Design, synthesis, and evaluation of water-soluble morpholino-decorated paclitaxel prodrugs with remarkably decreased toxicity

Article abstract of DOI:10.1016/j.bmcl.2016.06.012

Novel water-soluble paclitaxel prodrugs were designed and synthesized by introducing morpholino groups through different linkers. These derivatives showed 400–20,000-times greater water solubility than paclitaxel as well as comparable activity in MCF-7 an

Full text of DOI:10.1016/j.bmcl.2016.06.012

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of water-soluble morpholino-
decorated paclitaxel prodrugs with remarkably decreased toxicity
Siliang Feng a, Kuncheng Chen a,b, Chenhong Wang a, Xifeng Jiang a, Huajin Dong a, Zehui Gong a,
Keliang Liu a,
⇑
a State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China
b School of Pharmaceutical Sciences, Central South University, 172 Tongzipo Road, Changsha 410013, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel water-soluble paclitaxel prodrugs were designed and synthesized by introducing morpholino
groups through different linkers. These derivatives showed 400–20,000-times greater water solubility
than paclitaxel as well as comparable activity in MCF-7 and HeLa cell lines. The prodrug PM4 was tested
in the S-180 tumor mouse model, with paclitaxel as the positive control. The results showed that PM4
had comparable antitumor activity as paclitaxel, with tumor inhibition of 54% versus 56%, and remark-
ably decreased toxicity. The survival rate of treated mice was 8/8 in the PM4 group, compared to 3/8
in the paclitaxel group.
Received 25 February 2016
Revised 24 May 2016
Accepted 6 June 2016
Available online xxxx
Keywords:
Paclitaxel prodrugs
Morpholino group
Anticancer
Ó 2016 Elsevier Ltd. All rights reserved.
Water-soluble derivatives
Paclitaxel has been widely used in the clinic as an antitumor
drug for lung, breast, and ovarian cancers.1 It is a natural antimi-
crotubule diterpenoid isolated from the bark of the pacific yew tree
(Taxus brevifolia).2 However, due to its poor water solubility, pacli-
taxel is dissolved in dehydrated ethanol and Cremophor EL for clin-
ical use, which causes serious side effects associated with
hypersensitivity.3,4 Numerous attempts to improve the water solu-
bility of paclitaxel have been done by conjugating paclitaxel to
some hydrophilic molecules, such as amino acids,5,6 sugars,7–9
malic acid,10 polyethylene glycol,11–13 dextran,14 heparin,15 and
sulfonate.16 Although most of these derivatives have much better
water solubility than paclitaxel, some problems still remain, such
as low stability, limited improvement in solubility, decreased
activity, and high toxicity.
Morpholine is a hydrophilic molecule that could possibly
improve the water solubility when introduced into paclitaxel,
especially when the amino group is salified. Moreover, upon enter-
ing tumor tissues, the morpholino group would be protonated at
the slightly acidic extracellular pH of tumors (6.5–7.2).17–19 This
would promote the interactions of prodrugs containing mor-
pholino groups with negatively charged cell membranes and accel-
erate their endocytosis by tumor cells. Our previous work found
that morpholino-decorated polymeric micelles exhibit higher cel-
lular uptake at lower pH values (6.5–7.0).20 Therefore, the toxicity
of morpholino compounds to normal tissues, where the extracellu-
lar pH is 7.4, might be decreased.
Here, we report the design, synthesis, and evaluation of a series
of new paclitaxel prodrugs by introducing morpholino groups
through different linkers (Fig. 1). The primary aim of this work
was to study the influence of morpholino groups on improving
the water solubility of insoluble drugs like paclitaxel. Second, we
wanted to examine whether the administration of morpholino
derivatives of paclitaxel would prolong the survival time of
tumor-bearing mice by decreasing the drug toxicity to normal tis-
sues. In addition, the influence of different linkers on the stability
and activity of the derivatives was studied. The linker of the ester
bond in PM1 and the carbamate bond in PM2 may lead to different
release rates of paclitaxel, possibly resulting in activity variation. In
PM3, paclitaxel was conjugated to 4-(2-aminoethyl)morpholine
(AEM) through a succinyl group; while in PM4, two AEM groups
were introduced to reinforce the influence of the morpholino
group. In PM4, a disulfide linker was incorporated because it has
been reported that cleavage would occur only after cellular entry
of the conjugate after encountering a high glutathione concentra-
tion (typically 15 mM intracellular compared to 15 lM extracellu-
lar).21 The thiol resulting from glutathione cleavage has been
reported to cyclize into the proximate carbonyl group of the linker,
subsequently leading to the release of free paclitaxel.
