NQO1-Selective Activated Prodrug of Triptolide: Synthesis and Antihepatocellular Carcinoma Activity Evaluation

Article abstract of DOI:10.1021/acsmedchemlett.8b00404

Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis. Due to its poor response to conventional chemotherapy drugs, the prognosis for its survival is the worst. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an attractive ant

Full text of DOI:10.1021/acsmedchemlett.8b00404

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Letter
An NQO1-selective Activated prodrug of Triptolide: Synthesis
and Anti-Hepatocellular Carcinoma Activity Evaluation
Meilin Liu, Wei Song, Xi Du, Jingtian Su, Kang Dong, Yong Chen, and Zhihong Peng
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00404 • Publication Date (Web): 27 Nov 2018
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An NQO1-selective Activated Prodrug of Triptolide: Synthesis
and Anti-hepatocellular Carcinaoma Activity Evaluation
Meilin Liua,‡, Wei Songa,‡, Xi Dua, Jingtian Sua, Kang Donga, Yong Chena,*, Zhihong Penga,*
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aSchool of Life Sciences; Hubei Province Key Laboratory of Biotechnology of Chinese Traditional
Medicine; National & Local Joint Engineering Research Center of High-throughput Drug Screening
Technology; Hubei University, Wuhan 430062, China
KEYWORDS: CX-23, triptolide (TP), anti-hepatocellular carcinoma, NAD(P)H:quinone oxidoreductase 1 (NQO1)
ABSTRACT: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis. Due to its poor response to
conventional chemotherapy drugs, the prognosis for its survival is the worst. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an
attractive anticancer target due to its overexpression in HCC. Although triptolide (TP) possesses potent anti-tumor activity, its
clinical practice is greatly limited due to its general toxicities and narrow therapeutic window. Herein, we develop an NQO1-
selective activated TP analog, named CX-23, which exhibited antiproliferation of HepG2 over normal hepatocytes in vitro. In vivo
study shows that CX-23 can not only prevent the hepatocellular carcinoma progression but also migrate the liver and kidney
toxicity. These findings indicate that NQO1 may serve as a targeted delivery system to release an antitumor reagent and CX-23 may
be a promising lead for developing targeted anti-hepatocellular carcinoma drugs.
Primary liver cancer is one of the most common malignant
tumors of the digestive tract in the clinic, 90% of which are
hepatocellular carcinoma (HCC). Epidemiological surveys
show that approximately 500,000 patients worldwide die of
HCC every year, ranking fourth in the world for cancer
mortality (1). For patients with early liver cancer, liver
transplantation and resection are the preferred treatments for
liver cancer, but most patients have reached late stage or
metastasis at the time of diagnosis and cannot meet surgical
criteria. For patients who cannot undergo surgery,
chemotherapy is one of the important treatments for liver
cancer(2, 3). Chemotherapy can not only control the growth and
spread of tumors, but also transform inoperable tumors into
operable tumors and increase the cancer cure rate. Conventional
drugs used for anti-hepatocellular carcinoma are cytotoxic
drugs, among which the more commonly used cytotoxic drugs
are doxorubicin, cisplatin, docetaxel, and fluorouracil (4).
These drugs are more toxic and less effective. Sorafenib is the
first-line anti-hepatocellular carcinoma drug approved by Food
and Drug Administration, however, some patients still have
treatment failure or tolerability in clinical use. Compared with
the control group, the sorafenib treatment group only prolonged
the life of the patient for two months(5). Therefore, there is
urgent need for anti-hepatocellular carcinoma reagents
discovery and development.
at the nanomolar (nM) levels. Moreover, TP can circumvent
tumor drug-resistant tumor cells and sensitize cancer cells to
other chemotherapeutic agents(11-13). The xeroderma
pigmentosum type B (XPB/ERCC3) subunit (ATPase activity)
of Transcription factor II Human (TFIIH) was generally
accepted the principal TP target. TP can inhibit the ATPase
activity of XPB by covalently binding with the thiol of Cys342
of XPB, which initiate transcription by RNA polymerase II
(RNAPII) and nucleotide excision repair, offers a consistent
mechanism that can be used to interpret most of the known
pharmacologic activities of TP(14). Although triptolide has
great potential as an antitumor agent, its application in the clinic
is restricted due to its liver and kidney toxicities (15-17). The
most common side effects of TP are observed in the liver and
kidney, where the toxicities are mainly induced by the
accumulation of parent drug(18).
