Development of novel water-soluble photocleavable protective group and its application for design of photoresponsive paclitaxel prodrugs

Article abstract of DOI:10.1016/j.bmc.2008.04.022

A novel coumarin-based highly water-soluble photocleavable protective group was designed and synthesized, and then this photosensitive protecting group was used to design paclitaxel prodrugs. These novel paclitaxel conjugates demonstrated excellent water solubility, over 100 mg mL^-1. Thus, the use of a detergent in the formulation can be omitted completely, even at high doses. Phototaxel 11 released the parent drug, paclitaxel, quickly and efficiently by minimal tissue-damaging 365 nm UV light irradiation at low power, while laser activation at 355 nm led to extensive decomposition of the prodrug. The carbamate-type prodrug, phototaxel 11, was stable in the dark prior to activation, whereas carbonate-type phototaxel 9 demonstrated poor stability under aqueous conditions. For such prodrugs, tumor-tissue targeting after administration could be achieved by selective light delivery, similar to that used in photodynamic therapy. In addition, newly designed coumarin derivative 8 can be applied in organic chemistry as a photosensitive protective group and for the design of caged compounds.

Full text of DOI:10.1016/j.bmc.2008.04.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5389–5397
Development of novel water-soluble photocleavable
protective group and its application for design
of photoresponsive paclitaxel prodrugs
Mayo Noguchi,a Mariusz Skwarczynski,a Halan Prakash,b Shun Hirota,b,c
Tooru Kimura,a Yoshio Hayashia,d 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
bGraduate School of Materials Science, Nara Institute of Science and Technology, Nara 630-0192, Japan
cPRESTO, JST, Kawaguchi, Saitama 332-0012, Japan
dSchool of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan
Received 4 March 2008; revised 9 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—A novel coumarin-based highly water-soluble photocleavable protective group was designed and synthesized, and then
this photosensitive protecting group was used to design paclitaxel prodrugs. These novel paclitaxel conjugates demonstrated excel-
lent water solubility, over 100 mg mLꢀ1. Thus, the use of a detergent in the formulation can be omitted completely, even at high
doses. Phototaxel 11 released the parent drug, paclitaxel, quickly and efficiently by minimal tissue-damaging 365 nm UV light irra-
diation at low power, while laser activation at 355 nm led to extensive decomposition of the prodrug. The carbamate-type prodrug,
phototaxel 11, was stable in the dark prior to activation, whereas carbonate-type phototaxel 9 demonstrated poor stability under
aqueous conditions. For such prodrugs, tumor-tissue targeting after administration could be achieved by selective light delivery,
similar to that used in photodynamic therapy. In addition, newly designed coumarin derivative 8 can be applied in organic chemistry
as a photosensitive protective group and for the design of caged compounds.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The introduction of paclitaxel (1,1 Fig. 1) in clinical
O
cancer chemotherapy has markedly improved patient
O
O
survival time.2–4 Similar to most anticancer agents,
dose-limiting toxicity corresponding to paclitaxel’s side
effects and poor water solubility are significant draw-
backs. Its sparing water solubility requires coinjection
of a detergent, Cremophor EL, which causes hypersensi-
tivity reactions, and patients receiving this drug require
premedication.5 To overcome these problems, the pro-
drug strategy is promising.6,7 Tumor-targeting prodrugs
are designed to achieve a high local concentration of
antitumor drugs to decrease undesired side effects
caused by non-tissue selectivity of the drugs.8 Water-sol-
OH
R2
NH
O
O
O
H
O
O
O
HO
O
R1
O
1 (paclitaxel): R1 = H, R2 = Bz 2 (isotaxel): R1 = Bz, R2 = H
O
O
O
N
HCl
phototaxel 3: R1 = Bz, R2 =
Keywords: Water-soluble photocleavable protective group; Prodrug of
paclitaxel; Taxol; O–N intramolecular acyl migration.
O
*
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
Figure 1. Structures of paclitaxel (1), isotaxel (2), and phototaxel 3.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.022

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M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
uble prodrugs are considered to improve water solubility
of the parent drug and to avoid the use of toxic deter-
gents in the drug formulation.9 Additionally, according
to the general nature of prodrugs, the pharmacokinetic
profile of paclitaxel was also improved while reducing
the side effects. However, the improved effectiveness of
prodrugs over paclitaxel, often demonstrated in preclin-
ical study, has not been confirmed in clinical trials.6 This
issue suggests that the development of novel prodrug
strategies for paclitaxel is still needed.
DECM-caged compounds have been reported to be
water-soluble, non-toxic, thermally stable, and rapidly
photolyzed by visible light.27–29 Nonetheless, phototaxel
3 was completely insoluble in water,26 which would
cause serious problems in the practical application of
such an agent.
Thus, we decided to develop a new photocleavable pro-
tective group for the design of water-soluble photore-
sponsive prodrugs. This group can be also applied for
caged compound design and in organic chemistry, espe-
cially in green chemistry and native chemical ligation
where water-soluble protective groups are highly desired.
Herein, we report a new coumarin-based highly water-
soluble photocleavable protecting group (8, Scheme 1)
that was applied for the design of two new phototaxels
(9 and 11, Scheme 1), one of which demonstrated high
water solubility, high stability in the dark and quick re-
lease of the parent drug upon UV light irradiation.
