Synthesis and photochemical properties of photoactivated antitumor prodrugs releasing 5-fluorouracil

Article abstract of DOI:10.1039/b417734g

A new family of antitumor prodrugs (1-3) of 5-fluorouracil (5-FU) possessing photolabile 2-nitrobenzyl chromophores have been designed and synthesized to investigate the efficiency and mechanism of photoactivated 5-FU release upon UV-irradiation at λ

Full text of DOI:10.1039/b417734g

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A R T I C L E
Synthesis and photochemical properties of photoactivated antitumor
prodrugs releasing 5-fluorouracil†
Zhouen Zhang, Hiroshi Hatta, Takeo Ito and Sei-ichi Nishimoto*
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering,
Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, 615-8510, Japan.
E-mail: nishimot@scl.kyoto-u.ac.jp
Received 24th November 2004, Accepted 20th December 2004
First published as an Advance Article on the web 12th January 2005
A new family of antitumor prodrugs (1–3) of 5-fluorouracil (5-FU) possessing photolabile 2-nitrobenzyl
chromophores have been designed and synthesized to investigate the efficiency and mechanism of photoactivated
5-FU release upon UV-irradiation at kex = 365 nm. The photoactivated prodrug 3 derived from conjugation of 2 with
a tumor-homing cyclic peptide Cys-Asn-Gly-Arg-Cys (CNGRC) was so designed as to manifest a tumor-targeting
function.
In this study an attempt was made to design and synthesize
Introduction
a new family of photoactivated 5-FU prodrugs (1–2) that
Most antitumor drugs show poorly selective cytotoxicity which
are not subject to hydrolysis, based on our experience from
causes considerable limitations on their clinical applications.
the development of radiation-activated prodrugs.6 Focusing on
Several strategies for developing prodrugs have been explored
further sophistication, we conjugated prodrug 2 with a tumor-
to achieve better therapeutic effects and/or fewer side effects
homing cyclic peptide Cys-Asn-Gly-Arg-Cys (CNGRC)11 to
of antitumor drugs,1–2 but there are still a number of problems
generate the first prototype compound 3 for a tumor-targeting
which need to be overcome. Most prodrugs releasing antitumor
photoactivated antitumor prodrug. In view of the functionality
drugs by hydrolysis usually exhibit poor stability and produce
of the peptide CNGRC that recognizes a tumor marker of spe-
various side effects on normal cells.1,3 For enzyme-activated
cific aminopeptidase N (APN/CD13) isoform over-expressing
prodrugs undergoing enzymatic hydrolysis, findings of tumor-
on the surface of tumor blood vessels,11 the photoactivated
specific enzymes and methods for selective delivery of enzymes
prodrug 3 is expected to be accumulated in tumor tissues with
high selectivity. In addition, a lysine structure was incorporated
into the linker moiety in prodrug 3 to enhance the water
solubility. Herein, we report the synthesis and photochemical
or enzymatic genes to tumor tissues are still key subjects of
investigation.4–5
Previously, we proposed a different class of antitumor pro-
drugs such as 1-(5ꢀ-fluoro-6ꢀ-hydroxy-5ꢀ,6ꢀ-dihydrouracil-5ꢀ-yl)-
reactivity of prodrugs 1–3 that effectively released 5-FU upon
5-fluorouracil, 1-(2ꢀ-oxocycloalkyl)-5-fluorouracils, and 3-(2ꢀ-
UV irradiation at kex = 365 nm, and demonstrate the mechanism
of photoactivated 5-FU release from 1 in both acetonitrile and
aqueous solutions as investigated by means of nanosecond laser
flash photolysis (LFP).
oxopropyl)-5-fluoro-2ꢀ-deoxyuridine that are activated to release
5-fluorouracil (5-FU) or 5-fluoro-2ꢀ-deoxyuridine (5-FdUrd) by
hydrated electrons (eaq−) generated in water radiolysis under
hypoxic conditions.6 Unfortunately, due to competition with
endogenous electron acceptors in the tumor tissues, these
radiation-activated prodrugs produced less effective amounts of
5-FU or 5-FdUrd under practical conditions of limited radiation
6d,e,7
doses applicable to cancer radiotherapy.
