Site-specific and far-red-light-activatable prodrug of combretastatin A-4 using photo-unclick chemistry

Article abstract of DOI:10.1021/jm400139w

Although tissue-penetrable light (red and NIR) has great potential for spatiotemporally controlled release of therapeutic agents, it has been hampered because of the lack of chemistry translating the photonic energy to the cleavage of a chemical bond. Recently, we discovered that an aminoacrylate group could be cleaved to release parent drugs after oxidation by SO and have called this photo-unclick chemistry . We demonstrate its application to far-red-light-activated prodrugs. A prodrug of combretastatin A-4 (CA4) was prepared, CMP-L-CA4, where CMP is dithiaporphyrin, a photosensitizer, and L is an aminoacrylate linker. Upon irradiation with 690 nm diode laser, the aminoacrylate linker of the prodrug was cleaved, rapidly releasing CA4 (>80% in 10 min) in CDCl₃. In tissue culture, it showed about a 6-fold increase in its IC₅₀ in MCF-7 after irradiation, most likely because of the released CA4. Most significantly, CMP-L-CA4 had better antitumor efficacy in vivo than its noncleavable (NC) analog, CMP-NCL-CA4. This is the first demonstration of the in vivo efficacy of the novel low-energy-light-activatable prodrug using the photo-unclick chemistry.

Full text of DOI:10.1021/jm400139w

Article
pubs.acs.org/jmc
Site-Specific and Far-Red-Light-Activatable Prodrug of
Combretastatin A‑4 Using Photo-Unclick Chemistry
Moses Bio,† Pallavi Rajaputra,†,‡ Gregory Nkepang,†,‡ Samuel G. Awuah,† Abugafar M. L. Hossion,†
and Youngjae You*,†,‡
†Department of Pharmaceutical Sciences and ‡Department of Chemistry and Biochemistry, University of Oklahoma, Oklahoma City,
Oklahoma 73117, United States
S
* Supporting Information
ABSTRACT: Although tissue-penetrable light (red and NIR)
has great potential for spatiotemporally controlled release of
therapeutic agents, it has been hampered because of the lack of
chemistry translating the photonic energy to the cleavage of a
chemical bond. Recently, we discovered that an aminoacrylate
group could be cleaved to release parent drugs after oxidation
by SO and have called this “photo-unclick chemistry”. We
demonstrate its application to far-red-light-activated prodrugs.
A prodrug of combretastatin A-4 (CA4) was prepared, CMP−L−CA4, where CMP is dithiaporphyrin, a photosensitizer, and L is
an aminoacrylate linker. Upon irradiation with 690 nm diode laser, the aminoacrylate linker of the prodrug was cleaved, rapidly
releasing CA4 (>80% in 10 min) in CDCl3. In tissue culture, it showed about a 6-fold increase in its IC50 in MCF-7 after
irradiation, most likely because of the released CA4. Most significantly, CMP−L−CA4 had better antitumor efficacy in vivo than
its noncleavable (NC) analog, CMP−NCL−CA4. This is the first demonstration of the in vivo efficacy of the novel low-energy-
light-activatable prodrug using the photo-unclick chemistry.
cleaved by SO that is generated from the photosensitization
(light + photosensitizer). However, there are still some key
limitations in this strategy for broader application. These
include limited SO-cleavable linkers (to vinyl diether and vinyl
thiodiether), facile synthetic tools for the cleavable linkers,
regeneration of parent drugs, and demonstration of function-
ality using in vivo systems. Recently, we discovered the “photo-
unclick chemistry” of aminoacrylate, which overcomes all of
these issues (Figure 1).15
INTRODUCTION
■
A major problem of treatment with anticancer chemotherapy
drugs is that such treatment is often toxic to noncancerous cells,
creating systemic side effects. Various strategies, including
tumor-targeted drug delivery, target site-activated prodrugs, and
combination therapy, have been proposed to minimize such
systemic side effects.1−5 These strategies have a shared goal of
keeping the systemic concentration of the drug lower than its
toxic level while the drug concentration at tumor sites is kept
above the effective concentration.
