10-Boronic acid substituted camptothecin as prodrug of SN-38

Article abstract of DOI:10.1016/j.ejmech.2016.03.063

Malignant tumor cells have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H₂O₂), supporting the hypothesis that a prodrug could be activated by intracellular H₂O₂ and lead to a potential antitumor therapy. In this study, the 7-ethyl-10-boronic acid camptothecin (B1) was synthesized for the first time as prodrug of SN-38, by linking a cleavable aryl carbon-boron bond to the SN-38. Prodrug B1 selectively activated by H₂O₂, converted rapidly to the active form SN-38 under favorable oxidative conditions in cancer cells with elevated levels of H₂O₂. The cell survival assay showed that prodrug B1 was equally or more effective in inhibiting the growth of six different cancer cells, as compared to SN-38. Unexpectedly, prodrug B1 displayed even more potent Topo I inhibitory activity than SN-38, suggesting that it was not only a prodrug of SN-38 but also a typical Topo I inhibitor. Prodrug B1 also demonstrated a significant antitumor activity at 2.0 mg/kg in a xenograft model using human brain star glioblastoma cell lines U87MG.

Full text of DOI:10.1016/j.ejmech.2016.03.063

Accepted Manuscript
10-Boronic acid Substituted Camptothecin as Prodrug of SN-38
Lei Wang, Shao Xie, Longjun Ma, Yi Chen, Wei Lu
PII:
S0223-5234(16)30246-X
10.1016/j.ejmech.2016.03.063
EJMECH 8488
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2016
Revised Date: 22 March 2016
Accepted Date: 23 March 2016
Please cite this article as: L. Wang, S. Xie, L. Ma, Y. Chen, W. Lu, 10-Boronic acid Substituted
Camptothecin as Prodrug of SN-38, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.063.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
0-Boronic acid Substituted Camptothecin as Prodrug of SN-38
a
b
a
b,
a,
Lei Wang‡ , Shao Xie‡ , Longjun Ma , Yi Chen * and Wei Lu *
a
School of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai 200062, P. R. China
b
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China
*
Corresponding authors:
E-mail: wlu@chem.ecnu.edu.cn; Tel.: +86-21-62602475 (W. Lu);
E-mail: ychen@simm.ac.cn; Tel.: +86-21-50801552 (Y. Chen).
‡
These authors contributed equally.
Abstract
Malignant tumor cells have been found to have high levels of reactive oxygen
species such as hydrogen peroxide (H O ), supporting the hypothesis that a prodrug
2
2
could be activated by intracellular H O and lead to a potential antitumor therapy. In
2
2
this study, the 7-ethyl-10-boronic acid camptothecin (B1) was synthesized for the first
time as prodrug of SN-38, by linking a cleavable aryl carbon−boron bond to the
SN-38. Prodrug B1 selectively activated by H O , converted rapidly to the active form
2
2
SN-38 under favorable oxidative conditions in cancer cells with elevated levels of
H O . The cell survival assay showed that prodrug B1 was equally or more effective
2
2
in inhibiting the growth of six different cancer cells, as compared to SN-38.
Unexpectedly, prodrug B1 displayed even more potent Topo I inhibitory activity than
SN-38, suggesting that it was not only a prodrug of SN-38 but also a typical Topo I
inhibitor. Prodrug B1 also demonstrated a significant antitumor activity at 2.0 mg/kg
in a xenograft model using human brain star glioblastoma cell lines U87MG.
Keywords: boric acid; hydrogen peroxide; prodrug; camptothecin; antitumor.
1