Synthesis: The synthetic routes of the designed conjugates are
given in Schemes 1–4. PM1 was obtained by directly reacting
2-morpholineacetic acid with paclitaxel through an ester bond in
⇑
Corresponding author. Tel.: +86 10 6816 9363; fax: +86 10 6821 1656.
E-mail address: keliangliu55@126.com (K. Liu).
http://dx.doi.org/10.1016/j.bmcl.2016.06.012
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Feng, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.012

2
S. Feng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
AcO
O
OH
BzHN
Ph
O
O
OR
HO
BzO
H
O
AcO
Paxlitaxel: R=H
O
H
N
PM1: R=
O
PM3: R=
PM4: R=
N
N
O
O
O
O
O
O
H
N
H
N
S
N
O
S
N
H
PM2: R=
O
N
O
O
NH
O
N
O
Figure 1. Structures of paclitaxel and its four prodrugs modified by morpholino groups.
AcO
AcO
O
O
OH
BzHN
Ph
O
OH
BzHN
Ph
O
a
O
O
O
O
OH
HO
BzO
HO
BzO
H
H
O
O
AcO
AcO
N
O
PTX
PM1
Scheme 1. Synthesis of PM1. Reagents and conditions: (a) 2-morpholineacetic acid, EDCI, DMAP, DCM, rt.
AcO
AcO
AcO
O
O
O
OH
OH
OH
BzHN
Ph
O
BzHN
Ph
O
BzHN
Ph
O
a
b
O
O
O
O
OH
O
O
O
HO
BzO
HO
BzO
HO
BzO
H
H
H
O
O
O
O
HN
AcO
AcO
AcO
N
O
NO2
1
PM2
PTX
Scheme 2. Synthesis of PM2. Reagents and conditions: (a) 4-nitrophenyl chloroformate, pyridine, DCM, rt; (b) 4-(2-aminoethyl)morpholine, DMAP, DCM, rt.
AcO
AcO
AcO
O
O
O
OH
OH
OH
BzHN
Ph
O
BzHN
Ph
O
BzHN
Ph
O
a
b
O
O
O
O
OH
O
O
O
HO
BzO
HO
BzO
HO
H
H
H
O
O
O
BzO
AcO
AcO
AcO
O
O
OH
HN
N
O
PTX
PM3
2
Scheme 3. Synthesis of PM3. Reagents and conditions: (a) succinic anhydride, pyridine, rt; (b) 4-(2-aminoethyl)morpholine, EDCI, HOBt, DMF, rt.
the presence of 1-(3-dimethylaminopropyl)-3-ethylcardodiimide
hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in
dichloromethane (DCM) at room temperature (rt). The reaction
was completed in approximately 10 h. Next, DCM was evaporated,
Please cite this article in press as: Feng, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.012

S. Feng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
AcO
O
O
OH
BzHN
Ph
O
O
S
HO
S
a
b
c
O2N
O
O
S
S
SH
S
O
O
HO
N
N
HO
BzO
O
H
O
AcO
S
N
6
3
5
4
AcO
O
O
OH
BzHN
Ph
O
O
H
N
H
N
O
N
O
N
O
N
H
N
H
Trt
Trt
OH
O
d
S
e
HS
f
S
O
HO
O
O
H
O
O
O
O
BzO
O
AcO
O
HN
HN
N
O
N
O
O
H
N
N
S
N
H
S
O
O
HN
N
O
PM4
8
7
9
Scheme 4. Synthesis of PM4. Reagents and conditions: (a) 2-20-dithiodipyridine, DMAP, MeOH, rt; (b) 4-nitrophenyl chloroformate, pyridine, DCM, rt; (c) PTX, DMAP, DCM,
rt; (d) AEM, EDCI, HOBt, DCM, rt; (e) 5% EDT/TFA, rt; (f) 6, DMF, rt.
and the mixture was dissolved in a 50% acetonitrile/water solution
before it was purified to give a white powdery solid. To obtain
PM2, paclitaxel was first reacted with 4-nitrophenyl chloroformate
in the presence of pyridine in DCM to get compound 1. Then com-
pound 1 was reacted with AEM in the presence of DMAP in DCM to
obtain PM2. PM3 was prepared in two steps. First, paclitaxel was
reacted with succinic anhydride in dry pyridine to obtain com-
pound 2, which was then reacted with AEM in the presence of EDCI
and HOBt in DCM to obtain PM3. PM4 was synthesized in six steps.