With that goal, photodynamic therapy (PDT) provides an
excellent foundation for inspiring a new strategy for treating
tumors. In PDT, nontoxic photosensitizers are activated by
tissue-penetrable light (630−800 nm in the red and NIR range)
to locally generate short-lived reactive oxygen species (ROS)
such as singlet oxygen (SO)6−8 to ablate cancer cells while
causing only minimal side effects. The primary factor for the
selective tumor ablation is the focused irradiation on tumors. In
PDT, the tissue-penetrable light can be used as an excellent tool
for providing spatiotemporally controlled release of toxic
chemical species.
While UV and short-visible light have been extensively
studied for spatiotemporally controlled release of bioactive
molecules (called “caged compounds”),9,10 application of the
tissue-penetrable light has been extremely limited because of
the lack of chemistry that can translate the photonic energy to
chemical bond cleavage. Recently, a “smart” strategy was
proposed that takes advantage of the photosensitization and
unique chemistry of SO.11−14 Electron-rich olefins can be
Figure 1. Release of a drug from tissue-penetrable-light-activatable
prodrug via photo-unclick chemistry.
Combretastatins are well-known tubulin-binding agents that
were isolated from Combretum caffirum.16,17 Combretastatin A-
4 (CA4) is one of the most important natural molecules that
strongly inhibits tubulin polymerization.18 The cis isomer is
biologically active while the trans isomer has little or no
activity.19 The cis isomer has a very similar configuration to
colchicine (Figure 2a), and it binds to the colchicine binding
site of β-tubulin.20,21 We chose CA4 for a prodrug design
Received: January 29, 2013
© XXXX American Chemical Society
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binding conformation inside the colchicine binding small
pocket of β-tubulin.
On the basis of our previous studies, we hypothesized that
this novel prodrug strategy could be used to achieve antitumor
effects in animal models by locally releasing active anticancer
agents upon irradiation. We present our results here. We
prepared the CA4 prodrug (CMP−L−CA4) and two
pseudoprodrugs (CMP−NCL−CA4 and CMP−L−Rh) (Fig-
ure 2). Core-modified porphyrin (CMP) was selected as a
photosensitizer to generate SO using tissue-penetrable far-red
light (690 nm). CMP−NCL−CA4 was prepared as an analog
of CMP−L−CA4 that cannot release CA4. CMP−L−Rh was
used as a special fluorescence probe that emits bright
rhodamine fluorescence only after cleavage of the linker. We
report on the synthesis of these compounds, the cleavage of the
linker of CMP−L−CA4 in CDCl3, and the in vitro and in vivo
biological activities of these prodrugs.
Figure 2. (a) Structures of CA4 and colchicine. Schematic
representation of prodrug (b) CMP−L−CA4 and (c) pseudoprodrugs
CMP−NCL−CA4, and (d) CMP−L−Rh (CMP = core-modified
porphyrin, L = aminoacrylate linker, CA4 = combretastatin A-4, NCL
= noncleavable linker).
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of the prodrugs involved three or
four facile and high-yielding reactions (Scheme 1). CA4 was
esterified with 2-propynoic acid using DCC and DMAP at 0 °C
to give compound 1. Compound 2 (L-CA4) was synthesized in
94% yield through a click (yne−amine reaction) reaction of
compound 1 and 4-piperidinemethanol. Then, CMP−L−CA4
was synthesized by esterification of CMP-COOH and 2 in 69%
yield. The synthesis of CMP−NCL−CA4 also involved four
simple and high-yield steps. The phenolic group of CA4 was
because of its size and simple structure with a phenolic group
and because of its potent in vitro cytotoxicity with a nanomolar
IC50. In addition, we expected that the prodrug of CA4 should
have significantly lower cytotoxic activity because of the bulky
PS and linker at the 3′-position of CA4, disturbing the CA4
a
Scheme 1. Synthesis of CMP−L−CA4 and CMP−NCL−CA4
a
Reagents and conditions: (i) 2-propynoic acid, DCC, DMAP, rt, 24 h, 73%; (ii) 4-piperidinemethanol, THF, rt, 30 min, 94%; (iii) 1,3-
dibromopropane, anhydrous K2CO3, acetone, reflux, 12 h, 84%; (iv) N-Boc-piperazine, anhydrous K2CO3, anhydrous DMF, rt, 8 h, 87%; (v)
compound 4, TFA, anhydrous DCM, rt, 1 h; (f) compound 2 (L-CA4), DCC, DMAP, anhydrous DCM, rt, 24 h, 69%; (vi) compound 5 (NCL-
CA4), DCC, DMAP, anhydrous DCM, rt, 24 h, 74%.
