Development of a novel near-infrared fluorescent theranostic combretastain A-4 analogue, YK-5-252, to target triple negative breast cancer

Article abstract of DOI:10.1016/j.bmc.2017.02.046

The treatment of triple negative breast cancer (TNBC) is a significant challenge to cancer research. The lack of hormone receptors limits the treatment options available to patients with this diagnosis, forcing them to endure prolonged radiation and chemotherapy. Anti-angiogenesis is a chemotherapeutic strategy that targets the vasculature of tumors. Combretastatin A-4 (CA-4) is a well-known vasculature-disrupting agent, which has been shown to effectively kill a variety of cancers through inhibition of tubulin polymerization. Due to its toxicity, small molecule analogues of CA-4 have been sought out. We have designed a novel dual action CA-4 prodrug, YK-5-252, which releases the drug through a disulfide bond cleavage mechanism and contains a near-infrared (NIR) fluorophore, which allows fluorescence monitoring of cleavage. This disulfide linkage causes CA-4 to become effective only when released by glutathione (GSH) reducing the toxicity of the drug while simultaneously releasing the NIR fluorophore. Therefore the prodrug, YK-5-252, represents a novel CA-4 analogue which has reduced toxicity and can be used for theranostics imaging.

Full text of DOI:10.1016/j.bmc.2017.02.046

Accepted Manuscript
Development of a novel near-infrared fluorescent theranostic Combretastain
A-4 analogue, YK-5-252, to target triple negative breast cancer
Yali Kong, Jacqueline Smith, Kongwen Li, Jake Cui, John Han, Shujie Hou,
Milton L. Brown
PII:
S0968-0896(16)31013-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.046
BMC 13576
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 October 2016
15 February 2017
21 February 2017
Please cite this article as: Kong, Y., Smith, J., Li, K., Cui, J., Han, J., Hou, S., Brown, M.L., Development of a novel
near-infrared fluorescent theranostic Combretastain A-4 analogue, YK-5-252, to target triple negative breast cancer,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.046
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Development of a novel near-infrared fluorescent theranostic Combretastain A-4
analogue, YK-5-252, to target triple negative breast cancer
Yali Kong†1, Jacqueline Smith†1, Kongwen Li§2, Jake Cui§3, John Han§4, Shujie Hou1, Milton L.
Brown*1
†: Authors contributed equally; §: Authors contributed equally
1Department of Oncology, Center for Drug Discovery, Georgetown University Medical Center,
2
3970 Reservoir Road, Washington D.C, 20057; Fairfax High School, 3501 Rebel Run, Fairfax
VA 22030; 3Thomas Jefferson High School for Science and Technology, 6560 Braddock Road,
4
Alexandria, 22312; James Madison High School, 2500 James Madison Drive, Vienna, VA
22181
* To whom correspondence and reprints should be addressed: Milton L. Brown, Department of
Oncology and Drug Discovery Program, Lombardi Comprehensive Cancer Center, Georgetown
University Medical Center, 3970 Reservoir Road, Washington D.C, 20057.
Tel.: 202-687-8605. E-mail: mb544@georgetown.edu
Abstract
The treatment of triple negative breast cancer (TNBC) is a significant challenge to cancer
research. The lack of hormone receptors limits the treatment options available to patients with
this diagnosis, forcing them to endure prolonged radiation and chemotherapy. Anti-angiogenesis
is a chemotherapeutic strategy that targets the vasculature of tumors. Combretastatin A-4 (CA-
4) is a well-known vasculature-disrupting agent, which has been shown to effectively kill a
variety of cancers through inhibition of tubulin polymerization.
Due to its toxicity, small
molecule analogues of CA-4 have been sought out. We have designed a novel dual action CA-
4 prodrug, YK-5-252, which releases the drug through a disulfide bond cleavage mechanism
1

