Synthesis and biological evaluation of paclitaxel and vorinostat co-prodrugs for overcoming drug resistance in cancer therapy in vitro

Article abstract of DOI:10.1016/j.bmc.2019.02.046

Paclitaxel (PTX) is the first-line treatment drug for breast cancer. However, drug resistance after a course of treatment and low selectivity restricted its clinical utility sometimes. In this study, we successfully bound PTX and vorinostat (SAHA) to form

Full text of DOI:10.1016/j.bmc.2019.02.046

Accepted Manuscript
Synthesis and biological evaluation of paclitaxel and vorinostat co-prodrugs for
overcoming drug resistance in cancer therapy in vitro
Shuangxi Liu, Kaili Zhang, Qiwen Zhu, Qianqian Shen, Qiumeng Zhang, Jiahui
Yu, Yi Chen, Wei Lu
PII:
S0968-0896(18)31861-3
https://doi.org/10.1016/j.bmc.2019.02.046
BMC 14782
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 November 2018
18 February 2019
20 February 2019
Please cite this article as: Liu, S., Zhang, K., Zhu, Q., Shen, Q., Zhang, Q., Yu, J., Chen, Y., Lu, W., Synthesis and
biological evaluation of paclitaxel and vorinostat co-prodrugs for overcoming drug resistance in cancer therapy in
vitro, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.046
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Graphical Abstract
In vitro hydrolysis, co-prodrug 1a could release PTX and SAHA more efficiently and was further prepared to nanomicelles
with mPEG2000-PLA1750
a nanomicelles had a comparable or even better cytotoxicity than PTX especially in the drug-resistant MCF-7/ADR cells.
.
1
Synthesis and biological evaluation of
paclitaxel and vorinostat co-prodrugs for
overcoming drug resistance in cancer
therapy in vitro
Leave this area blank for abstract info.
a,1
b,c,1
a
b
a
a
b,
Shuangxi Liu , Kaili Zhang , Qiwen Zhu , Qianqian Shen , Qiumeng Zhang , Jiahui Yu , Yi Chen *, Wei
a,
Lu *
a
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular
Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, P. R. China.
b
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, P. R. China
c
University of Chinese Academy of Sciences, NO.19A Yuquan Road, Beijing 100049, P.R. China
*
Corresponding authors: Tel: +86 21 5080 6600; Email: ychen@simm.ac.cn (Y. Chen);
Tel: +86 21 6223 8771; Email: wlu@chem.ecnu.edu.cn (W. Lu)
1
These authors contributed equally to this work.

Bioorganic & Medicinal Chemistry
Synthesis and biological evaluation of paclitaxel and vorinostat co-prodrugs for
overcoming drug resistance in cancer therapy in vitro
a,1
b,c,1
a
b
a
a
b,*
Shuangxi Liu , Kaili Zhang , Qiwen Zhu , Qianqian Shen , Qiumeng Zhang , Jiahui Yu , Yi Chen ,
Wei Lu
a,*
a
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China
Normal University, 3663 North Zhongshan Road, Shanghai 200062, P. R. China.
b
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai institute of Materia Medica, Chinese Academy of Sciences, Shanghai
2
01203, P. R. China
c
University of Chinese Academy of Sciences, NO.19A Yuquan Road, Beijing 100049, P.R. China
Corresponding authors: Tel: +86 21 5080 6600; Email: ychen@simm.ac.cn (Y. Chen);
*
Tel: +86 21 6223 8771; Email: wlu@chem.ecnu.edu.cn (W. Lu)
1
These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Paclitaxel (PTX) is the first-line treatment drug for breast cancer. However, drug resistance
after a course of treatment and low selectivity restricted its clinical utility sometimes. In this
study, we successfully bound PTX and vorinostat (SAHA) to form co-prodrugs based on the
synergistic anticancer effects. The PTX-SAHA co-prodrugs were conjugated by glycine (1a) and
succinic acid (1b) respectively and the former have shown better activity in cytotoxicity, cell
cycle arrest and western-blot experiments. Therefore, 1a was further prepared to nanomicelles
with mPEG2000-PLA1750 as the carrier by using thin film method. PTX-SAHA co-prodrug
nanomicelles were spherical with a particle size of 20-100 nm. In vitro drug release test showed
Available online
1
a nanomicelles had sustained release effect, which could reduce the resistance of PTX. In vitro
Keywords:
PTX
SAHA
co-prodrug
nanomicelles.
cytotoxicity was evaluated by SRB assay in HCT-116 cells, MCF-7 and drug-resistant MCF-
7/ADR cells. The results showed 1a nanomicelles had comparable or even better cytotoxicity
than PTX especially in the MCF-7/ADR cells. All the results suggested that PTX-SAHA co-
prodrug nanomicelles were promising treatment for PTX resistance cancer.
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
Nowadays, cancer imposes great threat on people's lives with
drug resistance and further limits its application in clinical. The
main cause of resistance was that the variation of microtubule
structure can alter intracellular drug level and signal transduction,
incidence of various cancer has become higher and lack of
eventual cure. The commonly used treatments in the clinical
treatment of cancers include surgery, chemotherapy, radiotherapy
4
thereby avoiding the apoptosis pathways . Therefore, it’s
particularly important to increase drug accumulation in tumor
tissues and reduce their drug resistance. Histone deacetylase
inhibitors (HDACIs) have shown great prospect for a good deal
of cancers by using alone or combining with traditional
anticancer drugs. Treatment of cancer with HDACIs has
multiple-effects such as inhibition of cell proliferation, induction
1
and biological therapy . Generally speaking, traditional
chemotherapy has made a great contribution in cancer therapy
but remains limited by inferior biocompatibility, unexpected
systemic cytotoxicity and drug resistance of cancer cells.