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alkylated with 1,3-dibromopropane under the basic condition
to give compound 3. Compound 5 (NCL-CA4) was
synthesized via the substitution of bromo group by N-Boc-
piperazine under basic conditions and deprotection of the Boc
group using TFA. Finally, CMP−NCL−CA4 was synthesized
by the esterification of CMP-COOH and 5 using DCC and
DMAP at 0 °C in 74% yield.
Dark Toxicity and Phototoxicity. The dark toxicity was
determined to confirm whether the addition of CMP−L or
CMP−NCL to CA4 effectively reduced the cytotoxicity of CA4
(Figure 4). Indeed, the cytotoxicities of the prodrugs were
Effect of CMP−L−CA4 and CMP−NCL−CA4 on
Tubulin Polymerization. A tubulin polymerization assay
was conducted to examine whether the two prodrugs had
significantly lower activity in the inhibitory activity for tubulin
polymerization than the activity of CA4 because of the bulky
groups (CMP-L and CMLP-NCL). The tubulin polymerization
assay data revealed enhanced fluorescence emission as the
tubulin polymerized. Paclitaxel and CA4 were used as positive
control drugs (a polymerization enhancer and an inhibitor,
respectively) in addition to the negative control group (Figure
3). Without any drug (negative control group), tubulin
Figure 4. Dark and phototoxicity of CMP−L−CA4 and CMP−NCL−
CA4 and dark toxicity of CA4.
much lower than that of CA4 itself: IC50D = 9 nM (CA4), 164
nM (CMP−L−CA4), 1802 nM (CMP−NCL−CA4), a
reduction by 18- and 200-fold, respectively. It seemed that
the bulky groups, CMP−L and CMP−NCL, reduced the
cytotoxic activity of CA4, presumably through the interference
of its binding to tubulin, as estimated by the modeling study.
Interestingly, the CMP−NCL group was more effective than
the CMP−L group in masking the activity of CA4.
Phototoxicity was then determined to confirm the enhanced
cell damage after the irradiation by the release of CA4 from
CMP−L−CA4. Theoretically, two possible mechanisms could
damage the cells by [CMP−L−CA4 + hν]: either via a
photodynamic effect (i.e., SO from CMP moiety + hν) and/or
via the released CA4. On the other hand, only a photodynamic
effect could damage cells by [CMP−NCL−CA4 + hν]. A 6-fold
increase was observed after irradiation in CMP−L−CA4 (IC50D
= 164 nM → IC50P = 28 nM), but an increase by 1.7-fold
increase was observed in CMP−NCL−CA4 (IC50D = 1802 nM
→ IC50P = 1063 nM). It seemed that the photodynamic effect
(the cytotoxicity of SO) was smaller than the cytotoxic effect of
the released CA4 in [CMP−L−CA4 + hν].
The other interesting observation was that the shape of the
dose−response curve of [CMP−L−CA4 + hν] was different
than that of [CA4 + no hν]. If the released CA4 was the only
cause of the cytotoxicity of [CMP−L−CA4 + hν], they should
be same. At this point, it is not clear what caused the difference,
and this remains to be investigated in the following studies.
A Bystander Effect by CMP−L−CA4. A bystander effect
(killing neighboring cells) was found, which means cellular
damage was caused by the released CA4, and not by SO, in
[CMP−L−CA4 + hν] (Figure 5). SO, the toxic species in
PDT, has a very short half-life (∼40 ns) in aqueous media and
its diffusion distance is estimated to be between 20 and 200
nm.6−8 Thus, PDT cannot generate a bystander effect. In other
words, the SO generated in one cell could not damage
neighboring cells. To examine the bystander effect of CMP−
L−CA4, we irradiated only the left half of each well and then
visualized the center of the whole well (covering parts of both
the irradiated and unirradiated half) to determine the number
Figure 3. Effects of 3 μM paclitaxel, CA4, CMP−L−CA4, or CMP−
NCL−CA4 on tubulin polymerization: (a) one data set of
representative kinetic traces (the other two data are reported in
Figure S7 of the Supporting Information) and (b) inhibition of tubulin
polymerization by CA4, CMP−L−CA4, or CMP−NCL−CA4 after 1
h incubation at 37 °C (mean SD of three experiments).
