Novel drug isolated from mistletoe (1: E,4 E)-1,7-bis(4-hydroxyphenyl)hepta-1,4-dien-3-one for potential treatment of various cancers: Synthesis, pharmacokinetics and pharmacodynamics

Article abstract of DOI:10.1039/d0ra03674a

(1E,4E)-1,7-Bis(4-hydroxyphenyl)hepta-1,4-dien-3-one (DHDK) is a novel curcuminoid analogue isolated from mistletoe. DHDK exhibits better anti-tumour activity, higher bioavailability and superior stability than curcumin. DHDK is difficult to isolate from Viscum coloratum, but it can be synthesised. MTT (methylthiazolyldiphenyl tetrazolium bromide) assay was used to evaluate the in vitro cytotoxic activity of synthesised DHDK on 12 cancer cell lines. Results showed that DHDK exhibited excellent potential as an anticancer agent, especially for breast and lung cancer. Efficacy was further evaluated in vivo by using MCF-7 breast cancer models. DHDK showed a dose-dependent relationship without weight reduction, mortality growth inhibition or tissue toxicity. Pharmacokinetics and tissue distribution statistics were determined by LC-ESI-MS/MS. This work provided preliminary data on this natural compound and could open up new prospects for changing related parameters to improve drug efficacy.

Full text of DOI:10.1039/d0ra03674a

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Novel drug isolated from mistletoe (1E,4E)-1,7-
bis(4-hydroxyphenyl)hepta-1,4-dien-3-one for
potential treatment of various cancers: synthesis,
pharmacokinetics and pharmacodynamics†
Cite this: RSC Adv., 2020, 10, 27794
*
Jing Hong, Lin Meng, Peipei Yu, Ceng Zhou, Zhaoyan Zhang, Zhiguo Yu, Feng Qin
*
and Yunli Zhao
(1E,4E)-1,7-Bis(4-hydroxyphenyl)hepta-1,4-dien-3-one (DHDK) is a novel curcuminoid analogue isolated
from mistletoe. DHDK exhibits better anti-tumour activity, higher bioavailability and superior stability than
curcumin. DHDK is difficult to isolate from Viscum coloratum, but it can be synthesised. MTT
(methylthiazolyldiphenyl tetrazolium bromide) assay was used to evaluate the in vitro cytotoxic activity of
synthesised DHDK on 12 cancer cell lines. Results showed that DHDK exhibited excellent potential as an
anticancer agent, especially for breast and lung cancer. Efficacy was further evaluated in vivo by using
MCF-7 breast cancer models. DHDK showed a dose-dependent relationship without weight reduction,
mortality growth inhibition or tissue toxicity. Pharmacokinetics and tissue distribution statistics were
determined by LC-ESI-MS/MS. This work provided preliminary data on this natural compound and could
open up new prospects for changing related parameters to improve drug efficacy.
Received 24th April 2020
Accepted 9th July 2020
DOI: 10.1039/d0ra03674a
rsc.li/rsc-advances
effects, its low water solubility, fast degradation and low
bioavailability hinder its potential medical applications.9
1. Introduction
Extracts of Viscum coloratum (Kom.) Nakai (mistletoe) exhibit
numerous biological activities. Extracts of this whole plant are
used for treatment of various tumours in Europe,10 such as
breast,11 bladder12 and lung cancers.13 Mistletoe extract is
administered as a supportive treatment in cancer therapy in
numerous patients.14 Mistletoe (Viscum album) preparations
consist of active ingredients, such as mistletoe lectins and vis-
cotoxins, which show anti-tumour properties by causing cell
cycle delay or arrest and inducing apoptosis,15 inuencing
tumour angiogenesis16 and exerting immune-potentiating
activities that may strengthen the host defence system against
cancer.17 The components of mistletoe demonstrate anti-
multidrug resistance potential in vitro.18 We obtained (1E,4E)-
1,7-bis(4-hydroyphenyl)hepta-1,4-dien-3-one (DHDK, Fig. 1),
a new diaryl heptanone from V. coloratum.19 DHDK is a new
curcuminoid analogue that has better anti-tumour activity,
higher bioavailability and better stability than curcumin.20
Cancer is a life-threatening problem, and 1.35 billion new cases
are reported annually worldwide. It is the second leading cause
of death globally, and was responsible for an estimated 9.6
million deaths in 2018.1 Breast cancer is the most common
malignancy in females in numerous countries.2 Many types of
cancer treatments, such as surgery, chemotherapy and radiation
therapy, are available. Some people with cancer undergo only
one treatment, but most patients are subjected to a combina-
tion of treatments. However, severe side effects and increasing
multidrug resistance have limited the clinical application of
such treatments.3,4 In this regard, new drugs must be developed.
Natural products have been the focus of research because of
their advantages of unique structure, abundant biological
activity and multidrug resistance elimination. Curcumin is
a polyphenol naturally generated from the rhizome of Curcuma
longa L, which has long been used in traditional Chinese herbal
medicine for treatment of various diseases. Curcumin has
attracted much attention for its pharmacological activities,
especially its anti-tumour ability with various mechanisms.5–8
Although curcumin has anti-tumour efficacy and few side
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
E-mail: jinghong93@163.com; menglin777@163.com; Yupeipei_555@163.com;
zhouceng33@163.com; zhaoyanz86@163.com; zhiguo-yu@163.com; yunli76@163.
com; qf-1998@163.com
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra03674a
Fig. 1 (1E,4E)-1,7-Bis(4-hydroyphenyl)hepta-1,4-dien-3-one.