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1
. Introduction
Natural products have been the source of most of the active ingredients of
medicines. This is widely accepted to be true when applied to drug discovery in
olden times’ before the advent of high-throughput screening and the post-genomic
‘
era: more than 80% of drug substances were natural products or inspired by a natural
compound [1]. In the area of cancer, over the time frame from around the 1940’s to
2
007, of the 155 small molecules, 73% are other than synthetic, with 47% actually
being either natural products or directly derived therefrom [2]. What is noteworthy is
that lots of natural products and its derivatives contain the phenol hydroxyl as the key
pharmacophore, such as combretastatin A-4 (CA-4, 1, Fig. 1), etoposide (2, Fig. 1)
and SN-38 (the active metabolite of irinotecan, 3, Fig. 1).
O
O
O
O
O
MeO
MeO
HO
HO
O
OH
O
N
N
O
OMe
OMe
OH
O
OH
O
OMe
1
2
3
OH O
MeO
Figure 1. Representative natural products which contain the phenol hydroxyl.
Boron is an essential trace element for plants that occurs naturally in the
environment. There is increasing evidence that boron may be important for animals
and humans, as it is in plants [3]. Boron has a wide range of applications in chemistry,
material science, energy research, and electronics, as well as in life science. The drug
®
bortezomib (Velcade ), a drug in clinical use with boron as an active element, was
approved in 2003 as a proteasome inhibitor for the treatment of multiple myeloma and
non-Hodgkins lymphoma [4].
It is well established that alkyl- or aryl boronic acid and their esters can be easily
oxidated by H O [5]. Moreover, cancer cells are known to exhibit elevated intrinsic
2
2
oxidative stress [6-8]. The increased amounts of reactive oxygen species (ROS) can
be a therapeutic advantage, because it is an exclusive feature of cancer cells [9-10].
Therefore, it would be advantageous to develop novel anticancer prodrugs that can be
activated by the high level of ROS in cancer cells. The most common ROS include
2

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-
hydrogen peroxide (H O ), superoxide anions (O ), and hydroxyl radical. Among
2
2
2
these, H O has a pivotal role because it is a stable ROS and generated from nearly all
2
2
sources of oxygen radicals [11]. These factors make H O an ideal candidate as a
2
2
target to develop new ROS-inducible prodrugs with high selectivity to cancer cells. In
addition, the cell surface is coated with a dense forest of polysaccharides known as
the glycocalyx [12]. Boronic acids readily form boronate esters with the 1, 2- and 1,
3
-diols of saccharides [13], including those in the glycocalyx [14-19]. Boronates have
attributes that enhance drug selectivity toward tumor cells [20]. Meanwhile, Boronic
acids and esters do not appear to have intrinsic toxicity issues, and the end product,
boric acid, is considered nontoxic to humans [21].
Recently, various boron-based derivatives of 4-hydroxytamoxifen (4-6, Fig. 2) were
studied for the treatment of tamoxifen-resistant breast cancer. These prodrugs share
common boron-aryl carbon bonds and are converted into the active form
4
-hydroxytamoxifen under favorable oxidative conditions in breast cancer. These
bioisosteres were found to inhibit the growth of two breast cancer cell lines, MCF-7
and T47D, to a comparable or greater extent than that achieved by
4
-hydroxytamoxifen [22]. Moreover, Peng’s group has also shown that the prodrug of
nitrogen mustard coupled with an arylboronate (7 and 8, Fig. 2) can be triggered by
H O to release active drugs that can kill cancer cells [23-24].
2
2
N
N
N
HO
O
B
O
B
O
KF B
O
3
O
OH
4
5
6
HO
OH
O
N
O
O
B
B
O
O
O
O
N
B
O
O
N
O
Cl
Cl
O
N
Me
Cl
Br
Me
Cl
Br
OH O
7
8
9
Figure 2. Structures of representative boron-based compounds.
3