5
-6
of cell-cycle arrest and apoptosis . Suberoylanilide hydroxamic
acid (SAHA, Zolinza/ vorinostat) is an oral HDACI which has
For all the contemporary problems, drug combination
therapies have been widely exploited to decrease these unwanted
side-effects and enhance therapeutic effects simultaneously.
Combination chemotherapy has improved the efficacy of cancer
7
strong anticancer effects in hematological tumors . Recently,
several studies have demonstrated that SAHA could potentiate
PTX-induced antitumor effects against some human cancer cells
2
therapy notably as many instances had exhibited . Paclitaxel
(
lung cancer cells, ovarian cancer cells, endometrial cancer cells,
(PTX) is one of the most widely used clinical anticancer drugs
8-10
etc.)
.
which has a unique microtubule resistance mechanism and
generally used for breast cancer, ovarian cancer and other types
of cancer . However, the long-term use of PTX can develop into
Despite these advantages, sometimes the cancer therapy with
combination drugs can be severely restricted by some issues.
3

PTX is an intravenous preparation and SAHA is an oral
preparation which mean they may not reach the target site
concurrently. And the physiochemical properties of PTX and
SAHA are quite different that may cause the interaction
complicated. Besides, SAHA always hydrolyze to some inactive
metabolisms like carboxylic acid derivative especially in
2. Results and discussion
2
.1. Synthesis of the PTX-SAHA co-prodrug
The 1a were synthesized using a four-step procedure as
illustrated in scheme 1 with CH Cl as solvent. Firstly, the
2
2
compound 2 (SAHA) was activated to an active intermediate 3
by using CDI and then reacted with the tert-butyl protected
glycine to obtain compound 4. Subsequently, cleavage of the tert-
butyl group from compound 4 was performed using 30% TFA in
CH Cl , resulting in compound 5. Finally, compound 5 was
1
1
plasma .In that case, restructuring and modifying SAHA is
essential for enhancing the stability of SAHA. To date, several
strategies have been investigated to improve the stability of
1
2
SAHA, including clickable pH-responsive prodrug , selective
1
3
14
2
2
enzymatic cleavable prodrug , thiol-sensitive prodrug , redox-
reacted with PTX via an esterification reaction to produce the
product 1a.
1
5
responsive prodrug , and so on. Based on the previous
researches, we conjugated PTX and SAHA with a nontoxic linker
to form a “co-prodrug” for combined administration of PTX and
SAHA (Fig. 1). In this way, we expected to obtain the synergistic
effect of PTX and SAHA by reconverting to the two original
anticancer drugs concurrently under particular conditions.
The 1b were synthesized using a different way from 1a as
illustrated in scheme 2. PTX was reacted with succinic anhydride
to generate compound 6, which was catalyzed by DMAP and
pyridine. Compound 2 (SAHA) was activated to a active
intermediate 3 by using CDI and then reacted with the compound
6
to produce the other terminal product 1b.
The progress of the reaction was monitored by thin layer
chromatography (TLC). All reactions processed with good
yields and purified to high purity. Each compound was confirmed
1
13
by H NMR. The final products were confirmed by C NMR,
mass spectrometry and HPLC.
Fig. 1. Design of the PTX-SAHA co-prodrugs.
By conjugating the two drugs together in a cleavable way, we
enhanced the stability of SAHA and reduced the drug resistance
after cellular uptake of the PTX-SAHA co-prodrugs. We
connected the hydroxamic acid group of SAHA and the 2′-OH of
PTX to form a co-prodrug, and further formed the co-prodrug
Scheme 1. Reagents and conditions: (a) CDI, CH
2
Cl
2
, rt; (b) H-Gly-tBu,
, rt.
1
6
nanomicelles with mPEG2000-PLA1750 as the carrier . Glycine
Gly) and succinic acid were used as the linker of PTX-SAHA
rt; (c) 30% TFA in CH Cl , 0°C; (d) EDCI, HOBT, CH
2
2
2
Cl
2
(
co-prodrugs. The release mechanism of co-prodrugs was that the
carbonic ester connected with SAHA simply hydrolyzed through
hydrolysis or enzymolysis firstly. Then, the release rate of PTX
mainly depended on the stability of the rest part. It has been
reported that PTX-2′-Gly is unsteady when it’s exposed to water
1
7-18
and the other one is much steadier under same condition
study examined the stability of co-prodrugs and in vitro
. This
cytotoxicity in human colorectal cell line HCT-116, breast
cancer cell lines MCF-7 and MCF-7/ADR, as well as cell cycle
analysis. All these results showed that 1a released free PTX and
SAHA efficiently and had better cytotoxicity than 1b.
Furthermore, PTX-SAHA nanomicelles could enhance the
stability of 1a co-prodrug and prolong the retention time in blood
stream through accumulating in tumor site by way of enhanced
permeation and retention (EPR) effect. Comparing free PTX and
1
a co-prodrug, PTX-SAHA co-prodrug nanomicelles
considerably enhanced the anticancer efficacy and efficiently
reversed PTX resistance in cancer therapy.
Scheme 2. Reagents and conditions: (a) DMAP, pyridine, CH
2 2
Cl , rt; (b)
CDI, CH Cl , rt; (c) compound 6, rt.
2
2

2
.2. HPLC analysis of PTX-SAHA co-prodrugs
HPLC with fluorescence detection was used to identify the
hydrolysis of co-prodrugs and formation of PTX, SAHA and
other intermediates from the co-prodrugs. Formic acid buffer was
used in the mobile phase to serve as the ion-pairing reagent, and
to serve as the major buffer component. The retention times for
SAHA and PTX were 8.42 min and 13.81 min, respectively. In
addition, the retention times for compound 6, 1a and 1b were
Fig. 3. (A) Degradation of the co-prodrugs in human and mouse plasma;
(B) PTX released from the co-prodrugs in human and mouse plasma. HP:
human plasma; RP: mouse plasma.