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The isolation of natural DHDK from V. coloratum is difficult CDDP but was less than 11 mmol Lꢁ1 (3.25 mg mLꢁ1). The activity
and complicated. To our knowledge, methods for synthesis of of DHDK against different cancer cell lines indicated its
this compound have not been reported. Thus, we developed potential as an anticancer agent. In addition, DHDK showed
a total synthesis route in eight steps by using 3-(4-hydrox- stronger potency against the four human breast cancer cell lines
yphenyl)propanoic acid and hydroxybenzaldehyde to provide than the positive control CDDP. The IC50 value of the SKBR3 cell
raw materials for further amplifying the industrial and clinical line was as low as 1.71 mmol Lꢁ1 (Table 1). The curves of the
production of DHDK. The pharmacokinetics and pharmacody- inhibitory effect of DHDK and cisplatin on the growth of
namics of DHDK have also not been investigated. The current different tumour cell lines are shown in Fig. 2.
study involved the preliminary evaluation of DHDK for drug
2.2.2 Cytotoxicity against normal cell lines. The toxicity of
efficacy in vivo and in vitro and the determination of its phar- different concentrations of DHDK on normal cell lines (LO2
macokinetics and tissue distribution. Curcumin has been human normal liver cells, GES-1 human normal gastric
widely studied in different elds. We infer that DHDK would epithelial cells, BEAS-2B human normal lung epithelial cell) was
obtain greater attention than curcumin given it's great advan- investigated (Table 2). The results showed that DHDK has little
tages in terms of it's chemical and physical properties. Results inhibitory effect on normal cells, and the inhibition rate of each
can provide important basic laboratory database support for group is less than 5%, which is much smaller than the inhibi-
further clinical or other studies.
tion rate of DHDK on cancer cells at the same concentration.
This indicates that DHDK has certain selectivity for the toxic
effects of cancer cells and normal cells. Inhibition rate of
different DHDK concentrations to different normal cell lines are
shown in Table 3 (Fig. 3–5).
2. Results and discussion
2.1. Synthesis of DHDK
Inspired by the synthesis of curcumin, our strategy involved the
synthesis of DHDK by aldol reaction. Our route began with
commercially available 3-(4-hydroxyphenyl)propanoic acid and
hydroxybenzaldehyde. The processes were shown in Scheme 1.
3-(4-Hydroxyphenyl)propanoic acid and methanol were used to
2.3. Drug efficacy in vivo
All of the test groups showed inhibition of tumour proliferation
with the descending order of 40 mg kgꢁ1 (high-dose group),
20 mg kgꢁ1 (medium-dose group) and 10 mg kgꢁ1 (low-dose
group), the results were shown in Fig. 6. A high dosage was
more effective than a low dosage. The inhibition rates deter-
mined as tumour weight in 40, 20 and 10 mg kgꢁ1 groups were
47.85%, 35.22% and 28.44% respectively, which were lower
than that positive control group (64.10%) (Table 4). The tumour
weight and tumour inhibition rate of each administration
group and cisplatin group were signicantly lower than that of
the blank group (p < 0.01). The tumour weight and tumour
inhibition rate of each administration group were signicantly
lower than that of the cisplatin group (p < 0.01). From the
Fig. 6(b) and Table S2,† we can see that DHDK groups have
stable weight during the nine days, while the positive control
group has an obvious weight loss. From this phenomenon we
can infer that DHDK might have better drug safety than
cisplatin. Overall, 40 mg kgꢁ1 DHDK exhibited the highest
tumour inhibition rate compared with the other two groups.
generate compound
2 (3-(4-hydroxyphenyl)-propanoic acid
ꢀ
methyl ester) at 35 C. Compound 2 and hydroxybenzaldehyde
were protected with bromomethyl methyl ether to produce
compound 3 (4-(methoxymethoxy)-benzene propanoic acid
methyl ester) (1H NMR spectra shown in Fig. S1†) and
compound 6 (4-(methoxymethoxy)benzaldehyde), respectively.
Compound
5
(4-(methoxymethoxy)-benzenepropanal) was
afforded by the oxidation of compound 4 (4-(methoxymethoxy)-
benzene propanol) (1H NMR spectra shown in Fig. S2†).
Compound 4 was produced by the reduction of compound 3
with lithium aluminium hydride. The condensation of
compound
(methoxymethoxy)-phenyl]-but-3-en-2-one). Compound 8 (1,7-
bis(4-(methoxymethoxy)phenyl)hepta-1,4-dien-3-one) was
6 and acetone afforded compound 7 (3-[4-
generated by the condensation of compound 5 and 7. Finally,
the treatment of compound 8 with HCl furnished the desired
product 1 (1H NMR, 13C NMR, MS are shown in Fig. S3–S5†).
