Long conjugated 2-nitrobenzyl derivative caged anticancer prodrugs with visible light regulated release: Preparation and functionalizations

Article abstract of DOI:10.1039/c2ob25701g

A series of anticancer prodrugs with different chemical functional groups were prepared, in which the styryl conjugated 2-nitrobenzyl derivatives were introduced as the phototrigger to regulate the drug (chlorambucil) release. Compared to the common 4,5-dimethoxy-2-nitrobenzyl caged compounds, most of the prodrugs exhibited large and redshifted one-photon absorption within the visible range. One-photon excitation for the drug release was studied by measuring UV-vis absorption, FT-IR, and HPLC spectra, which suggested that chlorambucil was released effectively and precisely by manipulating external light conditions. And the introduction of different functional groups made this type of prodrug a good platform to further react with some typical drug carriers and to further form excellent visible light responsive drug delivery systems. Moreover, the drug also could be effectively released under the excitation of two-photon at 800 nm with comparable photorelease efficiencies. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25701g

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PAPER
Long conjugated 2-nitrobenzyl derivative caged anticancer prodrugs with
visible light regulated release: preparation and functionalizations†
Chunyan Bao,a Ming Jin,*b Bo Li,c Yaodong Xu,a Jingyan Jina and Linyong Zhu*a
Received 10th April 2012, Accepted 17th May 2012
DOI: 10.1039/c2ob25701g
A series of anticancer prodrugs with different chemical functional groups were prepared, in which the
styryl conjugated 2-nitrobenzyl derivatives were introduced as the phototrigger to regulate the drug
(chlorambucil) release. Compared to the common 4,5-dimethoxy-2-nitrobenzyl caged compounds,
most of the prodrugs exhibited large and redshifted one-photon absorption within the visible range.
One-photon excitation for the drug release was studied by measuring UV-vis absorption, FT-IR, and
HPLC spectra, which suggested that chlorambucil was released effectively and precisely by manipulating
external light conditions. And the introduction of different functional groups made this type of prodrug a
good platform to further react with some typical drug carriers and to further form excellent visible light
responsive drug delivery systems. Moreover, the drug also could be effectively released under the
excitation of two-photon at 800 nm with comparable photorelease efficiencies.
Among the reported photoactivated groups, 2-nitrobenzyl
derivatives (ONB) have gained wide acceptance and a lot of
Introduction
Recently, the design and synthesis of stimuli-responsive prodrugs
that exhibit reduced toxicity in health tissues and can be trans-
formed into pharmacologically active agents specifically within
the tumor regions have aroused considerable interest for the
development of modern chemotherapy of malignant tumors.1–3
Among the stimuli-responsive strategies for prodrugs, light exhi-
bits a predominant advantage. It is an external stimulus that
could allow a very mild activation of sensitive molecules and
provides a greater selectivity in terms of control over the
moment and the location of drug-release without physically dis-
turbing organized biological systems. To date, considerable
effort have been put into preparing photoactived prodrugs.4 For
example, we recently reported two types of photoactived pro-
drugs based on coumarin as phototrigger5 or N-methyl-4-picoli-
nium as photolabile group by photo-induced electron transfer
(PET) with semiconductor quantum dots (QDs).6
caged biologically active compounds that have been prepared
belong to this series4b,c,7,8 due to their versatile modification and
well-known photolysis mechanism.9 However, most of the
known ONB-caged chromophores showed UV absorption, which
is the major drawback and limitation for the practical application,
since the presence of UV light is damaging to cells as it also pro-
vides poor penetration due to light scattering and absorbance by
intrinsic biological chromophores.10 Recently, the Jullien and
Bolze groups reported that the elongation of the conjugation
backbone for the nitro group and the introduction of D–π–A
backbone would redshift the maximum wavelength of absorp-
tion, and enlarge the corresponding molar absorption coefficient
as well as the two-photon absorption (TPA) cross section.11
Therefore, the present paper would utilize the styryl-2-nitroben-
zyl (SNB) platform as cage group to prepare chlorambucil pro-
drugs photoactivated either by one-photon visible light or two-
photon NIR light.
Moreover, to reduce systemic toxicity and enhance therapeutic
efficacy, the photoactivated prodrugs always need to incorporate
with carriers prior to the actual applications, e.g. nanoscale car-
riers promise both prolonged circulation time due to the nano-
scale size and selective tumor accumulation via the enhanced
permeability and retention (EPR) effects. The widely used
carriers mainly contained biocompatible macromolecules,12 inor-
ganic nanoparticles,4c,5,7c liposomes,13 and protein nanocompo-
sites.14 To achieve this aim, linker chemistry is developed
between the photoactivated prodrugs and carriers because it pro-
vides more precise control in terms of the density and orientation
of the caged biomolecules and forms a stable linkage under
aShanghai Key Laboratory of Functional Materials Chemistry, Institute
of Fine Chemicals, East China University of Science and Technology,
Shanghai 200237, P. R. China. E-mail: linyongzhu@ecust.edu.cn;
Fax: +86-21-64253742
bInstitute of Functional Polymer Materials, School of Materials Science
and Engineering, Tongji University, Shanghai 200237, P. R. China.