1
3.63 min, 14.62 min and 15.06 min, respectively. The others
metabolic intermediate of SAHA such as N-phenylotanedi-
amide and 8-aniline-8-oxooctanoic acid didn’t produced.
2
.5. Cytotoxicity of PTX-SAHA co-prodrugs
2
.3. In vitro degradation and release of PTX-SAHA co-prodrugs
in PBS
PTX would become nontoxicity after esterification of the
hydroxyl group at the C-2’ position and SAHA would lose
activity after the hydroxamic acid was sealed. The cytotoxicity
was principally depending on the reconversion of PTX and
SAHA. For the two kinds of co-prodrugs, the rank order of
hydrolysis rate and PTX/SAHA reconversion rate was1a>1b.
The cancer cells were incubated with SAHA, PTX, mixture of
PTX and SAHA, co-prodrug 1a or 1b for 24h to identify if the
co-prodrug has the synergistic anticancer effects. Consistent with
the stability, 1a exhibited much more cytotoxic towards all three
cells than 1b. In addition, the IC50 of 1a is a little bit better than
PTX alone against MCF-7/ADR which demonstrates that the co-
prodrug 1a may have greater effect against drug-resistant cells
The hydrolysis studies of 1a and 1b were first performed in
PBS at pH 6.0 and 7.4 at 37°C. The disappearance of 1a/1b and
the formation of PTX/SAHA were quantified using HPLC. The
percentage of remaining 1a/1b and converted PTX/ SAHA was
plotted as a function of time (Fig. 2). The degradation curves in
Fig. 2A showed that the two co-prodrugs exhibited greater
stability under the slightly acidic physiological environment (pH
.0). There were over 85% 1a and 1b remained 24 hours later in
pH 6.0 PBS. Besides, 1b was much more stable than 1a so that
the reconversion of SAHA and PTX were little. The half life (t )
of 1a was 2 h at pH 7.4 and the reconversion of PTX and SAHA
reached the highest at 48 h were 50.3% and 56.8%, respectively.
These results illustrated in Fig. 2 indicated that the 1a can release
active anticancer drugs well for synergistic effects.
6
1/2
(Table 1).
Table 1. In Vitro Cytotoxicity Assay Data Summary
IC50nM (mean± SD)
Compound
SAHA
HCT-116
MCF-7
939.97 ± 98.02
<1.5
MCF-7/ADR
1413.40 ± 110.26
>10000
1617.76 ± 201.37
2.61 ± 1.67
PTX
PTX+SAHA 1.65 ± 0.85
<1.5
369.23 ± 16.63
1384.45 ± 83.88
4019.76 ± 143.35
1
1
a
9.55 ± 3.35
1.5
b
144.12 ± 17.09
62.35±3.36
The data are presented as average ± standard error (n=3).
2
.6. Cell cycle analysis
To investigate the cellular level of the synergistic effects, we
have studied the cell cycle variation of HCT-116 cells，MCF-7
and MCF-7/ADR cells treated with PTX/ SAHA mixture, PTX,
SAHA, co-prodrug 1a and 1b. Cell cycle analysis in these cells
indicated that the mixture of PTX and SAHA can induce a
marked G2/M cell cycle arrest after 24 h when compared to
untreated cells (Fig. 4). Both SAHA and PTX induced cell arrests
in the G2/M phase. SAHA induced the G2/M cell cycle arrest in
a concentration dependent manner in HCT-116 and MCF-7 cells,
while there was almost none effect in MCF-7/ADR cells. The 1a
has equivalent effects with PTX in HCT-116 and MCF-7 cells. In
addition, there was more induction of G2/M arrest in MCF-
Fig. 2. (A) Degradation of the co-prodrugs in PBS (pH7.4) and PBS
(
(
(
pH6.0); (B) PTX released from the co-prodrugs in PBS (pH7.4) and PBS
pH6.0); (C) SAHA released from the co-prodrugs in PBS (pH7.4) and PBS
pH6.0).
2
.4. In vitro degradation and release of PTX-SAHA co-prodrugs
in plasma
The hydrolysis studies of 1a and 1b were then performed in
human plasma and mouse plasma at 37 °C. The disappearance of
a/1b and the formation of PTX/SAHA were quantified using
7
/ADR cells exposed to 1a than PTX. Hence, the small gap in
cell cycle arrest between co-prodrug 1a and PTX is reasonable
and further indicates the effective hydrolyze of 1a to release PTX
and SAHA concurrently for collaborative treatment in tumor
cells.
1
HPLC. The percentage of remaining 1a/1b and converted PTX/
SAHA was plotted as a function of time (Fig. 3). The two co-
prodrugs had a short t1/2 nearly about 0.5h in both human and
mouse plasma. Besides, the hydrolysis rate of them was faster in
mouse plasma due to the difference of carboxylesterase.
Similarly, as the PBS stability above, the 1b was more stable and
had less reconversion of PTX than 1a. The highest reconversion
rates of PTX from 1a were up to 74.1% and 85.8% in human and
mouse plasma, respectively. The peak of SAHA was cross with
some substance in the plasma so that the conversion rate of
SAHA hadn’t listed out.
2
.7. Western blot analysis
On the basis of the cytotoxicity and cell cycle arrest experiments
in HCT-116, MCF-7 and MCF-7/ADR cells, we next further
determined the HDAC inhibitor activities of the co-prodrugs.
