Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel Chemotherapy

Article abstract of DOI:10.1021/acs.jmedchem.5b01971

Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several cancers, including ovarian, breast, pancreatic, and nonsmall cell lung cancer. However, it causes systemic side effects when administered parenterally. Photodynamic therapy (PDT) is a new strategy for treating local cancers using light and photosensitizer. Unfortunately, PDT is often followed by recurrence due to incomplete ablation of tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker. Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically reduced. However, once illuminated with far-red light, the prodrug effectively killed SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue penetrable and clinically useful far-red light, which kills the cancer cells through the combined effects of PDT and site-specific PTX chemotherapy.

Full text of DOI:10.1021/acs.jmedchem.5b01971

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Article
Far-red Light-activatable Prodrug of Paclitaxel for the Combined Effects
of Photodynamic Therapy and Site-specific Paclitaxel Chemotherapy
Pritam Thapa, Mengjie Li, Moses Bio, Pallavi Rajaputra, Gregory
Nkepang, Yajing Sun, Sukyung Woo, and Youngjae You
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01971 • Publication Date (Web): 14 Mar 2016
Downloaded from http://pubs.acs.org on March 15, 2016
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Far-red Light-activatable Prodrug of Paclitaxel for the Combined Effects of
Photodynamic Therapy and Site-specific Paclitaxel Chemotherapy
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Pritam Thapa, Mengjie Li, Moses Bio, Pallavi Rajaputra, Gregory Nkepang, Yajing Sun,
*
Sukyung Woo, Youngjae You
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City,
OK, 73117
Key words: Combination Therapy, Aminoacrylate, Light-activatable Prodrug, Singlet
Oxygen, Photodynamic Therapy, Chemotherapy, Paclitaxel
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ABSTRACT
Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several
cancers, including ovarian, breast, pancreatic, and non-small cell lung cancer. However,
it causes systemic side effects when administered parenterally. Photodynamic therapy
(PDT) is a new strategy for treating local cancers using light and photosensitizer.
Unfortunately, PDT is often followed by recurrence, due to incomplete ablation of
tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of
PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker.
Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically
reduced. However, once illuminated with far-red light, the prodrug effectively killed
SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released
PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue
penetrable and clinically useful far-red light, which kills the cancer cells through the
combined effects of PDT and site-specific PTX chemotherapy.
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INTRODUCTION
Paclitaxel (PTX), first isolated from Taxus brevifolia bark, is one of the most important
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, 2
chemotherapeutic agents. This drug promotes tubulin polymerization and stabilizes the
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microtubules, preventing depolymerization. PTX is used as the first-line therapy for
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various cancers, including ovarian cancer, breast cancer,
and non-small-cell lung
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, 10
11-15
cancer,
and is used for both adjuvant and neoadjuvant chemotherapy.
Numerous
prodrug forms of PTX have been developed and studied. These prodrugs include enzyme
cleavable/self-immolative prodrugs, antibody-directed enzyme prodrugs, albumin
conjugates, and PAMAM dendrimer conjugates, to reduce systemic side effects from
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6-22
chemotherapy.
However, dose-limiting systemic side effects are still considered a
major problem accompanying the systemic exposure of PTX.
Photodynamic therapy (PDT) is based on the use of visible or near IR light and a
photosensitizer. PDT was approved for the treatment of several cancers, including
2
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bladder, lung, and esophageal cancers.
PDT works when light of an appropriate
wavelength activates the photosensitizer to produce reactive oxygen species, mainly
2
8
singlet oxygen. Singlet oxygen is a toxic species that can kill cells by apoptosis and/or
2
9
necrosis. However, disease recurrence stemming from the cells surviving after PDT
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0, 31
treatment is a significant problem,
which partly is due to the short half-life (~ 10-320
3
2, 33
ns) and limited diffusion distance (~10-55 nm) of singlet oxygen in cells.
Most
34-39
recently, singlet oxygen has been applied to prodrug activation.
We proposed the novel combined therapy of photodynamic and site-specific
chemotherapy using unique conjugates of photosensitizers and anticancer drugs to ablate
40-43
localized tumors without causing systemic side effects.
The two functional moieties
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are connected with a singlet oxygen cleavable linker. Upon activation of the
photosensitizer with deep tissue-penetrable visible and far-red light, the conjugate
generates singlet oxygen that triggers immediate PDT damage and then cleaves the
linker, releasing the chemotherapeutic drugs only at the site of illumination. Our study of
various singlet oxygen-cleavable linkers led to the discovery of an efficient
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aminoacrylate linker.
Utilizing this aminoacrylate linker, our group prepared
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conjugates of model drugs, including combretastatin-A4 (CA4)
and SN-38. Both of
these compounds have a phenolic group and effectively released parent drugs with visible
or far-red light illumination.
The goals of the present study were 1) to prepare a far-red light activatable
prodrug of PTX by conjugating PTX with Pc (silicon phthalocyanine, a fluorescence
photosensitizer) via our aminoacrylate linker, 2) to demonstrate its PTX release profiles,
and 3) to evaluate its in vitro activity using SKOV-3 ovarian cancer cells. There are some
reports of photoactivatable prodrugs in which UV and visible, near-IR light were used to
46-50
release PTX from various delivery forms.
However, our study is, to our knowledge,
the first to report the use of far-red light to release PTX from a prodrug via singlet
oxygen generation. This approach permits the combined treatment of PDT and site-
specific PTX chemotherapy for more complete ablation of cancer cells.