Photodynamic therapy (PDT) has been used for cancer
treatment for a long time. This technique is based on the
administration of a sensitizer devoid of mutagenic prop-
erties, followed by exposure of the pathological area to
a specific wavelength of light.10 Two types of photoreac-
tion mechanisms are invoked to explain photosensitizer
action: free radical generation by electron or proton
transfer, or singlet oxygen generation by energy transfer,
from light-activated photosensitizers. The FDA has
approved photosensitizing agents, porphyrin derivatives,
such as porfimer sodium or verteporfin, for use in PDT.11
2. Design and synthesis
Photolabile ‘caged’ compounds are biologically inert pre-
cursors of active molecules. Their biological activity is
masked by a covalently attached photocleavable group
which can be selectively removed upon light irradiation
to release parent bioactive molecules. Caged compounds
have found wide application in life sciences to monitor
biological processes.12–19 For example, we applied this
caged strategy for the controlled generation of intact
amyloid b peptide 1–42 (Ab 1–42) to study Alzheimer’s
disease. The photo-triggered Ab 1–42 isopeptide (‘click
peptide’) possessing a photocleavable 6-nitroveratryloxy-
carbonyl (Nvoc) group released parent Ab 1–42 through
photoirradiation by 355 nm light and subsequent sponta-
neous O–N intramolecular acyl migration reaction.20–22
Initially, we synthesized the reported DECM derivative
7-N,N-[bis(tert-butoxycarbonylmethyl)-amino]-4-hydroxy-
methyl coumarin (6), which has been used for the prep-
aration of water-soluble caged phosphates and which
allowed the photorelease of caged compound upon
one- and two-photon excitation.30,31 As depicted in
Scheme 1, compound 6 was prepared from commercially
available 7-amino-4-methyl coumarin (4), according to
the slightly modified procedure reported by Hagen and
coworkers.31 Thus, compound 4 was treated with an ex-
cess of bromoacetic acid tert-butyl ester in the presence
of diisopropylethylamine to yield 5 with monoalkylated
coumarin as a major by-product. Oxidation of 5 with
SeO2 and subsequent reduction with NaBH4, according
to the previously described method, gave alcohol
6.26,29,31 Then, we tried to synthesize phototaxels with
derivative 7 as a photosensitive group (data not shown).
Alcohol 6 was coupled at 4-position via a carbonate or
carbamate bond to the 20-hydroxy group of paclitaxel
or 30-amino group of 30-N-debenzoylpaclitaxel, respec-
tively. The attempt to obtain free carboxyl groups of
coumarin moiety in conjugates was unsuccessful.
Decomposition of conjugates was observed even under
very mild deprotection conditions.
Previously, we developed a unique synthetic water-solu-
ble paclitaxel prodrug, isotaxel (2), which produces pac-
litaxel through O–N intramolecular acyl migration.23–25
This prodrug, a 20-O-benzoyl isoform of paclitaxel,
showed promising results in higher water solubility
and appropriate kinetics for parent drug release, which
proceeded without additional functional auxiliaries re-
leased during conversion to the parent drug. However,
the lack of tumor-targeting properties of isotaxel can
still be considered a serious shortcoming for practical
application of such a prodrug. Thus, taking into consid-
eration three strategies: prodrug, photodynamic ther-
apy, and caged compound chemistry, we recently
reported the first photo-triggered paclitaxel prodrug,
phototaxel 3,26 which has a coumarin derivative conju-
gated via a 30-carbamate to an amino moiety of isotaxel
(2) (Fig. 1). Phototaxel 3: (a) was selectively activated by
visible light irradiation (430 nm) leading to cleavage of
coumarin, then (b) released paclitaxel via subsequent
spontaneous O–N intramolecular acyl migration reac-
tion.22 Tumor-tissue targeting for such a prodrug after
its administration could be achieved by selective light
delivery, for example, by utilizing an endoscope or optic
fibers. 7-N,N-Diethylamino-4-hydroxymethyl coumarin
(DECM) was chosen as the photolabile group since
Considering the problems with the final deprotection of
conjugates, we decided to apply prodrug design in which
final deprotection can be omitted. A few years ago, we
reported a prodrug of HIV protease inhibitors that pos-
sessed simple water-soluble tertiary amine moieties.32,33
Thus, by combining the structure of coumarin derivative
7 and N-[2-(dimethylamino)ethyl]succinamic acid,33 we
designed a new photosensitive group 8. Compound 8
was prepared from alcohol 6 via the cleavage of t-Bu
protective groups with TFA (trifluoroacetic acid) to af-
ford 7, and subsequent coupling of crude 7 with N,N-
dimethylethylenediamine by mixed-anhydride method
using isobutyl chloroformate. Coumarin derivative with
only one carboxylic group substituted was a major by-
product of coupling. Photosensitive protective group 8

M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
5391
OH
a
b, c
d
O
O
N
O
O
N
O
O
H2N
O
O
O
O
O
O
O
O
4
5
6
O
O
O
O
OH
OH
OH
NH
O
f
O
3 HCl
O
H
e
H
N
O
HO
O
HO
N
N
O
O
O
N
O
O
R1
H
N
O
O
O
OH
O
N
O
O
8
9
7
O
O
O
OH
R1
O
O
NH
O
O
O
N
O
g
H
R1
=
O
O
O
HO
O
O
R2
H
N
N
N
O
10, R2 = H
H
N
O
h
3 HCl
O
11, R2 = C6H5C(O)
Scheme 1. Synthesis of phototaxels 9 and 11. Reagents and conditions: (a) BrCH2COOt-Bu, i-Pr2EtN, NaI, CH3CN, reflux, 4 days, 68%; (b) SeO2, p-
xylene, reflux, 24 h; (c) NaBH4, EtOH, rt, 30 min, 61% over two steps; (d) TFA/CHCl3/H2O 74:25:1, rt, 20 min; (e) isobutyl chloroformate, N-
methylmorpholine, DMF, ꢀ30 ꢁC, 2 h, then (CH3)2NCH2CH2NH2, N-methylmorpholine, ꢀ15 ꢁC, 1 h, then rt overnight, 65% over two steps; (f) 8,
4-nitrophenyl chloroformate, DMAP, CH3CN/DMSO, rt, 5 h, then 1, DMAP, 22 h, then HCl, 63%; (g) 8, 4-nitrophenyl chloroformate, DMAP,
DMF, rt, 3 h, then 30-N-debenzoylpaclitaxel, DMAP, 4 h, then HCl, 59%; (h) benzoic acid, EDCÆHCl, DMAP, CH3CN, rt, 2 h, then HCl, 82%.
was activated by coupling to 4-nitrophenyl chlorofor-
mate in the presence of DMAP (4-(dimethylamino)pyr-
idine),34 and then allowed to react with paclitaxel (1) to
yield 9. The final product was purified by HPLC with a
binary solvent system, a linear gradient of CH3CN in
1% aqueous acetic acid, due to prodrug partial decom-
position when 12 mM HCl aqueous solution was used.