polymerization reached its maximum at about 20 min. On
the other hand, paclitaxel and CA4 had typical patterns of
kinetic traces of tubulin polymerization disruptors: an enhancer
(paclitaxel) and an inhibitor (CA4). Compared to CA4, both
prodrugs had little to no inhibitory ability on tubulin (Figure
3b). After 1 h, CMP−L−CA4 and CMP−NCL−CA4 showed
only limited inhibition of polymerization (6 and 9%,
respectively); CA4 completely inhibited tubulin polymerization
(100%, Figure 3b). Thus, the bulky groups (CMP−L and
CMP−NCL) may have reduced the binding activity of these
prodrugs to tubulin.
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irradiation was attributed to the localized release of fluorescent
rhodamine dye after the oxidative cleavage of the aminoacrylate
linker of the minimally fluorescent CMP−L−Rh. To our
knowledge, this is the first demonstration of the use of a
cleavable SO linker in an animal model.
Antitumor Efficacy. The antitumor effects of the prodrugs
after irradiation were evaluated by using a mouse tumor model,
BALB/c mice having sc tumors produced by injection of colon
26 cells; 1.5 h after ip injection of prodrugs, each tumor was
irradiated by a 690 nm diode laser (360 J/cm2 = 200 mW/cm2
for 30 min) on days 0, 1, and 2. The treatment conditions were
determined from pilot studies, although those conditions might
not have been the optimal conditions.
Figure 5. Fluorescence live cell images of the center of each well
treated with (a) control, (b) CMP−L−CA4 (50 nM), and (c) IY69 (5
μM). Only the left half of each well was irradiated with a 690 nm diode
laser (11 mW/cm2 for 15 min). At these concentrations, CMP−L−
CA4 and IY69 did not produce significant dark toxicity.
A number of interesting results were observed (Figure 7).
First, the animals in the nonirradiated group G4 [CMP−L−
of live cells. As expected, a bystander effect was found for
CMP−L−CA4: cells in the nonirradiated side were damaged as
much as the irradiated side (Figure 5b). On the other hand, a
potent CMP photosensitizer IY6922 damaged only the cells in
the irradiated half of the well (Figure 5c), resulting from the
damage caused by only SO. This clearly demonstrated that
CMP−L−CA4 killed the cells mostly by the released CA4.
In Vivo Optical Imaging with the FRET Probe. The
cleavage of the aminoacrylate linker was demonstrated by using
an optical in vivo imaging study with a FRET optical probe,
CMP−L−Rh (Rh = rhodamine) (Figure 6a,b). An irradiation-
time-dependent increase in fluorescence emission was observed
(Figure 6c). The emission of the irradiated spot became more
intense as the irradiation time increased from 10 to 30 min. The
corresponding increase in the fluorescence intensity upon
Figure 7. Tumor growth curves. Drug administration was once a day
on days 0, 1, and 2; hν = 360 J/cm2 with 690 nm; and six mice were
used per group, except three mice were used in the control group.
Error bars represent SE.
CA4 + CA4 (4 μmol/kg each)] had tumor growth similar to
that of the control animals (G5, P > 0.05). It seemed the
antitumor effects of these two compounds were minimal
without irradiation. Second, irradiation produced a significantly
better antitumor effect (G2 compared to G4, P < 0.05).