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(
a)
O
hv, 3O2
N
O
Drug
1
O2
+
HO
Drug
PS
N
PS-L-D
(b)
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NH
combretastatin A-4
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CA4)
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HO
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Phenolic OH
HO
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HO
Paclitaxel (PTX)
(c)
1O2
3O2
Immediate
PDT effect
combined
effect
Pc-(L-PTX)2
PTX
Sustained effect
Bystander effect)
(
PS: photosensitizer
L: singlet oxygen cleavable linker
D: drug
Pc: phthalocyanine
Figure 1. (a) Light-activatable prodrug via singlet oxygen-cleavable aminoacrylate
linker, (b) Structures of anticancer drugs, CA4, SN-38, and PTX, (c) Schematic
presentation of singlet oxygen-cleavable prodrugs of PTX expressing the combined
effects of PDT and site-specific PTX chemotherapy.
RESULTS AND DISCUSSION
Design and synthesis of cleavable and non-cleavable PTX prodrugs, 4 (Pc-(L-
2 2
PTX) ) and 5 (Pc-(NCL-PTX) ). We designed two PTX prodrugs with a fluorescence
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photosensitizer, silicon phthalocyanine (Pc; Scheme 1). Compound 4 is the singlet
oxygen-cleavable PTX prodrug. Thus, it was expected to produce combined effects of
PDT and site-specific chemotherapy (Fig. 1c). However, compound 5 is a pseudo-
prodrug of 4 that does not release PTX, even after illumination. Thus, compound 5 was
expected to produce only PDT effects after illumination. For the conjugation of PTX with
Pc, the most reactive hydroxyl group, 2'-OH, was selected due to its critical role for the
binding of PTX to tubulin. There have been several reports of the importance of 2'-OH
for the cytotoxic effects of PTX; 2'-OH also plays a critical role in stabilizing tubulin
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polymerization by binding with the D26 region within the microtubule binding site.
By modifying 2'-OH, we could attenuate the activity of PTX. This activity attenuation is
an essential characteristic for estabilishing prodrug character.
The two PTX prodrugs were successfully synthesized through a facile and short
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scheme (Scheme 1), following our previous methods with a minor modification.
For
5
5
the synthesis of 4, first PTX intermediate 1 was prepared by the Steglich esterification,
in which PTX was reacted with propiolic acid in the presence of DCC and DMAP to
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2
obtain 1 in 86% yield. Compound 1 was then reacted with Pc intermediate 2 in THF to
produce compound 4 as a blue solid in 46% yield. Similarly, 5 was obtained by the
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preparation of PTX intermediate 3, according to the previously reported method,
and
conjugating it with Pc intermediate 2. In brief, PTX was reacted with succinic anhydride
in the presence of pyridine to obtain compound 3. Using an amide coupling reaction,
compound 3 was conjugated with Pc intermediate 2 to give compound 5 as a blue solid in
5
7% yield.
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O
R =
N
R
O
N
O
O
O
NH
NH
O
2'
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OH
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H
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O
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H
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N
R
O
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PTX
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O
NH
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N
NH
O
O
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N
O
N
O
N
N
O
N
N
iii
OH
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N
N
Si
5 (Pc-(NCL-PTX)2)
N
2
Si
N
N
O
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HO
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H
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N
OH
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O
N
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N
O
3
O
O
N
N
R
NH
O
Scheme 1. Synthesis of compound 4 and 5; Reagents and Conditions: i) Propiolic acid,
DCC, DMAP, THF, 0 °C, 3 h, 86%; ii) 2, THF, 0 °C, 1.5 h, 46%; iii) Succinic anhydride,
Dichloromethane, r.t., 24 h, 93%; and iv) 2, EDC·HCl, HOBt, Et
°C for 0.5 hà r.t. for 3.5 h, 57%.
3
N, Dichloromethane, 0
UV/Vis Absorption and Fluorescence Emission Spectra, and LogD7.4. To
evaluate the potential for UV-Vis detection and fluorescence imaging of these two
prodrugs, the UV/Vis absorption and fluorescence emission of both prodrugs were
determined in 5% DMSO in ACN (Fig. 2). UV/Vis absorption spectra (Fig. S13) showed
a sharp Q-band at 672 nm for both prodrugs. This is a typical peak for the Pc
chromophore. The graph plotted between the concentration and absorbance at the Q-band
exhibited linearity following the Beer Lambert law in the tested concentrations, ranging
from 0.5 to 8 µM. The molar absorption co-efficiency at 672 nm was 192,600 and
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51,000 M cm for 4 and 5, respectively, which is high and thus can be useful for UV-
Vis detection in the far-red region. These results indicate that the conjugation of Pc with
two PTXs with these linkers had no negative effect on the absorption properties of the Pc
chromophore. Similarly, the fluorescence spectra showed the λflu at 678 nm and 677 nm
for 4 and 5, respectively. This finding indicates that conjugation did not significantly shift
the emission of Pc fluorophore in the prodrugs. The absorption peak at 285 nm of 4 is
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attributed to the aminoacrylate linker. These two prodrugs have appropriate absorption
and emission profiles for fluorescence imaging in vitro and in vivo. The prodrugs were
also lipophilic. Partition coefficients, as denoted by logD7.4, were > 3.1 for both
compounds 4 and 5.
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abs (nm) [ε (M ·cm )]
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(76,100) (63,300) (31,200) (192,600)
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(82,700) (41,400) (251,000)
Compound
(nm)
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677
logD7.4
> 3.1
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672
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5
-
> 3.1
Figure 2. UV/Vis (a) and fluorescence (b) spectra of 4 and 5 in 5% DMSO/ACN, (c)
Spectral and logD7.4 values of 4 and 5.