2.2. Cytotoxicity in vitro
2.2.1 Cytotoxicity against cancer cell lines. DHDK showed
cytotoxic activity in the micromolar range on 12 cancer cell
lines. In particular, DHDK showed higher potency than CDDP
2.4. Determination of biochemical indexes in serum and
plasma
(cis-platinum) against four different human breast cancer cell The results of the measured biochemical indicators are shown
lines (SKBR3, MDA-MB231, MCF-7 and MDA-MB453) and in the Table 5. The results showed that the level of ALT in each
human skin malignant melanoma cell line SKMEL-28. The IC50 administration group was slightly lower than those in the blank
value of the ve cancer cell lines treated with DHDK were group, but there was no signicant difference between the
signicantly lower than that of cells treated with CDDP. There administration groups and the blank group. There was no
was no signicant difference in IC50 value between the DHDK signicant difference between the levels of BUN, CRE and AST
group and CDDP group on colon cancer cell (HT-29) and lung in each administration group and that in the blank group. This
cancer cell (Calu-3). The IC50 of other tumour cells treated with result initially indicated that DHDK has no potential liver and
DHDK was signicantly higher than that of cells treated with kidney toxicity.
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Scheme 1 Reagents and conditions: (a) CH3OH, H2SO4, 4 h, 35 ꢀC (b) DIPEA, DCM, acetone, MOMBr, 2 h, reflux (c) LiAlH4, dry THF, 3 h, rt (d)
DMP, DCM, NaHCO3, 4 h, 0 ꢀC (e) MOMBr, K2CO3, acetone, 5 h, reflux (f) H2O, 10% NaOH, acetone, rt (g) LDA, THF, 5 h, ꢁ78 ꢀC (h) HCl
(6 mol Lꢁ1), methanol, 15 min, 60 ꢀC, reflux.
was observed in the nude mice of the positive control group
(Fig. 7). This phenomenon has also been reported in other
related studies.21–23 The tumour slices are shown in Fig. 8. A
small amount of punctate nucleolysis and a low degree of
necrosis were detected in the low-dose group. Analysis of the
2.5. Pathological tissue sections
The tissue slices of female nude mice are shown in Fig. 7, 8 and
S6–S10.† No toxicity was observed in tissues exposed to different
dosages of DHDK. This nding could be due to the fact that
DHDK does not accumulate in tissues. Myocardial bre fracture
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Table 1 The IC50 values (mmol Lꢁ1) of DHDK and CDDP against cancer Table 3 Inhibition rate of different DHDK concentrations to different
cell linesa
normal cell lines
IC50, mmol Lꢁ1
Inhibition rate (%)
Cell line
Cancer type
DHDK
Cisplatin
Group
LO2
GES-1
BEAS-2B
SKBR3*
MDA-MB231*
MCF-7*
MDA-MB453*
Calu-3
A-549*
SKMEL-28*
HT-29
DU-145*
A375*
SKOV3*
N87*
Breast
Breast
Breast
Breast
Lung
Lung
Skin
Colon
Prostate
Melanoma
Ovary
1.71 ꢂ 0.10
4.49 ꢂ 046
9.09 ꢂ 0.46
10.42 ꢂ 0.92
6.65 ꢂ 0.42
8.01 ꢂ 0.44
9.26 ꢂ 0.57
4.79 ꢂ 0.54
4.62 ꢂ 0.22
6.64 ꢂ 0.40
9.59 ꢂ 0.44
10.81 ꢂ 0.50
3.04 ꢂ 0.17 Control group
12.81 ꢂ 0.58 0.14 mmol$Lꢁ1
11.16 ꢂ 0.60 0.42 mmol$Lꢁ1
15.43 ꢂ 0.83 1.26 mmol$Lꢁ1
7.39 ꢂ 0.25 3.8 mmol$Lꢁ1
3.03 ꢂ 0.24 11.3 mmol$Lꢁ1
12.94 ꢂ 0.96 34.0 mmol$Lꢁ1
4.71 ꢂ 0.17 Blank group
2.82 ꢂ 0.14
0
0
0
1.36
1.51
1.87
2.08
2.57
4.17
—
0.17
0.53
1.06
1.35
3.53
4.85
—
0.059
0.50
0.83
0.86
3.10
4.82
—
5.28 ꢂ 0.18
4.15 ꢂ 0.30
Stomach
8.35 ꢂ 0.30
a
IC50: half maximal inhibitory concentration; * signicant difference
compared to corresponding cisplatin group p < 0.05.
Fig. 3 Inhibition rate of different DHDK concentrations to LO2 human
normal liver cell.
Fig. 2 Curves of the inhibitory effects of DHDK and cisplatin on the
growth of different tumour cell lines.
cancer cells. Overall, based on the analyses of tissue slices and
efficacy in vivo, DHDK exhibits anti-tumour effects, and poten-
tial advantages in drug safety over cisplatin.