Inhibition of HDAC is usually indicated by tubulin acetylation
state. The cells were incubated with PTX, SAHA, the mixture of
PTX and SAHA, 1a and 1b for 10h and 24h. In addition, cells

without any treatment were used as control. Compared to
treatment of SAHA or 1b alone, Western blot analysis showed
hyperacetylation of tubulin after treating with 1a or PTX. The
results indicated that co-prodrug 1a exhibited greater efficacy in
inhibiting HDAC towards HCT-116, MCF-7 and MCF-7/ADR
cells (Fig. 5).
nanomicelles showed negative potential at around －11.5 mV,
suggesting the nanomicelles were capable of lengthening the
circulation time in tumor because nanomicelles with negative
surface charges are inclined to agglomerate due to interaction
with protein.
The morphology of the nanomicelles was measured by TEM,
as shown in Fig. 6B. It can be seen from the figure that the 1a
nanomicelles are spherical structure with a particle size of 20-
1
00nm, which is consistent with the results of the dynamic light
scattering (DLS) test.
Fig. 6. (A) Size and size distribution of 1a nanomicelles; (B) transmission
election microscopy (TEM) of 1a nanomicelles.
Fig. 4. Cell cycle analysis treated with PTX/SAHA mixture, PTX, SAHA, co-
prodrug 1b and 1a.(A) HCT-116 cells; (B) MCF-7cells; (7) MCF-7/ADR
cells.
2
.9. Colloidal stability of nanomicelles
The colloidal stability of the nanomicelles was investigated by
measuring the variation of mean particle size. The nanomicelles
were diluted with PBS and placed at 25 °C for 48 h. The results
demonstrated that the nanomicelles are stable at this condition
with no obvious variation of the mean particle size and PDI was
observed as shown in Fig. 7.
Fig. 5. Effect of individual compounds on tubulin depolymerization of HCT-
1
16, MCF-7 and MCF-7/ADR cells.
2
.8. Characterization of co-prodrug nanomicelles
The EE (%) and DL (%) of the co-prodrug nanomicelles are
5.6% ± 0.2% and 19.1% ± 0.1%, respectively.
Fig. 7. Variation of the mean particle size of 1a nanomicelles at 25 °C in
PBS (means ± SD, n=3).
9
Unlike blood vessels of healthy tissue, tumor blood vessels
2
.10. In vitro drug release from the nanomicelles in PBS
have enhanced permeability and retention (EPR) effect allowing
the size between 20-200 nm particles through. Therefore, Particle
size plays a vital role for nanomicelles that affects their in vivo
performance and pharmacokinetics. The mean particle size was
A long retention time of nanomicelles in the bloodstream is
often a prerequisite for successful accumulation in tumor. The 1a
nanomicelles were more stable than the original compound in
PBS7.4 as shown in Fig. 8. PTX and SAHA released slowly and
reached the balance within 48 h. The highest release amount of
PTX and SAHA were 12.34% and 40.42%, respectively. The
7
2.5 nm, as shown in Fig. 6A. And the main hydrodynamic
diameter of the nanomicelles was between 20 and 100 nm. These
results illustrated that the nanomicelles suffice for passing
through the tumor blood vessels. Besides, the zeta potential of

release amount of compound 1a was 3%, indicating that it can be
rapidly reconverted into the original drug when released from the
micelles. In general, the compound nanomicelles have obvious
sustained release effect, which can reduce the resistance of PTX
and play a better anti-tumor effect.
nanomicelles with the advantages of anti-drug resistance are
potential to be a new generation of synergistic therapy.
4
. Experimental
4
.1. Materials and methods
1
13
H and C nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker DRX-400 MHz spectrometer (400 and101
MHz, respectively) using CDCl , or DMSO-d as solvents with
3
6
TMS as an internal standard. Chemical shifts were reported as d
ppm) and spin-spin coupling constants as J (Hz) values. The
(
mass spectra (MS) were recorded on a Finnigan MAT-95 mass
spectrometer. Column chromatography was performed with silica
gel (200–300 mesh). All of the starting materials are
commercially available and were used without further
purification.
Vorinostat (SAHA) was synthesized before. Paclitaxel (PTX)
was purchased from Shanghai biocompound medical technology
Co., Ltd. BOC-glycine, Succinic anhydride, 4-Dimethyl-
aminopyridine (DMAP), 1,1’-carbonyldiimidazole(CDI) and 1-
Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCI) were all obtained from Energy Chemical Reagent Co.
(Shanghai ,China). All other chemical reagents used were of
analytical grade quality.
Fig. 8. Accumulative drug release of drugs from namomicellers at 37 °C.
2
.11. Cytotoxicity of nanomicelles
Naturally, your paper should start with a concise and
informative title. The inhibitory activity of compounds 1a
nanomicelles, 1a, SAHA, PTX, the mixture of PTX and SAHA
and mPEG2000-PLA1750 were incubated against the above cells
using the SRB assays (Table 2). The material mPEG2000-PLA1750
was found has no cytotoxicity. Compared with the original
compound 1a, the inhibition activity of 1a nanomicelles had
increased a lot. In addition, the inhibitory activity of 1a
nanomicelles was equal even better than that of PTX especially
in the MCF-7/ADR cells. These results showed that
4
.2. Synthesis of the PTX-SAHA co-prodrugs
4.2.1. Synthesis of compound 4
Under the protection of nitrogen, Compound 2 (SAHA) (1g,
3.78mmol) was dispersed in CH Cl (20 mL), and then CDI
2
2
(0.61g, 3.78mmol) was added. After stirring for 1 h at room
tempetature to form active intermediate 3, BOC-glycine (0.5g,
3.78 mmol) was added and stirred for 10h continually. The
reaction mixture was diluted with CH Cl and washed with 0.2M
1
ananomicelles could reconvert the PTX and SAHA in a slow
way and effective concentrations was achieved at the same time.