As an alternative
Results and discussion
molecular design, attention should be given to photoactivated
prodrugs with a variety of photolabile protected groups, the
triggering reactivity of which can be well controlled by selecting
the wavelength of exciting light. Photolabile compounds have
been widely applied to organic syntheses and biological inves-
tigations, since they can effectively release various active agents
with outstanding spatial and temporal precision upon con-
trolled photolysis.8 Inheriting such advantages, photoactivated
prodrugs are not only applicable to surface cancer treatment,
but also fitted even to deep-seated cancer therapy employing
endoscopes or optical fibers as used in photodynamic therapy.9
Nevertheless, there are few studies on the development of
photoactivated antitumor prodrugs. Moreover, all of the current
photoactivated prodrugs releasing phosphoamide mustard, 5-
FdUrd or leucyl-leucine methyl ester10 were constructed based
on labile linkages of phosphate, carbamate or ester that readily
undergo hydrolysis.
Synthesis
Photoactivated 5-FU prodrugs 1–2 were synthesized using a
similar method as for the preparation of 1-(2ꢀ-oxocycloalkyl)-5-
6b
fluorouracils reported previously. As outlined in Scheme 1(a),
2,4-bis(trimethylsilyloxy)-5-fluoropyrimidine was refluxed for
4 h with 2-nitrobenzyl bromide or 4-bromomethyl-3-nitroben-
zoic acid in acetonitrile under nitrogen in the dark. After cooling
at room temperature, the reaction mixture was treated with
methanol to give prodrugs 1 and 2 (see ESI†).
For the first prototype of a tumor-targeting photoactivated
prodrug, compound 3 conjugated with a tumor-homing pep-
tide CNGRC was synthesized as outlined in Scheme 1(b).
The protected peptide KGCNGRC-resin was manually pre-
pared by a standard Fmoc solid-phase synthesis method, us-
ing O-(7-azabenzotriazol-1-yl)-N,N,Nꢀ,Nꢀ-tetramethyluronium
hexafluorophosphate (HATU) and N,N-diisopropylethylamine
(DIPEA) as the activators. The resulting protected peptide
KGCNGRC-resin was condensed with compound 2, fol-
lowed by cleavage and disulfide cyclization with a trifluoro-
acetic acid (TFA)–trimethylsilyl chloride (TMSCl)–anisole–
dimethylsulfoxide (DMSO) system in a one-pot procedure12 to
produce prodrug 3.
† Electronic supplementary information (ESI) available: Experimental
procedures for photoprodrugs 1 and 2; Figures S1–S3: transient spectra
and kinetic plots for laser flash photolysis of photoprodrug 1. See
http://www.rsc.org/suppdata/ob/b4/b417734g/
5 9 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 5 9 2 – 5 9 6
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Scheme 1 (a) Syntheses of prodrugs 1 and 2; (b) synthesis of prodrug 3.
Photoactivation efficiency of 5-FU release
When solutions of 1 and 2 in acetonitrile–H2O (3 : 1 v/v) and an
aqueous solution of 3 were allowed to stand overnight at 37 ◦C
in the dark, neither decomposition of the prodrugs nor release
of 5-FU could be detected by analytical HPLC. In contrast,
UV irradiation at kex = 365 nm of these solutions resulted
in 5-FU release. Although the 2-nitrobenzyl chromophores
showed UV absorption maxima kmax at wavelengths around
265 nm, the longer wavelength of kex = 365 nm in the UV-
A (320–400 nm) was employed in this study because UV-B
(290–320 nm) and UV-C (100–290 nm) with wavelengths below
320 nm induce direct effects such as sub-lethal and lethal damage
of DNA/RNA bases in both normal and tumor cells.13 As shown
in Fig. 1, the photochemical conversion to 5-FU was efficient
and highly selective for all of the prodrugs. Apparently, prodrug
1 showed about 1.4 times higher photoactivation efficiency than
the carboxylated analog 2. This is mainly due to the UV spectral
characteristic that the absorption maximum in acetonitrile–H2O
Fig. 1 Photolysis of prodrugs 1 and 2 in acetonitrile–H2O (2 : 1, v/v)
and 3 in H2O at kex = 365 nm: decrease of prodrugs ((᭺) 1; (ꢁ) 2; (ꢀ) 3)
and 5-FU release from prodrugs ((᭹) 1; (ꢁ) 2; (ꢂ) 3).
selectivity) of 5-FU. This result greatly encourages further inves-
tigation of tumor-targeting photoactivated prodrugs conjugated
with various tumor-homing peptides by photolabile linkers.