pathological sections showed point necrosis and dissolved
nucleus in the medium-dose group. A large amount of tumour
necrosis with nuclear envelope breakdown and dissolved
nucleus was observed in the high-dose group. Moreover, a large
amount of tumour cell necrosis with nucleolysis was observed
in the positive control group. The results provide further
evidence that DHDK shows efficacy against anti-MCF-7 breast
2.6. Pharmacokinetic study
Pharmacokinetic parameters were determined by the two-
compartmental analysis of plasma concentration versus time
Table 2 OD values of different DHDK concentrations to different
normal cell lines
OD value (mean ꢂ SD)
Group
LO2
GES-1
BEAS-2B
Control group
0.14 mmol$Lꢁ1
0.42 mmol$Lꢁ1
1.26 mmol$Lꢁ1
3.8 mmol$Lꢁ1
11.3 mmol$Lꢁ1
34.0 mmol$Lꢁ1
Blank group
0.69 ꢂ 0.0091
0.69 ꢂ 0.013
0.68 ꢂ 0.039
0.68 ꢂ 0.022
0.68 ꢂ 0.031
0.68 ꢂ 0.0066
0.67 ꢂ 0.025
0.42 ꢂ 0.0014
0.71 ꢂ 0.022
0.71 ꢂ 0.0031
0.71 ꢂ 0.0098
0.71 ꢂ 0.0051
0.70 ꢂ 0.0035
0.69 ꢂ 0.0097
0.68 ꢂ 0.0075
0.042 ꢂ 0.0020
0.66 ꢂ 0.0086
0.66 ꢂ 0.0017
0.66 ꢂ 0.0028
0.66 ꢂ 0.0027
0.66 ꢂ 0.0010
0.64 ꢂ 0.0028
0.63 ꢂ 0.0030
0.041 ꢂ 0.0005
Fig. 4 Inhibition rate of different DHDK concentrations to GES-1
human normal gastric mucosal cell.
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Table 4 Tumour weight and inhibition rate of various groupsa
Inhibition rate
(IR, %)
Group
Tumour weight (mean)
A
B
C
D
E
834.36 ꢂ 41.00
—
597.04 ꢂ 38.19**
540.53 ꢂ 63.76**
435.13 ꢂ 140.10**#
299.53 ꢂ 109.60**#
28.44**##
35.22**##
47.85**##
64.10**
a
(A) Negative control group, (B) low-dose group, (C) medium-dose
group, (D) high-dose group, (E) positive control. ** Signicant
difference compared to A, p < 0.01; ## signicant difference compared
to (E), p < 0.01.
Fig. 5 Inhibition rate of different DHDK concentrations to BEAS-2B
human normal lung epithelial cell.
has a much shorter half-life than cisplatin, indicating a higher
metabolic rate and/or higher affinity with red blood cells than
cisplatin. The distribution volume of DHDK is extraordinarily
less than the average blood volume of a rat. This may be because
the initial plasma concentration drops so fast that the C0 value
and its error are too large, which leads to the calculated V1 being
too small. This also shows that DHDK might have a strong
affinity with blood cells and has a tendency to quickly distribute
in blood cells. Above all, DHDK might have stronger anti-tumor
effect and lower renal toxicity than cisplatin. We need to further
study the changing process of DHDK in the body in detail and
optimize pharmacokinetic parameters based on this process.
For example, we could use structural modication or drug
delivery systems to optimize pharmacokinetic parameters, such
as ABI-007 paclitaxel nanoparticles of American Biological
Company,25 which can signicantly improve the pharmacoki-
netic parameters to obtain better pharmacodynamics. PD
showed that the tumour inhibition rates of DHDK in 40 mg
by using DAS2.0. The results indicated that DHDK belongs to
the group of fast metabolites. The main pharmacokinetic
parameters are listed in Tables 6 and S3.† At the beginning, the
plasma concentration of DHDK rapidly decreased, the curve
was shown in Fig. 9. Compared with cisplatin, DHDK has poor
pharmacokinetic parameters. We obtained the pharmacoki-
netic parameters of cisplatin from ref. 24, the main pharma-
cokinetic parameters were shown in Table 7. The dosage of
DHDK is 1.4 times higher than cisplatin, but the ‘relative
bioavailability’ of DHDK was only 1.28% of cisplatin. Despite
the low AUC(0–t) of DHDK, the anti-tumour effect was proved to
be good according to the efficacy evaluation in vivo (tumour
inhibition rate of dose 40 mg kgꢁ1 was 47.85% on mice).
Therefore, we can optimize the pharmacokinetic behavior to
gain a more satisfying AUC. DHDK has a slower clearance rate
than cisplatin, which means less excretion through the proto-
type, so DHDK is less toxic in the kidney than cisplatin. DHDK
Fig. 6 (a) Mean tumour volume–time curve of various groups after tail vein injection of DHDK solution. (b) Mean weight–time curve of various
groups after tail vein injection of DHDK solution. (c) Tumour picture of various groups obtained after the mice were sacrificed ((A) negative
control group, (B) low-dose group, (C) medium-dose group, (D) high-dose group, (E) positive control group, n ¼ 6).