As a great promising an anticancer drug lead, several
analogues of TP have been developed (Figure S1).
Noteworthily all the analogues of TP in clinical development
were adopted prodrug strategies and keep the intact core
structure of TP. PG490-88 (omtriptolide), an esterification of
14-OH group of TP by a succinate acid, failed in phase I clinical
trial for its slow and incomplete biotransformation in vivo(19).
It indicated the modification of the 14-OH TP impede the
enzymatic cleavage due to the hindrance of stereoselectivity.
Minnelide is a phosphonooxymethyl derivative of TP and the
latest TP analog which is under the phase Ⅱ clinical trial.
Minnelide can be metabolized by alkaline phosphatase and then
spontaneously releases TP to carry out the antitumor activity
(20). Therefore, simple esterification of 14-OH of TP may be
not a good method, but spontaneously transformation is an
effective way to achieve the successful goal.
Triptolide (TP) is a di-terpene tri-epoxide compound purified
from Tripterygium Wilfordii Hook F (TWHF) (6) and possesses
the ability to treat diseases such as tumor(7), immune system
disorders(8), diseases of central nervous system (9) and
cardiovascular (10). Among the pharmacological effects, its
antitumor activity attracted the most attention of researchers. In
vitro studies have shown the high potential antiproliferation of
TP against all 60 NCI human tumor cells with the concentration
Due to on-target toxicity of TP, it is necessary to develop a
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cancer targeted pro-drug approach. NAD(P)H:quinone
XPB makes it an ideal prodrug which can be converted to TP
by cellular NQO1 but devoid of undesirable toxicity prior to
entry into cells.
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oxidoreductase 1 (NQO1) is a cytosolic reductase and a
homodimeric flavoprotein that utilizes NAD(P)H to catalyze
the two-electron reduction of a broad range of substrates most
notably quinones(21). NQO1 is an attractive anticancer target
for its higher levels expressed in many solid tumors (22). In
hepatocellular carcinoma, the positive rate and highly positive
rate of NQO1 protein were 86.42% and 58.97% in HCC tissues,
and were significantly higher than in either adjacent nontumor
tissues or normal liver tissues. Consistently, the five years
survival rates of the cancer patients with over-expressed NQO1
are lower compared with those with low NQO1 levels(23, 24).
Several classes of bio-reductive compounds that can undergo
NQO1 reduction to active species have been developed (25-27).
Mitomycin C is present in a clinically used antitumor antibiotic.
Other quinones such as β-lapachone, EO9, AZQ and RH1 that
show improved properties over mitomycin have not entered
clinical research(22). Alternative strategy is that the antitumor
reagents was selectively activated by NQO1 in cancer cells.
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We have designed and screened TP analogs which contains
quinone propionic acid structure (Figure S2). The resulting
complexes were scored using the AutoDock Vina and the
highest scoring compound (CX-23) was selected for further
analysis. It can be clearly seen that quinone moiety of CX-23
binds with the residues of the NQO1 active site and flavin
adenine dinucleotide (FAD) which is required for NQO1
catalytic activity (Fig. 1A). The leaving group triptolide is left
outside the substrate binding pocket, suggesting that the
interaction of quinone with NQO1 active site is not affected by
the introduction of triptolide to quinone.
CX-23 contains a propionic acid, as a linkage between
quinone and the C14 hydroxy group which is the most reactive
and chemical modified functional group in TP. Reduction of the
quinone propionic acid esters or amides bearing three methyl
groups would lead to the corresponding hydroquinones in
which severe steric repulsion between three methyl groups
promotes rapid lactonization and release of an alcohol or amine.
Several the quinone propionic acid compounds have been
investigated for reductase-activated prodrugs such as those
based on mustard and oxindoles for anticancer therapy (28).