Irradiation improved the antitumor effects of CMP−L−CA4,
possibly because of the released CA4 and the PDT effect (CMP
moiety + hν). Third, G3 group [CMP−NCL−CA4 + CA4 +
hν] had a significant (P < 0.05) delay in tumor growth when
compared to the tumor growth in the control group G5 (P <
0.05). This may have resulted from the PDT effect of [CMP−
NCL−CA4 + hν], because we did not see such a significant
tumor growth delay caused by CA4 itself at similar dose in our
pilot studies. These data are consistent with the data from a
previous report of a minimal CA4-induced antitumor effect
even at a very high dose (506 μmol/kg).23 In addition, we saw
signs of necrosis on the treated tumors, which is a typical sign
of a PDT effect. Last, but most important, animals in the G1
group [CMP−L−CA4 (2 μmol/kg) + hν] experienced a
significantly (P < 0.05) superior antitumor effect than animals
in the G3 group that mimicked a combination therapy [PDT
with CMP−NCL−CA4 plus a systemic CA4 (2 μmol/kg each)
+ hν]. This was most likely caused by the released CA4.
Interestingly, we did not see any significant loss of body weight
in animals from any group (Figure S8, Supporting Informa-
tion), which is one of the indicative signs for acute systemic
toxicity.
Figure 6. (a) The structure of the FRET probe (CMP−L−Rh), (b)
the procedure for in vivo optical imaging, and (c) optical images of the
mouse after irradiation for 0 (i), 10 (ii), 20 (iii), and 30 (iv) min with a
690 nm diode laser 1.5 h postinjection of CMP−L−Rh. *scale bar
unit: fluorescence arbitrary unit.
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solvent was removed by evaporation. The crude product was purified
CONCLUSION
■
by using column chromatography with ethyl acetate:hexane (8:2) to
1
We have demonstrated the application of photo-unclick
chemistry in an anticancer prodrug from its synthesis to its in
vivo antitumor effects. The prodrug was readily prepared via
facile reactions with mild reaction conditions and high yields.
The aminoacrylate linker was cleaved fast by the irradiation
with tissue-penetrable far-red light (690 nm), releasing the
anticancer drug, CA4. The released CA4 effectively damaged
neighboring cells through bystander effects. The cleavage of the
aminoacrylate linker was also confirmed in mouse tissue by
using an in vivo optical imaging with a FRET molecular probe.
Most significantly, we demonstrated that the prodrug CA4
(CMP−L−CA4), which was minimally active in vivo, had an
enhanced antitumor effect without any significant signs of acute
toxicity. As far as we know, this is the first demonstration of the
use of site-specific release of anticancer drug via photo-unclick
chemistry in an animal model.
give a yellow oil (compound 4, 539 mg, 87%): H NMR (300 MHz,
CDCl3) δ 1.45 (s, 9H), 1.91 (m, 2H), 2.46 (s, 4H), 2.56 (t, J = 7.3 Hz,
2H), 3.41 (s, 4H), 3.69 (s, 6H), 3.82 (d, J = 2.4 Hz, 6H), 3.88 (t, J =
6.1 Hz, 2H), 6.43 (d, J = 12.3 Hz, 1H), 6.47 (d, J = 12.3 Hz, 1H), 6.51
(s, 2H), 6.76 (d, J = 7.8 Hz, 1H), 6.84 (s, 2H); HRMS ESI (m/z)
calcd for C30H42N2O7 ([M + H]+) 543.2992, found 543.3066.
Compound 5 (NCL-CA4). Compound 4 (425 mg, 0.78 mmol)
was dissolved in dry DCM (6 mL). After TFA (0.06 mL) was added to
the solution at 0 °C, it was stirred under nitrogen for 1 h. The reaction
mixture was then concentrated under vacuum and used directly in the
next step without purification.
CMP−NCL−CA4. The title compound was prepared according to
the method described for compound CMP−L−CA4 by employing
CMP-COOH (70 mg, 0.10 mmol), compound 5 (85 mg, 0.19 mmol),
DCC (78 mg, 0.38 mmol), and DMAP (23 mg, 0.19 mmol) to give a
1
reddish purple solid (compound 9, 164 mg, 74%): H NMR (300
MHz, CDCl3) δ 2.08 (t, J = 5.6 Hz, 2H), 2.62 (m, 6H), 3.72 (m, 4H),
3.78 (s, 3H), 3.86 (s, 3H), 3.88 (s, 6H), 4.16 (t, J = 6.5 Hz, 2H), 4.98
(s, 2H), 6.54 (d, J = 11.8 Hz, 1H), 6.74 (s, 2H), 6.89 (d, J = 8.2 Hz,
1H), 6.96 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 9.8 Hz, 1H), 7.13 (s, 1H),
7.41 (d, J = 8.0 Hz, 2H), 7.84 (s, 9H), 8.21 (d, J = 8.3 Hz, 2H), 8.26
(s, 6H), 8.71 (m, 4H), 9.72 (m, 4H); HRMS ESI (m/z) calcd for
C71H62N4O7S2 ([M + H]+) 1147.4060, found 1147.4128.
