Novel hybrids of natural oridonin-bearing nitrogen mustards as potential anticancer drug candidates

Article abstract of DOI:10.1021/ml500141f

A series of novel hybrids from natural product oridonin and nitrogen mustards were designed and synthesized to obtain more efficacious and less toxic antitumor agents. The antiproliferative evaluation showed that most conjugates were more potent than their parent compounds oridonin and clinically used nitrogen mustards against four human cancer cell lines (K562, MCF-7, Bel-7402, and MGC-803). Furthermore, the representative compounds 16a-c exhibited antiproliferative activities against the multidrug resistant cell lines (SW620/AD300 and NCI-H460/MX20). It was shown that the most effective compound 16b possesses a strong inhibitory activity with an IC₅₀ value 21-fold lower than that of oridonin in MCF-7 cells and also exhibits selective cytotoxicity toward the cancer cells. Intriguingly, compound 16b has been demonstrated to significantly induce apoptosis and affect cell cycle progression in human hepatoma Bel-7402 cells.

Full text of DOI:10.1021/ml500141f

Letter
pubs.acs.org/acsmedchemlett
Novel Hybrids of Natural Oridonin-Bearing Nitrogen Mustards as
Potential Anticancer Drug Candidates
Shengtao Xu,† Lingling Pei,† Chengqian Wang,† Yun-Kai Zhang,‡ Dahong Li,†,§ Hequan Yao,†
Xiaoming Wu,† Zhe-Sheng Chen,*,‡ Yijun Sun,∥ and Jinyi Xu*,†
†State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia
Xiang, Nanjing 210009, China
‡College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens, New York 11439, United States
§Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education and School of Traditional Chinese Materia
Medica, Shenyang Pharmaceutical University, 103 Wen Hua Road, Shenyang 110016, China
∥Drug Screening Center, Nanjing Key Gen Biotech. Co., Ltd., Nanjing 210012, China
S
* Supporting Information
ABSTRACT: A series of novel hybrids from natural product
oridonin and nitrogen mustards were designed and synthe-
sized to obtain more efficacious and less toxic antitumor
agents. The antiproliferative evaluation showed that most
conjugates were more potent than their parent compounds
oridonin and clinically used nitrogen mustards against four
human cancer cell lines (K562, MCF-7, Bel-7402, and MGC-
803). Furthermore, the representative compounds 16a−c
exhibited antiproliferative activities against the multidrug
resistant cell lines (SW620/AD300 and NCI-H460/MX20). It was shown that the most effective compound 16b possesses a
strong inhibitory activity with an IC50 value 21-fold lower than that of oridonin in MCF-7 cells and also exhibits selective
cytotoxicity toward the cancer cells. Intriguingly, compound 16b has been demonstrated to significantly induce apoptosis and
affect cell cycle progression in human hepatoma Bel-7402 cells.
KEYWORDS: Oridonin, antiproliferative activities, nitrogen mustards, combination principle, drug-resistant, apoptosis
ancer is a major global health problem, representing the
1
Csecond-leading cause of death worldwide.
Effective
anticancer agents with novel scaffolds or new mechanisms of
action are urgently needed for the highly aggressive and drug-
resistant cancer.
Nitrogen mustards are among the DNA alkylating agents that
are most widely used in cancer chemotherapy.2 However, the
lack of drug-specific affinity toward cancer cells necessitates the
use of a large dose of a drug to achieve high local concentration,
systemic toxicity leading to many side effects, and the failure of
chemotherapy due to acquired drug resistance limited the use
of nitrogen mustards in clinics.3 In this regard, it is necessary to
carry out chemical modifications on the nitrogen mustards to
acquire new agents with enhanced anticancer activity, reduced
systemic toxicity, and high selectivity for chemotherapy of
Figure 1. Structures of chlorambucil and available anticancer drugs
designed by using combination principle.
cancer.4
Combination principle is used widely in drug design.5 There
have been a great deal of reports describing the hybrids
between natural products and anticancer drugs,6 in which the
individual molecules are commonly connected via a chemically
stable linker.7 For example, prednimustine (Figure 1), the
prednisolone ester of chlorambucil, not only exhibits a similar
profile to that of chlorambucil in murine tumors8 but also
demonstrates activity against chlorambucil-resistant tumors.
