Development of first photoresponsive prodrug of paclitaxel

Article abstract of DOI:10.1016/j.bmcl.2006.06.030

A prodrug of paclitaxel which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (O-acyl isoform of paclitaxel) has been synthesized. The prodrug was selectively converted to isotaxel by visible light irradiation (430 nm) with the c

Full text of DOI:10.1016/j.bmcl.2006.06.030

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4492–4496
Development of first photoresponsive prodrug of paclitaxel
Mariusz Skwarczynski,a Mayo Noguchi,a Shun Hirota,b,c
Youhei Sohma,a,b Tooru Kimura,a Yoshio Hayashia,* and Yoshiaki Kisoa,*
aDepartment of Medicinal Chemistry, Center for Frontier Research in Medicinal Science and 21st Century COE Program,
Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
bDepartment of Physical Chemistry, 21st Century COE Program, Kyoto Pharmaceutical University,
Yamashina-ku, Kyoto 607-8412, Japan
cPRESTO, JST, Kawaguchi, Saitama 332-0012, Japan
Received 9 May 2006; accepted 9 June 2006
Available online 27 June 2006
Abstract—A prodrug of paclitaxel which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (O-acyl isoform
of paclitaxel) has been synthesized. The prodrug was selectively converted to isotaxel by visible light irradiation (430 nm) with the
cleavage of coumarin. Finally, paclitaxel was released by subsequent spontaneous O–N intramolecular acyl migration.
Ó 2006 Elsevier Ltd. All rights reserved.
A large number of anticancer agents have been devel-
oped in recent decades. However, these agents have very
little or no specificity for the target tumor tissues which
leads to systemic toxicity. Among them, paclitaxel
(1,TaxolÒ) is considered to be one of the most impor-
tant drugs in cancer chemotherapy. However, this agent
also has low tumor selectivity.
explain photosensitizer action: free radical generation
by electron or proton transfer, or singlet oxygen
generation by energy transfer, from light activated photo-
sensitizers. To date, the FDA has approved a photosensi-
tizing agent, porphyrin derivative, called PhotofrinÒ for
the use in PDT.6
Photoactivation also affords a useful technique in life
science to monitor biological processes by using so-called
‘caged’ compounds.7–12 These compounds are artificial
molecules whose biological activity is masked by a cova-
lently attached photocleavable group which can be selec-
tively removed upon light activation to release parent
bioactive molecules. Recently, we applied this caged strat-
egy for a controlled generation of intact amyloid b peptide
1–42 (Ab1–42) for the study of Alzheimer’s disease in
combination with an isopeptide method to mask biologi-
cal features of Ab1–42. A synthesized phototriggered
Ab1–42 isopeptide (‘click peptide’) possessing a photo-
cleavable 6-nitroveratryloxycarbonyl (Nvoc) group affor-
ded intact Ab1–42 with a quick and one-way conversion
reaction through photoirradiation by 355 nm light and
subsequent spontaneous O–N intramolecular acyl migra-
tion reaction.13
To overcome this problem, prodrug strategy is specially
promising.1,2 A lot of prodrugs of paclitaxel have already
been designed to specifically deliver them to the tumor tis-
sues with a site-specific chemical delivery system. In addi-
tion, macromolecular prodrugs showed targetable
properties due to enhanced permeability and retention
(EPR) effect, and monoclonal antibodies (mAbs) were
used as a vehicle to deliver 1 selectively to the tumor tis-
sues. Some of them exhibited very promising properties;
however, none of them is available in clinical use.3,4
Photodynamic therapy (PDT) is used for cancer treat-
ment. This technique is based on the administration of a
sensitizer devoid of mutagenic properties, followed by
the exposure of the pathological area to visible light.5
Two types of photoreaction mechanisms are invoked to
Taking all those three strategies (prodrug, photodynamic
therapy, and caged compound chemistry) into consid-
eration, we designed a photoresponsive targeting
prodrug of paclitaxel 1, namely phototaxel 2, which
has a coumarin derivative conjugated to an amino group
Keywords: Taxol; Tumor targeting prodrug; Photoresponsive;
Coumarin; O–N intramolecular acyl migration; Visible light
irradiation.
*
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.030