120
100
80
60
40
20
0
PTX
PM4
PTX released from PM4
First, compound
6
was prepared as described previously.22
Compound 9 was then prepared by first coupling AEM with
compound 7, followed by removal of the triphenylmethyl group.
Compound 6 was then reacted with compound 9 in dimethylfor-
mamide (DMF) to obtain PM4. All four prodrugs were purified by
reverse-phase C4 silica gel column chromatography and lyophi-
lized in the form of acetic acid salts.
0
10
20
30
40
50
With the purified prodrugs in hand, their water solubility was
measured. The results showed that their solubility in water was
remarkably improved to at least 400 times greater than that of
paclitaxel (Table 1). Among them, PM4, with two morpholino
groups, showed the highest water solubility of up to 5 mg/mL,
which is 20,000 times greater than that of paclitaxel. The solubili-
ties of PM2 and PM3 were both around 0.5 mg/mL. Although PM1
had the lowest water solubility of 0.1 mg/mL, it was still much
higher than that of paclitaxel.
Time(h)
Figure 2. Stability of PTX and PM4 in mouse plasma (*Detected drug concentration
divided by total concentration).
400
incubated at 37 °C. At the desired time points 100
removed and quenched with 300 l acetonitrile. The mixture was
l
l) were added to fetal bovine serum (FBS, 3600
ll) and
l aliquots were
l
l
The chemical stability of these four derivatives was then evalu-
centrifuged for 5 min at 10,000 rounds per minute. The super-
natant was analyzed by HPLC. The results showed that paclitaxel
ated.22 Briefly, solutions of the derivatives in DMSO (0.5 mM,
Table 1
The water solubility and cytotoxicity of paclitaxel conjugates in two tumor cell lines
Compd
Water solubility* (mg/mL)
IC50 (nM)
MCF-7 (nM) 48 h
MCF-7 (nM) 72 h
HeLa (nM) 48 h
HeLa (nM) 72 h
Paclitaxel
PM1
PM2
PM3
PM4
0.0002524
3.10
1.25
3.49
>1000
0.74
4.07
23.29
49.20
>1000
19.82
20.65
12.62
22.95
>1000
14.55
14.01
0.1
0.5
0.5
5
11.96
>1000
4.23
7.45
*
Equivalent dose of paclitaxel.
Please cite this article in press as: Feng, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.012

4
S. Feng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
In vivo activity of paclitaxel and its prodrugs against an S-180 ascites tumora
Group
Doseb (mg/kg)
Average tumor weight (g)
Tumor inhibitionc (%)
Body weight changesd (%)
No. of deaths
Control
Paclitaxel
PM4
—
25
25
7.35
3.34
3.43
—
56
54
ꢁ2.8
ꢁ28.1
ꢁ16.1
1/8
5/8
0/8
a
b
c
Prodrugs and paclitaxel were given every other day (iv ꢀ 4). Treatment was initiated 24 h after implantation.
Equivalent dose of paclitaxel.
Tumor inhibition = (1 ꢁ average tumor weight of treated group/average tumor weight of control group) ꢀ 100.
d
Body weight changes = (average body weight of mice at the tenth day/average body weight of mice at the first day ꢁ 1) ꢀ 100.
could be released from PM1, PM3 and PM4 while no paclitaxel
released from PM2 was detected (Fig. S1). In PM1 and PM4, about
40% paclitaxel was released in 15 h while in PM3, paclitaxel was
completely released in about 5 h. PM2 degraded nearly completely
in 4 h. However, no paclitaxel was detected, probably, due to the
high stability of the carbamate bond (Fig. S2).
Biological evaluation: The cytotoxicity of paclitaxel and the pro-
drugs was detected in two tumor cell lines, including a human
breast cancer (MCF-7) cell line and a human cervical cancer (HeLa)
cell line. Cells were seeded at a density of 4 ꢀ 103 cells/well in 96-
well plates 24 h before treatment. Paclitaxel and the prodrugs were
then added to the cells and incubated for 48 h or 72 h, respectively.