Also included within this group are estramustine phosphate
sodium and uramustine (Figure 1).9
Received: April 8, 2014
Accepted: May 28, 2014
© XXXX American Chemical Society
A
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Natural diterpenoids with unique chemical skeletons and
interesting activities have attracted our attention and aroused
our curiosity for intensive study. Oridonin (1) is a widely
distributed ent-kaurene in the Rabdosia plants and has recently
attracted much attention due to its extensive biological
activities.10 Although oridonin exhibited unique, safe, broad
antitumor activity, the development of oridonin for cancer
therapy was hampered by its relatively moderate potency.
Therefore, developing novel oridonin derivatives by rational
modification to improve its anticancer activity profile is in
urgent need.11,12
Scheme 2. Synthesis of Melphalan Derivatives 7, 8, 10, and
11 and Benzoic Acid Mustard 15
a
In our previous studies, we disclosed that some novel 1-O-
and 14-O-derivatives of oridonin showed excellent cytotox-
icities against several human cancer cell lines in vitro and in vivo.
It was found that the modification on the 14-hydroxy of
oridonin did not diminish the cytotoxicities against a panel of
human cancer cell lines.13 These findings prompted us to
introduce different kinds of nitrogen mustards to the 14-
hydroxy of oridonin, including chlorambucil, melphalan, and
formylmerphalan. In addition, the benzoic acid mustard was
also chosen due to its low toxicity.14,15
Herein, in this presentation, we describe the design and
synthesis of a series of novel oridonin-coupled nitrogen
mustard conjugates. It is hoped that the oridonin/nitrogen
mustard hybrids could exhibit potent synergistic anticancer
effect, leading to a higher antiproliferative efficacy, a broader
therapeutic scope, and a lower systemic toxicity.
a
Reagents and conditions: (a) Boc2O, TEA, dioxane, rt, 3 h, 95% for
compound 7; Ac2O, HCOOH, 50 °C, 5 h, 67% for compound 8; (b)
SOCl2, MeOH, reflux, 12 h, 93%; (c) corresponding anhydrides,
DMAP, DCM, rt, 18 h, 93% for compound 10; 24 h, 91% for
compound 11; (d) ethanol, H2SO4, reflux, 5 h, 94%; (e) ethylene
oxide, H2O, HCOOH, rt, 24 h, 81%; (f) (i) POCl3, 50 °C, 0.5 h; (ii)
10% HCl, 12 h, 81% over two steps.
First, the oridonin analogues (2 and 5) were synthesized as
shown in Scheme 1 according to our previous protocols.16 The
a
Scheme 1. Synthesis of Oridonin Analogues 2 and 5
nitrogen mustard conjugates (16a−f, 17a−c, and 18a−c) were
obtained through the synthetic route outlined in Scheme 3.
The oridonin-coupled nitrogen mustard derivatives were
screened for their in vitro antiproliferative activities against four
human cancer cell lines, human leukemia K562 cells, breast
adenocarcinoma MCF-7 cells, human hepatocellular carcinoma
Bel-7402 cells, and MCG-803 human gastric cancer cells, by the
standard MTT methods, and the results are summarized in
Table 1. The IC50 values revealed that all the target compounds
were more potent than positive control drug chlorambucil and
melphalan whatever the cell line considered, and most of the
conjugates exhibited more potent inhibitory activities against
four cancer cell lines than their parent oridonin. As shown in
Table 1, although melphalan showed more potent cytotoxicity
than chlorambucil and benzoic acid mustard, compounds 16a
and 16d−f incorporated oridonin with melphalan showed less
activity than compounds 16b−c that are incorporated with
chlorambucil or benzoic acid mustard. Considering compounds
16a, 17a, and 18a, varying only the substitution on C-1
position of oridonin, the IC50 values of 16a were higher than
those of 17a and 18a in all tested cell lines. However, when
conjugated with chlorambucil or benzoic acid mustard, the
modification on the C-1 position of oridonin had no impact on
the cytotoxicities of these conjugates; 16b−c, 17b−c, and
18b−c all had relatively high antiproliferative activities against
four cancer cell lines. It is interesting that the benzoic acid
mustard has less cytotoxicity, while when incorporated with
oridonin, conjugate 16b with OH at C-1, is the most potent
hybrid among all the target compounds, with the IC50 values at
1.12 0.07, 0.68 0.02, 0.50 0.01, and 1.09 0.14 μM for
K562, MCF-7, Bel-7402, and MGC-803, respectively.