M. Skwarczynski et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4492–4496
4493
of isotaxel (3,O-acyl isoform of paclitaxel).14–16 This
OH
prodrug was expected to: (a) be selectively activated by
visible light irradiation (430 nm) leading to cleavage of
coumarin, then (b) release paclitaxel via subsequent
spontaneous O–N intramolecular acyl migration reac-
tion (Fig. 1). 7-N,N-Diethylamino-4-hydroxymethyl
coumarin (5, DECM) was chosen as a photolabile group
since DECM-caged compounds have been reported to
be water-soluble, thermally stable, and rapidly photo-
lyzed by visible light.9,17,18
a, b
c
N
O
O
N
O
O
4
5
N
O
O
O
O
O
NH
O
OH
O
d
As depicted in Scheme 1, 7-N,N-diethylamino-4-
hydroxymethyl coumarin 5 was prepared from commer-
cially available 7-N,N-diethylamino-4-methyl coumarin
4 according to the procedure reported by B. Giese and
coworkers.18 Compound 5 was activated by coupling
to 4-nitrophenyl chloroformate in presence of DMAP
(4-(dimethylamino)pyridine),12 then allowed to react
with 30-N-debenzoylpaclitaxel, which was synthesized
by a previously described method,14,19 to afford 6. Finally,
benzoylation of the 20-hydroxyl group with benzoic
acid by the EDC-DMAP method (EDC,1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide), and following
HPLC purification with ion-exchange by elution with
12 mM aq HCl gave phototaxel 2 as a HCl salt.20
O
2
O
O
H
O
O
OH
HO
O
O
6
Scheme 1. Syntheses of phototaxel 2. Reagents and conditions: (a)
SeO2, p-xylene, reflux, 24 h; (b) NaBH4, EtOH, rt, 4 h, 49% over two
steps; (c) 4-nitrophenyl chloroformate, DMAP, CH2Cl2, rt, 6 h, then
30-N-debenzoylpaclitaxel, DMAP, 20 h, 40%; (d) benzoic acid,
EDCÆHCl, DMAP, CHCl3, rt, 6 h, then HCl, 70%.
Previously reported DECM-caged compounds demon-
strated good water-solubility;9,17,18 however, prodrug 2
had lower water-solubility (<0.00025 mg mLꢀ1) than
parent drug 1. Therefore, to study the kinetics of photo-
conversion, 2 was dissolved in a mixture of 0.1 M phos-
phate buffer (PB, pH 7.4) and methanol (1:1, v/v) to
simulate physiological conditions21 and to obtain con-
centration of prodrug enough high for the clear detec-
tion in further evaluations (20–50 lM). Prodrug 2 was
photoirradiated with a diode laser (Melles Griot-85
BTL 010 laser, 430.6 nm, 10 mW) at 15 °C. The absorp-
tion spectra were taken immediately after irradiation
(Fig. 2A). Photolysis of DECM-caged compound is
expected to produce compound 5,9,18 which can be ob-
served as a shift of long-wavelength absorption maxi-
mum to the lower wavelength (compounds 5 and 2
had intensive maxima at 385 and 393 nm, respectively).
Indeed, we observed not only the shift but also a signif-
icant reduction of absorption intensity. This suggested
that 2 was photolytically cleaved and the released cou-
marin derivative was partially decomposed. This obser-
vation was further confirmed by HPLC analysis
(Fig. 2B), which was performed after irradiation fol-
lowed by incubation at rt for at least 1 h to induce com-
plete O–N intramolecular acyl migration to the parent
paclitaxel (the t1/2 value of migration of isotaxel 3 to re-
lease 1 was 15.1 min under physiological conditions).14–16
On the HPLC charts three signals were identified by
mass spectrometry analysis and confirmed with indepen-
dently synthesized compounds as 5 (rt = 20.5 min), 1
(rt = 28.5 min), and 2 (rt = 36.0 min). Thus, time-depen-
dent parent drug release from phototaxel 2 was indicat-
ed and quantitative analysis of prodrug kinetics upon
photoirradiation (Fig. 3) showed that paclitaxel was re-
leased with 69% yield after 30 min; however, only 20%
of 5 was recovered. Prodrug 2 was stable in the dark
in phosphate-buffered saline (PBS, pH 7.4) at 4 °C for
at least 1 month and as a solid state for at least 4 months
at ꢀ20 °C.
N
HCl
O
O
O
O
O
O
OH
O
NH
O
O
O
O
H
O
O
HO
O
O
O
2, phototaxel
irradiation
430 nm
O
O
O
OH
O
O
H2N
O
O
H
O
HO
O
O
O
O
3, isotaxel
O-N acyl migration
O
O
O
OH
O
O
O
N
O
H
O
H
OH
HO
O
O
O
1, paclitaxel
Figure 1. Releasing mechanism of paclitaxel 1 from its photorespon-
sive prodrug 2.

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M. Skwarczynski et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4492–4496
Figure 2. (A) Absorption spectra (250–500 nm) of prodrug 2 solution in PB(0.1 M)/MeOH (1:1) before (red line) and after irradiation (430.6 nm,
10 mW) for desired period of time (other lines) at 15 °C; (B) HPLC charts for prodrug 2 subjected to above irradiation conditions followed by
incubation at rt to induce migration, detected at 230 nm (line colors are corresponding to irradiation time in the same manner as for absorption
spectra).
Extensive literature searches revealed that photolabile
prodrug strategy has not yet been proposed for paclit-
axel, although there are a few reports that demonstrated
utilization of caged paclitaxel derivatives (activated by
UV irradiation) for molecular biology related study.22,23
Thus, herein a novel approach for developing a photore-
sponsive tumor targeting paclitaxel prodrug is demon-
strated throughout the design and synthesis of
phototaxel 2. Prodrug 2 is not expected to be active pri-
or to photoconversion, based on previous SAR studies
on paclitaxel derivatives.24,25 Namely, it is known that
both extensive modifications of the N-acyl moiety
and masking of the 20-OH group restrain paclitaxel
activity.25 Upon visible light irradiation (430.6 nm,10 mW)
this prodrug released isotaxel 3 (half-life of prodrug
was 4.8 min, Fig. 3), and subsequent spontaneous O–N
intramolecular acyl migration (t1/2 = 15.1 min)14–16
formed intact paclitaxel 1. This delay of parent drug
release after irradiation (related to migration of benzoyl
Figure 3. Time course of photolysis of prodrug 2 and release of 5 and 1
in 0.1 M PB/MeOH (1:1) at 430.6 nm. The percentage was determined
by HPLC.