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Camptothecin (CPT) is a highly effective anti-tumor and antibiotic alkaloid that
was first isolated by Wall and Wani in 1966 [25]. Its antitumor activity is induced by
directly binding to topoisomerase I (Topo I), which results in interference with the
catalytic cycle of DNA-Topo I and stabilization of the DNA-Topo I binary complex
[
26-27]. According to the previous studies of CPT derivatives, the SAR had
summarized that hydroxyl substitution 10-position enhance their antitumor activity,
such as topotecan, 10-hydroxy-CPT and SN-38 (the active metabolite of irinotecan,
which gained FDA approval in 1994) [28]. Although a number of camptothecin
derivatives were synthesized, but boronic acid substituted analogs have not been
reported [29-42]. Kim et al. developed a new theranostic prodrug (9, Fig. 2), which
contains a H O -induced cleavable boronate ester for activating a fluorescent moiety
2
2
(coumarin) and releasing a potent anticancer drug, SN-38 [43]. This prodrug reacted
with physiological levels of H O , resulting in dissociation of the conjugates and
2
2
delivery of chemotherapeutic agent, providing a powerful new tool for cancer therapy.
In this study, we report 7-ethyl-10-boronic acid camptothecin (B1, Scheme 1) for
the first time, it is anticipated that upon entering cancer cells, the prodrug B1 will
undergo facile oxidization reaction to release SN-38 that can kill cancer cells.
OH
B
HO
O
O
HO
N
N
H O
2
2
N
N
O
O
B1
SN-38
OHO
Scheme 1. Design of prodrug B1 and H O -promoted release of SN-38.
OHO
2
2
2
. Results and discussion
2
.1. Chemistry
Detailed synthetic strategy to 7-ethyl-10-boronic acid camptothecin B1 was
illustrated in Scheme 2. According to published procedures, SN-38 was first
converted into triflate 10 in high yield (95%) [44], then triflate 10 was borylated using
palladium catalyzed Miyaura chemistry [45]. Finally, boronate ester 11 was oxidated
to give boronic acid B1 in a 50% yield [46].
4

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O
OH
B
TfO
O
B
O
O
HO
O
N
N
N
a
b
c
SN-38
N
N
N
O
O
O
1
0
11
B1
OHO
OHO
OHO
Scheme 2. Reagents and conditions: (a) N,N-bis(trifluoromethylsulfonyl)aniline, TEA, DMF, rt,
8%; (b) Pd(dppf)Cl , KOAc, bis(pinacolato)diboron, 1,4-dioxane, 80°C, 80%; and (c) NaIO ,
9
2
4
NH OAc, acetone : water = 1 : 1, rt, 50%.
4
2
.2. H O and ROS selectivity test
2 2
With the prodrug B1 available, firstly, B1 was investigated its ability to release
SN-38 by treating different concentration of H O in phosphate buffer (pH=7.4).
2
2
SN-38’s transformation was analyzed via high performance liquid chromatography. In
the presence of H O , SN-38 was observed at a concentration of H O as low as 0.5
2
2
2
2
mM (22%), in a concentration-dependent manner. This clearly shows that the prodrug
B1 can be activated by H O to form SN-38 (Fig. 3a). As H O is not the only ROS in
2
2
2
2
biological system, we also studied the inducible activity of B1 toward other ROS,
-
such as tert-butylhydroperoxide (TBHP), hypochlorite (OCl ), hydroxyl radical and
tert-butoxy radical. The results shown that B1 was highly selective for H O over
2
2
other ROS. In the presence of H O , compounds B1 displayed efficient SN-38 release
2
2
(100%), while less than 8% release of SN-38 were observed with other ROS (Fig. 3b).
a)
b)
Figure 3. (a) Concentration dependence of compounds B1 (1 mM) for SN-38 release upon H O2
2
activation (0.5, 1.0, 2.0, 4.0, 8.0 and 10 mM, respectively); (b) Release of SN-38 induced by B1 (1
mM) upon treatment with various ROS at 10 mM.
2
.3. Relative drug release by MCF-7 cells
To determine if the prodrug B1 undergo oxidative cleavage by intracellular
hydrogen peroxide present at elevated concentration levels in the cancer cells, we
5