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Table 5 The values of ALT, ALT, BUN, CRE in various groups
Groups
BUN (mmol Lꢁ1) mean ꢂ SD
CRE (mmol Lꢁ1) mean ꢂ SD
AST (U Lꢁ1) mean ꢂ SD
ALT (U Lꢁ1) mean ꢂ SD
Blank
9.14 ꢂ 0.084
10.16 ꢂ 1.35
9.74 ꢂ 0.47
9.86 ꢂ 0.33
86.37 ꢂ 5.97
78.85 ꢂ 3.90
87.97 ꢂ 7.63
82.97 ꢂ 2.97
51.11 ꢂ 2.88
50.90 ꢂ 3.04
50.93 ꢂ 0.81
52.98 ꢂ 3.07
37.82 ꢂ 6.80
39.18 ꢂ 0.81
39.70 ꢂ 2.75
38.41 ꢂ 0.67
Low dose group
Medium dose group
High dose group
kgꢁ1, 20 mg kgꢁ1, and 10 mg kgꢁ1 were 47.85%, 35.22%, and content in other tissues. The clearance rates followed the order
28.44%, respectively. The tumour inhibition rate of cisplatin of kidney > spleen > heart > lung > liver (shown in Table 8).
group (5 mg kgꢁ1) was 64.10%. The dosage of DHDK high-dose
group was 8 times higher than cisplatin group and aer dose
conversion, the AUC(0–t) of DHDK high-dose group was 7.3% of 3. Materials and methods
cisplatin group, but the tumour inhibition rate of DHDK in the
high-dose group was 74.6% of the cisplatin group. This data
further suggested that DHDK might have a better anti-cancer
effect than cisplatin by improving pharmacokinetic parame-
3.1. Chemicals and reagents
Methanol, tetrahydrofuran, hydrochloride, triethylamine, for-
mic acid, acetonitrile, anhydrous ethanol and dimethyl sulph-
oxide of HPLC grade were purchased from Concord Technology
(Tianjin, China). Sulfuric acid, anhydrous ether, potassium
bicarbonate and bromomethyl methyl ether were obtained from
ters in vivo.
2.7. Tissue distribution
Tianjin Kaixin Chemical Industry (Tianjin, China). Lithium
The tissue distribution of DHDK at different time intervals is aluminum hydride, anhydrous sodium sulphate, sodium
shown in Fig. 10 and Table S4.† The DHDK concentration in hydroxide, sodium chloride, N,N-diisopropylehylamine and
different tissues in rats followed the order of lung > liver > heart N,N-lithium diisopropylamine were acquired from Tianjin Fuyu
> kidney > spleen. However, the concentration in the tissues was Fine Chemical (Tianjin, China). Anhydrous potassium
low and rapidly declined except for in the kidney and spleen, carbonate, phosphoric acid and isopropanol were supplied by
consistent with the results of the pharmacokinetic study. The Comio Chemical Reagent (Tianjin, China). Petroleum ether,
concentration of DHDK in the lung tissue was higher than that ethyl acetate, dichlorimethane, methyl tertiary butyl ether
in the other tissues. This phenomenon is consistent with the in (MTBE), methanol, anhydrous alcohol and acetone were
vitro cytotoxicity result in Calu-3 lung cancer cell line. Therefore, supplied by Shandong Yuwang Industry (Shandong, China).
the application of DHDK in lung cancer treatment can be Normal saline, Tween 80 and 95% ethanol were bought from
further developed. For example, DHDK spray could be devel- Shenyang Weilong Trade Limited (Shenyang, China). Puried
oped to increase its concentration in the lung and reduce the water was purchased from Shenyang Pharmaceutical University
Fig. 7 Heart histopathology of various groups obtained after the mice were sacrificed ((A) negative control group, (B) low-dose group, (C)
medium-dose group, (D) high-dose group, (E) positive control group).
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Fig. 8 Tumour histopathology of various groups obtained after the mice were sacrificed ((A) negative control group, (B) low-dose group, (C)
medium-dose group, (D) high-dose group, (E) positive control group).
Table 6 Main pharmacokinetic parameters of DHDK after tail vein
injection of 14 mg kgꢁ1 DHDK solution (n ¼ 6)a
Section. Curcumin was purchased from Chengdu Chroma
Biotechnology (Chengdu, China). All other chemical reagents
used were of analytical grade. Methylthiazolyldiphenyl tetrazo-
lium bromide (MTT) and cis-platinum were acquired from
Sigma-Aldrich (St. Louis, MO, USA).
Parameters
Unit
Mean
SD
t1/2a
h
0.064
0.021
t1/2b
h
1.79
459.47
534.32
0.037
0.047
193.20
202.71
0.009
AUC0/t
AUC0/N
V1
mg Lꢁ1
mg Lꢁ1
L kgꢁ1
h
h
3.2. Synthesis of DHDK
The total synthesis route is shown in Scheme 1.
CL
L kg hꢁ1
0.008
0.002
3-(4-Hydroxyphenyl)-propanoic acid methyl ester 2. 3-(4-
Hydroxyphenyl) propanoic acid (2.8 g, 16.67 mmol) was dis-
solved in MeOH (40 mL), concentrated sulphuric acid (0.4 mL)
was added dropwise and allowed to react at 35 ꢀC for 4 h. A
portion of the methanol was removed by concentration under
reduced pressure. 10 mL of water was added to the solution and
adjusted to pH 6 by adding saturated sodium bicarbonate. The
mixture was extracted with EtOAc three times, washed with
brine, dried with anhydrous Na2SO4 and concentrated in
a rotary evaporator, the resulting product (2.8 g, white powder)
was pure enough for the next step (95% yield).
a
SD: standard deviation.
and further puried by passing through a 0.22 mm lter. 3-(4-
Hydroxyphenyl)propanoic acid and hydroxybenzaldehyde were
obtained from Liaoning Benyuan Pharmaceutical (Benxi,
China). DHDK was synthesised by Shenyang Pharmaceutical
University, Pharmaceutical Analysis Teaching and Research
4-(Methoxymethoxy)-benzene propanoic acid methyl ester 3.