The novel class of tumor-targeting photoactivated anticancer
prodrugs reported herein show resistance to hydrolysis could be
easily accumulated within tumor tissue and then controlled to
effectively release active antitumor agents by applied light. So
these prodrugs could selectively work within antitumor tissues
and minimize side effects on normal tissues. These prodrugs also
show higher efficiency and selectivity to release active agents in
comparison to radiation-activated prodrugs.6
max
(3 : 1 v/v) (kabs = 264 nm) of 2 bearing a carboxylic group
max
was slightly blue-shifted relative to that of 1 (kabs = 267 nm).
On the other hand, the KGCNGRC-conjugated prodrug 3
showed about 6 times smaller efficiency of photoactivated 5-FU
release in aqueous solution (kmax = 262 nm) than prodrug 1 in
acetonitrile–H2O (3 : 1 v/v). Prodrug 3 produced some unknown
byproducts in the photolysis, while still showing high efficiency
and selectivity of 5-FU release: 365 nm UV irradiation of 3
for 6 hours resulted in 80% decomposition and 60% yield (75%
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 5 9 2 – 5 9 6
5 9 3

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Nanosecond laser flash photolysis (LFP) of prodrug 1
Scheme 3 illustrates a proposed mechanism by which prodrug
1 is photoactivated to release 5-FU via a series of reaction steps
in acetonitrile–H2O (2 : 1 v/v) solution at various pH values. The
observed rate constant ka/b for the ring-closure of A (or A−) is
strongly pH-dependent and is more accelerated with increasing
acidity in the pH range <10 (see Fig. S3(a) in ESI†) Two
sigmoidal pH–rate profiles are clearly seen in Fig. S3(a), which
correspond to the acid–base equilibrium constants for A/A−
(pKa/a− ≈ 3.8) and A−/A2− (pKa−/a2− ≈ 10), respectively (see
Scheme 3). The rate of ring-closure from A2− to B2− occurring
at pH > 10 seems to be extremely small (ka/b ≈ 20 s−1). In
the wide pH range of 1 to 12, ka/b is about 50–10000 times
A mechanistic study on the photoactivated release of 5-FU
was performed by nanosecond 266 nm LFP of prodrug 1
(0.1 mM) in both Ar-saturated acetonitrile and aqueous acetoni-
trile solutions. An intense fluorescence emission was observed
immediately after 266 nm laser flash excitation of prodrug 1
in acetonitrile. Along with the fluorescence decay, buildup of
a transient with kmax = 410 nm was observed (see Fig. S1(a)
in ESI†). By reference to a previous study,14 this transient is
attributable to an intermediate of aci-nitro tautomer (A) formed
by photoinduced intramolecular hydrogen transfer, as shown in
Scheme 2. The decay of intermediate A is well represented in
terms of first-order kinetics with a rate constant of ka/b = (1.4
0.1) × 104 s−1 (see Fig. S1(b) in ESI†). The absorption of transient
A decayed to the baseline level (see the inset of Fig. S1(b)) on
a sub-ms timescale without leaving any residual absorption at
wavelengths above 300 nm. Subsequently, slow buildup of a
new transient was observed with absorption at around 320 nm
to attain its maximum value 2 s after LFP (see Fig. S1(a)
and (c)). This transient may be assigned to an intermediate
C generated from the preceding intermediate B that shows no
greater than kb/c and kb/c is about 2.5–10 times greater than kc/4
.
Obviously, formation of C involves acid catalysis at pH < 7,
while decomposition of C is weakly catalyzed by acid at pH < 3
and by base at pH > 7 (see Fig. S3(b)).
Conclusion
A new family of photoactivated 5-FU prodrugs 1–3 with pho-
tolabile 2-nitrobenzyl chromophores could release quantitative
14c,d
amounts of 5-FU in solution upon UV irradiation at kex
=
absorption at wavelengths above 300 nm (see Scheme 2).
365 nm, while being quite stable in the dark. A photoactivation
mechanism by which the representative prodrug 1 releases 5-
FU was clarified by means of nanosecond LFP. The prodrug
3 derived from conjugation of 2 with a typical tumor-homing
cyclic peptide CNGRC is a prototype for the first tumor-
targeting photoactivated antitumor prodrug. In view of the
evidence that the prodrug 3 was effectively photoactivated to
release 5-FU, further investigations on the synthesis of related
compounds and evaluation of their anti-tumor activity are in
progress.