Then, the product was converted to HCl salt via lyoph-
ilization using only a slight excess of 1 N HCl. Prodrug
11 was synthesized in a similar manner. Compound 8
was activated by coupling to 4-nitrophenyl chlorofor-
mate in the presence of DMAP, then allowed to react
with 30-N-debenzoylpaclitaxel, which was synthesized
by a previously described method.23,35 Finally, benzoyl-
ation of the resultant carbamate 10 with benzoic acid by
the EDC–DMAP method (EDC, 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide), followed by HPLC purifi-
cation with ion exchange by elution with 1% AcOH
and then salt exchange with 1 N HCl gave phototaxel
11 as an HCl salt (Scheme 1).
(0.00025 mg mLꢀ1),25 and at least 200-fold higher than
that of isotaxel (0.45 mg mLꢀ1). The water solubility
of prodrugs with coumarin derivative 8 drastically in-
creased in comparison to phototaxel 3 and was even
greater than that of water-soluble prodrug 2. Thus, the
use of a detergent for the formulation of 9 or 11 can
be omitted even with a high dose of prodrugs.
Because we expected that carbonate prodrug 9 can be
less stable than carbamate 11, before studying the parent
drug release, the stability of prodrugs was examined.
Prodrug 9 showed poor stability in the dark, 20% of
paclitaxel was released in phosphate-buffered saline
(PBS, pH 7.4, 37 ꢁC) after 8 h of incubation, and only
18% of the prodrug remained when 9 was stored at
4 ꢁC for 13 days, while in the solid form, the prodrug
was stable at ꢀ20 ꢁC for at least 4 months. Thus, com-
pound 9 can be water-soluble prodrug of 1 but is not
an ideal candidate for selectively activated prodrug since
it decomposed under physiological conditions. In con-
trast, prodrug 11 was stable under physiological condi-
tions (PBS, pH 7.4, 37 ꢁC, 8 h) as well as under
storage conditions (PBS, pH 7.4, 4 ꢁC, for at least 1
month) and in a solid state at ꢀ20 ꢁC for at least 4
months. Thus, for further detailed study, only photot-
axel 11 was chosen (Fig. 2).
3. Results and discussion
The water solubility of 9 and 11 was determined as a va-
lue higher than 100 mg mLꢀ1, which was at least
400,000-fold higher than that of paclitaxel

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M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
irradiation
1, paclitaxel
phototaxel 9
irradiation
O-N acyl
migration
phototaxel 11
2, isotaxel
Figure 2. Releasing mechanism of paclitaxel (1) from its photorespon-
sive prodrugs 9 and 11.
The high water solubility of new phototaxel 11 allowed
us to study the kinetics of photoconversion under phys-
iological conditions in phosphate-buffered saline (PBS,
pH 7.4), in contrast to phototaxel 3, for which an organ-
ic solvent was necessary for prodrug solubilization.26
Prodrug 11 was irradiated with UV pulses (355 nm,
10 Hz, 5 mJ), at 23 ꢁC. This irradiation wavelength
was chosen as coumarin derivative 8 had intensive max-
ima at 353 nm and prodrug 11 at 361 nm. Thus, modifi-
cation of the coumarin moiety in prodrug 3 (DECM has
intensive maxima at 385 nm)26 caused a shift in the
absorption maximum to a shorter wavelength in com-
pound 7 (376 nm)31 and even a further shift in 8
(353 nm). The reason for such a shift may be related
to the electron-withdrawing properties of N-substituted
groups in coumarin. Irradiation at wavelengths above
300 nm is less likely to be absorbed by the biological
environment.36 Thus, photoirradiation with long-wave-
length UV light (UV-A) has been mainly used for pro-
drug activation.37–43 UV-A is already used clinically in
the treatment of atopic dermatitis44,45 and in photoche-
motherapy (PUVA therapy).46 In comparison to visible
or infrared light irradiation (above 600 nm) used in stan-
dard photodynamic therapy, UV-A light has signifi-
cantly lower tissue penetration.47–49 On the other
hand, therapy with red light is associated with side ef-
fects such as burning pain and heating due to thermal
irradiation. Buchczyk and coworkers demonstrated that
the application of UV-A to photodynamic therapy can
be highly promising.50 In addition, many promising
new prodrug strategies target only a subset of cells in tu-
mors, and their therapeutic utility is associated with ‘by-
stander effects, by which cell killing extends from
targeted cells to untargeted tumor cells in the vicin-
ity.51–54 Thus, a prodrug does not need to be activated
by light at the whole tumor site as a drug can usually dif-
fuse within tumor tissue.