E-mail: mingjin@tongji.edu.cn; Fax: +86-21-69580143
cKey Laboratory of Polar Materials and Devices, Ministry of Education,
Department of Electronic Engineering, East China Normal University,
Shanghai, 200241, P. R. China
†Electronic supplementary information (ESI) available: Detailed
synthesis procedures and characterizations for the intermediate com-
pounds, UV-vis and HPLC spectra for the final new prodrugs. See DOI:
10.1039/c2ob25701g
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Scheme 2 The schematic synthesis procedures for the prodrugs 1–7.
Regeants and conditions: a, H2SO4 (85%), guanidinium nitrate, at
0–5 °C; b, H2SO4/H2O, NaNO2, 0 °C for 10 min, then KI at 0 °C for
30 min, rt for 3 h; c, BPO/NBS, CCl4, reflux for 36 h; d, Na2CO3,
acetone/water, reflux for 48 h; e, K2CO3/KI, bromoethane, acetone,
reflux; f, Ph3P+CH3Br−, t-BuOK, THF, rt; g, 12, Pd(OAc)2/N(OEt)3,
110 °C for 12 h; h, chlorambucil, DCM/DCC/DMAP, rt for 12 h; i,
p-hydroxybenzaldehyde 13, PPTS/3,4-2H-dihydropyran/DCM, rt; j,
TFA, DCM, rt; k, 3-bromopropyne or 1,4-dibromobutane, K2CO3/
acetone or acetonitrile; l, tert-butyl 2-bromoacetate/K2CO3/acetonitrile,
rt for 24 h, then TFA/DCM; m, methacryloyl chloride, N(Et)3/DCM,
0 °C, 12 h; n, p-hydroxybenzaldehyde, 2-bromo-1,1-dimethoxyethane
K2CO3/KI/DMF, 100 °C overnight; o, TFA/DCM, rt for 2 h.
Scheme 1 Structures of designed SNB based prodrugs 1–7.
in vivo conditions. Herein, we designed and synthesized a series
of styryl conjugated 2-nitrobenzyl-caged (SNB) chlorambucil
prodrugs (1–7) with different functional linkage (as shown in
Scheme 1) that expected to conjugate with carriers. The success-
ful controllable drug release regulated by either one-photon
visible light or two-photon NIR laser light suggested that the
prepared model prodrug served well as a photo-cage for the drug
whose release can be regulated precisely by controlling the
irradiation conditions. And the different chemical functionaliza-
tion was hopeful to provide a new promising strategy for conju-
gating the prodrugs with carriers to further form excellent photo-
responsive drug delivery system.
acid, azide, amino, hydroxyl, and thiol contained carriers by
simple esterification, amidation, efficient “click chemistry”, and
thiol-ene reactions, respectively.15 Especially, the introduction of
methacrylate was also expected to copolymerization in some
biocompatible macromolecules besides the conjugation with
thiol-containing carriers by thiol-ene reaction.
Results and discussion
Design and synthesis of SNB prodrugs
The synthetic methods for the prodrugs were described in
Scheme 2. Prodrug 1 was obtained by the esterification of
chlorambucil with benzyl alcohol 16 in the presence of dicyclo-
hexylcarbodiimide (DCC) and 4-N,N-dimethylaminopyridine
(DMAP) in 78.7% yield. The latter was obtained by Heck reac-
tion between 4-ethoxystyrene (compound 15) and 5-iodo-2-nitro-
benzyl alcohol (compound 12) in the presence of a palladium
catalyst.16 Compound 15 was synthesized in two steps from
4-hydroxylbenzaldehyde by (1) alkylation with bromoethane
to provide compound 14; (2) reduction by Witting reaction.
Compound 12 was obtained in four steps from m-toluidine by
(1) nitration utilising guanidinium nitrate in 85% sulfuric acid as
a nitrating agent;17 (2) diazotation with sodium nitrite followed
by nucleophilic substitution with iodide to provide 4-iodo-2-
methyl-1-nitrobenzene 10; (3) bromination by N-bromosuccin-
imide (NBS) to afford 4-iodo-2-bromomethyl-1-nitrobenzene 11;
(4) hydrolysis to 4-iodo-2-hydroxymethyl-1-nitrobenzene 12.