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analyzed the cell culture media for concentrations of the prodrug and the active form,
SN-38. Using HPLC, we were able to separate, identify, and quantify the prodrugs
and its active product. As shown in Figure 4, the boronic acid prodrug B1 was
observed at 12.87 min, while the desired active drug form, SN-38, eluted at 8.85 min.
The relative peak areas were measured at 59% (prodrug B1) and 41% (SN-38),
respectively, indicating that nearly half of the prodrug has been converted to SN-38 in
the media after incubation with MCF-7 cells for 48 h.
Figure 4. Relative concentrations of prodrug and its oxidative product, SN-38, as determined by
HPLC after incubation with MCF-7 cells.
2
.4. Cytotoxicity studies
Following, prodrug B1 was assessed in vitro antitumor activities against human
colon cancer cell lines HCT-15 and HT-29, human breast cancer cell lines MCF-7 and
MDA-MB-231, human brain star glioblastoma cell lines U87MG and U251, with
SN-38 as the reference compound. As shown in Table 1, compound B1 exhibited
significant in vitro cytotoxic activities against these six tested tumor cell lines,
displayed a comparable (MCF-7, U251) or greater (HT-29) cytotoxic activity than that
of SN-38, indicating that the 10-boric acid groups of the SN-38 might influence the
cytotoxic activities of the new derivative.
Table 1. Cytotoxicity assay against HCT-15, HT-29, MCF-7, MDA-MB-231, U87MG and U251
cell lines.
IC (
ꢀ
M)
5
0
compounds
HCT-15 HT-29
MDA-MB-231 MCF-7
U87MG
0.047
U251
0.036
0.021
B1
0.018
0.008
0.047
0.054
4.284
1.970
0.628
0.515
SN-38
0.030
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2
.5. Topo I inhibition studies
As shown in Figure 5, prodrug B1 was also tested for its inhibitory effects on Topo
I, using CPT and SN-38 as the reference drug. Compound B1 significantly inhibited
Topo I at 100 ꢀM, even more potent inhibitory activity than SN-38. The above result
suggested that this novel boric acid substituted derivative was also a typical Topo I
inhibitor.
Figure 5. Topo-I-mediated supercoiled pBR322 DNA relaxation was inhibited by B1 compared to
the reference compound CPT and SN-38 at 100 ꢀM.
In general, anticancer drugs increase ROS and increase oxidative stress in cancer
cells to kill the cancer cells. Herein, as prodrug of SN-38, compound B1 displayed
significant in vitro cytotoxic activity and Topo I inhibitory activity. One possible
explanation was that compound B1 itself might kill cancer cells to increase ROS and
oxidative stress. Meanwhile, the increasing ROS activated B1 to release SN-38 in turn,
to enhance anticancer activity.
2
.6. Antitumor activity of B1 in vivo
Finally, the effect of prodrug B1 on inhibiting tumor growth was evaluated in
xenograft model. The xenograft model antitumor assay was performed using human
brain star glioblastoma cell lines U87MG, with irinotecan (CPT-11) being used as the
reference drug (Table 2). Prodrug B1 was administered intraperitoneally (i.p.) at 0.5
and 2.0 mg/kg doses three times a week to the end (q3w/14). Only at doses of 2.0
mg/kg, it exhibited a weakly relative tumor increment rate (T/C) against that achieved
with CPT-11 at a dose of 15 mg/kg (28.10 vs. 8.39), at doses of 0.5 mg/kg, it lost of
inhibiting tumor proliferation activity. On the basis of Student’s t-test evaluation, B1
at 2.0 mg/kg (P < 0.01) exhibited a significant antitumor activity in vivo without overt
signs of symptoms or an anaphylactic reaction.
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Table 2. Assessment of antitumor efficacy of compound B1 in vivo.
a
b
c
Group
Dose (mg/kg)
Letha toxicity
BWC (%)
RTV (mean±SD)
T/C (%)
control
CPT-11
-
0/12
0/6
0/6
0/6
23.12
15.88
2.11
16.69±7.52
1.40±0.32**
4.69±1.92*
-
15
8.39
2
0
.0
.5
28.10
105.99
B1
12.69
17.69±11.39*
a
Number of dead mice/total number of mice.
b
Percentage of mouse body-weight change (BWC) after drug treatment: BWC%=(mean BWfinal day/BWfirst day × 100)
－
100.
c
*
P﹤0.01, **P﹤0.001.
3
. Conclusion
In conclusion, to our best knowledge, the 7-ethyl-10-boronic acid camptothecin B1
was synthesized for the first time, as a prodrug by linking a cleavable aryl
carbon−boron bond to the SN-38. The utility of this prodrug was based on the fact
that it can be converted rapidly to the active form under favorable oxidative
conditions in cancer cells with elevated levels of hydrogen peroxide. The cell survival
assay showed that the prodrug was equally or more effective in inhibiting the growth
of six different cancer cells, as compared to SN-38. Unexpectedly, prodrug B1
displayed even more potent Topo I inhibitory activity than SN-38, suggesting that it
was not only a prodrug of SN-38 but also a typical Topo I inhibitor. Prodrug B1 also
demonstrated a significant antitumor activity at 2.0 mg/kg in a xenograft model using
human brain star glioblastoma cell lines U87MG. On the basis of these positive results,
our study demonstrated the feasibility of using boron chemistry as a prodrug strategy
for SN-38, and this strategy would be applied to the research and development of
natural products.
4
. Experimental Section
4
.1. Chemistry
All reagents and solvents were purchased from the suppliers and purified/dried if
1
anhydrous one was necessary. The H NMR spectra was recorded on Bruker-400 (400
1
MHz) using TMS as internal standard. H chemical shifts were reported as δ (ppm)
and spin-spin coupling constants as J (Hz) values. The mass spectra (MS) were
8