a
Reagents and conditions: (a) Jones reagent, acetone, 0 °C, 10 min,
88%; (b) 2,2-dimethoxypropane, acetone, TsOH, 56 °C, 20 min, 85%;
(c) Ac2O, TEA, DMAP, rt, 2 h, 95%; (d) 10% HCl, THF (1:1), rt, 30
min, 98%.
oxidation of 1 with Jones reagent afforded ketone 2. The
treatment of 1 with 2,2-dimethoxypropane in the presence of
TsOH provided ketal 3 in 85% yield. Compound 3 upon
reaction with Ac2O/DMAP/TEA led to acetylated compound 4
in the yield of 95%. Deprotection of 4 gave the corresponding
alcohol 5 in almost quantitative yield. Then the nitrogen
mustard intermediates were prepared as depicted in Scheme 2.
The selective protection of the primary amine of melphalan (6)
afforded Boc-protected melphalan (7) or formylmerphalan (8).
Esterification of 6 carried out in dry methanol in the presence
of SOCl2 led to ester 9 in quantitative yield, and subsequent
reaction of 9 with succinic anhydride or glutaric anhydride
provided melphalan derivatives 10 and 11, respectively. The
benzoic acid mustard (15) was prepared according to the
literature procedure.14 At last, the target oridonin-coupled
B
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ACS Medicinal Chemistry Letters
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MX20 cells, representative compounds 16a−c, which are
coupled with melphalan, benzoic acid mustard, and chlor-
ambucil, respectively, were selected to test their antiproliferative
activities against the drug-resistant and parental sensitive cells
by using MTT methods. As shown in Table 2, oridonin had
moderate anticancer activities against both sensitive and drug-
resistant cells. Unfortunately, when conjugated with melphalan,
the antiproliferative activity of compound 16a decreased,
especially on the NCI-H460/MX20 cells, which did not exhibit
any cytotoxicity even at a concentration of 100 μM. This result
indicated that 16a may be a substrate of multidrug transporter
BCRP (breast cancer resistance protein, also called ABCG2).
To our delight, compounds 16b and 16c, the 14-OH modified
oridonin ester of benzoic acid mustard or chlorambucil, not
only displayed potent antiproliferative activities against cancer
cells but also demonstrated activities against drug-resistant
cancer cells. Particularly, hybrid 16b exhibited the most potent
antiproliferative activities against SW620, SW620/AD300,
NCI-H460, and NCI-H460/MX20 cells with IC50 values at
1.96 0.11, 1.86 0.06, 2.35 0.14, and 2.91 0.12 μM,
respectively. Therefore, it clearly illustrated that multidrug
transport P-glycoprotein (P-gp/ABCB1) did not affect
antiproliferative activities of these compounds. As a result, the
design of hybrids from natural product and nitrogen mustards
appears to be a viable strategy to address the problem of MDR
in cancer therapy.
Scheme 3. Synthesis of Oridonin Nitrogen Mustard
Derivatives 16a−f, 17a−c, and 18a−c
a
Nonselective cytotoxicity is also the main effect that limits
the use of optimal doses in most conventional chemo-
therapeutic regiments.19 In an attempt to estimate the toxicity
of conjugates 16b and 16c compared with chlorambucil or
benzoic acid mustard, we tested the toxicities of the compounds
1, 15, 16b, 16c, and chlorambucil on the human normal liver
cells L-02 and human liver cancer cells Bel-7402. Cell apoptosis
induced by these compounds is both concentration and time
dependent; the survival curves of 16b and 16c in Bel-7402 and
L-02 cells after a 72 h treatment showed that 90.10% kill was
achieved for 16b in Bel-7402 at the concentration of 2.5 μM,
but only 25.1% human normal liver L-02 cells were killed at this
concentration. For compound 16c, 90.4% Bel-7402 cells were
a
Reagents and conditions: (a) 7, 8, 10, 11, 15, chlorambucil, EDCI,
DMAP, DCM, rt, 12−24 h, 60%−82%; (b) 10, 15, chlorambucil,
EDCI, DMAP, DCM, rt, 9−16 h, 71%−83%; (c) 10, 15, chlorambucil,
EDCI, DMAP, DCM, rt, 12−18 h, 72−78%.