Prodrug 2 was also synthesized similar as for the prodrug
1 except 3,4-dihydro-2H-pyran was used to protect and finally
release of the hydroxyl group.18 Prodrugs 3–6 were prepared
from prodrug 2 by different alkylation and esterification,
Thinking about molecular engineering to extend the maximum
absorption wavelength and increase the molar absorptivity,
elongation of the conjugated π-system or an increase in the
power of the donor and acceptor side groups for ONB photo-
trigger was required. Although the introduction of the stilbene
decreased the photolysis quantum yield Φ,11 the advantages –
visible light absorption and suitable two-photon sensitivity –
made them still regarded as an excellent photo-cage, especially
for bioactive systems. Here, a styrene group was introduced to
increase the length of the 2-nitrobenzyl conjugated system and
unfunctionalized ethoxyl was used as the electron-pushing group
for prodrug 1 which would be treated as model prodrug in our
paper to investigate photochemical properties, such as the
UV-Vis, FT-IR, and HPLC spectra for the photorelease of
the combined chlorambucil drug. Furthermore, considering the
incorporation with carriers, several functional groups including
hydroxyl, alkynyl, carboxylic acid, alkyl bromide, methacrylate,
aldehyde (see prodrug 2–7) were introduced to form pre-
functional prodrugs. The introduction of these functional groups
were proposed to conjugated with the corresponding carboxylic
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Table 1 Photochemical properties of prodrugs 1–7a
λmax
Prodrugs (nm)
εmax
ελ = 400 nm
Φchem
Φchem × ελ
(×104)b (×104)c
(×10−4 d
)
= 400 nme
1
2
3
4
5
6
7
376
374
369
372
375
351
372
2.33
2.17
2.22
2.46
2.06
2.27
2.27
1.73
1.61
1.03
1.56
1.51
0.51
1.20
6.7
5.2
11.4
3.2
6.0
15.4
7.3
11.6
8.4
11.7
5.0
9.1
7.9
8.8
a All experiments were done in acetonitrile solution with concentration
of 10−4 M. b Molar absorptivity at maximum absorption wavelength.
c Molar absorptivity at 400 nm. d Photolysis quantum yield for the
consumption of prodrugs upon the irradiation of 400 nm (10 mw cm−2).
e The overall photolysis efficiency: the photolysis quantum yield and
molar absorptivity at 400 nm.
Scheme 3
prodrugs.
A
schematic presentation for photocleavage of the
Fig. 1 Normalized absorption (dashed) and fluorescence spectra (λex
380 nm) (solid) of prodrug 1 in solvents of different polarity.
=
respectively. Prodrug 7 was obtained by the similar synthesis
procedure as for prodrug 1 and 2 excepting 2-bromo-1,1-
dimethoxyethane was used to react with 4-hydroxylbenzalde-
hyde, and finally aldehyde substituted prodrug was generated by
TFA-mediated deacetalization.19
Photophysical and photolysis results
The absorption properties of the prodrugs are summarized in
Table 1. Except for prodrug 6 (electron-drawing substitution), the
maximum absorption of the prodrugs is located around 370 nm
and the absorption band extended up to 450 nm which is red-
shifted about 20 nm compared with the popular 4,5-dimethoxy-2-
nitrobenzyl phototriggers, as well as the molar absorption coeffi-
cient increased four times.11a,20 It suggested that the introduction
of donating power and π-conjugated stilbene prolonged the
absorption wavelength and enlarged the molar absorption coeffi-
cient, as well as provided the opportunity for the one-photon
photorelease by visible light. Due to the similar absorption proper-
ties for the different substitutions, prodrug 1 was selected as the
model to investigate the concrete photophysical and photochemi-
cal properties in the following experiments as mentioned above.
First, the normalized UV-vis and fluorescence spectra of
prodrug 1 in various solvents of increasing polarity are shown in
Fig. 2 One-photon irradiation (λ ≥ 400 nm, 120 mW cm−2) of a sol-
ution of prodrug 1 in acetonitrile (10−4 M). (a) Evolution of the UV-vis
absorption spectra of the solution as a function of time (t (min) = 0, 1, 5,
10, 15, 20, 25, 30, 35, 40). (b) Evolution of the IR spectra of the sol-
ution as a function of time (t (min) = 0, 5, 15, 30), the sample was pre-
pared by dropping the solution on a KBr pellet and tested after drying
under vacuum.
Fig. 1. It is found that the polarity influenced the position of the
longest absorption and emission peaks.21 Then, drug release
experiments of prodrug 1 were proceeded by exposing an aceto-
nitrile solution (10−4 M) to the visible light (λ ≥ 400 nm).