The cell viability was determined by an MTS assay. The results
showed that all derivatives except for PM2 had comparable cyto-
toxicity to paclitaxel, with IC50 values ranging from 0.6- to 3.9-fold
of that of paclitaxel (Table 1). As predicted, the IC50 values
decreased as the incubation time proceeded from 48 h to 72 h,
indicating that an adequate exposure time is necessary for the
drugs to kill the tumor cells effectively. Thus, the parent paclitaxel
could be released effectively from these prodrugs. In contrast, PM2
solution group (control), only one of the eight mice died, possibly
due to the fast proliferation of the tumor cells.
To further investigate the reason for the greatly reduced toxicity
of PM4, the stability of PM4 in plasma was studied. PM4 was incu-
bated in mouse plasma at 37 °C for 48 h, and the concentrations of
PM4 and paclitaxel released at different times were detected. The
results showed that PM4 degraded almost completely in first
10 h and only around 50% paclitaxel was released (Fig. 2).
However, when incubated with 10 mM dithiothreitol, paclitaxel
was completely released from PM4 in 10 min (Fig. S3). These
results demonstrate that paclitaxel could be sufficiently released
from PM4 after entering cells with high concentration of glu-
tathione. Different from paclitaxel, PM4 may be taken up faster
in the slightly acidic tumor surroundings than in normal tissues
for the introduction of morpholino groups, leading to reduced tox-
icity to normal tissues.
In summary, we report the design, synthesis, and evaluation of
a series of novel water-soluble paclitaxel prodrugs that contain
morpholino groups. All the derivatives possessed much better
water solubility and all but PM2 exhibited an equivalent in vitro
activity compared to the parent paclitaxel in MCF-7 and HeLa cells.
The linkers that conjugated the morpholino groups to the parent
paclitaxel had an important influence on the solubility and stabil-
ity of the derivatives, which may have affected their activity and
the release of paclitaxel. The optimal prodrug PM4 was adminis-
tered intravenously to mice bearing ascites tumors and showed
equivalent tumor inhibition compared to the parent paclitaxel,
with remarkably decreased toxicity. These morpholino-decorated
paclitaxel prodrugs may have great potential for further develop-
ment. This strategy and methodology may also be applied to the
design of water-soluble prodrugs of other anticancer drugs.
did not kill either cell line, even at a concentration of 1 lM. This
result may be due to the carbamate bond, which made the prodrug
too stable to release paclitaxel effectively. This was in accordance
with the result of the chemical stability evaluation.
Water-soluble PM4 was given intravenously to mice at different
doses to evaluate its acute toxicity. Mice administered PM4 at a
dose of 90 mg/kg (equivalent dose of paclitaxel) were still alive
after 1 week. It has been reported that the maximum tolerated
dose of paclitaxel is approximately 30 mg/kg,23 and we found that
when paclitaxel was given at a dose of 60 mg/kg, the mice died
within 24 h. Therefore, PM4 appears to be less toxic than the par-
ent paclitaxel and was chosen to carry out further activity evalua-
tion in vivo.
Acknowledgment
The in vivo activity evaluation was performed on mice bearing
an ascites tumor. S-180 cells were implanted subcutaneously in
mice, and 24 h later, PM4 dissolved in 5% glucose solution was
administered intravenously to the mice. Paclitaxel dissolved in cre-
mophor/ethanol (50%/50%) diluted 10 times by 5% glucose solution
was used as the positive control, and 5% glucose solution was used
as the negative control. The prodrug was given every other day for
a total of four times, and the mice alive at the tenth day were
weighed and sacrificed. The tumors were retrieved and weighed,
and the tumor inhibition was calculated. A summary of the prelim-
inary results is given in Table 2.
This work was financially supported by The National Key
Technologies R&D Program for New Drugs of China
(2012ZX09301003-004).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.06.
012.
As shown in Table 2, at a dose of 25 mg/kg, the tumor inhibition
of paclitaxel was 56%, which is very close to that of PM4, which
was 54%, meaning that the in vivo activity of the prodrug was
equivalent to that of the parent paclitaxel. And as expected, judg-
ing from the body weight changes and the number of deaths, the
toxicity of PM4 seemed to be much lower than that of the parent
paclitaxel. In the paclitaxel group, the average body weight of
the mice decreased by as much as 28.1%; while in the PM4 group,
it was only 16.1%. Moreover, at the end of the experiment, only
three out of eight mice survived in the paclitaxel group; while in
the PM4 group, all eight mice were alive. In the 5% glucose aqueous
References and notes
1. de Weger, V. A.; Beijnen, J. H.; Schellens, J. H. M. Anti-Cancer Drugs 2014, 25,
488.