Table 2. In Vitro Cytotoxicity Assay Data Summary
IC50nM(mean± SD)
2
2
HCl, dried Na SO , filtered. The filtrate was concentrated under
2
4
reduced pressure. The residue was purified using silica gel
column chromatography with CH Cl and MeOH to give 4 (1.05
Compound
HCT-116
20.73 ± 5.06
3.15 ± 0.59
>30000
MCF-7
MCF-7/ADR
619.96 ± 192.27
393.95 ± 193.91
>30000
2
2
1
g, 66%) as a white solid. m.p.126-128°C。 H NMR (400 MHz,
1
1
a
19.83 ± 7.94
2.88 ± 0.70
>30000
DMSO) δ 11.41 (s, 1H), 9.84 (s, 1H), 8.01 (t, J = 5.8 Hz, 1H),
a nanomicelles
7
.58 (d, J = 7.8 Hz, 2H), 7.27 (t, J = 7.9 Hz, 2H), 7.01 (t, J = 7.3
Hz, 1H), 3.68 (d, J = 6.1 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.06
t, J = 7.2 Hz, 2H), 1.54 (dd, J = 16.0, 6.9 Hz, 4H), 1.41 (s, 9H),
mPEG2000-PLA1750
(
1
9
130.15 ±
0.73
2030.83 ±
369.92
+
SAHA
7298.57 ± 963.80
1.29 (s, 4H)；MS (ESI): m/z Calcd for C H N O [M+H] :
4
2
1
31
3
6
22.2, found: 422.1.
PTX
≤3.99
1.51 ± 0.46
18.51 ± 10.39
1998.28 ± 719.39
1180.62 ± 73.86
4
.2.2. Synthesis of compound 5
SAHA+PTX
8.89 ± 4.29
Compound 4 (1.05 g, 2.49 mmol) was dissolved in 30%TFA
in CH Cl (10 mL) at 0 °C for 0.5h. After stirring for 1.5 h at
2
2
room temperature, the reaction mixture was concentrated directly
under reduced pressure. The residue was purified using silica gel
column chromatography with CH Cl and MeOH to give 5 (90%)
3
. Conclusion
We have shown novel PTX-SAHA co-prodrugs which were
2
2
1
as a white solid. m.p.143-145°C. H NMR (400 MHz, DMSO) δ
conjugated by glycine or succinic acid. The biological
1
7
2.68 (s, 1H), 11.41 (s, 1H), 9.84 (s, 1H), 7.98 (t, J = 5.8 Hz, 1H),
.58 (d, J = 7.8 Hz, 2H), 7.28 (t, J = 7.9 Hz, 2H), 7.01 (t, J = 7.4
assessment of 1a was much better than 1b and therefore 1a was
further formed the co-prodrug containing nanomicelles with
polymer mPEG2000-PLA1750 as the carrier. We followed up with
cellular testing in previous cell lines to determine the effects of
anticancer activity. In these follow-up assays, we inferred that the
nanomicelles can be used in the treatment of drug-resistant MCF-
Hz, 1H), 3.71 (d, J = 6.0 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 2.06 (t,
1
3
J = 7.2 Hz, 2H), 1.54 (dd, J = 16.6, 6.8 Hz, 4H), 1.29 (s, 4H). C
NMR (101 MHz, DMSO) δ 171.12, 169.95, 155.43, 139.31,
1
2
28.58, 122.86, 118.99, 42.29, 36.33, 31.86, 28.28, 24.96,
+
4.63；MS (ESI): m/z Calcd for C H N O [M+H] : 366.1,
1
7
23
3
6
7
/ADR by releasing PTX and SAHA concurrently and maintain
found: 366.2.
effective concentration for a long while in the tumor site. The 1a
co-prodrug nanomicelles possessed several superiorities,
including (1) prolonged blood circulation and increased
accumulation at the tumor site; (2) sufficiently intracellular drug
release, which are contributed to remarkably PTX-resistance
reversion efficacy in vitro. The PTX-SAHA co-prodrug and its’
4
.2.3. Synthesis of compound 1a
To a solution of compound 5 (0.213g, 0.586 mol) in CH Cl
2
2
(
(
(
10 mL) were added EDCI (0.112 g, 0.586 mmol) and HOBT
0.079 g, 0.586 mmol). After stirred for 0.5 h at 0 °C, paclitaxel
0.506 g, 0.586 mmol) was added and stirred for 15h at room
temperature. The mixture was diluted with CH Cl and washed
2
2

with water, dried over Na SO and evaporated to dryness to give
crude product, which was further purified by silica gel column
170.47, 170.35, 169.94, 168.29, 167.42, 167.00, 142.68, 137.96,
136.84, 133.76, 133.63, 132.79, 131.95, 130.23, 129.22, 129.07,
128.99, 128.72, 128.60, 127.34, 126.96, 124.25, 119.81, 84.43,
81.03, 79.06, 77.35, 77.24, 77.03, 76.72, 76.42, 75.60, 75.04,
74.67, 72.10, 71.93, 58.46, 53.24, 45.61, 43.14, 37.06, 35.55,
35.33, 32.38, 29.71, 28.95, 28.09, 27.93, 27.13, 26.76, 25.05,
24.55, 22.66, 22.06, 20.85, 14.83, 9.62；HRMS (ESI): m/z
2
4
chromatography with CH Cl and MeOH to give 1a (0.132 g,
2
2
1
1
8%) as a white solid. m.p.147-149°C。 H NMR (400 MHz,
CDCl ) δ 11.02 (s, 1H), 9.86 (s, 1H), 8.09 (t, J = 15.5 Hz, 4H),
3
7
6
7
6
5
1
3
2
3
1
1
1
1
8
7
3
2
.76 – 7.64 (m, 3H), 7.55 (dd, J = 16.7, 8.0 Hz, 5H), 7.45 (d, J =
.7 Hz, 3H), 7.35 (q, J = 7.8 Hz, 4H), 7.28 (s, 1H), 7.24 (s, 1H),
.13 (t, J = 6.8 Hz, 1H), 7.05 (t, J = 7.3 Hz, 2H), 6.89 (s, 1H),
.21 (s, 1H), 5.95 (s, 1H), 5.78 (s, 1H), 5.57 (d, J = 6.7 Hz, 1H),
.35 (d, J = 8.0 Hz, 1H), 4.93 (d, J = 9.7 Hz, 1H), 4.33 (d, J =
6.9 Hz, 1H), 4.24 (d, J = 8.5 Hz, 1H), 4.12 (t, J = 9.4 Hz, 1H),
.92 (s, 1H), 3.54 (d, J = 5.2 Hz, 1H), 3.14 (d, J = 5.8 Hz, 1H),
.88 (s, 1H), 2.57 (s, 1H), 2.25 (s, 9H), 2.16 (s, 3H), 1.81 (d, J =
1.4 Hz, 6H), 1.45 (d, J = 14.5 Hz, 3H), 1.38 – 1.18 (m, 8H),
+
Calcd for C H N O [M+Na] : 1222.4725, found: 1222.4724.