Based on the photolysis mechanism for ONB phototriggers,11a
prodrug 1 was photolyzed to yield the parent chlorambucil and
corresponding 2-nitroso-5-styrylbenzaldehyde derivatives as
photo byproducts (as shown in Scheme 3). Fig. 2a and 2b
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displayed typical evolution of the UV-vis and FT-IR spectra as a
function of time upon illumination of visible light (λ ≥ 400 nm,
120 mW cm−2). In Fig. 2a, a rapid decrease in absorbance
around 376 nm was observed, which suggested the photolysis of
SNB was proceeded effectively and gradually. And the appear-
ance of isosbestic points at 246, 269, 324, and 431 nm indicated
a relatively clean photochemical reaction for prodrug 1 upon
visible light irradiation. In the FT-IR spectra shown in Fig. 2b,
the signal of the characteristic peaks belonging to NO2 groups at
1578 cm−1 and 1334 cm−1 were reduced and a new peak at
1695 cm−1 belonging to nitrosoaldehyde appeared and increased
as a function of irradiation time, which further confirmed the
drug release proceeded well upon illumination of visible light.
To check the kinetics of the photorelease process, HPLC
analysis was performed. As illustrated in Fig. 3a, the release pro-
cesses progressed effectively for prodrug 1 upon the irradiation
with visible light (λ ≥ 400 nm, 120 mW cm−2), and the
maximum release of chlorambucil reached 68% after 50 min
exposure. The incomplete photolysis for prodrug 1 may be attrib-
uted to an internal filtering effect from the photo-byproduct.
Precise control of the photolytic release was demonstrated by
monitoring the progress of chlorambucil release after periods of
exposure to light and dark conditions, as shown in Fig. 3b. The
distinctive “stepped” profile revealed that the drug release only
proceeded under light conditions, thus realizing “light-regulated
precise release”. The photolysis was further studied in PBS
buffer solution containing 50% acetonitrile. As shown in Fig. 3c,
the photorelease was also effectively carried out, although the
release rate was a little slower than that in acetonitrile. This may
be attributed to the relatively bad solubility for prodrug in PBS
solution. The photorelease stability was also validated with the
high remaining rate of the prodrugs (larger than 95%) by
keeping the prodrug solution in the dark for 48 h. All these
results indicated that this kind of SNB-based phototriggers were
stable enough for the biological application and would act as the
photo-responsive linker between drug and carrier, and the drug
release could be precisely controlled by manipulating external
light conditions.
The sensitivity of prodrug 1 to two-photon excitation was also
studied and quantified as the product of TPA cross-section δa
and the uncaging quantum yield Φ. The δa value was determined
to 20 GM by using a 80 fs-pulsed, mode-locked Ti:sapphire
laser at 800 nm by the method of Z-scan technique. Photorelease
experiment with two-photon excitation for prodrug 1 was
measured by HPLC and graphed as a function of time as shown
in Fig. 4. It showed that the release of chlorambucil was pro-
gressed effectively and it reached 20% after 2 h with a ∼0.2 mm
diameter beam spot. Although the overall photorelease
efficiency, δaΦ, was not high (about 14 mGM at 800 nm), there
were some successful examples to support the biological appli-
cations, even at the single-cell level.8a,22
Fig. 3 (a) The time course of drug release for prodrug 1 under visible
light irradiation (λ ≥ 400 nm, 120 mW cm−2) determined by HPLC
analysis. (b) The partial progress for the release of chlorambucil from
prodrug 1 in light and dark conditions. “ON” indicates the beginning of
light irradiation; “OFF” indicates the ending of light irradiation. (c) The
comparison for time course of drug release of prodrug 1 in different
solvent under the same irradiation conditions.
Conclusions
We synthesized a new series of SNB caged prodrugs coupled
with various functional groups. Upon either one or two-photon
excitation, chlorambucil could be released in a controlled way
using visible or NIR light at 800 nm, which avoided the use of
UV light and is more suitable for biological applications. In par-
ticular, the introduction of different chemical linkage provided
opportunity for the prodrugs to further conjugate with different
biocompatible carriers, which provided
a new promising
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evaporated. The obtained crude was purified by silica gel chrom-
atography (hexane–ethyl acetate, 8 : 1) to afford a yellow solid
Prodrug 1 (460 mg, 78.7% yields). 1H NMR (400 MHz,
CDCl3), δ (ppm): δ = 8.10 (d, J = 8.6 Hz, 1H), 7.59 (s, 1H),
7.52 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.18 (d, J =
16.3 Hz, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 16.3 Hz,
1H), 6.91 (d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.5 Hz, 2H), 5.52
(s, 2H), 4.05 (q, J = 7.0 Hz, 2H), 3.66 (t, J = 7.2 Hz, 4H), 3.58
(t, J = 7.2 Hz, 4H), 2.59 (t, J = 7.4 Hz, 2H), 2.46 (t, J = 7.4 Hz,
2H), 2.03–1.93 (m, 2H), 1.43 (t, J = 7.0 Hz, 3H). 13C NMR
(100 MHz, CDCl3), δ (ppm): δ = 159.6, 145.2, 143.9, 137.7,
133.1, 128.7, 128.4, 127.0, 126.0, 125.4, 123.8, 114.8, 63.6,
62.8, 14.8. ES-HRMS (m/z): calcd for C31H35 Cl2N2O4,
585.1926 [M + H]+; found, 585.1923.