2. Wani, M. C.; Talyor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc.
1971, 93, 2325.
3. Weiss, R. B.; Donehower, R. C.; Wiernik, P. H.; Ohnuma, T.; Gralla, R. J.; Trump,
D. L.; Baker, J. R., Jr; Van Echo, D. A.; Von Hoff, D. D.; Leyland-Jones, B. J. Clin.
Oncol. 1990, 8, 1263.
4. Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Eur. J. Cancer 2001, 37,
1590.
5. Paradis, R.; Page, M. Anticancer Res. 1998, 18, 2711.
Please cite this article in press as: Feng, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.012

S. Feng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
6. Mathew, A. E.; Mejilano, M. R.; Nath, J. P.; Himes, R. H.; Stella, V. J. J. Med. Chem.
1992, 35, 145.
7. Lin, Y. S.; Tungpradit, R.; Sinchaikul, S.; An, F. M.; Liu, D. Z.; Phutrakul, S.; Chen,
S. T. J. Med. Chem. 2008, 51, 7428.
8. Mikuni, K.; Nakanishi, K.; Hara, K.; Hara, K.; Iwatani, W.; Amano, T.; Nakamura,
K.; Tsuchiya, Y.; Okumoto, H.; Mandai, T. Biol. Pharm. Bull. 2008, 31, 1115.
9. Liu, D. Z.; Sinchaikul, S.; Reddy, P. G.; Chang, M. Y.; Chen, S. T. Bioorg. Med. Chem.
Lett. 2007, 17, 617.
10. Damen, E.; Wiegerinck, P.; Braamer, L.; Sperling, D.; Vos, D.; Scheeren, H. W.
Bioorg. Med. Chem. 2000, 8, 427.
11. Greenwald, R. B.; Pendri, A.; Bolikal, D. J. Org. Chem. 1995, 60, 331.
12. Feng, X.; Yuan, Y. J.; Wu, J. C. Bioorg. Med. Chem. Lett. 2002, 12, 3301.
13. Rodrigues, P. C. A.; Scheuermann, K.; Stockmar, C.; Maier, G.; Fiebig, H. H.;
Unger, C.; Mulhaüpt, R.; Kratz, F. Bioorg. Med. Chem. Lett. 2003, 13, 355.
14. Nakamura, J.; Nakajima, N.; Matsumura, K.; Hyon, S. H. Anticancer Res. 2010, 30,
903.
16. Zhao, Z. Y.; David, G. I.; Kingston, D. J. Nat. Prod. 1991, 54, 1607.
17. Ko, J. Y.; Park, S.; Lee, H.; Koo, H.; Kim, M. S.; Choi, K.; Kwon, I. C.; Jeong, S. Y.;
Kim, K.; Lee, D. S. Small 2010, 6, 2539.
18. Min, K. H.; Kim, J. H.; Bae, S. M.; Shin, H.; Kim, M. S.; Park, S.; Lee, H.; Park, R.
W.; Kim, I. S.; Kim, K.; Kwon, I. C.; Jeong, S. Y.; Lee, D. S. J. Controlled Release
2010, 144, 259.
19. Lee, E. S.; Shin, H. J.; Na, K.; Bae, Y. H. J. Controlled Release 2003, 90, 363.
20. Zhang, Y.; Wang, C. H.; Xu, C.; Yang, C. P.; Zhang, Z. Q.; Yan, H. S.; Liu, K. L. Chem.
Commun. 2013, 7286.
21. Dubikovskaya, E. A.; Thorne, S. H.; Pillow, T. H.; Contag, C. H.; Wender, P. A.
PNAS 2008, 105, 12128.
22. Seligson, A. L.; Terry, R. C.; Bressi, J. C.; Douglass, J. G.; Sovak, M. Anti-Cancer
Drugs 2001, 12, 305.
23. Vrudhula, V. M.; Macmaster, J. F.; Li, Z.; Kerr, D. E.; Senter, P. D. Bioorg. Med.
Chem. Lett. 2002, 12, 3591.
24. Vyas, D.; Wong, H.; Crosswell, A. R.; Casazza, A. M.; Knipe, J. O.; Mamber, S. W.;
Doyle, T. W. Bioorg. Med. Chem. Lett. 1993, 3, 1357.
15. Park, I. K.; Kim, Y. J.; Tran, T. H.; Huh, K. M.; Lee, Y. K. Polymer 2010, 51, 3387.
Please cite this article in press as: Feng, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.012