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recorded on a Finnigan MAT-95 mass spectrometer. Melting points were taken on a
Fisher–Johns melting point apparatus, uncorrected and reported in degrees
Centigrade. HPLC analyses were performed on an HP 1100 series LC system.
4
4
.2. General procedure for the synthesis of compound B1
.2.1. 7-ethyl-10-(trifluoromethylsulfonyloxy)camptothecin (10)
To a solution of SN-38 (1.0 g, 2.5 mmol) and N, N-bis(trifluoromethylsulfonyl)aniline
(1.3 g, 3.6 mmol) in anhydrous dimethyl formamide (5 mL) was added triethylamine
o
(
1.0 mL, 7.5 mmol) and the reaction mixture was stirred under argon at 60 C for 3 h.
The reaction mixture was cooled and concentrated under reduced pressure to remove
the solvents. The crude product was purified by flash chromatography to give
o
1
compound 10 (1.27 g, 95%). White solid, m.p. 242–244 C; H NMR (400 MHz,
CDCl ) δ 8.34 (d, J = 9.3 Hz, 1H), 7.97 (d, J = 2.2 Hz, 1H), 7.72 (s, 1H), 7.65 (dd, J =
3
9
.3, 2.2 Hz, 1H), 5.72 (d, J = 16.4 Hz, 1H), 5.48 – 5.09 (m, 3H), 4.27 (s, 1H), 3.19 (q,
J = 7.5 Hz, 2H), 2.00 – 1.80 (m, 2H), 1.42 (t, J = 7.6 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H).
1
3
C NMR (101 MHz, CDCl ) δ 173.77, 157.62, 153.52, 150.27, 148.27, 148.01,
3
1
9
4
46.27, 146.16, 133.57, 128.27, 127.38, 123.74, 120.51, 119.54, 117.32, 115.74,
8.79, 72.93, 66.34, 49.48, 31.81, 23.37, 14.12, 7.94.
.2.2. 7-ethyl-10-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)camptothecin (11)
The compound 10 (550 mg, 1.0 mmol), powdered KOAc (300 mg, 3.0 mmol), and
bis(pinacolato)diboron (320 mg, 1.1 mmol) were added to a sealed tube that had been
purged with argon. A solution of Pd(dppf)Cl (80 mg, 0.1 mmol) in degassed
2
anhydrous dioxane (10 mL) was added, and the reaction mixture was stirred under
o
argon at 80 C for 12 h. The reaction mixture was cooled and filtered through a short
celite pad. The filtrate was concentrated under reduced pressure to remove the
solvents. The crude product was purified by flash chromatography to give compound
o
1
1
1 (0.4 g, 80%). Faint yellow solid, m.p. 263–265 C; H NMR (400 MHz, CDCl ) δ
3
8
.59 (s, 1H), 8.22 – 8.13 (m, 2H), 7.67 (s, 1H), 5.75 (d, J = 16.3 Hz, 1H), 5.35 – 5.23
(m, 3H), 3.81 (s, 1H), 3.27 (q, J = 7.7 Hz, 2H), 1.96 – 1.82 (m, 2H), 1.49 – 1.37 (m,
1
3
1
1
5H), 1.03 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 174.11, 157.83, 152.81,
3
51.11, 150.31, 147.22, 146.69, 135.12, 131.38, 129.78, 126.95, 126.41, 118.73,
9