6
4
72
4
19
4
.3. HPLC analysis of PTX-SAHA co-prodrugs
Chromatographic detection was carried out by using Agilent
200 high performance liquid chromatography (HPLC, Agilent,
1
PaloAlto, CA) with a Diamonsil C18 (250 mm×4.6 mm) column.
The detection was conducted at 254 nm (UV detector) with the
column temperature at room temperature. The sample volume for
each injection was 50 μL. The gradient elution was applied with
a mobile phase of acetonitrile (ACN) containing 0.1% formic
acid and water containing 0.1% fomic acid with a constant flow
rate of 1mL/min: 0–15 min (5% ACN to 95% ACN), 15–25 min
1
3
.10 (d, J = 14.0 Hz, 7H). C NMR (101 MHz, CDCl ) δ 202.42,
3
71.22, 171.03, 170.76, 170.39, 167.96, 167.58, 165.85, 154.72,
41.04, 137.25, 135.31, 133.16, 132.91, 131.31, 130.91, 129.13,
28.18, 127.86, 127.59, 127.49, 126.69, 126.19, 123.06, 118.95,
3.18, 80.20, 77.74, 76.34, 76.22, 76.02, 75.70, 75.17, 74.60,
3.65, 71.24, 69.81, 57.23, 53.96, 45.26, 41.88, 36.03, 34.80,
3.54, 31.59, 28.68, 27.33, 27.00, 25.52, 24.28, 21.47, 20.61,
0.14, 13.53, 8.64；HRMS (ESI): m/z Calcd for C H N O ,
(95% ACN). An external standard method was established by
HPLC in order to determine the content of each compound.
6
4
72
4
19
+
4
.4. In vitro drug release of PTX-SAHA co-prodrugs in PBS
[
M+Na] : 1223.4689, found: 1223.4688.
4
.2.4. Synthesis of compound 6
PTX-SAHA co-prodrug solutions with concentration of 10
Under the protection of nitrogen, paclitaxel (0.1 g, 0.117
mmol) was dissolved in CH Cl (20 mL), and then pyridine
μM/L were diluted to 50 μM/mL by 10 mM PBS buffer (pH
7.4/6.0). After incubation at 37 °C for 0.5 h, 1 h, 2 h, 3 h, 4 h, 6
h,12 h and 24 h, the areas of PTX-SAHA co-prodrugs and other
species were detected by HPLC as described in Section 4.3. Then,
each content was calculated by the standard concentration curve,
which was obtained by an external standard method.
2
2
(
0.044 g, 0.433 mmol), succinic anhydride (0.016 g, 0.163 mmol)
and DMAP (0.014 g, 0.117 mmol) were added. After stirred for
3h at room temperature, the mixture was poured into water,
adjusted to slightly acidic pH and extracted with CH Cl . The
1
2
2
organic layer was washed with brine, dried over Na SO and
filtered. The filtrate was concentrated under reduced pressure.
The residue was purified using silica gel column chromatography
2
4
4
.5. In vitro drug release of PTX-SAHA co-prodrugs in plasma
PTX-SAHA co-prodrug solutions with concentration of
with CH Cl and MeOH to give 6 (0.065 g, 58%) as a white
2
2
100μM/L were diluted to 100μM/mL by plasma (human / mouse).
After incubation at 37°C for 0.5h, 1h, 2h, 3h, 4h, 5h and 6h,50μL
reaction mixture was quenched by 50µL cold ACN and
centrifuged at 30000 rpm×5 min. The supernatant was detected
by HPLC as described in Section 4.3. Then, each compound was
calculated by the concentration-area standard curve, which was
obtained by an external standard method.
1
solid.m.p.178-180°C。 H NMR (400 MHz, CDCl ) δ 8.13 (d, J
3
=
7.6 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H),
7
9
1
1
8
2
.52 (d, J = 7.3 Hz, 3H), 7.38 (t, J = 15.8 Hz, 7H), 7.05 (d, J =
.2 Hz, 1H), 6.25 (dd, J = 18.3, 9.3 Hz, 2H), 5.99 (d, J = 8.7 Hz,
H), 5.68 (d, J = 6.7 Hz, 1H), 5.53 (s, 1H), 4.97 (d, J = 9.3 Hz,
H), 4.48 – 4.39 (m, 1H), 4.31 (d, J = 8.3 Hz, 1H), 4.19 (d, J =
.3 Hz, 1H), 3.80 (d, J = 6.7 Hz, 1H), 2.66 (dd, J = 11.7, 6.8 Hz,
H), 2.57 (d, J = 6.9 Hz, 3H), 2.44 (s, 3H), 2.36 (dd, J = 15.1, 9.3
4
.6. Cytotoxicity of 1a and 1b co-prodrugs
Hz, 1H), 2.18 (d, J = 21.7 Hz, 4H), 1.88 (d, J = 24.8 Hz, 5H),
The in vitro Cytotoxicity assay of 1a and 1b was evaluated
1
.67 (s, 3H), 1.20 (t, J = 25.3 Hz, 6H)；MS (ESI): m/z Calcd for
against human colorectal cell line HCT-116, breast cancer cell
lines MCF-7 and drug-resistant breast cancer cell lines MCF-
7/ADR using an SRB assay. Briefly, the cells
+
C H NO [M+H] : 954.3, found: 954.4.