Figure 3. HPLC charts for compound 8, prodrug 11, and paclitaxel (1)
subjected to UV light irradiation (355 nm, 10 Hz, 5 mJ) detected at
230 nm.
of taxoids would be more effective without risking diffu-
sion from the irradiation site. In addition, prodrug 11 is
not expected to be active prior to photoconversion,
based on previous SAR studies on paclitaxel
derivatives.57,58
The recovery yield of paclitaxel (1) after 4 min of irradi-
ation was 24%, significantly smaller than that of previ-
ously reported phototaxel 3 (69%).26 This low recovery
was probably related to the observed partial decomposi-
tion of prodrug 11 upon UV light irradiation (pulse la-
ser, 355 nm, 10 Hz, 5 mJ), because the coumarin
moiety of the prodrug was highly unstable under irradi-
ation conditions. After 0.5 min of irradiation of couma-
rin derivative 8, its complete decomposition was
observed (Fig. 3), while 1 was almost stable under these
photoirradiation conditions, and only a small amount of
paclitaxel decomposition (about 2%) was observed after
8 min of irradiation (Fig. 3). In contrast, when prodrug
11 was irradiated with a light of a UV lamp (365 nm,
6 W, UV Ray Lamp, Model UV-LS, Ishii Laboratory
Work Co., Ltd), instead of an intense light of a pulse la-
ser, the recovery yield was much higher (69%), retaining
a fast rate of prodrug conversion to the parent drug.
Thus, 50% of the reaction was completed after
4.0 0.6 min under the above continuous irradiation
conditions (Figs. 4 and 5 ). HPLC analysis indicated
one noteworthy minor by-product formation under
these irradiation conditions; however, we were not able
to characterize it (signal at 14 min, Fig. 4). This by-
product might be formed from coumarin 8, as release
of the photosensitive group 8 from 11 was very poor
Upon UV light irradiation (pulse laser, 355 nm, 10 Hz,
5 mJ), prodrug 11 quickly released isotaxel (2) and sub-
sequent spontaneous O–N intramolecular acyl migra-
tion (t1/2 = 15.1 min)23–25 formed paclitaxel (1) (Fig. 3).
In comparison, prodrug 9 can release paclitaxel without
any delay (Fig. 2). However, as 9 released the parent
drug in a non-specific manner prior to photoactivation,
it could not be employed as a selectively activated pro-
drug of paclitaxel. The delay of parent drug release from
11 after irradiation (related to migration of the benzoyl
group) is supposed to be short enough to avoid interme-
diate (2) diffusion from the tumor tissue before the
parent drug is released. Moreover, we recently demon-
strated faster O–N intramolecular acyl (or alkoxycar-
bonyl55,56
taxoids.22,23,35 Phototaxoids derived from these types
)
migration in other highly potent

M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
5393
that of the laser. Thus, the choice of an appropriate UV
source is highly important for the practical application
of water-soluble protective group 8. Nonetheless, even
longer wavelengths could be used for prodrug activa-
tion, because 11 absorbs light up to the visible light
wavelength (see Supplementary data Figure 1S). In
addition, coumarin derivative 8 may also be cleaved
with IR light two-photon photoirradiation in the same
manner as derivative 7.31
4. Conclusion
We designed and synthesized a coumarin-based novel
highly water-soluble protective group. This masking
group can also be applied to organic chemistry to
protect hydroxy or amine moiety, in caged compound
chemistry,and finally in photoresponsive prodrug
strategies. Herein, this photosensitive coumarin deriv-
ative was used in the design of paclitaxel prodrugs.
The carbamate-type prodrug, phototaxel 11, released
the parent drug, paclitaxel, quickly with minimal tis-
sue-damaging 365 nm UV-A light irradiation at low
power and was stable prior to activation, whereas
carbonate-type prodrug 9 demonstrated poor stability
under aqueous conditions. Both prodrugs demon-
strated excellent water solubility and thus can be
easily formulated for biological study. We also dem-
onstrated that the choice of appropriate irradiation
conditions is the key issue in light-triggered prodrug
strategies.
Figure 4. HPLC charts for prodrug 11 subjected to UV light
irradiation (365 nm, 6 W) detected at 230 nm.
100
80
60
%
1
11
40
5. Experimental
5.1. Materials
20
0
0
20
40
Time (min)
60
Reagents and solvents were obtained from Wako Pure
Chemical Industries Ltd (Osaka, Japan), Nacalai Tes-
que (Kyoto, Japan), Kanto Chemical Co. Inc. (Tokyo,
Japan), and Aldrich Chemical Co. Inc. (Milwaukee,
WI), and were used without further purification. Col-
umn chromatography was performed on Merck
107734 silica gel 60 (70–230 mesh). TLC was performed
using Merck Silica gel 60 F254 precoated plates. Analyt-
ical HPLC was performed using a C18 reverse phase
column (4.6 · 150 mm; YMC Pack ODS AM302) with
a binary solvent system: a linear gradient of CH3CN
Figure 5. Time course of photolysis of prodrug 11 and release of 1 in
10 mM PBS at 365 nm. The percentage was determined by HPLC.
Data are the average ( standard error) of three assays.
(with 11% yield after 1 h of irradiation). Paclitaxel was
stable under these conditions (less than 2% decomposi-
tion after 1 h) and, in contrast to pulse laser application,
coumarin derivative 8 alone was completely stable for at
least 15 min under UV irradiation. Thus, the reason for
the non-quantitative yield of 1 release could be decom-
position of the prodrug and/or non-specific absorption
of compounds on the surface of experimental tubes, as
was suggested in the case of prodrug 3.26 The higher
yield and purity of paclitaxel release via irradiation with
a UV lamp can be related to weaker UV lamp energy
compared to the pulse laser as well as its longer wave-
length of irradiation, 365 nm compared to 355 nm by
the laser. Thus, the 355-nm pulse laser is the third har-
monic of the YAG laser and exhibits a very intense pulse
light of a single wavelength. The wavelength cannot be
adjusted to 365 nm. The UV lamp has a relatively broad
wavelength and its center wavelength is 365 nm. The
intensity of the UV lamp is much weaker compared with
in 0.1% aqueous TFA at a flow rate of 0.9 mL minꢀ1
,
detected at 230 nm. Preparative HPLC was carried
out on a C18 reverse phase column (20 · 250 mm;
YMC Pack ODS SH343-5) with a binary solvent sys-
tem: a linear gradient of CH3CN in 12 mm aqueous
HCl or 1% aqueous acetic acid at a flow rate of
5.0 mL minꢀ1, detected at 230 nm. Solvents used for
HPLC were of HPLC grade. All other chemicals were
of analytical grade or better. 1H NMR spectra were ob-
tained using a 400 MHz Varian UNITY INOVA
400NB spectrometer with TMS as an internal standard
at 25 ꢁC. FAB-MS was performed on a JEOL JMS-
SX102A spectrometer equipped with the JMA-
DA7000 data system. EI-MS was performed on a JEOL
JMS-GCmate spectrometer.