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9
8.34, 84.51, 72.91, 66.52, 49.59, 31.76, 25.09, 23.15, 14.53, 7.97.
4
.2.3. 7-ethyl-10- boronocamptothecin (B1)
To a solution of compound 11 (400 mg, 0.8 mmol) in acetone:water = 1:1 (10 mL)
was added NH OAc (125 mg, 1.6 mmol) and NaIO (500 mg, 2.4 mmol), then the
4
4
reaction mixture was stirred at room tempereture for 24 h. The reaction mixture was
concentrated under reduced pressure to remove acetone, the aqueous phase was
extracted with chloroform: isopropanol=3: 1 and concentrated to afford crude produce,
which was further purified by column chromatograpgy to give pure B1 (200 mg,
o
1
5
8
1
0%). Faint yellow solid, m.p.>300 C; H NMR (400 MHz, DMSO) δ 8.76 (s, 1H),
.45 (s, 2H), 8.17 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.34 (s, 1H), 6.53 (s,
H), 5.44 (s, 2H), 5.34 (s, 2H), 3.26 (q, J = 7.6 Hz, 2H), 1.94 – 1.80 (m, 2H), 1.35 (t,
13
J = 7.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). C NMR (101 MHz, DMSO) δ 172.52,
1
1
56.87, 152.27, 150.04, 149.46, 146.29, 146.02, 134.69, 130.70, 128.46, 127.95,
25.89, 119.05, 96.85, 72.42, 65.32, 49.53, 30.36, 22.26, 14.14, 7.78; HRMS (ESI)
+
m/z calcd for C H BN O [M+H] : 421.1571; found: 421.1574.
2
2
22
2
6
4
.3. H O and ROS selectivity test
2 2
ROS induced drug release was assessed at pH of 7.4 using HPLC assays. Prodrug
stock solutions were prepared in DMSO at concentrations of 10 mM. Aliquots of 10
ꢀ
L of DMSO prodrug stocks was mixed with 50 mM H O (1ꢀL, 2 ꢀL, 4ꢀL, 8ꢀL,
2 2
1
6ꢀL and 20ꢀL, respectively) and appropriate amount of phosphate buffer (pH=7.4)
to give a final volume of 100 ꢀL. The reaction was incubated at 37 °C for 8 h and
quenched by an equal volume of Sodium thiosulfate solution (50 mM), then subjected
to HPLC analysis. Reactive oxygen species were administered to reaction tube as
follows. H O , tert-butylhydroperoxide (TBHP), and hypochlorite (NaOCl) were
2
2
delivered from 30%, 70%, and 14.5% aqueous solutions respectively. Hydroxyl
2
+
radical (•OH) and tert-butoxy radical (•OBu) were generated by reaction of 1mM Fe
with H O or TBHP, respectively.
2
2
4
.4. Relative drug release by MCF-7 cells
For measurement of drug release by breast cancer cells, MCF-7 cells were incubated
-5
with B1 at 10 M. After 2 days, the cell culture media were collected, centrifuged to
1
0