M. Skwarczynski et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4492–4496
4495
group) is supposed to be short enough to avoid the
intermediate (3) diffusion from the photoirradiated site
before the parent drug is released. Moreover, we recently
demonstrated faster O–N intramolecular acyl migra-
tion in other highly potent taxoids.14,15,19 For example,
prodrug of canadensol (30-N-isopropylcarbonyl-30-N-
debenzoylpaclitaxel) had a t1/2 value of 4.3 min under
physiological conditions.15,19 20-O-Benzyloxycarbonyl-
30N-debenzoyl-paclitaxel, prodrug design based on the
O–N intramolecular alkoxycarbonyl migration reac-
tion,26 exhibited even instantaneous conversion to a par-
ent carbamate-type taxoid (t1/2 < 1 min).14 Phototaxoids
derived from these types of taxoids would be more effec-
tive without risking diffusion from the irradiation site.
subsidy from MEXT (Ministry of Education, Culture,
Sports, Science and Technology), and the 21st Century
Center of Excellence Program ‘Development of Drug
Discovery Frontier Integrated from Tradition to Prote-
ome’ from MEXT. M.S. is grateful for the Postdoctoral
Fellowship of JSPS. Y.S. is grateful for the Research
Fellowship of JSPS for Young Scientists.
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The observed recovery yield (69%) of paclitaxel 1 in the
photo-triggered conversion of 2 in HPLC analysis is in
agreement with previous reports on caged compounds
(typical released yield was 40–70%).7,27,28 This relatively
moderate recovery might be related to partial decompo-
sition of prodrug 2 or intermediate 3 due to their pho-
toinstability, as we observed low recovery of coumarin
derivative 5 from prodrug 2 contrary to a previous
report18 (Figs. 2 and 3). However, no major byproduct
formation was detected by HPLC, and paclitaxel 1
was almost stable under the photo-irradiation condi-
tions used for conversion of prodrug 2, that is, only a
small amount of paclitaxel decomposition (about 2%)
was observed by photo-irradiation for 0.5 h (data not
shown). Another reason could be non-specific absorp-
tion of compounds on the surface of experimental tubes.
DECM 5 has been chosen as a photolabile group, as
much less expensive and simple-to-use light sources are
available for experiments in the visible wavelength re-
gion. In spite of intensive maxima of DECM-caged
compounds are in UV-range (around 390 nm), these
compounds have been activated by visible lights (even
by irradiation at 436 nm).9,18 Moreover, during our ini-
tial experiments on prodrug 2 irradiated with UV pulses
(355 nm, 10 Hz, 5–20 mJ), extensive decomposition of 2
was observed (data not shown). In contrast, irradiation
at 430.6 nm showed to be effective for triggering parent
drug release without any major decomposition of pro-
drug or parent drug.
In conclusion, we designed and synthesized a new photo-
responsive paclitaxel prodrug based on an idea that com-
bined both photodynamic cancer therapy and caged
chemistry. The prodrug, phototaxel 2, released parent
drug, paclitaxel, with a reasonable conversion time by
visible light irradiation suggesting that this strategy is
practically applicable for wide range of anticancer agents
to develop new photoresponsive prodrugs. This would ex-
pand the current photodynamic therapy which is depen-
dent on photosensitizers (porphyrin derivatives) that
generatefree radicals or singlet oxygen as cytotoxic spices.
20. 1H NMR (CDCl3, 400 MHz): d = 8.03 (d, J = 4.5 Hz, 2H),
7.97 (d, J = 6.6 Hz, 2H), 7.61 (t, J = 6.8 Hz, 1H), 7.52–7.21
(m, 10H), 7.02 (br s, 1H), 6.51–6.38 (m, 3H), 6.30 (s, 1H),
6.00 (br s, 2H), 5.67 (d, J = 7.3 Hz, 1H), 5.66–5.61 (m,
2H), 4.27, 4.07 (2d, J = 14.7 Hz, 2H), 4.97 (d, J = 8.1 Hz,
1H), 4.46 (dd, J = 6.5, 10.9 Hz, 1H), 4.24, 4.21 (2d,
J = 8.7 Hz, 2H), 3.83 (d, J = 7.0 Hz, 1H), 3.39 (br s, 4H),
2.62–2.51 (m, 1H), 2.45 (s, 3H), 2.36–2.30 (m, 1H), 2.24 (s,
3H), 2.19–2.07 (m, 1H), 2.00 (s, 3H), 1.92–1.85 (m, 1H),
Acknowledgments
This research was supported in part by the ‘Academic
Frontier’ Project for Private Universities: matching fund

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HRMS (FAB+): calcd for C62H66N2O14 [M++Na]:
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