5394
M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
5.2. Chemistry
for 2 h. Then N,N-dimethylethylenediamine (184 lL,
1.69 mmol) and N-methylmorpholine (159 lL, 1.45
mmol) were added, the solution was slowly warmed up
to ꢀ15 ꢁC, stirred 1 h, slowly warmed up to room tem-
perature, and stirred overnight. The reaction mixture
was directly applied to preparative HPLC, which was
eluted with 12 mm aqueous HCl over 10 min, and then
a linear gradient of 0–25% CH3CN in 12 mm aqueous
HCl over 25 min at a flow rate of 5 mL/min. The desired
fraction was collected and lyophilized to give 8 as an
HCl salt (176 mg, 0.316 mmol, 65%). 1H NMR
(CD3OD, 400 MHz): d = 7.52 (d, 3J(H,H) = 9.0 Hz,
1H, CH), 6.59 (dd, 4J(H,H) = 2.5 Hz, 3J(H,H) = 8.9 Hz,
5.2.1. 7-[Bis( tert-butoxycarbonylmethyl)amino]-4-meth-
31
ylcoumarin (5).
7-Amino-4-methylcoumarin (4.4 g,
25 mmol), NaI (3.8 g, 25 mmol), diisopropylethylamine
(21.9 mL, 126 mmol), and bromoacetic acid tert-butyl
ester (37.1 mL, 0.25 mol) in 120 mL dry CH3CN were
refluxed for 4 days under an argon atmosphere. The
mixture was cooled to room temperature, filtered, and
the solvent was removed under reduced pressure. The
residue was dissolved in AcOEt, washed with water
and brine, dried over MgSO4, and concentrated in va-
cuo. The resulting oil was purified by silica-gel column
chromatography (AcOEt/hexane 1:3) to yield 5 (6.90 g,
17.1 mmol, 68%). 1H NMR (CDCl3, 400 MHz):
d = 7.42 (d, 3J(H,H) = 8.8 Hz, 1H, CH), 6.52 (dd,
4
1H, CH), 6.44 (d, J(H,H) = 2.5 Hz, 1H, CH), 6.30 (s,
4
1H, CH), 4.77 (d, J(H,H) = 1.3 Hz, 2H, CH2), 4.36 (s,
4H, CH2), 3.64 (t, 3J(H,H) = 5.6 Hz, 4H, CH2), 3.32
(t, 3J(H,H) = 5.6 Hz, 4H, CH2, partially overlapping
with signal from CD3OD), 2.93 (s, 12H, CH3). EI MS:
calcd for C22H33N5O5 [M +]: 447.2481, found:
447.2476. Anal. Calcd for C22H36Cl3N5O52H2O, C:
44.56, H: 6.80, N: 11.81, found: C: 44.77, H: 6.62, N:
11.71. Purity was 99% (HPLC analysis at 230 nm).
3
4J(H,H) = 2.6 Hz, J(H,H) = 8.8 Hz, 1H, CH), 6.45 (d,
4
4J(H,H) = 2.6 Hz, 1H, CH), 6.02 (d, J(H,H) = 1.3 Hz,
1H, CH), 4.06 (s, 4H, CH2), 2.35 (d, J(H,H) = 1.3 Hz,
4
3H, CH3), 1.48 (s, 18H, CH3). EI MS: calcd for
C22H29NO6 [M+]: 403.1995, found: 403.2001. Anal.
Calcd for C22H29NO6, C: 65.49; H: 7.24; N: 3.47, found:
C: 65.45, H: 7.26, N: 3.55. Purity was 95% (HPLC anal-
ysis at 230 nm).
5.2.4. 7-[Bis-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoeth-
yl]amino]-4-[(20-O-paclitaxel)carbonyloxymethyl] couma-
rin hydrochloride (9). The whole procedure was carried
out in the dark. Compound 8 (16 mg, 0.031 mmol), 4-
nitrophenyl chloroformate (7.8 mg, 0.038 mmol), and
4-(dimethylamino)pyridine (7.5 mg, 0.062 mmol) in
4 mL of dry acetonitrile were stirred at room tempera-
ture for 3 h under an argon atmosphere. For complete
solubilization, 1 mL of DMSO was added and the mix-
ture was stirred for an additional 2 h. Then, another
5.2.2. 7-[Bis(tert-butoxycarbonylmethyl)amino]-4-(hydro-
31
xymethyl)coumarin (6).
Selenium dioxide (0.43 g,
3.8 mmol) and 5 (1.03 g, 2.55 mmol) in 25 mL p-xylene
were refluxed with vigorous stirring under an argon
atmosphere. After 24 h, the mixture was filtered and
concentrated under reduced pressure. The dark brown
residual oil was dissolved in ethanol (30 mL), then so-
dium borohydride (48.3 mg, 1.28 mmol) was added,
and the solution was stirred for 1 h at room tempera-
ture. Thereafter, the suspension was carefully hydro-
lyzed with 1 M HCl (2 mL), diluted with H2O, and
partially concentrated under reduced pressure to remove
EtOH. The resulting mixture was extracted with AcOEt.