Compound 2 (2.8 g, 15.54 mmol) and N,N-diisopropylethyl-
amine (3.5 mL, 21.65 mmol) were dissolved in anhydrous
dichloromethane, stirred for 30 min and 50 mL of acetone and
1.7 mL of bromomethyl methyl ether (21.65 mmol) were added.
The mixture was heated to reux, monitored by TLC and cooled
Table 7 Main pharmacokinetic parameters of DHDK and cisplatin
Parameters
Unit
DHDK
Cisplatin
t1/2a
t1/2b
AUC0/t
V1
h
h
0.064
1.79
459.47
0.008
0.037
0.3
23
36 000
9.2
0.23
mg Lꢁ1
L kg
h
L kg hꢁ1
Fig. 9 Mean plasma concentration–time curves of DHDK after the tail
vein injection of 14 mg kgꢁ1 DHDK solution (n ¼ 6).
CL
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Fig. 10 (a) Mean tissue concentration–time curves of DHDK after tail vein injection of 14 mg kgꢁ1 DHDK solution (n ¼ 6). (b) Mean DHDK
concentrations in various tissues at indicated time points after tail vein injection of 14 mg kgꢁ1 DHDK solution (n ¼ 6).
to room temperature. The mixture was washed with dilute The layers were washed with saturated ammonium chloride,
hydrochloric acid (20 mL, 1.0 mol Lꢁ1) and water, extracted with combined the organic layers, dried with anhydrous sodium
EtOAc three times, dried with anhydrous Na2SO4 and concen- sulphate and concentrated in a rotatory evaporator. The
trated in a rotary evaporator. The resulting crude product was resulting crude residual was puried using a silica gel column
puried by silica gel column chromatography (5% ethyl acetate (3% ethyl acetate in hexane) to give 2.22 g of compound 5 as
in hexane) to give 2.93 g of compound 3 as white crystals (84% colorless transparent oily liquid (93% yield).
yield).
4-(Methoxymethoxy)-benzene
4-(Methoxymethoxy)-benzaldehyde 6. Hydroxybenzaldehyde
Lithium (2.5 g, 20.47 mmol) and anhydrous potassium carbonate (14 g,
propanol
4.
aluminium hydride (1.5 g, 40.18 mmol) was dispersed in 81.89 mmol) were added to acetone (100 mL) and stirred for
tetrahydrofuran (50 mL) at 0 ꢀC. Lithium aluminium hydride in 30 min. Bromomethyl methyl ether (2 mL, 26.61 mmol) was
tetrahydrofuran (10 mL) was added dropwise to compound 3 then added. Aer heating to reux for 5 h, the mixture was
(2.93 g, 13.05 mmol) tetrahydrofuran solution. Aer stirring at cooled to room temperature and ltered. The ltrate was
room temperature for 3 h, 10 mL of ethyl acetate was slowly extracted with EtOAc three times, washed with diluted hydro-
added dropwise and quenched by adding 10 mL of water. The chloric acid (20 mL, 1.0 mol Lꢁ1), washed with water twice,
pH was adjusted to 6 by adding diluted hydrochloric acid. The dried with anhydrous Na2SO4 and concentrated in vacuo. Aer
mixture was extracted with EtOAc three times. The organic conrmation by TLC, the resulting product (3.06 g, light yellow
layers were combined, washed with brine, dried with anhydrous liquid) was pure enough for next step without further purica-
Na2SO4 and concentrated in a rotary evaporator. The resulting tion (90% yield).
product (2.41 g, colorless oily liquid) was pure enough for the
next step (94% yield).
(E)-4-(4-(Methoxymethoxy)phenyl)but-3-en-2-one
Compound 6 (3.06 g, 18.41 mmol) was dissolved in acetone (30
7.
4-(Methoxymethoxy)-benzenepropanal 5. DMP (1,1,1-tri- mL), water (5 mL) and aqueous 10% NaOH (15 mL) were
acetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one) (10.4 g, 24.56 sequentially added at 0 ꢀC with nitrogen protection and allowed
mmol), compound 4 (2.41 g, 12.28 mmol) and sodium bicar- to react at 25 ꢀC. The reaction was monitored by TLC. The
bonate (2.09 g, 24.56 mmol) were sequentially added to DCM mixture was extracted by EtOAc three times. The organic layers
(30 mL). The mixture was stirred at 0 ꢀC for 4 h and monitored were combined, washed with brine, dried with anhydrous
via TLC. Sodium thiosulphate was used to quench the reaction. Na2SO4 and concentrated in a rotary evaporator. The resulting
Table 8 Main pharmacokinetic parameters of DHDK after tail vein injection of DHDK solution (mean ꢂ SD, n ¼ 5)a
Parameters
AUC0/N
Unit
Heart
Liver
Spleen
Lung
Kidney
mg Lꢁ1
h
413.54 ꢂ
2453.46 ꢂ 424.52
5.846 ꢂ 1.02
206.81 ꢂ 84.27
81.66 ꢂ 45.73
1627.09 ꢂ 298.71
8.89 ꢂ 1.97
57.23 ꢂ 32.28
244.93 ꢂ 9.95
364.13
CL
L kg hꢁ1
50.93 ꢂ 26.52
a
SD: standard deviation.