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Stability of Prodrugs 4 and 5 under Dark Conditions. Because there are two
ester bonds in both prodrugs (Scheme 1), we wanted to evaluate their stability against
hydrolysis in complete medium in the dark. The prodrugs in the medium were quantified
at various time points (t = 2-168 h; Fig. 3) using HPLC. The results were compared with
those of the initial sample (t = 0). Compound 4 was stable for 96 h with a less than 7%
decrease (Fig. 3c). Similarly, 5 was stable for 24 h with only 4% loss. After 96 h, 14% of
the prodrug compound was lost. The presence of the saturated ester bonds in 5 may
account for its marginally lower stability (p = 0.21) than 4, which has the conjugated
ester bond. Overall, the hydrolysis of these two prodrugs in the media was not rapid
without illumination.
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ꢀ
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ꢀ
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Figure 3. HPLC Chromatograms of 4 (a) and 5 (b); a: DMSO alone, b: extracted from
the medium without any prodrug, c: prodrug in 5% DMSO in ACN (10 µM), d - i: the
prodrug extract from the medium (10 µM) at 0, 2, 24, 48, 96, and 168 h, respectively, (c)
Remaining prodrug (%) at various time points from three experiments (* p < 0.05).
Tubulin Polymerization Effect of 4 and 5. The two prodrugs were anticipated to
be significantly less active than PTX. Since PTX promotes tubulin polymerization to
make microtubules, we tested 4 and 5 with a tubulin polymerization assay kit. In this
assay, fluorescence increases represent increased tubulin polymerization. We expected
that the introduction of bulky groups (Pc-L and Pc-NCL) to 2'-OH of PTX (Scheme 1),
which is important for tubulin binding, would reduce the tubulin polymerization activity
of PTX. Indeed, both compounds did not show enhancement in tubulin polymerization,
unlike PTX (Fig. 4a). With PTX, the tubulin polymerization was rapid and reached a
maximum at ~16 min. Combretastatin A-4 (CA4), a tubulin polymerization
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inhibitor,
almost completely prevented the polymerization for 1 h. However, the
curves of 4 and 5 were similar to that of the control, reaching a plateau at ~ 40 min. Even
at higher concentrations (10x), these prodrugs did not show any significant enhancement
in tubulin polymerization (Fig. 4b). The activity of PTX for tubulin polymerization was
successfully masked in both prodrugs. Next, we explored the tubulin polymerization
effect of irradiated samples of compounds 4 and 5 (Fig. S15). As anticipated, we
observed that the tubulin polymerization effect of irradiated sample 4 was significantly
increased, while there was not much change for the irradiated sample 5. This further
supports our finding that there was a significant release of PTX from the prodrug 4 upon
irradiation with far-red light.
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Figure 4. (a) Effects of 4 and 5 at 3 µM on tubulin polymerization, and (b) Effects on
tubulin polymerization at higher concentrations of prodrugs (1x = 3 µM and 10x = 30
µM). Control = vehicle only.
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Photocleavage of 4 and 5 in the Complete Medium. We first wanted to
determine if the intact PTX could be released from prodrug 4 upon illumination, because
the release of secondary alcohol from the aminoacrylate linker had not yet been
confirmed and the double bond (at C11 and C12) of the PTX could be susceptible to
oxidation by singlet oxygen. We also wanted to determine the rate of PTX release from
the prodrugs. PTX was quantified after illumination of these prodrugs with 690 nm laser
2
light at 5.6 mW/cm . The prodrug samples (10 µM) were prepared in DMEM with 5%
FCS. The prodrug solution (0.5 mL) was placed in a clear glass beaker (3-cm diameter)
to increase the area of the illumination. The entire beaker was illuminated with the laser
from above for 10, 20, and 30 min. PTX was then extracted with dichloromethane,
quantified by HPLC, and compared with the standard PTX solution.
We were able to detect intact PTX from the illuminated solution of compound 4.
A peak at the same retention time with standard PTX itself was observed from the
illuminated prodrug solution at 11 min (Fig 5b). We further confirmed the peak with
high-resolution mass spectroscopy after collecting the peak fraction from HPLC (Fig.
S14). The release of PTX by the illumination was rapid. After the illumination for 10 and
3
0 min, about 83% and 93% of PTX was released from the prodrug (Fig. 5 inset).
Extraction efficiency (EE) of PTX from the complete media was 74 ± 6%. However, we
could not detect PTX from the illuminated sample of 5 due to the absence of the singlet
oxygen-cleavable linker (Fig. 5f).
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Time (min)
Figure 5. HPLC chromatograms of (a) 2.5% DMSO in ACN; (b) PTX (20 µM) in the
2
media; (c-e) 4 (10 µM) in the media illuminated with 690 nm at 5.6 mW/cm for 10, 20,
and 30 min; (f) 5 (10 µM) in the media illuminated for 30 min; (Inset) PTX release (%)
after illumination of 4.
Subcellular Localization Compared with Tubulin Tracker Green. Tubulin
polymerization assay showed that the prodrugs 4 and 5 did not promote tubulin
polymerization. We assumed that these two prodrugs do not specifically bind to
polymerized tubulin inside cells due to the steric bulkiness at 2'-OH. To confirm the
tubulin polymerization assay results in cells, we performed a subcellular localization
study with the confocal microscope. The prodrugs have an excellent fluorophore (Pc) that
can be easily visualized through fluorescence imaging. To specifically stain polymerized
tubulin (microtubules) inside cells, tubulin tracker green was used. SKOV-3 cells were
incubated with either prodrug. Then, tubulin tracker green was added 45-60 min before
images were captured. We first obtained three sequential images: bright-field (i) à
tubulin tracker green fluorescence (ii) à prodrug fluorescence image (iii) (Fig. 6). Then,
we overlapped the images of tubulin tracker green with 4 or 5 (iv).