Formation of C is also supported by the concomitant growth
of a very weak absorption at 740 nm, which is characteristic
of nitroso compounds.15 The intermediate C (see Fig. S1(c))
showed first-order kinetic growth and decay with the respective
rate constants of kb/c = 1.2 0.1 s−1 and kc/4 = 0.13 0.02 s−1 to
generate the final products 5-FU and 2-nitrosobenzaldehyde (4)
14c,d
(Scheme 2).
These products were confirmed by HR-FABMS
in a separate experiment with the 365 nm UV irradiation of 1
(1 mM) in Ar-saturated acetonitrile solution.
The transient absorption spectrum of aci-nitro tautomer A
with kmax = 410 nm was also observed upon LFP of 1 in
acetonitrile–H2O (2 : 1 v/v) solution, as in bulk acetonitrile
solution, while kmax was quickly shifted to a longer wavelength
of 460 nm (Fig. S2(a) and S1(b)). This red-shift is attributed to
ionization of A to A− with the first-order kinetic rate constant of
ka/a− = (1.0 0.1) × 106 s−1 (see Scheme 3). Similar ionization
processes were also observed in the LFP of several 2-nitobenzyl
compounds in aqueous solution.14 This conclusion is also in
accord with observations in the LFP of 1 in acetonitrile–H2O
(2 : 1 v/v) solution at various pH values. While the absorption
with kmax = 410 nm was observed in acidic solutions (pH ≤
2.5) until it decayed to baseline and subsequent buildup at
around 320 nm appeared (see Fig. S2(c)), the absorption with
kmax = 460 nm appeared quickly after the LFP in neutral and
basic solutions (see Fig. S2(d)). The deprotonated intermediate
A− formed in acetonitrile–H2O (2 : 1 v/v) solution was more
stable than the protonated intermediate A formed in acetonitrile.
Experimental
General
Thin-layer chromatography was performed on a silica gel coated
glass sheet (Silica Gel 60 F254 TLC plates, Merck & Co.). Wakogel
C-300 silica gel (45–75 lm, Wako Pure Chemical Industries Ltd.)
was used for column chromatography. HPLC was performed on
a Hitachi D-7000 HPLC system. An Interstil ODS-3 column
(φ 4.6 mm × 250 mm, GL Science Inc., Japan) was used for
analytical HPLC. Prodrugs and photoproducts were eluted with
acetonitrile–H2O mixed solvents at a flow rate of 0.6 mL min−1
and detected by UV absorbance at 220 nm. A preparative
Interstil ODS-3 column (φ 10 mm × 250 mm, GL Science
Inc., Japan) was used for preparative HPLC. Products were
eluted with a linear gradient of acetonitrile (10.8–50%, 30 min)
containing 0.05% TFA at a flow rate of 3.0 mL min−1 and
detected by UV absorbance at 260 nm. 1H NMR and 13C NMR
spectra were recorded on a JEOL JNM-EX-400 (400 MHz) or a
JNM-AL300 (300 MHz) spectrometer. Fast atom bombardment
mass spectrometry (FAB MS) was performed on a JEOL
JMS-SX102A mass spectrometer, using glycerol matrix or 3-
nitrobenzyl alcohol (NBA) matrix.
Following the first-order kinetics with ka/b = (1.4
0.1) ×
103 s−1, A− underwent ring-closure to B− that is transparent
at wavelengths above 300 nm. Subsequently, slow ring-opening
of B− into C (kmax = 320 nm) occurred with kb/c = 1.1
0.1 s−1, followed by decomposition of C to release 5-FU and
2-nitrosobenzaldehyde with kc/4 = 0.13 0.02 s−1 (see Fig. S2(a)
and Scheme 3).
Scheme 2 Proposed mechanism of photoactived 5-FU release from prodrug 1 in acetonitrile.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 5 9 2 – 5 9 6
5 9 4

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condensed with compound 2 (30 mg, 0.1 mmol) under activation
by HATU and DIPEA. After filtration and washing with
N,N-dimethylformamide (5 × 4 mL) and dichloromethane
(4 × 4 mL), the resulting protected 5-FU–KGCNGRC-resin
was treated with a mixture of trimethylsilyl chloride (TMSCl,
0.22 mL)–anisole (0.3 mL)–trifluoroacetic acid (TFA, 26 mL)
at room temperature. After 1 hour, dimethylsulfoxide (DMSO,
3.8 mL, 300 equivalent) was added to the reaction mixture which
was then stirred for another 1 h at 4 ◦C.12 After removal of the
resin by filtration, ice-cooled dry diethyl ether (80 mL) was added
to the filtrate. The precipitate was collected by centrifugation
and washed with ice-cooled dry diethyl ether (4 × 20 mL).