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product was puried by silica gel column chromatography to 10 min until the crystals were completely dissolved. Absorbance
give 3.19 g of compound 7 as white crystals (84% yield). (A value) was determined and processed using the GraphPad
1,7-Bis(4-(methoxymethoxy)-phenyl)hepta-1,4-dien-3-one 8. Prism v5.01 soware. Results were used to draw the dose–
Compound 7 (3.19 g, 18.41 mmol) was dissolved in THF (50 mL) response inhibition curves of the different tumour cell lines.
and cooled to ꢁ78 ꢀC. The system was dried and protected with IC50 values were then calculated.
nitrogen, slowly added with lithium diisopropylamide (19.33
mmol) and allowed to react for 1 h. Compound 5 (2.22 g, 11.43
mmol) in tetrahydrofuran (10 mL) was then slowly added to the
mixture, allowed to react for 5 h, quenched with water and
extracted with EtOAc three times. The organic layers were
combined, washed with brine, dried with anhydrous Na2SO4
and concentrated in a rotary evaporator. The crude residue
(reddish brown oily liquid) was used in the next step without
further purication (36% yield).
3.4. Preparation of DHDK micelles
DHDK micelles were prepared by thin-lm hydration. Specic
amounts of DHDK and Tween 80 were dissolved in 3 mL of
ethꢀanol. The solution was evaporated by rotary evaporation at
50 C to obtain a thin lm. The lm was dispersed with 0.9%
normal saline solution at 50 ꢀC and vortexed for 5 min to obtain
DHDK micelles.
(1E,4E)-1,7-Bis(4-hydroxyphenyl)hepta-1,4-dien-3-one
1.
3.5. Drug efficacy in vivo
Compound 8 was dissolved in methanol (50 mL), hydrochloric
acid (10 mL, 6 mol Lꢁ1) was quickly added at 60 ꢀC and allowed
3.5.1 Establishment of MCF-7 breast cancer model. The
to react for 15 min. The mixture was extracted with EtOAc three human breast cancer cell MCF-7 was trypsinised and centri-
times, washed with brine, dried with anhydrous Na2SO4 and fuged at 1000 rpm for 5 min. The supernatant was removed, and
concentrated in a rotary evaporator. The resulting product was the residue was washed twice with PBS and centrifuged again.
puried by open silica gel column chromatography and octa- The supernatant was removed, and the residue was resus-
decyl silyl chromatography to give 0.67 g of compound 1 as pended in the culture medium. Finally, the residue was
yellow powder (55% yield, 96% purity).
adjusted to a concentration of 5 ꢃ 107 cells per mL with phys-
iological saline and injected (0.2 mL, 1 ꢃ 107) into the ante-
brachium right axillary of a nude mouse. Tumour formation
time, growth, inltration and metastasis were observed and
3.3. Drug efficacy in vitro
3.3.1 Cell culture information. Calu-3, A-549 (human lung recorded in detail.
cancer), A375 (human skin melanoma), SKMEL-28 (human skin
3.5.2 Dosage regimen. Thirty female nude mice were
melanoma), DU-145 (human prostate carcinoma), N87 (human randomly divided into ve groups (n ¼ 6 per group). DHDK
gastric epithelial), SKBR3, MDA-MB231, MCF-7, MDA-MB453 micelles were injected into nude mouse models of well-
(human breast adenocarcinoma), SKOV3 (human ovarian established MCF-7 xenogra tumour every day for 9 days.
cancer) and HT-29 (human colorectal carcinoma) cell lines were Dosages of 10, 20 and 40 mg kgꢁ1 were administered to mice in
obtained from the American Type Culture Collection (ATCC). low-, medium- and high-dosage groups, respectively. The posi-
The cells were cultured in DMEM or RPMI-1640 media at 37 ꢀC tive group (cisplatin) was established by administering 5 mg
in an atmosphere of 5% CO2 and 100% relative humidity under kgꢁ1 cisplatin to the same conditioned female nude mice via the
sterile conditions to maintain continuous logarithmic growth. tail vein for 9 days. The blank control group consisted of mice
The media were supplemented with 10% heat-inactivated fetal that were not given any drug. Tumour volume and mouse
bovine serum and 1% penicillin/streptomycin.
weight were calculated and measured during the experiments.
3.3.2 MTT cell proliferation assay. With CDDP as positive
control, MTT assay was used to determine the inhibition rate of
DHDK on the proliferation of the 12 tumour cell lines and 3
normal cell lines. The anti-tumour spectrum of DHDK was then
initially screened. The cells in the logarithmic growth phase (5
ꢃ 104 cells per mL, 100 mL) were separately seeded in 96-well
at-bottomed microculture plates (approximately 4000–5000
cells per well). The edges of the wells were lled with sterile PBS.