The organic phase was washed with H2O and brine,
dried over MgSO4, and concentrated in vacuo. The
resulting oil was purified by silica-gel column chroma-
portion
of
4-(dimethylamino)pyridine
(1.9 mg,
0.015 mmol) and paclitaxel (6.6 mg, 0.0077 mmol) were
added. The reaction mixture was stirred at room tem-
perature for 22 h, quenched with 1 mL of MeOH, and
applied to preparative HPLC, which was eluted with
10% CH3CN in 1% aqueous AcOH over 5 min, and then
a linear gradient of 30–50% CH3CN in 1% aqueous
AcOH over 20 min at a flow rate of 5 mL/min. The de-
sired fraction was collected and lyophilized to give 9 as
an AcOH salt (7.0 mg, 0.0049 mmol). Then, the product
was lyophilized from a mixture of CH3CN (2 mL) and
water (1 mL) with 1 N HCl (17.2 lL, 0.0172 mmol) to
afford 9 as an HCl salt (7.0 mg, 0.00487 mmol, 63%).
1H NMR (CD3OD, 400 MHz): d = 8.11–8.09 (m, 2H,
CH), 7.82–7.79 (m, 2H, CH), 7.68–7.64 (m, 1H, CH),
tography (AcOEt/hexane 1:3) to yield
6 (0.65 g,
1.55 mmol, 61%). 1H NMR (CDCl3, 400 MHz):
d = 7.28 (d, 3J(H,H) = 9.0 Hz, 1H, CH), 6.48 (dd,
3
4J(H,H) = 2.7 Hz, J(H,H) = 8.9 Hz, 1H, CH), 6.44 (d,
4
4J(H,H) = 2.6 Hz, 1H, CH), 6.37 (t, J(H,H) = 1.4 Hz,
1H, CH), 4.78 (d, J(H,H) = 1.3 Hz, 2H, CH2), 4.06 (s,
4
4H, CH2), 1.49 (s, 18H, CH3). EI MS: calcd for
C22H29NO7 [M+]: 419.1944, found: 419.1939. Anal.
Calcd for C22H29NO7, C: 62.99, H: 6.97, N: 3.34, found:
C: 62.87, H: 6.84, N: 3.37. Purity was 97% (HPLC anal-
ysis at 230 nm).
3
7.59–7.41 (m, 10H, CH), 7.29 (br t, J(H,H) = 7.3 Hz,
1H, CH), 6.59 (dd, 4J(H,H) = 2.6 Hz, 3J(H,H) = 9.0 Hz,
1H, CH), 6.44 (d, J(H,H) = 2.4 Hz, 1H, CH), 6.37 (s,
4
1H, CH), 6.21 (s, 1H, CH), 6.04 (br t, 3J(H,H) = 8.5 Hz,
1H, CH), 5.86 (d, J(H,H) = 6.4 Hz, 1H, CH), 5.62 (d,
3
3
5.2.3. 7-[Bis-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoeth-
yl]amino]-4-(hydroxymethyl) coumarin hydrochloride (8).
Deprotection of compound 6 was achieved in an identi-
cal manner as reported by Hagen et al.,31 and the desired
product 7 was used for the next step without any purifi-
cation. Compound 7 (148 mg, 0.482 mmol) in 15 mL
dehydrated DMF was cooled down to ꢀ30 ꢁC under
an argon atmosphere. N-Methylmorpholine (159 lL,
1.45 mmol) and isobutyl chlorocarbonate (187 lL,
1.45 mmol) were added, and the solution was stirred
3J(H,H) = 7.3 Hz, 1H, CH), 5.58 (d, J(H,H) = 6.4 Hz,
1H, CH), 5.44, 5.38 (2d, J(H,H) = 15.3 Hz, 2H, CH2),
2
3
4.97 (br d, J(H,H) = 7.7 Hz, 1H, CH), 4.38, 4.33 (2d,
3
2J(H,H) = 19.4 Hz, 4H, CH2), 4.25 (dd, J(H,H) = 6.7,
11.1 Hz, 1H, CH), 4.18 (s, 2H, CH2), 3.77 (d,
3
3J(H,H) = 7.1 Hz, 1H, CH), 3.63 (t, J(H,H) = 5.3 Hz,
4H, CH2), 3.33–3.27 (t, 4H, CH2, overlapping with sig-
nal from CD3OD), 2.92 (s, 12H, CH3), 2.57–2.50 (m,
1H, CH2), 2.38 (s, 3H, CH3), 2.20–2.12 (m, 1H, CH2),
2.17 (s, 3H, CH3), 1.91–1.80 (m, 2H, CH2), 1.77 (d,

M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
5395
4J(H,H) = 1.1 Hz, 3H, CH3), 1.65 (s, 3H, CH3), 1.13 (s,
3H, CH3), 1.11 (s, 3H, CH3). HRMS (FAB+): calcd For
C70H83N6O20 [M++H]: 1327.5662 found: 1327.5654.
der an argon atmosphere. Then, the reaction mixture
was applied directly to preparative HPLC, which was
eluted with 10% CH3CN in 1% aqueous AcOH over
20 min, and then a linear gradient of 10–50% CH3CN
in 1% aqueous AcOH over 40 min at a flow rate of
5 mL/min. The desired fraction was collected and lyoph-
ilized to give 11 as an AcOH salt (6.0 mg, 0.0042 mmol).