The quinine can be reduced by NQO1 and the propionic acid
can facilitate the spontaneously transformation to release TP
(Figure 1C), which can covalently bind to the XPB and exert its
pharmacological activities.
Figure 1. (A) the best pose of CX-23 bound NQO1 (ΔE= -12.2
kcal/mol) using AutoDock Vina; The image was visualized
using Pymol. Graphical representation of CX-23-NQO1
complex, TP Complex represented as capped sticks. FAD is
expressed in blue. (B) synthesis of CX-23; (C) proposed
metabolic and transformed pathways of CX-23 activated by
NQO1.
We evaluated the effects of CX-23 and TP on the proliferation
of HepG2 and normal hepatocytes. CX-23 presents
a
comparable antitumor cell activity with IC50 value of 54.3 nM
and TP retained the considerable antitumor cell activity with
IC50 value of 43.3 nM (Figure S4B). In contrast, in the case of
normal hepatocytes with undetectable levels of NQO1, the IC50
value was 540 nM, a value 9.89-fold above its anti-HepG2
activity and 14.3-fold higher than TP with the values of 37.7
nM (Figure S4C), which means a wide therapeutic index for
CX-23. The levels of NQO1 activities both in normal
hepatocytes and HepG2 cells were studied. The results show
that the value of NQO1 activity is 361 ± 84 nmol /min/mg
protein in HepG2 cells lysates, but undetected NQO1 activity in
normal hepatocyte lysates.
To synthesis of CX-23, hydroquinone was first converted into
CX-21 upon the reaction with methyl β, β-dimethylacrylate in
the presence of methanesulfanic acid. CX-22 was synthesized
using
NBS
and
esterified
(DCC)
with
TP
and
involving
N, N-
dicyclohexylcarbodiimide
dimethylaminopyridine (DMAP) to yield CX-23 (Figure 1B).
The structure of the CX-21, CX-22 and CX-23 were confirmed
by mass spectrometry and NMR spectrometry (Figure S3).
First, we studied if CX-23 was targeted to XPB. The effect of
CX-23 on XPB ATPase activity was evaluated (Figure S4A).
CX-23 had no inhibitory effects on the XPB ATPase activity,
whereas TP, which had been proved to target the XPB presented
a higher inhibitory, indicating CX-23 cannot directly bind with
XPB. Due to the same target for the toxicity and
pharmacological activity of TP, the inability of CX-23 to inhibit
To determine whether CX-23 depended on NQO1-activated for
its antiproliferation, we test CX-23 in combination with a
NQO1 inhibitor, dicoumarol(29), which alone shows no effect
on the HepG2 with the concentration up to 20 µM. From Figure
S4B, we can see that CX-23 show the antiproliferation of
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HepG2 when sole use. However, when in combination with
according for xenobiotic oxidation, indicating the carboxyl
ester hydrolase-resistance of CX-23 and non-oxidized by
CYP450 enzymes.
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dicoumarol, the antitumor efficacy of CX-23 has lost partly,
suggesting that dicoumarol can impede the antiproliferation of
HepG2 by CX-23 and CX-23 may depend on NQO1 to exert its
anti-hepatocellular carcinoma activity.
We have set up the analytical method which showed good
linearity, selectivity, and reproducibility and this method (Table
S1, S2) can be applied for the pharmacokinetics study of CX-
23 (Figure 2D, Table S3). After a single intraperitoneal (ip)
dose at 1.0 mg/kg, CX-23 was absorbed quickly with the
absorption half-life value of 1.63 min and the time to reach the
maximum plasma concentration (tmax) is 3 min with a
maximum concentration of 243 µg/L. CX-23 was also
distributed quickly with the distribution half-life value of 15.1
min. The ratios of AUCliver / AUCplasma and AUCkidney / AUCplasma
are less than 5%, which means less compound exposure in liver
and kidney. The plasma concentration of CX-23 was sustained
above IC50 for 40 min and declined slowly to 1.38 µg/L at 2h.
After 2h, the levels of CX-23 in plasma was under detectable.
The terminal half-life was 154 min, indicating the parent drug
was eliminated quickly without any cumulation in vivo.