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(ai)
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Figure 6. Confocal images of SKOV-3 cells after incubation with 4 (a), 5 (b), at 0.5 µM
for 23 h. Tubulin tracker green (1 µM) was added to all wells 45-60 min before taking
images; (i) bright-field image, (ii) excitation at 488 nm and emission filter at 500 - 550
nm, (iii) excitation at 633 nm and emission filter at 650 - 800 nm, (iv) overlap of ii and
iii.
Nearly homogenous staining was observed throughout the cytosol in both images
6
2
(
aii and bii). This is a typical staining pattern of tubulin tracker green. However, 4
showed uneven and vesicular localization pattern around the nuclei (Fig. 6aiii), similar to
63, 64
lysosomal or endosomal staining patterns.
Emission of 5 from the cells was weak, but
showed a pattern similar to that of 4. Tubulin tracker green and the two prodrugs showed
very different subcellular localization patterns as observed in the enlarged image (Fig.
S16) Thus, we concluded that these two prodrugs did not specifically bind to
microtubules inside cells, consistent with the results from the tubulin polymerization
assay.
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The lower emission of 5 might be due to the lower uptake or quenching of the
fluorescence by aggregation. Both prodrugs had limited solubility in aqueous solution
(
logD7.4 > 3.1). Thus, we dissolved the prodrugs in DMSO for cellular studies. During the
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photo and dark toxicity experiments, we had no problems using the DMSO stock
solutions. However, the initial subcellular localization study using the DMSO stock
solutions was unsuccessful for both prodrugs, presumably due to aggregation. To
improve the solubility in the media, we used Tween 80 (1%) in dextrose (5%) solution
for the dilutions, which improved the fluorescence signal of both prodrugs in the confocal
imaging. However, the emission of 5 was much lower than that of 4. Although the logD7.4
of these prodrugs were not distinguishable, compound 5 seemed to have a lower
solubility in aqueous medium than 4. Compound 5 showed much lower emission than 5
at equimolar concentrations in both Tween 80 (1%)/dextrose (5%) solution and DMEM
(Fig. S17).
Stability of 4 and 5 with the SKOV-3 Cells. To see the impact of the cells on
PTX release during the in vitro study, stability of the two prodrugs in the SKOV-3 culture
media was determined in the dark. The two prodrugs were quantified by HPLC after 24
and 72 h incubation in the cell culture medium. The retention times of 4 and 5 were 32
and 21 min in the HPLC chromatograms, respectively (Fig. 3). The calibration curves
suggested a linear relationship between the peak area and concentration in the range of
0
8
.1 to 5 µM (70 µL injection, Fig. 7a). The equation of the regression line is y = 0.513x +
2
2
9.22 (r =0.995) and y = 0.316x + 73.28 (r =0.994) for 4 and 5, respectively. Total
recovered percentiles of 4 and 5 from the culture medium and cells are shown in Figure
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b. Small reductions of 4.9% for 4 and 3.1% for 5, were found after 3 day incubation.
The data suggest that both prodrugs are stable in the dark for at least 3 days, even with the
cells.
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r2 = 0.9944
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Conc (nM)
(b)
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Incubation time (h)
ꢀ
Figure 7. (a) Calibration curves of 4 and 5 based on HPLC. (b) Recovered 4 and 5 from
the cell culture medium (cells + culture medium) 24 and 72 h after the addition of
prodrugs (500 nM).
Dark and Phototoxicity. Based on the above results - the masking of the tubulin
polymerization effect of PTX, efficient photo-release of PTX from 4, and good stability
in the culture medium - we expected low dark toxicity for both prodrugs and high
phototoxicity of 4 due to the released PTX by the illumination. Cells were treated with
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PTX, 4, or 5. Then, cell survival was determined using MTT, with or without
2
illumination using a 690-nm diode laser at 5.6 mW/cm for 30 min.
PTX itself showed potent cytotoxicity against SKOV-3 cells with IC50 of 4.7 nM
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(
red lines in Fig. 8), which is close to the reported data. However, both prodrugs 4 and 5
showed dramatically reduced dark toxicity (black lines) with IC50 of 910 nM and 1279
nM, 190- and 270- fold reductions from the IC50 of PTX, respectively. Once illuminated,
both prodrugs showed strong cytotoxicity (green lines) with IC50 of 4 and 5 being 3.9 and
24 nM. Since 5 cannot release PTX upon illumination and it is stable in the culture
medium, its phototoxicity should have come from its PDT effect through singlet oxygen.
5
seems to be a powerful photosensitizer. The most potent phototoxicity of 4 might come
from the combined effects of PDT and released PTX chemotherapy.
To evaluate the contribution of the photo-released PTX in the media to the
phototoxicity of 4 and 5, the culture medium was replaced with fresh medium, called
washing. Washing can remove products of the illumination from the media except those
in side cells, e.g., released PTX in the media. In both prodrugs, the washing of the parent
medium significantly reduced the phototoxicity (blue lines in Fig. 8): IC50 of 89 and 53
nM for 4 and 5, respectively. While there was an ~2-fold reduction in IC50 for 5, an ~23-
fold reduction was observed for 4. The dramatic decrease in the phototoxicity of 4 should
have come from the removal of the released PTX from the medium. This finding supports
the combined contributions of PDT and released PTX to the phototoxicity of 4.