(E)-5-(4-Hydroxystyryl)-2-nitrobenzyl 4-(4-(bis(2-chloroethyl)-
amino)phenyl)butanoate (Prodrug 2). In the dark, yellow solid
20 (600 mg, 0.94 mmol) was dissolved in 20 mL dichloro-
methane and was treated with TFA (1.0 g, 8.8 mmol) at room
temperature for 24 hours. Then, the solvent was evaporated and
prodrug 2 was obtained as a yellow solid 390 mg (74.6%) after
silica gel column chromatography (hexane–ethyl acetate, 6 : 1).
1H NMR (400 MHz, CDCl3), δ (ppm): δ = 8.14 (d, J = 8.6 Hz,
1H), 7.60 (s, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 8.2 Hz,
2H), 7.18 (d, J = 16.3 Hz, 1H), 7.08 (d, J = 8.2 Hz, 2H),
6.96 (d, J = 16.3 Hz, 1H), 6.86 (d, J = 8.2 Hz, 2H), 6.62 (d,
J = 8.2 Hz, 2H), 5.55 (s, 2H), 5.26 (s, 1H), 3.68 (t, J = 7.0 Hz,
4H), 3.60 (t, J = 7.0 Hz, 4H), 2.61 (t, J = 7.4 Hz, 2H), 2.48 (t,
J = 7.4 Hz, 2H), 2.05–1.96 (m, 2H). 13C NMR (100 MHz,
CDCl3), δ (ppm): δ = 173.1, 156.6, 145.6, 143.5, 133.1, 132.9,
129.7, 128.7, 126.7, 126.0, 125.6, 123.8, 115.9, 112.5, 63.2,
53.7, 40.4, 34.0, 33.6, 26.7. ESI-HRMS (m/z): calcd for
C29H31Cl2N2O5, 557.1610 [M + H]+; found, 557.1609.
Fig. 4 Time course of two-photon excited drug release from prodrug 1
at 800 nm (0.60 mJ cm−2 per pulse). Sample (10−4 M) was irradiated in
acetonitrile solution.
platform for designing and preparing intelligent drug delivery
system with precise modulation by manipulating external light
conditions.
Experimental
General
Materials. All reagents were purchased from commercial
available sources such as Aldrich, TCI or Fisher and used
without further purification. Tetrahydrofuran (THF) was distilled
over sodium/benzophenone. Acetonitrile and dichloromethane
(CH2Cl2) were distilled from calcium hydride before use, and
Et3N was redistilled from and dried over KOH pellets.
(E)-2-Nitro-5-(4-(prop-2-yn-1-yloxy)styryl)benzyl
4-(4-(bis-
Characterization. Proton and carbon nuclear magnetic reso-
nance spectra (1H, 13C NMR) were recorded on a Bruker Avance
400 MHz spectrometer. Chemical shifts were reported in parts
per million (ppm) downfield from the Me4Si resonance which
was used as the internal standard when recording 1H NMR
spectra. Mass spectra were recorded on a Micromass GCTTM
and a Micromass LCTTM. Absorption spectra were recorded on
a Shimadzu UV-2550 UV-vis spectrometer. FT-IR spectra were
obtained on a Nicolet 380 FT-IR spectrometer with KBr pellets.
The steady-state fluorescence experiments were performed on a
Varian Cary Eclipses fluorescence spectrometer. Samples for
absorption and emission measurements were measured using
1 cm × 1 cm quartz cuvettes. Reversed-phase HPLC using a
BetaBasic-18 column was analyzed by Agilent technologies
1200 series.