The crude product was purified by preparative HPLC (Rt =
19.8 min) and freeze-dried to give a fluffy pale yellow powder of
5-FU–KGCNGRC conjugate 3 (3.1 mg, 15% calculated based
on the Fmoc-Cys(Acm)-PEG-PS-resin), as identified by FAB-
HRMS (positive mode, glycerol matrix): m/z 1026.3320 [MH+],
C38H53N15O14FS2 requires 1026.3322.
Nanosecond laser flash photolysis (LFP)
The nanosecond spectroscopic LFP experiment for prodrugs 1–
3 was performed with a Unisoku TSP-601 flash spectrometer. A
Continuum Surelite-I Nd–YAG (Q-switched) laser with fourth
harmonic generation at 266 nm was used for the nanosecond
flash excitation. Further details of the laser flash system have
6c
been described previously.
Solutions of 1 (0.1 mM) in acetonitrile or acetonitrile–H2O
(2 : 1 v/v) were deaerated by argon bubbling prior to the laser
flash photolysis. In the pH–rate profile experiment, solutions
of 1 (0.1 mM) in acetonitrile–H2O (2 : 1 v/v) at various
pH values were prepared using 10 mM phosphate buffer with
acidification by 0.001–0.1 M perchloric acid or basification by
0.001–0.1 M sodium hydroxide. The first-order rate constants
ka/b were evaluated from the decay of the absorption at 410 nm
(pH ≤ 3.8) or 460 nm (pH ≥ 3.5), while kb/c and kc/4 were
determined from the growth or decay of the absorption at
320 nm, respectively.
UV irradiation of photoprodrugs and products analysis
A 25 W ultraviolet transilluminator (kex = 365 nm, Model TFL-
40, UVP Inc.) was used for steady-state UV irradiation. For a
product study, Ar-saturated solution of 1 (1 mM) in acetonitrile
was photoirradiated at kex = 365 nm for 20 min and the final
products of 5-FU and o-nitrosobenzaldehyde 4 were determined
by FAB-HRMAS (positive mode, glycerol matrix): for 5-FU,
m/z 131.0252 [MH+], C4H4O2N2F requires 131.0251; for 4, m/z
136.0404 [MH+], C7H6O2N requires 136.0399.
Solutions of 1 (1 mM) in acetonitrile–H2O (3 : 1 v/v), 2
(1 mM) in acetonitrile–H2O (3 : 1 v/v) and 3 (1 mM) in H2O were
irradiated at kex = 365 nm in air at 37 ◦C, while the corresponding
◦
control solutions were kept overnight in the dark at 37 C. At
various time intervals, an aliquot (10 lL) was sampled and
examined by analytical HPLC. A mixed solvent of acetonitrile–
H2O (1 : 1 v/v) with HCl (pH 3) was used as eluent in the
HPLC analyses for 1 and 2, while a linear gradient elution with
acetonitrile–H2O mixed solvents (5.9–50%, 30 min) containing
0.05% (v/v) trifluoroacetic acid (TFA) was applied for the HPLC
analysis of 3. The concentrations of 1–3 and released 5-FU
were quantified from the HPLC peak areas, using the respective
calibration curves prepared by reference to authentic samples.
Scheme 3 Proposed mechanism of photoactived 5-FU release from
prodrug 1 in acetonitrile–H2O (2 : 1 v/v) solution.
1-(2ꢀ-Nitro-4ꢀ-carboxybenzyl)-5-fluorouracil–KGCNGRC
conjugate (3)
A protected peptide KGCNGRC-resin was manually prepared
by an Fmoc solid phase synthesis method, using Fmoc-
Cys(Acm)-PEG-PS resin (100 mg, 0.02 mmol) and 5 equivalent
Fmoc-amino acids, Arg(Pbf), Asn(Trt), Cys(Acm), Gly and
Lys(Boc), along with O-(7-azabenzotriazol-1-yl)-N,N,Nꢀ,Nꢀ-
tetramethyluronium hexafluorophosphate (HATU, 5 equiva-
lent) and N,N-diisoproylethylamine (DIPEA, 10 equivalent)
as activators. The protected peptide KGCNGRC-resin was
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5 9 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 5 9 2 – 5 9 6