We envision that this prodrug strategy will be applicable for
various clinical needs to reduce the systemic side effects during
local chemotherapy, such as localized neoadjuvant chemo-
therapy and localized adjuvant chemotherapy. In addition, this
prodrug strategy can be readily adapted to more advanced drug
delivery systems.
Tubulin Polymerization Assay. The fluorescence-based tubulin
polymerization was determined by using a kit supplied by
Cytoskeleton, Inc. (cat. # BK011P). Its basic principle is that the
fluorescence reporter increases as it is incorporated into microtubules
during the course of polymerization. The assay was performed
following the experimental procedure as described in version 2.1 of the
tubulin polymerization assay kit manual. Briefly, the test drug (final
concentration = 3 μM) was added to a mixture of tubulin and GTP in
a buffer solution, and the reaction mixture was incubated at 37 °C.
Fluorescence was monitored (excitation = 360 nm and emission = 450
nm) by using a fluorescence plate reader (SpectraMax Gemini EM,
Molecular device). Paclitaxel and CA4 were included in the assay as
positive controls in addition to the negative control (vehicle only).
Dark Toxicity and Phototoxicity. The cytotoxicities of CMP−
L−CA4 and CMP−NCL−CA4 were determined with or without
irradiation. MCF-7 cells were maintained in minimum essential
medium (α-MEM) supplemented with 10% bovine growth serum, 2
mM L-glutamine, 50 units/mL penicillin G, 50 μg/mL streptomycin,
and 1.0 μg/mL fungizone. MCF-7 cells (5000 cells/well) were seeded
on 96-well plates in the medium and then incubated for 24 h at 37 °C
in 5% CO2. Stock solutions (2 mM) were prepared in DMSO. The
stock solutions were further diluted with medium to get necessary final
concentrations. The diluted solution (10 μL) was then added to each
well (190 μL). The plates were incubated for 24 h and then removed
from the incubator.
EXPERIMENTAL SECTION
■
Synthesis. CA4,24 compound 1,25 compound 2 (L-CA4),25 CMP-
COOH,26 IY69,27 and CMP−L−Rh15 were synthesized as reported
previously. The purity of the biologically evaluated compounds
(CMP−L−CA4 and CMP−NCL−CA4) was confirmed by HPLC
to be >95% (Figures S13 and S14, Supporting Information).
CMP−L−CA4. To a stirred solution of CMP-COOH (200 mg, 0.28
mmol) and compound 2 (267 mg, 0.55 mmol) in dry DCM (15 mL)
was dropwise added a solution of DCC (228 mg, 1.1 mmol) and
DMAP (67 mg, 33.7 mmol) in dry DCM (20 mL). The reaction
mixture was then stirred at rt for 24 h. The solvent was removed under
reduced pressure to give the crude product that was then purified by
column chromatography by using ethyl acetate:hexane (6:4) as an
eluent to give a reddish purple solid (CMP−L−CA4) (452 mg, 69%):
1H NMR (300 MHz, CD2Cl2) δ 0.90 (m, 2H), 1.28 (m, 2H), 1.41 (m,
2H), 1.87 (m, 1H), 3.09 (s, 1H), 3.66 (s, 6H), 3.75 (d, J = 6.4 Hz,
6H), 4.22 (d, J = 5.2 Hz, 2H), 4.80 (d, J = 12.8 Hz, 1H), 4.96 (s, 2H),
6.44 (s, 2H), 6.52 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 6.97 (s, 1H), 7.09
(d, J = 6.2 Hz, 1H), 7.40 (d, J = 7.7 Hz, 2H), 7.50 (d, J = 12.8 Hz,
1H), 7.54 (br s, 9H), 8.27 (m, 8), 8.72 (m, 4H), 9.74 (m, 4H); HRMS
ESI (m/z) calcd for C73H62N3O9S2 ([M + H]+) 1188.3849, found
1188.3875.