Multidrug resistance (MDR) is one of the major reasons for
the failure of cancer chemotherapy.17 One of the major
mechanisms in cancer cells that give rise to MDR is the
overexpression of ATP-binding cassette (ABC) transporters.18
To investigate whether these nitrogen mustard-fused oridonin
derivatives are effective on drug-resistant ABCB1-overexpress-
ing SW620/AD300 and ABCG2-overexpressing NCI-H460/
a
Table 1. IC50 Values (μM) of Synthetic Oridonin Nitrogen Mustard Derivatives (16a−f, 17a−c, and 18a−c) against Human
b
Cancer Cell Lines
compd
K562
MCF-7
Bel-7402
MGC-803
oridonin
4.76 0.32
14.60 1.21
80.54 4.73
19.58 1.02
152.15 8.66
14.37 1.24
7.48 0.53
49.31 2.87
24.76 1.35
>200
5.69 0.37
chlorambucil
60.12 5.48
33.37 1.16
142.67 10.23
9.01 0.78
133.64 11.25
36.88 1.21
146.20 9.73
9.30 0.18
melphalan
benzoic acid mustard (15)
16a
16b
16c
16d
16e
16f
8.35 0.74
c
c
c
c
1.12 0.07
0.68 0.02
0.50 0.01
1.09 0.14
c
c
c
c
2.28 0.69
1.32 0.05
1.43 0.09
2.02 0.27
5.74 0.45
12.90 0.96
7.91 0.85
6.92 0.45
6.72 0.58
7.10 0.33
4.71 0.16
c
c
c
1.41 0.10
3.22 0.45
2.36 0.67
c
7.03 0.34
14.58 0.28
3.84 0.32
c
c
c
17a
17b
17c
18a
18b
18c
1.73 0.09
3.63 0.15
2.56 0.51
c
c
c
c
1.35 0.12
0.91 0.03
2.41 0.23
3.76 0.53
c
c
c
c
1.10 0.15
0.79 0.01
1.37 0.06
2.25 0.19
c
c
c
c
1.66 0.22
2.88 0.19
1.41 0.11
3.39 0.27
c
c
c
c
1.17 0.14
0.67 0.01
0.90 0.03
1.60 0.08
c
c
c
c
2.37 0.19
2.25 0.08
2.53 0.47
3.95 0.52
a
b
c
IC50: concentration that inhibits 50% of cell growth. MTT method: drug exposure was for 72 h (means SD, n = 3). *p < 0.001 vs oridonin
group.
C
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a
b
Table 2. IC50 Values (μM) of Representative Compounds 16a−c in the Drug-Resistant and Parental Sensitive Cells
compd
SW620
SW620/AD300
NCI-H460
NCI-H460/MX20
oridonin
16a
6.26 0.33
13.95 0.16
1.96 0.11
2.70 0.07
4.67 0.17
14.92 1.28
1.86 0.06
2.28 0.09
17.14 1.06
28.41 0.76
2.35 0.14
4.81 0.11
21.71 0.98
>100
16b
2.91 0.12
7.26 0.28
16c
a
b
IC50: concentration that inhibits 50% of cell growth. MTT cytotoxicity assay was assessed in pairs of parental and transporter-overexpressing cell
lines: SW620 and ABCB1-overexpressing SW620/AD300 cells; NCI-H460 and ABCG2-overexpressing NCI-H460/MX20 cells.
killed at 5 μM, while only 17.36% normal liver cells were killed.
As seen in Table 3, we found that conjugate 16b has
a
Table 3. IC50 Values (μM) of Compounds 1, 15, 16b, 16c,
and Chlorambucil against Human Liver Cancer Cells Bel-
7402 and Human Normal Liver Cells L-02
b
compd
Bel-7402
L-02
SI
oridonin
chlorambucil
15
7.48 0.53
49.31 1.87
>200
17.78 0.64
105.78 8.22
>100
2.38
2.14
16b
0.50 0.01
1.43 0.09
4.03 0.36
9.62 0.23
8.06
6.73
16c
a
b
IC50: concentration that inhibits 50% of cell growth. SI: selective
index (IC50 on normal cells/IC50 on tumor cells).