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Concentration (nM)
Figure 8. Dark and phototoxicity of 4 (a) and 5 (b) against SKOV-3 ovarian cancer cells,
PTX cytotoxicity, and prodrug phototoxicity.
CONCLUSIONS
We designed a light-activatable prodrug of PTX that can express both PDT and
chemotherapeutic effects at the illuminated area. The PTX prodrug 4 and its pseudo-
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prodrug 5 were readily prepared using a facile synthetic scheme with mild reaction
conditions and moderate yields. The activity of PTX was effectively shielded in the
prodrugs. However, once illuminated, 4 released a high yield of intact PTX, which
caused damage to cancer cells in addition to the immediate effects of PDT. Thus, in vivo
studies for preclinical optical imaging, toxicity, and antitumor efficacy determinations are
warranted. We are currently screening various formulations for an animal study to
improve the solubility of these highly lipophilic prodrugs.
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This study presents two key significant findings regarding light-activated
prodrugs. First, this is the first investigation demonstrating that PTX prodrug can be
activated using far-red light and can express both PDT and chemotherapeutic effects
simultaneously. PTX is one of the most successful chemotherapeutic drugs in the clinic,
and far-red can be easily adopted in the clinic due to its non-toxicity and tissue
penetrability. We believe this approach has excellent potential for clinical translation to
treat localized tumors. Second, this work demonstrates the applicability of our photo-
unclick chemistry (singlet oxygen-mediated release of aminoacrylate type-linkers to
drugs) with an aliphatic secondary alcohol. Thus far, we have primarily demonstrated
photo-unclick chemistry with phenolic compounds. These results show the applicability
of our strategy to broader drugs and bioactive compounds with an aliphatic alcohol,
which is a common functional group.
EXPERIMENTAL SECTION
Chemicals and equipment. The starting materials and reagents obtained from
Aldrich Chemical co., Fisher Scientific, and VWR were analytical or ACS grades and
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6
were used without further purification. HPLC grade solvents were purchased from
Pharmco-AAPER. The products in the syntheses were visualized on thin layer
chromatography (TLC plates, cat # Z193291, Sigma-Aldrich) under UV light. Column
chromatography was carried out in 40-63 mm ( 230-400 mesh) silica gel purchased from
SiliCycle Inc. (cat # R10030B). NMR spectra were recorded on a Varian 300 MHz
spectrometer. The data were analyzed using iNMR (Version 5.4.2) and the chemical
shifts were calibrated according to the residual solvent peaks. Chemical shifts were (δ)
recorded in ppm with coupling constants (J) in hertz (Hz). Agilent 1260 series HPLC
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system (Agilent Technologies, USA) was used to quantify PTX, Pc-(L-PTX)
2
, and Pc-
(
NCL-PTX) . A BDS Hypersil C18 column (250 × 4.6 mm, 5-µm particle size) was used
2
with a Pinnacle DB C18 guard column (10 × 4 mm, 5-µm particle size). HRMS was
analyzed using an ABSciex QSTAR Elite hybrid quadrupole/TOF mass spectrometer.
UV-Vis Lambda 25 (Perkin Elmer) and a 10-mm optical path length quartz cuvette were
used for UV-Vis absorption spectra. The fluorescence was monitored in an LS50B
spectrometer (Perkin Elmer).
Synthesis. Compound 2 (Scheme 1) was prepared according to the previously
4
2
reported method. HPLC revealed >95% purity of 4 and 5, which were used for
biological tests.
Compound 1. This compound was prepared based on the previous report with a
55
minor modification. We added propiolic acid (36.9 mg, 0.52 mmol, 1.5 equiv) to a
stirred solution of PTX (300 mg, 0.35 mmol, 1.0 equiv) in THF (5 mL) at 0 °C under N
2
gas. After stirring for few minutes, we slowly added DCC (73.2 mg, 0.35 mmol, 1.0
equiv) and DMAP (1.2 mg, 0.01 mmol, 0.03 equiv) dissolved in THF (2 mL) at 0 °C
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under N
2
gas. The reaction mixture was stirred at 0 °C for 30 min. Stirring continued for
4
h at room temperature. The precipitate was removed by filtration and washed with ethyl
acetate. The filtrate was concentrated to obtain a yellow viscous compound that was
purified by column chromatography using ethyl acetate and hexane (1:1 v/v) to produce
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f
273 mg (0.30 mmol, 86%) of compound 1 as a white solid. R = 0.37 (ethyl acetate/ n-
1
hexane = 2:1 v/v). H NMR (CDCl
3
, 300 MH
Z
) δ 8.13 (dd, J = 8.7, 1.5 Hz, 2H), 7.73
(dd, J = 8.4, 1.5 Hz, 2H), 7.63-7.58 (m, 1H), 7.54-7.37 (m, 10H), 6.87 (d, J = 9.3 Hz),
6.28 (br s, 1H), 6.27 (t, J = 8.7 Hz, 1H), 6.01 (dd, J = 9.3, 2.7 Hz), 5.68 (d, J = 6.9 Hz),
5
8
.57 (d, J = 2.7 Hz, 1H), 4.97 (dd, J = 9.6, 1.8 Hz, 1H), 4.47-4.41 (m, 1H), 4.31 (d, J =
.7 Hz, 1H), 4.20 (d, J = 8.4 Hz, 1H), 3.81 (d, J = 6.9 Hz, 1H), 3.04 (s, 1H), 2.61-2.51
(m, 2H), 2.44 (s, 3H), 2.44-2.35 (m, 1H), 2.23 (s, 3H), 2.23-2.16 (m, 1H), 1.91 (s, 3H),
13
1
2
1
7
.90-1.84 (m, 1H), 1.68 (s, 3H), 1.29 (br s, 1H), 1.25 (s, 3H), 1.13 (s, 3H); C NMR δ
03.8, 171.3, 169.8, 167.1, 167.0, 151.4, 142.6, 136.5, 133.7, 133.5, 132.9, 132.1, 130.2,
29.2, 129.1, 128.7, 128.7, 127.1, 126.52, 84.4, 81.1, 79.2, 77.5, 76.6, 75.5, 75.3, 75.1,
3.4, 72.3, 72.1, 58.5, 52.5, 45.52, 43.2, 35.5, 35.5, 26.8, 22.7, 22.2, 20.8, 14.8, 9.6.