For the Phototoxicity Study. The plate without a cover was gently
shaken on an orbital shaker (Lab-line, Barnstead International), to
ensure uniformity of the light during the irradiation, and irradiated by
a diode laser (690 nm, 5.6 mW/cm2) for 30 min.
Compound 3. To a solution of CA4 (1.20 g, 3.79 mmol) in
acetone (20 mL) were added anhydrous K2CO3 (1.57g 11.37 mmol)
and 1,3-dibromopropane (0.76 g, 3.79 mmol). The reaction mixture
was refluxed in an oil bath for 12 h. After the reaction, the K2CO3 was
removed by suction filtration and the solvent was removed under
reduced pressure to give the crude product, which was then purified by
using column chromatography with ethyl acetate:hexane (3:7) to give
a yellow oil (compound 3, 1.39 g, 84%): 1H NMR (300 MHz, CDCl3)
δ 2.26 (m, 2H), 3.56 (t, J = 6.5 Hz, 2H), 3.72 (s, 6H), 3.85 (s, 6H),
3.95 (t, J = 5.6 Hz, 2H), 6.49 (d, J = 12.0 Hz, 1H), 6.50 (d, J = 12.0
Hz, 1H), 6.53 (s, 2H), 6.79 (d, J = 7.9 Hz, 1H), 6.87 (s, 2H); HRMS
ESI (m/z) calcd for C21H25Br3O5 ([M + H]+) 437.0885, found
437.0964.
Compound 4. To a solution of N-Boc-piperazine (212 mg, 1.14
mmol) in dry DMF (10 mL) were added anhydrous K2CO3 (789 mg,
5.71 mmol) and compound 3 (500 mg, 1.14 mmol). The reaction
mixture was stirred at rt for 8 h. The K2CO3 was removed by suction
filtration and the solvent was removed under reduced pressure. The
residue was dissolved with water and extracted with ethyl acetate. The
combined organic layers were dried over anhydrous Na2SO4, and the
For the Dark Toxicity Study. Plates were kept in the dark for 30
min, and then returned to the incubator. Cell viability was determined
after 3 days by using the MTT assay. Briefly, a 10 μL solution of MTT
(10 mg in 1 mL PBS buffer) was added to each well and the plate was
incubated for 4 h. Then, the MTT solution was removed, the cells
were dissolved in 200 μL of DMSO, and the absorbance within each
well was measured at 570 nm with background subtraction at 650 nm.
The cell viability was then quantified by measuring the absorbance of
the treated wells compared to that of the untreated wells (controls)
and expressed as a percentage.
Bystander Effect by CMP−L−CA4. Colon 26 cells were seeded
at 5000 cells/mL/well in 24-well plates and then incubated for 24 h.
Stock solutions (2 mM in DMSO) were diluted with medium. The
diluted solutions (25 μL) were added to the wells (1 mL) to get the
appropriate final concentrations [CMP−L−CA4 (50 nM) and IY69 (5
μM)]. After 24 h incubation, the plates were irradiated from the
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bottom with a 690 nm diode laser at 11 mW/cm2 for 15 min. While
being irradiating, half of each well was blocked with a black masking
tape (THORLABS, Cat# T743-1.0) to protect cells on the other half
of each well. The irradiated plates were incubated for an additional 48
h. Then a Calcein AM live cell staining assay was performed. Briefly,
the cells were washed once with the Calcein AM wash buffer (1 mL),
and then fresh wash buffer (250 mL) and the working reagent (250
mL) were added to the wells. The cells were incubated for 30 min.
Fluorescent images were obtained with an Olympus IX51 inverted
microscope with green fluorescence channel to visualize live cells. All
images were taken at 10 × magnifications.
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-405-271-6593, ext. 47473. E-mail: youngjae-you@
ouhsc.edu.
■
Author Contributions
M.B. and Y.Y. designed the project. M.B. and Y.Y. wrote the
manuscript. M.B. and G.N. prepared the prodrugs and P.R.,
M.B., and S.G.A. carried out the biological evaluations of the
compounds. A.M.L.H. carried out the molecular modeling
study (unpublished data).