Then, the product was lyophilized from a mixture of
CH3CN (2 mL) and water (1 mL) with 1 N HCl
(13.4 lL, 0.0134 mmol) to afford 11 as an HCl salt
(6.0 mg, 0.0042 mmol, 82%). 1H NMR (CD3OD,
400 MHz): d = 8.68 (d, 3J(H,H) = 9.7 Hz, 1H, CH),
Anal. Calcd for C70H85Cl3N6O20Æ7H O, C: 53.79, H:
2
6.38, N: 5.38, found: C: 53.72, H: 6.19, N: 5.11. Purity
was 99% (HPLC analysis at 230 nm).
5.2.5. 7-[Bis-[2-[[2-(dimethylamino)ethyl]amino]-2-oxo-
ethyl]amino]-4-[[30-N-(30-N-debenzoylpaclitaxel)]carbon-
yloxymethyl] coumarin Hydrochloride (10). The whole
procedure was carried out in the dark. Compound 8
(15.2 mg, 0.0273 mmol), 4-nitrophenyl chloroformate
(5.5 mg, 0.0273 mmol), and 4-(dimethylamino)pyridine
(6.7 mg, 0.0546 mmol) in 2 mL of dry DMF were
stirred at room temperature for 3 h under an argon
atmosphere. Then, another portion of 4-(dimethyl-
amino)-pyridine (3.4 mg, 0.027 mmol) and 30-N-deben-
zoylpaclitaxel23,35 (6.8 mg, 0.0091 mmol) were added.
The reaction mixture was stirred at room temperature
for 4 h, and applied directly to preparative HPLC,
which was eluted with 10% CH3CN in 1% aqueous
AcOH over 5 min, and then a linear gradient of 25–
45% CH3CN in 1% aqueous AcOH over 20 min at a
flow rate of 5 mL/min. The desired fraction was col-
lected and lyophilized to give 10 as an AcOH salt
(7.2 mg, 0.0053 mmol). Then, the product was lyophi-
lized from a mixture of CH3CN (2 mL) and water
(1 mL) with 1 N HCl (17.6 lL, 0.0176 mmol) to afford
10 as an HCl salt (7.1 mg, 0.0053 mmol, 59%). 1H
NMR (CD3OD, 400 MHz): d = 8.08 (d, 3J(H,H) =
3
8.10–8.07 (m, 3H, CH), 7.64 (br t, J(H,H) = 7.4 Hz,
1H, CH), 7.56–7.42 (m, 9H, CH), 7.29 (br t,
3
3J(H,H) = 7.3 Hz, 1H, CH), 7.20 (d, J(H,H) = 9.0 Hz,
1H, CH), 6.46 (s, 1H, CH), 6.46–6.43 (dd, 1H, CH, par-
tially overlapping with previous signal), 6.35 (d,
4J(H,H) = 2.0 Hz, 1H, CH), 6.20 (br t, 3J(H,H) =
9.1 Hz, 1H, CH), 6.17 (s, 1H, CH), 5.67–5.59 (m, 3H,
CH), 5.39, 5.17 (2d, 2J(H,H) = 16.8 Hz, 2H, CH2),
5.00 (br d, 3J(H,H) = 8.2 Hz, 1H, CH), 4.37 (dd,
3J(H,H) = 6.6, 10.9 Hz, 1H, CH), 4.33 (br s, 4H, CH2),
2
4.17, 4.15 (2d, J(H,H) = 12.0 Hz, 2H, CH2), 3.86 (d,
3
3J(H,H) = 7.3 Hz, 1H, CH), 3.64 (t, J(H,H) = 5.3 Hz,
4H, CH2), 3.33–3.27 (t, 4H, CH2, overlapping with sig-
nal from CD3OD), 2.94 (s, 12H, CH3), 2.49 (s, 3H,
CH3), 2.47–2.43 (m, 1H, CH2), 2.34–2.26 (m, 1H,
CH2), 2.17 (s, 3H, CH3), 2.00 (s, 3H, CH3), 1.94–1.88
(m, 1H, CH2), 1.84–1.77 (m, 1H, CH2), 1.65 (s, 3H,
CH3), 1.13 (s, 6H, CH3). HRMS (FAB+): calcd For
C70H83N6O20 [M++H]: 1327.5662 found: 1327.5670.
Anal. Calcd for C70H85Cl3N6O20Æ3.5H2O, C: 56.05, H:
6.18, N: 5.60, found: C: 56.04, H: 6.18, N: 5.54. Purity
was higher than 98% (HPLC analysis at 230 nm).
3
6.8 Hz, 2H, CH), 8.01 (d, J(H,H) = 9.7 Hz, 1H, CH),
7.68–7.64 (m, 1H, CH), 7.52–7.39 (m, 7H, CH), 7.31–
7.25 (m, 2H, CH), 6.49 (dd, 4J(H,H) = 2.2 Hz,
3J(H,H) = 9.5 Hz, 1H, CH), 6.44 (s, 1H, CH), 6.37 (d,
4J(H,H) = 2.0 Hz, 1H, CH), 6.24 (br t, 3J(H,H) =
9.0 Hz, 1H, CH), 6.18 (s, 1H, CH), 5.61 (d,
5.3. Water solubility
2
3J(H,H) = 7.3 Hz, 1H, CH), 5.32, 5.17 (2d, J(H,H) =
16.4 Hz, 2H, CH2), 5.28 (d, 3J(H,H) = 3.7 Hz, 1H,
CH), 4.98 (dd, 3J(H,H) = 1.8, 7.9 Hz, 1H, CH), 4.64
(d, 3J(H,H) = 4.2 Hz, 1H, CH), 4.34 (br s, 5H, CH
and CH2), 4.16, 4.14 (2d, 2J(H,H) = 14.1 Hz, 2H,
CH2), 3.84 (d, 3J(H,H) = 7.3 Hz, 1H, CH), 3.63 (t,
3J(H,H) = 5.2 Hz, 4H, CH2), 3.34–3.26 (t, 4H, CH2,
overlapping with signal from CD3OD), 2.93 (s, 12H,
CH3), 2.48–2.41 (m, 1H, CH2), 2.41 (s, 3H, CH3),
2.33–2.27 (m, 1H, CH2), 2.18 (s, 3H, CH3), 2.00–1.94
(m, 1H, CH2), 1.94 (s, 3H, CH3), 1.83–1.76 (m, 1H,
CH2), 1.65 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.15 (s,
3H, CH3). HRMS (FAB+): calcd For C63H79N6O19
[M ++H]: 1223.5400 found: 1223.5409. Anal. Calcd for
The prodrugs (9, 11) were saturated in distilled water
and shaken vigorously. The saturated solutions were
sonicated for 15 min at 25 ꢁC and passed through the
centrifugal filter (0.45 lm filter unit, Ultrafreeꢂ-MC,
Millipore). The filtrate was analyzed, using RP-HPLC.