(2-chloroethyl)amino)phenyl)butanoate (Prodrug 3). In the
dark, yellow prodrug 2 (100 mg, 0.18 mmol) and potassium car-
bonate (50 mg, 0.36 mmol) were dissolved in acetone (20 mL)
at room temperature. After injecting 3-bromopropyne (26 mg,
0.22 mmol), the mixture was kept stirring at room temperature
for another 6 hours. Finally, the obtained mixture was filtered,
and concentrated. The crude product was purified by silica gel
column chromatography (hexane–ethyl acetate, 6 : 1) to afford
1
90 mg prodrug 3 as a yellow solid with 84.1% yield. H NMR
(400 MHz, CDCl3), δ (ppm): δ = 8.13 (d, J = 8.4 Hz, 1H), 7.61
(s, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.20
(d, J = 16.3 Hz, 1H), 7.07 (d, J = 7.9 Hz, 2H), 7.01 (d, J = 8.1
Hz, 2H), 6.98 (d, J = 16.3 Hz, 1H), 6.61 (d, J = 7.9 Hz, 2H),
5.54 (s, 2H), 4.74 (s, 2H), 3.67 (t, J = 7.0 Hz, 4H), 3.60 (t,
J = 7.0 Hz, 4H), 2.60 (t, J = 7.2 Hz, 2H), 2.55 (s, 1H), 2.46 (t,
J = 7.1 Hz, 2H), 2.04–1.95 (m, 2H). 13C NMR (100 MHz,
CDCl3), δ (ppm): δ = 172.9, 158.2, 145.7, 144.4, 143.4, 132.9,
130.3, 129.7, 128.4, 126.8, 126.0, 125.7, 124.4, 115.3, 112.2,
78.2, 75.8, 63.1, 55.9, 53.6, 40.5, 34.0, 33.6, 26.7. ES-HRMS
(m/z): calcd for C32H33Cl2N2O5, 595.1767 [M + H]+; found,
595.1766.
Preparation of prodrugs
(E)-5-(4-Ethoxystyryl)-2-nitrobenzyl 4-(4-(bis(2-chloroethyl)-
amino)phenyl)butanoate (Prodrug 1). Under protection of
argon, compound 16 (300 mg, 1.0 mmol) and chloroambucil
(335 mg, 1.1 mmol) were dissolved in dichloromethane at room
temperature in the presence of DCC (248 mg, 1.2 mmol) and
DMAP (12 mg, 0.1 mmol), the reaction was kept at rt for
12 hours. Then the mixture was filtered and the solvent was
(E)-2-(4-(3-(((4-(4-(Bis(2-chloroethyl)amino)phenyl)butanoyl)-
oxy)methyl)-4-nitrostyryl)phenoxy)acetic acid (Prodrug 4). In
the dark, a mixture of prodrug 2 (100 mg, 0.18 mmol), tert-butyl
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2-bromoacetate (60 mg, 0.31 mmol) and potassium carbonate
(50 mg, 0.36 mmol) in acetonitrile (20 mL) were stirred at room
temperature for 24 hours. The reaction mixture was filtered and
the solvent evaporated. The residual was purified by silica
gel chromatography (hexane–ethyl acetate, 4 : 1) to afford
prodrug 2 (68 mg, 0.12 mmol) with methacryloyl chloride
(26 mg, 0.25 mmol) in the presence of triethylamine (37 mg,
0.36 mmol) were dissolved in dichloromethane (20 mL) and
kept at 0 °C for 12 hours. The reaction mixture was filtered and
the solvent was evaporated in vacuum. The residual was purified
by silica gel chromatography (hexane–ethyl acetate, 4 : 1) to
afford prodrug 6 as a yellow solid (55 mg, 69.9% yield).
1H NMR (400 MHz, CDCl3), δ (ppm): δ = 8.13 (d, J = 8.6 Hz,
1H), 7.64 (s, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.4 Hz,
2H), 7.23 (d, J = 16.3 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H),
7.08 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 16.3 Hz, 1H), 6.60 (d, J =
8.4 Hz, 2H), 6.37 (s, 1H), 5.78 (s, 1H), 5.54 (s, 2H), 3.67 (t, J =
7.0 Hz, 4H), 3.59 (t, J = 7.0 Hz, 4H), 2.60 (t, J = 7.5 Hz, 2H),
2.47 (t, J = 7.4 Hz, 2H), 2.08 (s, 3H), 2.03–1.96 (m, 2H).
13C NMR (100 MHz, CDCl3), δ (ppm): δ = 172.9, 165.7, 151.3,
145.9, 144.4, 142.9, 135.7, 133.7, 133.0, 132.3, 129.7, 128.0,
127.0, 126.2, 126.0, 125.9, 122.2, 112.2, 63.1, 53.5, 40.5, 34.0,
33.6, 26.8,18.4. ES-HRMS (m/z): calcd for C33H35Cl2N2O6,
625.1872 [M + H]+; found, 625.1875.