In Vivo Optical Imaging Using the FRET Probe. The injection
solution of CMP−L−Rh was prepared by solubilizing it in Tween 80
(100 μL) with a mortar and pestle to get a viscous paste. The paste
was left for 4 h at rt. Then, the viscous paste was stirred well and
extracted into 5% dextrose solution. The solution was filtered through
a 0.2 μm sterile syringe filter. The concentration of the solution was
determined by diluting the formulation in DMSO and the absorbance
was measured by the absorbance of CMP group. The concentration
was calculated from the extinction coefficient of PS−L−Rh at 438 nm
in DMSO (ε = 287 000 M−1 cm−1) using the Beer−Lambert law.
One four-week-old female athymic nu/nu mouse (∼20 g, Charles
River Laboratories, Inc.) was used to investigate the cleavage of
CMP−L−Rh by irradiation. The mouse was imaged by using an IVIS
imaging system (Caliper Life Sciences), which consists of a
cryogenically cooled imaging system coupled to a data acquisition
computer running Living Image software. The mouse was irradiated
with a 690 nm diode laser at 500 mW/cm2 at the neck region 1.5 h
postinjection of the CMP−L−Rh (24 μmol/kg, ip). Fluorescence
images were taken after irradiation for 0, 10, 20, and 30 min. Before
taking the images, the mouse was anesthetized in an acrylic chamber
with 2.5% isoflurane/air mixture. The following parameters were used
to acquire images with Living imaging software: fluorescence mode,
exposure time, 5 s; binning, medium; F/Stop, 2; excitation, 535 nm;
and emission, 580 nm. During postprocessing, image counts were
adjusted to 3 × 104 au as minimum and 6.5 × 104 au as maximum
color scale.
Antitumor Efficacy Study. Four- to six-week-old BALB/c mice
(18−20 g, Charles River Laboratories, Inc.) were used for the murine
tumor model. The mice were implanted sc with 2 × 106 colon 26 cells
in PBS (100 μL) on the lower back-neck region. Tumor growth was
monitored with a digital caliper. Two dimensions, l and w (l, the
longest axis of tumor; w, the axis perpendicular to l), were used to
calculate tumor volume (lw2/2). Mice with a tumor of the diameter 3−
5 mm were used for the experiments.
Stock solutions in DMSO (4 or 8 mM) and further dilutions in 1%
Tween 80 and 5% dextrose solution to achieve final doses were as
follows: CMP−L−CA4, 4 mM → 2 or 4 μmol/kg; CMP−NCL−CA4
+ CA4, 4 mM → 2 μmol/kg; CMP−L−CA4 + CA4, 8 mM each → 4
μmol/kg. Samples were filtered using 0.2 μm sterile syringe filter. To
each mouse, 200 μL of sample was injected ip once a day on days 0, 1,
and 2. Then, 1 h 30 min later mice were anesthetized with ketamine 80
mg kg−1 and xylazine 6 mg kg−1, by ip injection. Tumors were
irradiated with a 690 nm diode laser at 200 mW/cm2 for 30 min (360
J/cm2). Tumor size was measured with the digital caliper every day
after the treatment.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Department of Defense
(Breast Cancer Research Program) under Award Number
W81XWH-09-1-0071. Views and opinions of and endorse-
ments by the authors do not reflect those of the U.S. Army or
the Department of Defense. Helpful discussion with Drs.
Michael Ihnat and Sukyung Woo is appreciated. We thank the
Peggy and Charles Stephenson Cancer Center for the use of
the Molecular Imaging Core, which provided the optical
imaging service.
ABBREVIATIONS USED
■
CA4, combretastatin A-4; CMP, core-modified porphyrin;
IY69, 5-phenyl-10,15-bis(4-carboxylatomethoxyphenyl)-20-(2-
thienyl)-21,23-dithiaporphyrin; L, aminoacrylate linker; NCL,
noncleavable linker; PDT, photodynamic therapy; ROS,
reactive oxygen species; SO, singlet oxygen.
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ASSOCIATED CONTENT
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S
* Supporting Information
A detailed description for general experimental conditions,
molecular modeling, effect of ROS quenchers on oxidation rate,
and spectral data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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