4. Hoechst 333258 staining
The nuclear morphological modifications were analyzed by
fluorescent microscopy using Hoechst staining. In this experiment,
Bel-7402 cells were seeded on 6-well plates, with 2 mL of medium.
Following 24 h of incubation, compound E2 was added at indicated
concentrations and cells were incubated again for a period of 48 h.
Cells were harvested by mild trypsinization, collected by centrifu-
gation and washed twice with PBS. Cells were then stained with
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