BOC-protected intermediate as
a yellow solid (97 mg,
78.2%).1H NMR (400 MHz, CDCl3), δ (ppm): δ = 8.12 (d, J =
8.5 Hz, 1H), 7.60 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.47 (d, J =
8.7 Hz, 2H), 7.19 (d, J = 16.3 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H),
6.98 (d, J = 16.3 Hz, 1H), 6.91 (d, J = 8.7 Hz, 2H), 6.61 (d, J =
8.6 Hz, 2H), 5.53 (s, 2H), 4.56 (s, 2H), 3.67 (t, J = 7.0 Hz, 4H),
3.60 (t, J = 7.0 Hz, 4H), 2.60 (t, J = 7.4 Hz, 2H), 2.46 (t, J =
7.4 Hz, 2H), 2.02–1.94 (m, 2H), 1.50 (s, 9H). 13C NMR
(100 MHz, CDCl3), δ (ppm): δ = 172.8, 167.7, 158.6, 145.7,
143.4, 133.0, 132.9, 130.0, 129.6, 128.5, 126.9, 126.0, 124.3,
115.0, 82.6, 65.6, 63.2, 54.4, 39.8, 34.0, 33.5, 33.4, 38.0, 26.7.
ES-HRMS (m/z): calcd for C35H41Cl2N2O7, 671.2291 [M + H] +;
found, 671.2291. Then, the obtained intermediate (80 mg,
0.12 mmol) was dissolved in dichloromethane (10 mL) in dark
and was treated with TFA (200 mg, 8.8 mmol) at room tempera-
ture for 24 h. Finally, prodrug 4 was obtained as a yellow solid
(50 mg, 68.2%) after silica gel column chromatography
(hexane–ethyl acetate, 2 : 1). 1H NMR (400 MHz, CDCl3),
δ (ppm): δ = 8.12 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.56 (d, J =
8.5 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 16.3 Hz, 1H),
7.07 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 16.3 Hz, 1H), 6.95 (d, J =
8.6 Hz, 2H), 6.61 (d, J = 8.6 Hz, 2H), 5.53 (s, 2H), 4.72 (s, 2H),
3.68 (t, J = 6.8 Hz, 4H), 3.59 (t, J = 6.8 Hz, 4H), 2.60 (t, J = 7.4
Hz, 2H), 2.46 (t, J = 7.4 Hz, 2H), 2.02–1.95 (m, 2H). 13C NMR
(100 MHz, CDCl3), δ (ppm): δ = 173.0, 158.0, 145.7, 144.3,
143.2, 132.9, 132.6, 130.5, 130.2, 129.7, 128.6, 126.9, 125.7,
124.8, 115.0, 112.3, 64.7, 63.2, 53.6, 40.5, 34.0, 33.5, 33.4,
26.7. ES-HRMS (m/z): calcd for C31H33Cl2N2O7, 615.1665
[M + H]+; found, 615.1667.
(E)-2-Nitro-5-(4-(2-oxoethoxy)styryl)benzyl 4-(4-(bis(2-chloro-
ethyl)amino)phenyl)butanoate (Prodrug 7). In the dark, com-
pound 24 (100 mg, 0.15 mmol) in dichloromethane (15 mL)
was treated with TFA (120 mg, 1 mmol) at room temperature for
2 hours, and target prodrug 7 was obtained as a yellow solid
1
(55 mg, 59.2% yield) after column chromatography. H NMR
(400 MHz, CDCl3), δ (ppm): δ = 9.88 (s, 1H), 8.13 (d, J =
8.5 Hz, 1H), 7.61 (s, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.49 (d, J =
8.5 Hz, 2H), 7.20 (d, J = 16.3 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H),
7.00 (d, J = 16.3 Hz, 1H), 6.92 (d, J = 8.5 Hz, 2H), 6.68 (d, J =
8.2 Hz, 2H), 5.54 (s, 2H), 4.63 (s, 2H), 3.68 (t, J = 6.6 Hz, 4H),
3.61 (t, J = 6.6 Hz, 4H), 2.61 (t, J = 7.3 Hz, 2H), 2.46 (t, J =
7.3 Hz, 2H), 2.02–1.94 (m, 2H). 13C NMR (100 MHz, CDCl3),
δ (ppm): δ = 198.6, 172.9, 158.1, 145.8, 143.2, 132.9, 132.7,
130.1, 129.8, 128.6, 126.9, 126.0, 125.7, 124.7, 114.9, 112.8,
72.7, 63.2, 53.9, 40.2, 34.0, 33.5, 26.7. EI-HRMS (m/z): calcd
for C31H32Cl2N2O6, 598.1637 [M]+; found, 598.1645.
(E)-5-(4-(4-Bromobutoxy)styryl)-2-nitrobenzyl
4-(4-(bis(2-
chloroethyl)amino)phenyl)butanoate (Prodrug 5). In the dark, a
mixture of prodrug 2 (100 mg, 0.18 mmol), 1,4-dibromobutane
(54 mg, 0.25 mmol) and potassium carbonate (50 mg,
0.36 mmol) in 25 mL acetonitrile was stirred and heated to 55 °C.