Compound 3. This compound was prepared based on the previous report with a
5
6, 57
minor modification.
At room temperature, we slowly added 3.5 mL pyridine to a
stirred solution of compound PTX (1.5 g, 1.75 mmol, 1.0 equiv) and succinic anhydride
(0.263 g, 2.64 mmol, 1.5 equiv) in dichloromethane (70 mL). The reaction mixture was
2
stirred at room temperature for 24 h under N gas. Then, the mixture was concentrated to
give a white solid. The residue was purified by silica gel column chromatography using
ethyl acetate and hexane as eluents (1:1 to 8:1 v/v) to produce 1.55 g (1.63 mmol, 93%)
1
of compound 3 as a white solid. R
f
= 0.21 (ethyl acetate/ n-hexane = 8:1 v/v). H NMR
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
CDCl
3
, 300 MH
Z
) δ 8.13 (d, J = 7.2 Hz, 2H), 7.73 (d, J = 7.2, 1.5 Hz, 2H), 7.63-7.58 (m,
1
5
H), 7.53-7.48 (m, 10H), 7.03 (d, J = 9.3 Hz), 6.28 (br s, 1H), 6.27 (t, J = 8.4 Hz, 1H),
.99 (dd, J = 9.0, 2.7 Hz, 1H), 5.68 (d, J = 6.6 Hz, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.97 (d,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 8.7 Hz, 1H), 4.46-4.40 (m, 1H), 4.31 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.4 Hz, 1H),
3.80 (d, J = 6.6 Hz, 1H), 2.77-2.50 (m, 6H), 2.43 (s, 3H), 2.44-2.31 (m, 1H), 2.21 (s, 3H),
2
3
.17-2.12 (m, 1H), 1.90 (br s, 4H), 1.83 (br s, 1H), 1.67 (s, 3H), 1.22 (s, 3H), 1.12 (s,
1
3
H); C NMR δ 203.8, 171.2, 171.0, 169.9, 167.9, 167.2, 167.0, 142.7, 136.8, 133.7,
133.5, 132.7, 132.0, 130.2, 129.1, 129.1, 128.7, 128.7, 128.5, 127.2, 126.5, 84.4, 81.1,
7
2
9.1, 76.4, 75.6, 75.1, 74.2, 72.1, 71.9, 58.5, 52.7, 45.6, 43.1, 35.5, 28.8, 28.4, 26.8, 22.6,
2.1, 20.8, 14.8, 9.6.
Compound 4. We slowly added compound 2 (80 mg, 0.10 mmol, 1.0 equiv)
dissolved in THF to a stirred solution of compound 1 (218 mg, 0.24 mmol, 2.4 equiv) in
dry THF (5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1.5 h. Then, THF
was evaporated to give a blue solid. The residue was purified by column chromatography
using ethyl acetate and n-hexane (2:1, v/v) followed by dichloromethane and methanol
f
(20:1, v/v) to yield 119 mg (0.045 mmol, 46%) of 4 as a blue solid. R = 0.21
1
(dichloromethane/ methanol = 20:1 v/v). H NMR (CDCl
3
, 300 MH
Z
) δ 9.63 (dd, J = 5.7,
3
.3 Hz, 8H), 8.34 (dd, J = 5.7, 3.0 Hz, 8H), 8.11 (d, J = 7.2 Hz, 4H), 7.75 (d, J = 7.2 Hz,
4H), 7.63-7.57 (m, 2H), 7.53-7.29 (m, 20H), 7.07 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 12.9
Hz, 2H), 6.29 (br s, 2H), 6.22 (t, J = 9.0 Hz, 2H), 5.84 (dd, J = 8.7, 4.5 Hz, 2H), 5.66 (d,
J = 6.9 Hz, 2H), 5.53 (d, J = 3.9 Hz, 2H), 4.97 (d, J = 9.9 Hz, 2H), 4.47-4.41 (m, 2H),
4
.31 (d, J = 8.7 Hz, 2H), 4.29 (d, J = 13.2 Hz, 2H), 4.19 (d, J = 8.4 Hz, 2H), 3.80 (d, J =
6
.9 Hz, 2H), 2.60-2.49 (m, 4H), 2.39 (s, 6H), 2.30-2.25 (m, 2H), 2.23 (s, 6H), 2.16 (br s,
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H), 2.09-2.01 (m, 2H), 1.93 (s, 6H), 1.87-1.82 (m, 2H), 1.67 (s, 8H), 1.25 (s, 6H), 1.13
1
3
(s, 6H), 0.25 (br s, 8H), -0.54 (br s, 4H), -1.93 (t, J = 4.8 Hz, 4H); C NMR δ 203.9,
1
1
71.3, 169.8, 169.2, 168.7, 167.0, 166.9, 152.7, 149.2, 143.2, 137.7, 135.8, 133.9, 133.7,
32.4, 131.8, 131.2, 130.2, 129.2, 128.8, 128.7, 128.6, 128.2, 127.1, 126.8, 123.7, 84.4,
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37
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43
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45
46
47
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49
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53
54
55
56
57
58
59
60
81.2, 80.9, 79.2, 77.2, 76.4, 75.6, 75.1, 72.7, 72.1, 71.2, 58.5, 56.6, 53.6, 53.2, 45.6, 43.1,
2
+
3
1
146 14
5.5, 26.8, 22.6, 22.1, 20.9, 14.8, 9.6; HRMS-ESI : m/z calcd. for [C144H N O32Si] :
2
+
305.4998, [M+2H] , found 1305.4999. Purity = 95% (HPLC Chromatogram, Fig. S11).