5
1
55
17
4
.2.5 Synthesis of compound 1b
Compound 4 (0.068 mmol) was synthesized as described
at logarithmic phase were seeded into 96-well plates
4
above. The compound 6(0.065 g, 0.068 mmol) was added and
stirred for 10h at room temperature. The reaction mixture was
poured into water and extracted with CH Cl . The organic layer
(1.0×10 cells per well) in 100μL culture medium. Cells were
treated in triplicate with gradient concentrations of test drugs and
incubated at 37°C for 72 h. The growth inhibitory effects on the
cell lines were measured with SRB assay subsequently. The drug
concentration required for 50% growth inhibition (IC50) of tumor
cells was determined from dose–response curves.
2
2
was washed with brine, dried over Na SO and filtered. The
2
4
filtrate was concentrated under reduced pressure. The residue was
purified using silica gel column chromatography with CH Cl and
2
2
MeOH to give 1b (0.046 g, 57%) as a white solid. m.p.144-
4
.7. Cell cycle assay
1
1
7
7
46°C. H NMR (400 MHz, CDCl ) δ 9.28 (s, 1H), 8.13 (d, J =
.4 Hz, 2H), 7.79 (d, J = 7.7 Hz, 2H), 7.62 (t, J = 7.1 Hz, 1H),
.58 – 7.34 (m, 12H), 7.29 (s, 3H), 7.08 (t, J = 7.2 Hz, 1H), 6.27
3
Representative treatment groups were harvested at 24 h. Cells
5
at logarithmic phase were seeded at 2ꢀ ×ꢀ 10 per cells in 6-well
plates and treated with compounds at the indicated concentration
or with vehicle as a control. After 24 h, these cells were washed
three times with cold PBS, treated with trypsin, neutralized with
nutrient solution, centrifuged and discarded the supernatent.
Following, these cells were washed with cold PBS for once, 300
µl PBS and 700 µl ethanol were added and fixed for overnight at
(s, 1H), 6.16 (t, J = 8.5 Hz, 1H), 5.95 (s, 1H), 5.66 (d, J = 6.8 Hz,
1
1
H), 5.49 (d, J = 4.2 Hz, 1H), 4.96 (d, J = 9.4 Hz, 1H), 4.42 (s,
H), 4.30 (d, J = 8.2 Hz, 1H), 4.18 (d, J = 8.3 Hz, 1H), 3.78 (d, J
=
7.0 Hz, 1H), 2.78 (dd, J = 23.6, 7.2 Hz, 4H), 2.58 – 2.48 (m,
2
2
H), 2.40 (s, 3H), 2.30 (t, J = 7.0 Hz, 2H), 2.22 (s, 3H), 2.12 (s,
H), 2.06 – 1.96 (m, 1H), 1.95 – 1.83 (m, 4H), 1.76 (s, 1H), 1.66
(
d, J = 3.2 Hz, 7H), 1.59 (s, 2H), 1.34 (s, 4H), 1.16 (d, J = 35.0
4
°C. Subsequently, the cells were centrifuged (450 rpm×5 min),
13
Hz, 6H). C NMR (101 MHz, CDCl ) δ 203.80, 171.68, 171.28,
discarded ethanol, washed with PBS and discarded supernatent.
3

At last, the cells were precipitated with 500 µl PBS containing 10
µg/mL RNase and 10 µg/mL PI, stained with propidium iodide
maintain the final concentration of the 1a co-prodrug
nanomicelles was 1 mg/mL and placed at room temperature for 2
days. The experiment was performed in triplet.
(10 μg/mL) for 30 min and then analyzed using Flowjo.
4
.8. Western blot analysis
4.11. In vitro drug release from 1a co-prodrug nanomicelles in
PBS
Cells at logarithmic phase were seeded in 6-well plates (3.0 ×
5
1
0 cells per well) and cultured overnight. Compounds were
Release of PTX, SAHA and 1a from the nanomicelles in PBS
(pH 7.4) was measured by RP-HPLC as described in Section 4.3.
The nanomicelles (2 mg/mL) was dissolved in PBS enclosed in
dialysis tube (MWCO2000). The dialysis tube was placed at 10
mL PBS solution kept in a THZ-C isothermal shaker at 37°C and
100 rpm. At the predetermined time point, 50μL of the sample
solution was withdrawn, and same amount of PBS was added
immediately. The accumulative drug release was measured by
RP-HPLC.
added to the wells and cells were incubated at 37 °C for 24 h.
Cells without any treatment were used as control. The cells were
washed with cold PBS three times, harvested and resuspended in
RIPA lysis buffer. The lysates were boiled for 10 min, mixed and
collected the protein. Proteins were separated on SDS-
polyacrylaminde gel and separated by electrophoresis. The
semidry method was used for transferring the proteins to NC
membrane. Following, the proteins were stained with ponceaux,
sealed for 1 h with confining liquid after marking the proteins.
After incubation with corresponding primary antibody at 4 °C
overnight, the membrane was eluted with eluent three times.
Following, membrane was incubated with secondary antibody
which marked with horse radish peroxidase (HRP) for 1 h at
room temperature, washed with TBST three times and visualized
using ChemiDoc MP detection system (Bio-Rad).