The in vivo anti-hepatocellular carcinoma activity and toxicity
of CX-23 were evaluated by using HepG2 xenograft nude mice
model. In preliminary experiment, we found there is no obvious
toxic symptoms of CX-23 after a 1.0 mg/kg ip dose and TP after
a 0.2 mg/kg ip dose. Accordingly, one week after HepG2 cell
inoculated, mice were dosed once daily by ip over a period of
two weeks. As shown in Figure 3, both TP and CX-23 caused
an effective reduction in tumor growth. The equimolar dosage
of CX-23 shows the similar antitumor effects (Figure.3A, 3B)
as TP. During the whole treatment period, there are no
significant body weight changes (Figure.3C) in the treated
animals, suggesting there is no or minimal toxicity.
A complementary assay was used to study the role of NQO1
in the transformation of CX-23 by investigating the metabolism
of CX-23 in HepG2 lysates and recombinant NQO1. The
metabolism half-life of CX-23 in HepG2 lysates is 7.27 min
(Figure S5A) and two major metabolites (TP and CX-21) are
identified (Figure 2A). Dicoumarol shows concentration-
dependent inhibition of CX-23 metabolism in NQO1 and the
transformation of CX-23 into TP was completely inhibited with
the dicoumarol concentration of 20 µM (Figure 2B). The
recombinant NQO1 can also transform CX-23 into TP and CX-
21 in the presence of NADPH with apparent Km and Vmax of
0.309 µM and 0.223 pmol/mg/min, respectively (Figure 2C).
Together, the results suggest that NQO1 can facilitate the
transformation of CX-23 into TP. The reduction of CX-23 is
NQO1-dependent and the products generated include CX-21
and TP, the latter being capable of inhibit ATPase activity.
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Figure 2. (A) the metabolism of CX-23 in HepG2 lysates with
the presence of NADPH; (B) metabolism of CX-23 by NQO1
with the presence of dicoumarol; (C) formation rates of TP in
recombinant NQO1 from CX-23; (D) pharmacokinetics of CX-
23 in mice after 1.0 mg/kg ip dose.
To confirm that the antitumor activity of CX-23 was due to
the activation by NQO1. We used a panel of primary and cancer
cell lines which has high expression of NQO1 to obtain a profile
of the general cytotoxicity of CX-23. For human keratinocytes
and human skin fibroblast, the average IC50 value was above
1.0 µM. In comparison, the IC50 value for Hela and A549 were
38.2 nM and 59.4 nM (Figure S4D), respectively, indicating
that CX-23 was more selective for the highly-expressed NQO1
tumor cells.
Figure 3. Anti-hepatocellular carcinoma activity of CX-23
compared with TP in nude mice. (A) tumor volume change
during the experiment; (B) tumor weight at the end timepoint;
(C) mice weight during the experiment; (D)images of tumor
tissues at the end timepoint. (**p < 0.01 as compared with
control; nꢀ=ꢀ8 mice/group). Student’s t test was performed for
the statistical analysis.
Most drugs do not have specific tissue distribution and are
easier to be eliminated in plasma and liver. Only a small number
of drugs enter the treatment site, which can cause less effective.
In this case, we carry out the metabolic stability of CX-23 both
in human plasma and human liver microsomes. After 1 h
incubation in vitro, the half-life of CX-23 in human plasma
(Figure S5B) and liver microsomes (Figure S5C) are above 1 h.
The plasma containing abundant carboxyl ester hydrolase and
the microsomes containing cytochrome P450 enzymes
To further determine the toxicity of CX-23, Alanine
aminotransferase (ALT), Aspartate aminotransferase (AST)
and creatinine, Blood urea nitrogen levels were used to evaluate
the liver and renal toxicity. As shown in Figure S6, there is no
difference for TP and CX-23 group in the levels of ALT and
AST, and no pathological abnormalities found in the liver
compared with the control group. However, the levels of
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creatinine and BUN in TP group are significantly increased
estimates of incidence and mortality worldwide for 36 cancers
in 185 countries. CA: a cancer journal for clinicians 2018.
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compared with control group (Figure 4), indicating abnormal
renal function. The histological examination (Figure 4B-E)
showed that t glomerular capillary loop lesions were observed
in TP treated group.