After keeping at this temperature for 5 hours, the reaction mixture
was filtered and the solvent was evaporated. The crude mixture
was purified by silica gel chromatography (hexane–acetate, 6 : 1)
General methods for the photolysis and drug release
One-photon photolytic reaction of the prodrugs 1–7 was per-
formed by irradiating the solution (10−4 M in acetonitrile or
acetonitrile-PBS solution (1 : 1)) with a CHFXM-500W lamp
(λ ≥ 400 nm with power of 120 mW cm−2). Between certain
time intervals, a small aliquot (20 μL) of the suspension was
taken out and analyzed by reversed-phase HPLC using a Beta-
Basic-18 column eluted with a mixture of 90% acetonitrile and
10% methanol for all prodrugs in acetonitrile solution except
prodrug 7 was eluted with 90% methanol and 10% acetonitrile
and prodrug 1 in acetonitrile/PBS solution was eluted with 90%
methanol and 10% water at a flow rate of 0.5 mL min−1. The
chromatogram was plotted by using absorbance detection at
254 nm.
The characterization of the two-photon absorption cross
section: two-photon absorption cross-sections were determined
by the method of Z-scan technique. The 800 nm pump source
was from the fundamental of a fs mode-locked Ti:sapphire laser
system (output beam ≈ 80 fs duration and 250 kHz repetition
rate). The laser was focused on a quartz cuvette with an optical
1
to afford prodrug 5 as a yellow solid (110 mg, 88.5%). H NMR
(400 MHz, CDCl3), δ (ppm): δ = 8.12 (d, J = 8.5 Hz, 1H),
7.61 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H),
7.20 (d, J = 16.3 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.98 (d, J =
16.3 Hz, 1H), 6.91 (d, J = 8.5 Hz, 2H), 6.61 (d, J = 8.4 Hz, 2H),
5.54 (s, 2H), 4.04 (t, J = 6.0 Hz, 2H), 3.68 (t, J = 6.3 Hz, 4H),
3.60 (t, J = 6.3 Hz, 4H), 3.51 (t J = 6.5 Hz, 2H), 2.61 (t, J = 7.5
Hz, 2H), 2.47 (t, J = 7.4 Hz, 2H), 2.13–2.05 (m, 2H), 2.03–1.94
(m, 4H). 13C NMR (100 MHz, CDCl3), δ (ppm): δ = 172.9,
159.6, 145.6, 144.4, 143.5, 133.1, 132.9, 130.4, 129.7, 128.9,
128.5, 126.7, 126.0, 125.6, 123.9, 114.9, 112.2, 67.0, 63.2, 53.6,
40.5, 34.0, 33.6, 33.4, 29.4, 27.8, 26.7. EI-HRMS (m/z): calcd for
C33H37BrCl2N2O5, 690.1263 [M + H]+; found, 690.1258.
(E)-5-(4-(Methacryloyloxy)styryl)-2-nitrobenzyl
4-(4-(bis(2-
chloroethyl)amino)phenyl)butanoate (Prodrug 6). In the dark,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5238–5244 | 5243

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path length of 1 mm. The sample was dissolved in CH3CN at a
concentration of 10−4 M. Two-photon absorption cross-section
(δ) was obtained as follows: by assuming a spatially and tem-
porally Gaussian profile for laser beam, the normalized energy
transmittance T(z), for two-photon absorption (2PA) can be
given as
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ð1
1
pffiffiffi
TðzÞ ¼
ln½1 þ q0 expðꢀx2Þꢁdx;
ð1Þ
πq0
ꢀ1
where q0 = βI0Leff, β is 2PA coefficient, Leff = [1 − exp(−αl)]/α,
α is linear absorption coefficient, and l is the sample path length.
The 2PA coefficient can be obtained by fitting the open aperture
Z-scan traces by using eqn (1). From the 2PA coefficient β, 2PA
cross section (δTPA) can be deduced using the relation δTPA
=
hνβ/N0, where N0 is the number density of prodrug molecules
dispersed in the solution.
The two-photon photolysis was performed by irradiating
with a 80 fs pulses mode-locked Ti:sapphire laser at 800 nm and
at a repetition rate of 250 kHz in a quartz cuvette. The NIR
beam was focused onto an ester suspension placed in a stirrer-
containing 10 × 10 mm cuvette. With a beam spot diameter of
approximately 0.20 mm, the excitation density used for prodrug
1 was 0.60 mJ cm−2 per pulse. The data were collected as
described as that for one-photon photolysis.
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This work was supported by NSFC (20902069, 20903039,
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5244 | Org. Biomol. Chem., 2012, 10, 5238–5244
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