4
.12. Cytotoxicity of 1a co-prodrug nanomicelles
The in vitro Cytotoxicity assay of 1a co-prodrug nanomicelles
was evaluated as described in section 4.6.
Acknowledgments
Authors greatly appreciated the support from Shanghai
Science and Technology Council No 16DZ2280100.
4
.9. Preparation and characterization of 1a co-prodrug
nanomicelles
Thin-film hydration method was used to prepare nanomicelles.
References and notes
7
5 mg compound 1a and 300 mg mPEG2000-PLA1750 copolymer
were dissolved in 12 mL acetonitrile. The mixture was
1. Ethun, C.G.; Bilen, M.A.; Jani, A.B.; Maithel, S.K.; Ogan, K.;
evaporated slowly under reduced pressure for 2 h and continued
to vacuum drying for 24 h to remove organic solvents completely.
The mixture skeleton was heated to 50°C until it turned to
transparent gel samples completely, then 15 mL ultrapure water
was added with vibration adequately. Then the light blue
emulsion was filtered with 0.22 μm microfiltration membrane,
Master, V.A. CA Cancer J. Clin. 2017, 67, 362-377.
2
.
Kumar, S.; Shweta, S. N.; Setty, M. M.; Arti. J. Drug Deliv. Ther.
012 , 2, 711-713.
2
3
4
.
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Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature. 1979, 277, 665-667.
Dowdy, S. C.; Jiang, S.J.; Zhou, X. C.; Hou, X. N.; Jin, F.;
Podratz, K. C.; Jiang, S.W. Mol. Cancer Ther. 2006, 5, 2767-
2776.
o
5. Huang,M.;Geng,M. Sci. China Life Sci. 2017, 60, 94-97.
lyophilized and stored at 4 C.
6
7
8
.
.
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Fallkenberg, K.J.; Johnstone,R.W. Nat. Rev. Drug Discov. 2014,
3, 673-691.
Duvic, M.; Vu, J. Expert Opin. on Inv. Drugs. 2007, 16, 1111-
120.
Cooper, A. L.; Greenberg, V. L.; Lancaster, P. S.; van Nagell, J.
R.; Zimmer, S. G.; Modesitt, S. C. Gynecol. oncol. 2007, 104,
596-601.
1
After the co-prodrug was extracted from the micelles by ACN,
the final concentration of co-prodrug was measured by RP-HPLC
analysis. The encapsulation efficiency (EE) and drug loading
1
(DL) of the co-prodrug nanomicelles were calculated by the
following equations:
9
10.
.
Modesitt, S.C.; Parsons, S.J., Gynecol. oncol. 2010, 119, 351-357.
Shi, Y.K.; Li, Z.H.; Han, X.Q.; Yi, J.H.; Wang, Z.H.; Hou, J.L.;
Feng, C.R.; Fang, Q.H.; Wang, H.H.; Zhang, P.F. et al. Cancer
Chemoth. Pharma. 2010, 66, 1131-1140.
EE (%) = Wencapsulated / Winput× 100%
DL (%) = Wencapsulated / Wtotal× 100%
1
1. Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez, B.;
Where, Winput is the amount of co-prodrug was added before
preparation, Wencapsulated is the amount of co-prodrug encapsulated
in the nanomicelles after purification and Wtotal is the total
amount of co-prodrug nanomicelles after purification.
Deprez-Poulain, R. J. Med. Chem. 2009, 52, 6790-6802.
12.
Delatouche, R.; Denis, I.; Grinda, M.; Bahhaj, F.; Baucher, E.;
Collette, F.; et al. European Journal of Pharma. Biopharm. 2013,
85, 862-872.
1
3. Thomas, M.; Rivault, F.; Tranoy-Opalinski, I.; Roche, J., Gesson,
The mean particle size, size distribution, polydispersity index
PDI) and zeta（ζ） potential were measured by dynamic light
scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments,UK).
The samples were prepared to a concentration of 1mg/mL with
distilled water.
J.P.; Papot, S. Bioorg. Med. Chem. Lett. 2007, 17, 983-986.
14. Daniel, K.B.; Sullivan, E.D.; Yao, C.; Chan, J.C.; Jennings, P.A.;
Fierke, C.A.; Cohen, S.M. J. Med. Chem. 2015, 58, 4812-4821.
5. Han, L.; Wang, T.; Wu, J.; Yin, X.; Fang, H.; Zhang, N. Int. J.
Nanomed. 2016, 11, 6003-6022.
(
1
1
1
6. Sankar, R.; Ravikumar, V. Biomed. Pharmacother.; 2014, 68, 865-
8
71.
7. Greenwald, R.B.; Hong, Z.; Reddy, P. J Org. Chem. 2003, 34,
894-4896.
The morphology of the nanomicelles was observed by
transmission electron microscopy (TEM) (JM-2100, Japanese).
The nanomicelles were diluted with distilled water and dropped
onto a 300 mesh copper grid coated with a thin carbon film. The
grids were dried at room temperature and observed by TEM
subsequently.
4
18. Wong, P.T.; Choi, S.K. Chem. Rev. 2015, 115, 3388-3432.
Supplementary Material
Supplementary data related to this article can be found at
4
.10. Colloidal stability of 1a co-prodrug nanomicelles
The colloidal stability of nanomicelles was investigated by
measuring variation of mean particle size and PDI of the
nanomicelles. The nanomicelles were diluted with PBS to
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Highlights

Design of PTX and SAHA co-prodrugs are based on the synergistic antitumor
effect.

In this study, we successfully bound PTX and SAHA to form co-prodrugs 1a and
1
b.


1a was further prepared to nanomicelles with mPEG2000-PLA1750 as the carrier.
PTX-SAHA co-prodrug nanomicelles are promising for treatment of drug-fast
cancer.