The water solubility was determined using a calibration
curve prepared from standard solutions containing
known quantities of prodrugs.
5.4. Photolysis of 1, 8, and 11 with a laser
The conversion profiles of 11 were determined in phos-
phate-buffered saline (10 mM PBS, pH 7.4). To 990 lL
of PBS (pH 7.4) was added 10 lL of solution, including
1, 8 and 11 (2 mM in DMSO), and the mixture was irra-
diated with UV pulses (355 nm, 10 Hz, 5 mJ) obtained
from the third harmonic of an Nd-YAG laser (contin-
uum, Minilite II; pulse energy, 5 mJ; pulse width, 5 ns)
at 23 ꢁC through a quartz cell. At the desired time
points, 1 mL of MeOH was added to the samples and
in the case of 11, the samples were kept in the dark at
room temperature for a few hours to induce complete
O–N intramolecular acyl migration to the parent paclit-
axel (1). Finally, 200 lL of the mixture was directly ana-
lyzed by RP-HPLC. HPLC was performed using a C18
(4.6 · 150 mm; YMC Pack ODS AM302) reverse phase
C63H81Cl3N6O19Æ8.5H O, C: 50.93, H: 6.65, N: 5.66,
2
found: C: 51.05, H: 6.54, N: 5.18. Purity was 99%
(HPLC analysis at 230 nm).
5.2.6. 7-[Bis-[2-[[2-(dimethylamino)ethyl]amino]-2-oxo-
ethyl]amino]-4-[[30-N-(20-O-benzoyl-30-N-debenzoylpaclit-
axel)] carbonyloxymethyl] coumarin hydrochloride (11).
The whole procedure was carried out in the dark. Com-
pound 10 (6.8 mg, 0.0051 mmol), benzoic acid (1.9 mg,
0.0153 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (2.9 mg, 0.0153 mmol), and 4-(dimethyl-
amino)pyridine (0.6 mg, 0.0051 mmol) in 2 mL of dry
acetonitrile were stirred at room temperature for 2 h un-

5396
M. Noguchi et al. / Bioorg. Med. Chem. 16 (2008) 5389–5397
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The conversion profiles of the 11 were determined in
phosphate-buffered saline (10 mM PBS, pH 7.4). To
297 lL of PBS (pH 7.4) was added 3 lL of solution
including, 1, 8 and 11 (2 mM in DMSO), and the mix-
ture was irradiated with a UV lamp (365 nm, 6 W, UV
Ray Lamp, Model UV-LS, Ishii Laboratory Work
Co., Ltd) at room temperature. At the desired time
points 300 lL of MeOH was added to the samples,
and in the case of 11, the samples were kept in the dark
at room temperature for a few hours to induce complete
O–N intramolecular acyl migration to the parent paclit-
axel (1). Finally, 200 lL of the mixture was directly ana-
lyzed by RP-HPLC. HPLC was performed using a C18
(4.6 · 150 mm; YMC Pack ODS AM302) reverse phase
column with a binary solvent system: linear gradient of
CH3CN (0–100%, 40 min) in 0.1% aqueous TFA at a
flow rate of 0.9 mL/min, detected at UV 230 nm.
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Acknowledgments
20. Taniguchi, A.; Sohma, Y.; Kimura, M.; Okada, T.; Ikeda,
K.; Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.;
Kiso, Y. J. Am. Chem. Soc. 2006, 128, 696.
21. Sohma, Y.; Taniguchi, A.; Yoshiya, T.; Chiyomori, Y.;
Fukao, F.; Nakamura, S.; Skwarczynski, M.; Okada, T.;
Ikeda, K.; Hayashi, Y.; Kimura, T.; Hirota, S.; Matsu-
zaki, K.; Kiso, Y. J. Pept. Sci. 2006, 12, 823.
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2813.
This research was supported in part by the ‘Academic
Frontier’ Project for Private Universities: matching fund
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. HP is grateful for the Postdoctoral
Fellowship of JSPS. We thank Dr. Z. Ziora for critical
reading of the manuscript.
23. Skwarczynski, M.; Sohma, Y.; Noguchi, M.; Kimura, M.;
Hayashi, Y.; Hamada, Y.; Kimura, T.; Kiso, Y. J. Med.
Chem. 2005, 48, 2655.
24. Sohma, Y.; Hayashi, Y.; Skwarczynski, M.; Hamada, Y.;
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Supplementary data
25. Hayashi, Y.; Skwarczynski, M.; Hamada, Y.; Sohma, Y.;
Kimura, T.; Kiso, Y. J. Med. Chem. 2003, 46, 3782.
26. Skwarczynski, M.; Noguchi, M.; Hirota, S.; Sohma, Y.;
Kimura, T.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem.
Lett. 2006, 16, 4492.
Absorption spectra of phototaxel 11, and spectral and
analytical data for compounds. This material is avail-
able online at www.sciencedirect.com. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2008.04.022.
27. Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P.
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