Compound 5. To a stirred solution of compound 2 (100 mg, 0.13 mmol, 1.0
equiv) in dry dichloromethane (20 mL) we slowly added compound 3 (298 mg, 0.31
mmol, 2.5 equiv) at 0 °C. Then we added EDC⋅HCl (67 mg, 0.35 mmol, 2.8 equiv),
HOBt hydrate (53 mg, 0.35 mmol, 2.8 equiv) and Et
3
N (31 mg, 0.31 mmol, 2.5 equiv) at
0
°C. The reaction mixture was stirred at 0 °C for 30 min. Stirring continued for 3.5 h at
room temperature. Then, dichloromethane was evaporated to give a blue solid. The
residue was purified by column chromatography using ethyl acetate and n-hexane (8:1,
v/v) followed by dichloromethane and methanol (40:1 to 20:1, v/v) to give 192 mg (0.072
1
mmol, 57%) of 5 as a blue solid. R
NMR (CDCl , 300 MH
.73 (dd, J = 8.1, 1.2 Hz, 4H), 7.62-7.59 (m, 2H), 7.54-7.24 (m, 20H), 7.16 (d, J = 8.1
Hz, 2H), 6.27 (br s, 2H), 6.17 (t, J = 9.0 Hz, 2H), 5.84 (dd, J = 7.5, 3.0 Hz), 5.66 (d, J =
.9 Hz, 2H), 5.39 (dd, J = 3.9, 1.5 Hz, 2H), 4.97 (d, J = 9.3 Hz, 2H), 4.45-4.39 (m, 2H),
4.31 (d, J = 8.4 Hz, 2H), 4.19 (d, J = 8.4 Hz, 2H), 3.78 (d, J = 6.9 Hz, 2H), 2.55-2.53 (m,
2H), 2.36 (s, 6H), 2.27 (br s, 8H), 2.26-2.00 (m, 6H), 2.21 (s, 6H), 1.89 (s, 6H), 1.81 (br
f
= 0.43 (dichloromethane/ methanol = 15:1 v/v). H
3
Z
) δ 9.63 (m, 8H), 8.34 (m, 8H), 8.11 (dd, J = 8.1, 1.2 Hz, 4H),
7
6
1
s, 2H), 1.67 (s, 6H), 1.26 (s, 6H), 1.12 (s, 6H), 0.34 (br s, 4H), 0.19 (br s, 4H), -0.62 (t, J
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1
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2
2
2
2
2
2
2
2
2
3
3
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5
5
5
5
5
5
5
6
13
=
4.8 Hz, 4H), -1.93 (t, J = 4.8 Hz, 4H); C NMR δ 203.8, 172.1, 171.2, 169.8, 168.4,
1
68.2, 167.2, 167.0, 149.2, 142.8, 137.2, 135.8, 133.7, 133.6, 132.7, 131.8, 131.1, 130.2,
29.2, 128.9, 128.7, 128.5, 128.4, 127.2, 126.7, 123.7, 84.4, 81.0, 79.1, 76.4, 75.6, 75.0,
4.1, 72.1, 71.6, 58.5, 56.8, 53.3, 51.4, 51.2, 45.5, 44.0, 43.1, 40.6, 35.5, 35.4, 28.9, 27.7,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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4
5
6
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8
9
0
7
2
+
150 14
26.8, 22.6, 22.1, 20.8, 14.8, 9.6; HRMS-ESI : m/z calcd. for [C146H N O34Si] :
2
+
1
335.5104, [M+2H] , found 1335.4337. Purity = 98% (HPLC Chromatogram, Fig. S12).
LogD7.4 determination. The partition coefficient in of n-octanol and phosphate
buffer (PBS, pH 7.4) for 4 and 5 was measured following the previously reported
43
method, with some modifications. In brief, the stock solution of the prodrugs was
prepared in DMSO (10 mM). The stock solution (10 mL) was added to n-octanol and
PBS (1 mL each) in a vial. The mixture was vortexed for 30 min. Then, the mixture was
kept in the dark for 3 h for phase separation. Once there was clear phase separation, both
layers were collected in different vials. Each layer (100 mL) was diluted to 1 mL of ACN
(
5% DMSO) and fluorescence spectra (excitation at 615 nm, emission at 680 nm) were
taken. We did not detect the signal in the PBS layer in either prodrug. We determined the
lowest concentration of the prodrugs that was detectable using the fluorimeter. The
logD7.4 value was then estimated using the ratio of concentration in n-octanol to lowest
detectable concentration.