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Figure 4. Effect of TP/CX-23 on kidney of nude mice. (A) the
toxicity evaluation of TP and CX-23 on serum biochemical
parameters. Mice was dosed every day for 15 days. * p < 0.05,
** p < 0.01 vs. control. (B) control group kidney section; (C)
kidney section of TP (0.2 mg/kg)-treated group; (D) kidney
section of CX-23 (0.3 mg/kg)-treated group; (E) kidney section
of CX-23 (1.0 mg/kg)-treated group. (H&E staining, X200)
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In conclusion, CX-23, a quinone propionic acid conjugated
TP and prodrug has shown excellent anti-hepatocellular
carcinoma activity, impeding the tumor cells growth
meanwhile reducing the kidney toxicity. CX-23 represents a
potent new targeted to NQO1-activated antitumor drugs.
Schwertfeger KL, Sangwan V, Banerjee S, Saluja AK, Griffith
TS. Triptolide enhances the tumoricidal activity of TRAIL
against renal cell carcinoma. FEBS J 2015;282(24):4747-
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ASSOCIATED CONTENT
Supporting Information
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Zheng Y, Zhang WJ, Wang XM. Triptolide with
potential medicinal value for diseases of the central nervous
system. CNS Neurosci Ther 2013;19(2):76-82.
10.
J, Sun H, Ding Y, Zhao P, Gong K. Triptolide protects rat
heart against pressure overload-induced cardiac fibrosis. Int J
Cardiol 2013;168(3):2498-2505.
The supporting Information is available free of charge on the
ACS Publications website.
Zhang Z, Qu X, Ni Y, Zhang K, Dong Z, Yan X, Qin
Applied methods, Figures S1-S6 and Table S1-S3.
11.
Zhu W, Li J, Wu S, Li S, Le L, Su X, Qiu P, Hu H,
AUTHOR INFORMATION
Corresponding Author
Yan G. Triptolide cooperates with Cisplatin to induce
apoptosis in gemcitabine-resistant pancreatic cancer. Pancreas
2012;41(7):1029-1038.
Email: zpeng1@nd.edu (Zhihong Peng); cy101610@qq.com
(Yong Chen)
12.
Chen Z, Sangwan V, Banerjee S, Chugh R, Dudeja
V, Vickers SM, Saluja AK. Triptolide sensitizes pancreatic
cancer cells to TRAIL-induced activation of the death receptor
pathway. Cancer Lett 2014;348(1-2):156-166.
Author Contributions
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Li Y, Hu S. Triptolide sensitizes liver cancer cell
‡ Meilin Liu and Wei Song have contributed equally.
Notes
lines to chemotherapy in vitro and in vivo. Panminerva Med
2014;56(3):211-220.
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D, Liu JO. Covalent modification of a cysteine residue in the
XPB subunit of the general transcription factor TFIIH through
single epoxide cleavage of the transcription inhibitor
triptolide. Angew Chem Int Ed Engl 2015;54(6):1859-1863.
15.
M, Huang Z. Activation of the farnesoid X receptor attenuates
triptolide-induced liver toxicity. Phytomedicine
2015;22(10):894-901.
He QL, Titov DV, Li J, Tan M, Ye Z, Zhao Y, Romo
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Jin J, Sun X, Zhao Z, Wang W, Qiu Y, Fu X, Huang
The study was supported by Science Foundation for the Youth
Scholars of Hubei Provincial Department of Education for
Science and technology research program (No.: Q2017007).
16.
Li XX, Du FY, Liu HX, Ji JB, Xing J. Investigation
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An NQO1-selective Activated prodrug of Triptolide: Synthesis and Anti-Hepatocellular
Carcinoma Activity Evaluation
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Meilin Liu a,‡, Wei Songa,‡, Xi Dua, Jingtian Sua, Kang Donga, Yong Chena,*, Zhihong Penga,*
aSchool of Life Sciences; Hubei Province Key Laboratory of Biotechnology of Chinese
Traditional Medicine; National & Local Joint Engineering Research Center of High-throughput
Drug Screening Technology; Hubei University, Wuhan 430062, China
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