Aer culture for 24 h, the cells were completely adhered to the
wall and the original culture solution was removed. The nal
concentrations of DHDK in the wells were as follows: 0.14, 0.41,
1.2, 3.7, 11.3 and 33.3 mmol Lꢁ1 in 200 mL of media. The positive
control was CDDP with concentrations of 0.14, 0.42, 1.26, 3.8,
11.3 and 34.0 mmol Lꢁ1. Each concentration in the treatment
groups as well as the blank and control groups were tested in
3.6. Determination of biochemical indexes in serum and
plasma
Twenty four female mice were randomly divided into four
groups (n ¼ 6 per group). DHDK micelles were injected into
mice every day for 9 days. Consistent with the dosing regimen in
vivo efficacy evaluation, the mice were killed on the last day aer
administration, and plasma and serum were taken. Take the
appropriate amount of mice serum and follow the instructions
in the kit to determine the AST, ALT and CRE in the serum. Take
the appropriate amount of mice plasma and follow the
instructions in the kit to determine the BUN in the plasma.
3.7. Pathological tissue sections
sextuplicate (n ¼ 6). Aer incubation for 72 h, the cells in each Histopathological technique is used for comprehensive obser-
well were added with 100 mL MTT (5 mg mLꢁ1). The plates were vation of specic tissue samples and the state of structure. In
incubated for 4 h, the supernatant was aspirated and 150 mL of more than 100 years of application and development, this
DMSO was added to each well. The plates were shaken for technique has become an important technology in life sciences
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and is the gold standard for disease diagnosis in clinical prac- analytical method performed was the same as that in the
tice. In the current study, we evaluated the conditions of the pharmacokinetics study.
pathological sections of different tissues. At the end of the 9 day
treatment, the tumour-bearing nude mice were anesthetised
and killed to collect fresh tissues (heart, liver, spleen, lung,
kidney, brain and tumour). The tissues were soaked and xed
with 10% formalin solution, embedded in paraffin and cut into
4 mm paraffin sections. The sections were subjected to HE
staining to detect the toxicity of DHDK.
3.10. Ethical statement
All in vivo experimental studies were performed in accordance
with the Guideline for Animal Experimentation of Shenyang
Pharmaceutical University and with the approval from the
Animal Ethics Committee of Shenyang Pharmaceutical Univer-
sity (Approval number: 211002000310215).
3.8. Pharmacokinetics in vivo
4. Conclusion
3.8.1 Dosage regimen and sample collection. Based on in
vivo and in vitro activities, DHDK micelle injection was con-
ducted for PK studies at the dosage of 14 mg kgꢁ1 by tail vein.
Prior to treatment, the rats were fasted for 12 h and allowed to
drink freely. Blood samples (0.3 mL) were collected from the
orbital venous plexus into a heparinised centrifuge tube at 0,
0.033, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 8 and 12 h. The blood
samples were centrifuged. Plasma was obtained and stored at
This study involves synthesis of DHDK, determination of its in
vitro and in vivo drug efficacy with respect to dosage and anal-
yses of tissue slices, pharmacokinetics and tissue distribution.
DHDK is a novel drug that can be completely synthesised by
asymmetric aldol condensation. DHDK has anti-tumour effect
in vitro, especially against breast and lung cancers. This study
optimized the dosage and the in vivo effect of DHDK was posi-
tively related to the dosage applied. The high-dose group
showed better antitumor effect than the other two dose groups
and was without toxicity. DHDK has little inhibitory effect on
normal cells, which indicates that DHDK has certain selectivity
for the toxic effects of cancer cells and normal cells. The results
of the measured biochemical indicators of AST, ALT, BUN and
CRE indicated that DHDK has no potential liver and kidney
toxicity. The result of tissue distribution showed that the drug is
easy to distribute in lung tissue, so the effect in treating lung
cancer may be need further research.
ꢀ
ꢁ80 C until analysis.
3.8.2 LC-ESI-MS/MS condition. LC-ESI-MS/MS (Waters I-
Class UPLC, Waters Xevo TQ-S) was used to determine plasma
concentration by using a Thermo Hypersil GOLD C18 column
(2.1 mm ꢃ 100 mm, 1.9 mm). The mobile phase consisted of
0.1% formic acid in water (A) and acetonitrile (B). The gradient
elution conditions were 0–2.5 min (A : B ¼ 55 : 45 to 15 : 85),
2.5–2.6 min (A : B ¼ 15 : 85 to 55 : 45) and 2.6–3.5 min (A : B ¼
55 : 45 to 55 : 45); injection volume of 2 mL; and ow rate of 0.3
mL minꢁ1. The scanning mode of MS was MRM (multi-reaction
monitoring). DHDK was determined with precursor ion at m/z
294.98 and product ion at m/z 106.67. Internal standard cur-
cumin was analysed with precursor ion at m/z 369.1 and product
ion at m/z 284.8.
Conflicts of interest
The authors declare no conict of interest.
3.8.3 Sample pretreatment. The rat plasma was thawed and
vortexed. The plasma (100 mL) was added with 10 mL of the
internal standard and 10 mL of methanol, vortexed for 1 min
and added with 2 mL of MTBE. The mixture was vortexed for
5 min and centrifuged at 12 000 rpm at 4 ꢀC for 5 min. The
supernatant was blow dried at 37 ꢀC, and the residue was
redissolved with 100 mL of the initial mobile phase (water :
acetonitrile ¼ 55 : 45) and vortexed for 3 min to dissolve. The
mixture was centrifuged at 12 000 rpm for 3 min at 4 ꢀC, and 2
mL of the supernatant was injected into the system.
Acknowledgements
The authors are grateful for the Natural Science Foundation of
Liaoning Province for funding toward nancial support (No.
20180551065)
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