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remove cells, and diluted to a fixed volume for relative quantitation by HPLC.
Chromatographic peak areas corresponding to the prodrug B1 and SN-38 were used
as concentrations in the media.
4
.5. Cytotoxicity assays
Cytotoxicity assays of prodrug B1 was performed on human colon cancer cell lines
HCT-15 and HT-29, human breast cancer cell lines MCF-7 and MDA-MB-231,
human brain star glioblastoma cell lines U87MG and U251, which were obtained
from the American Type Culture Collection (Manassas, VA). Cells (6,000–10,000) in
1
00ꢀL culture medium per well were seeded into 96-well microtest plates (Falcon,
CA). Cells were treated in triplicate with gradient concentrations of test drugs and
incubated at 37 °C for 72 h. The growth inhibitory effects on the cell lines were
measured with a sulforhodamine B (SRB; Sigma, St. Louis, MO) assay. The drug
concentration required for 50% growth inhibition (IC ) of tumor cells was
5
0
determined from dose–response curves.
.6. Assay of enzyme activity inhibiting Topo I
4
The Topo I we used was purchased from Amersham Biosciences. One unit is defined
as the amount of enzyme that completely relaxed 0.5 mg of supercoiled pBR322 DNA
in 20 ꢀL of the reaction mixture for 30 min at 37 °C. The reaction buffer contained 35
mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl , 5 mM spermidine, 0.01% bovine
2
serum albumin, 0.25 ꢀg supercoiled pBR322, and 2 units of Topo I in a total volume
of 20 ꢀL. Relaxation was induced at 37 °C for 30 min and terminated by the addition
of 1/10 volume of 10% SDS. The mixture was then treated with proteinase K at a
final concentration of 50 ꢀg/mL and incubated at 37 °C for 30 min. An equal volume
of chloroform:isoamyl alcohol (24:1) was used to extract the reaction mixture, and the
supernatant was then removed onto 1% agarose gel for analysis. Electrophoresis was
carried out in 1 × TAE at 4 V/cm for 2 h, and the gel stained with ethidium bromide
(0.5 ꢀg/mL), and photographed through a gel document system, ChemiGenius 2.
4
.7. Antitumor activity of B1 in vivo
A xenograft model antitumor assay was performed using human brain star
glioblastoma cell lines U87MG. When the tumor graft volume reached about 130
11

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3
mm , mice were randomly separated into four groups (n = 12 for control and n = 6 for
treatment). Prodrug B1 was administered intraperitoneally (i.p.) three times a week to
the end. These results were statistically significant (P < 0.001, Student’s t-test).
Acknowledgements
This work was supported by the grants of The National Natural Science Foundation
of China (No. 81172936 ).
References and notes
[
[
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Table 1. Cytotoxicity assay against HCT-15, HT-29, MCF-7, MDA-MB-231, U87MG
and U251 cell lines.
Table 2. Assessment of antitumor efficacy of compound B1 in vivo.
Figure 1. Representative natural products which contain the phenol hydroxyl.
Figure 2. Structures of representative boron-based compounds.
Figure 3. (a) Concentration dependence of compounds B1 (1 mM) for SN-38 release
upon H O activation (0.5, 1.0, 2.0, 4.0, 8.0 and 10 mM, respectively); (b) Release of
2
2
SN-38 induced by B1 (1 mM) upon treatment with various ROS at 10 mM.
Figure 4. Relative concentrations of prodrug and its oxidative product, SN-38, as
determined by HPLC after incubation with MCF-7 cells.
Figure 5. Topo-I-mediated supercoiled pBR322 DNA relaxation was inhibited by B1
compared to the reference compound CPT and SN-38 at 100 ꢀM.
Scheme 1. Design of prodrug B1 and H O -promoted release of SN-38.
2
2
Scheme 2. Reagents and conditions: (a) N,N-bis(trifluoromethylsulfonyl)aniline, TEA,
DMF, rt, 98%; (b) Pd(dppf)Cl , KOAc, bis(pinacolato)diboron, 1,4-dioxane, 80°C,
2
8
0%; and (c) NaIO , NH OAc, acetone : water = 1 : 1, rt, 50%.
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Highlights
ꢀ
ꢀ
A boronic acid substituted camptothecin (B1) was synthesized for the first time.
Compound B1 selectively activated by cancer cells with elevated levels of H O ,
2
2
converted rapidly to the hydroxy form and displayed potent antitumor activity.
ꢀ
This strategy would be applied to the research and development of natural
products.