Figure 2. (a) IC50 values (μM) of the hybrid (16b), oridonin, benzoic
acid mustard, and oridonin + benzoic acid mustard against the MCF-7
cells; (b) IC50 values (μM) of the hybrid (16c), oridonin,
chlorambucil, and oridonin + chlorambucil against the MCF-7 cells.
approximately 8-fold higher selectivity for Bel-7402 cells
compared with normal L-02 cells, which was even much higher
than that of oridonin. The data suggests that the hybrids from
natural product and nitrogen mustards would be safer for the
cancer patients receiving chemotherapy.
Drug combinations are widely used for the treatment of
cancer to increase the efficiency and reduce side effects.20
Oridonin has been reported to be combined with anticancer
drugs to treat the cancer patients in clinical practice.21 In order
to investigate whether conjugates 16b and 16c exhibited their
cytotoxicities alone or through the synergistic effect, we tested
the potency of equimolar concentration of oridonin, benzoic
acid mustard, or 16b alone, and the combination of equimolar
oridonin and benzoic acid mustard on MCF-7 cells. The results
showed that the combination of the oridonin and benzoic acid
mustard was not superior to conjugate 16b alone at any
concentration. The same phenomenon could also be observed
on the compound 16c (see Supporting Information Figure S1).
As shown in Figure 2, the IC50 values of 1:1 combination of
oridonin and benzoic acid mustard or chlorambucil was 11-fold
or 6-fold higher than that of 16b or 16c on MCF-7 cells,
respectively. These results indicated that hybrids 16b and 16c
were more potent than oridonin or an equimolar mixture of its
components nitrogen mustard and oridonin. Therefore, the
efficacy of 16b and 16c in breast cancer could not be mimicked
by the simple direct combination.
To determine whether the suppression of cell growth by
compound 16b is caused by a cell-cycle effect, the DNA
content of cell nuclei was detected by flow cytometry. As
shown in Figure 3, compound 16b could influence cell cycle
progression at low micromolar concentrations. When Bel-7402
cells were treated with different concentrations of compound
16b (0.125, 0.25, or 0.5 μM), the percentage of cells in G2
fraction decreased from 19.52% to 4.46% associated with a
percentage increase of G1-phase cells (39.66% to 51.98%, see
Supporting Information Table S1).
Figure 3. Effects of hybrid 16b on cell cycle of Bel-7402 cells.
To clarify whether the loss of cancer cell viability promoted
by conjugate 16b is associated with apoptosis, an annexin V-
FITC/propidium iodide (PI) binding assay was performed. Bel-
7402 cells were treated with vehicle alone or with various
concentrations (0.125, 0.25, or 0.5 μM) of compound 16b for
48 h and then stained with FITC-annexin V and propidium
iodide (PI). The percentages of apoptotic Bel-7402 cells were
determined by flow cytometry. As shown in Figure 4, conjugate
16b caused significant induction of apoptosis in a concen-
tration-dependent manner. When treated with 0.125, 0.25, and
0.5 μM compound 16b for 48 h; the percentages of apoptotic
cells were 10.77%, 22.99%, and 48.08% (Q2 + Q4),
respectively, compared with 5.21% of vehicle control. This
result demonstrated that the antiproliferative activity of hybrid
D
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ACKNOWLEDGMENTS
■
The authors thank Drs. S. E. Bates and R. W. Robey (NIH,
Maryland, USA) for the cell lines H460, H460/MX20, SW620,
and SW620/AD300. The authors thank COUP-US.com (New
Jersey, USA) for free editing of the article.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic methods and characterization of target compounds;
procedures for pharmacological activities. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*(J.X.) Tel: +86-25-83271299. E-mail: jinyixu@china.com.
*(Z.-S.C.) Tel: +1-718-990-1432. E-mail: orchenz@stjohns.
edu.
Funding
This work is supported by the National Natural Science
Foundation (No. 81373280) and Huahai Pharmaceutical
Graduate Innovation Fund (CX13B-002HH).
Notes
The authors declare no competing financial interest.
E
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