Release of PTX from the prodrugs after illumination. Extraction of PTX from
the complete medium: PTX solution (0.5 mL, 20 μM) in the complete medium prepared
from the DMSO stock was added to a centrifuge tube. Dichloromethane (5 mL) was
added to the tube and vortexed for 1 min, followed by centrifugation at 3000 rpm for 10
min for phase separation. The upper media layer was carefully removed using a pasteur
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pipette. The lower organic solvent layer was then concentrated with a nitrogen flush. The
residue was reconstituted in 100% ACN (0.5 mL), filtered through a 0.2-µm PTFE
membrane filter, and transferred into a vial. HPLC was performed using the isocratic
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60
2
mobile phase of 60% ACN and 40% 10 mM ammonium acetate in dH O (milliQ) at the
flow rate of 0.5 mL/min with an injection volume of 10 uL. Quantification of released
PTX from PTX prodrugs: Prodrug solution (0.5 mL, 10 µM) in complete medium, diluted
from the DMSO stock solution, was added to a 3-cm-diameter glass beaker and was
2
illuminated from above with a laser (690 nm, 5.6 mW/cm ) for 10, 20, or 30 min. We
used the beaker to increase the surface area of light exposure. Then, the illuminated
sample was transferred into a centrifuge tube by washing the beaker with
dichloromethane (5 mL). We followed the same extraction procedure for the PTX
sample, described above. HPLC conditions: Isocratic mobile phase of 60% ACN and
4
0% ammonium acetate (10 mM) in dH
2
O (milliQ) at a flow rate of 0.5 mL/min with an
injection volume of 10 µL.
Stability of 4 and 5 in the Complete Medium under Dark Conditions. A solution
(4 mL, 10 µM) of 4 or 5 prepared in the complete medium from the DMSO stock solution
was incubated at 37 °C in the dark. The sample (0.5 mL) was taken at various time points
(
0, 2 h, 24 h, 48 h, 96 h, and 168 h) and extracted with dichloromethane following the
same extraction procedure described for PTX. The extracted sample was reconstituted in
.5 mL of 5% DMSO in ACN and injected into HPLC. HPLC conditions: Isocratic
0
mobile phase of 90% ACN and 10% ammonium acetate (10 mM) in dH
flow rate of 0.5 mL/min with an injection volume of 10 µL.
2
O (milliQ) at a
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Stability of 4 and 5 with the Cells. On day 0, SKOV-3 cells (5000 cells/well)
2
were seeded on 96-well plates and then incubated at 37 °C in 5% CO . On day 1, the
solution (10 µL of 4 or 5 was added to each well (190 µL) to make a final concentration
of 500 nM. Both cells and medium were collected at 24 and 72 h after the addition of the
drugs, respectively. The medium was first collected into the centrifuge tube. The cells
were digested using trypsin (100 µL) and were then transferred to the centrifuge tube.
After washing with fresh medium (200 µL), all liquids were combined into a 10-mL
centrifuge tube. Dichloromethane (1 mL) was subsequently added to the tube. The
mixture was vortexed for 10 min and centrifuged at 3000 rpm for 30 min. The upper
layer was removed and the extraction was repeated once. The organic layers were
combined into a clean vial and dried with nitrogen. The extracts were then reconstituted
in 5% DMSO in ACN (200 µL). After sonication for 30 min, the samples were filtered
through a 0.2-µm PTFE membrane filter. The filtrate (70 µL) was injected into the HPLC
with the mobile phase consisting of a mixture of 90% ACN and 10% ammonium acetate
0
1
2
3
4
5
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7
8
9
0
1
2
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0
1
2
3
4
5
6
7
8
9
0
2
in 10 mM dH O (milliQ) at a flow rate of 0.5 mL/min. With a diode array detector
(DAD), all separations were detected at 675 nm. Calibration curves of 4 and 5 were
prepared with final concentrations of 0.1, 0.5, 1, 2, 5, and 10 µM in the complete
medium.
Tubulin Polymerization Assay. A tubulin polymerization assay kit (cat #
BK011P, Cytoskeleton, Inc.) was used to assess the effect of prodrugs on tubulin
polymerization. The experiment was carried out based on the product protocol as
described in version 3.0 of the tubulin polymerization assay kit manual. The basic
principle is that the increase in number of microtubules gives higher fluorescence due to
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the incorporation of a fluorescent reporter into microtubules. Briefly, the test compound 4
or 5 in DMSO was diluted with distilled water. In 96-well plates, a reaction mixture of
tubulin and GTP in a buffer solution was added to the test compound to give final
concentration of 3 µM. The reaction mixture was incubated at 37 °C. Fluorescence was
monitored (excitation = 360 nm and emission = 450 nm) every 2 min for 1 h. PTX and
CA4 were used as positive and negative controls, respectively. DMSO solution was used
as a vehicle=only control.
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Confocal Microscopic Imaging. SKOV-3 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.
All procedures were performed under minimal light. SKOV-3 cells were seeded at 1.0 -
5
1
.5× 10 cells/well in 24-well plates containing 12-mm diameter cover slips. Cells were
then incubated for 24 h using a Sanyo MCO-18AIC-UV incubator. The stock solution of
prodrug 4 or 5 diluted with medium to a concentration of 500 nM, was added to the
plates, and was incubated for 24 h. The cells were stained with tubulin tracker green 45-
6
0 min before imaging. Briefly, the medium was removed and the cover slips were
washed with HBSS. Tubulin tracker green in HBSS was added to make final
concentrations of 1 µM. The wells were incubated for 45-60 min at 37 °C in 5% CO
2
.
Before visualization under the microscope, cover slips were washed three times with
HBSS. Three random fields per treatment were imaged under the confocal microscope
(Leica SP2 confocal microscope). Images were captured with a 63x 1.2 NA water Corr
lens.
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