and contains a near-infrared (NIR) fluorophore, which allows fluorescence monitoring of
cleavage. This disulfide linkage causes CA-4 to become effective only when released by
glutathione (GSH) reducing the toxicity of the drug while simultaneously releasing the NIR
fluorophore. Therefore the prodrug, YK-5-252, represents a novel CA-4 analogue which has
reduced toxicity and can be used for theranostics imaging.
1. Introduction
Targeted-therapies are safe alternatives to chemotherapy and radiation in many breast
cancers. These treatments involve small molecule binding to hormone receptors, which
interfere with the signaling pathways that cause uncontrollable cell growth. However ten to
twenty percent of breast cancers diagnosed lack the three major receptors: Estrogen receptor
(ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). This
represents the most devastating prognosis, known as triple negative breast cancer
(TNBC).1 Due to the poor prognosis, there are no targeted therapies currently available against
TNBC. Methods to target proteins other than hormone receptors are needed as alternative
means to treat TNBC.
Inhibition of angiogenesis is an ideal approach to treat TNBC since it does not rely on
hormone receptor targeting. Angiogenesis is increased in cancer cells due to their fast growing
and aggressive nature therefore targeting this process is a way to selectively affect cancer cells.
Combretastatin A-4 (CA-4) is a tubulin-binding agent, which targets tumor vasculature, shuts off
blood flow in new vessels, and as a result, regress neovascularization and shrinkage of
tumors2,3. It acts by binding at or near the colchicine site of tubulin, which inhibits its assembly.
CA-4’s effectiveness as an anticancer treatment has been largely restricted by lack of solubility
and its harmful side effect. As a result, several small molecule analogues of CA-4 have been
developed.4-8 Our group embarked on improving the solubility of CA-4 by creating boronic acid
9-11
and other novel analogs
while others used different approaches to improve solubility. Most
2

notably the phosphate prodrug, CA-4P has been tested in clinical trials against a variety of
cancers.12-16 CA-4P is water soluble and cleaved by endogenous phosphatases to yield the
active compound CA-4. CA-4P was moderately effective in clinical trials however several grade
3 or 4 adverse events were experienced. Therefore novel prodrug strategies are still needed to
reduce the toxicity of cancer drugs.
One of the most promising enhancements of cancer treatment is the development of
selective drug delivery mechanisms that are capable of releasing treatments in response to
individual tumor microenvironments. These strategies reduce the harsh side effects of drugs by
selectively targeting the malignant cells. Novel nanoparticle and antibody-based therapies are
in clinical trials, however these methods have their own drawbacks such as lack of specificity or
targeting narrowly expressed proteins. Another promising mechanism for specific drug delivery
is glutathione (GSH)-mediated prodrug activation.17,18 Glutathione is an important antioxidant
that prevents oxidative stress to various parts of the cell.19,20 Cancer cells have up to 30-40
times higher glutathione concentration than healthy cells,21 therefore a prodrug strategy which
utilizes a disulfide bond linker is proposed to more rapidly release drugs in cancer cells than
normal cells. This glutathione (GSH)-mediated prodrug strategy is one of the most promising
drug delivery mechanisms proposed.
In order to ensure that these drug delivery strategies are successful, imaging agents are
often employed to monitor drug uptake; these agents are known as theranostic compounds.
Theranostics are equipped with both fluorescent biomarkers and small molecule drugs are
being actively investigated for their roles in cancer treatment. These drugs are designed to
improve drug delivery monitoring due to their ability to concurrently release their biomarker and
drug constituents. Most of the fluorophores used for routine cellular imaging are limited due to
short wavelength emission, tissue auto-fluorescence interference and low photostability.
However theranostics require the use of fluorophores that can be detected through tissue and
give low background signal in biological samples. Very few biological components fluorescence
3

in the near-infrared (NIR) region; therefore fluorophores which emit in this region are ideal for
theranostics. NIR photons can penetrate tissue deeply with little damage, and the NIR
wavelength range of 700-1000 nm escapes the range of tissue auto-fluorescence, allowing for
greater accuracy in detection.22-24 A fluorophore with both NIR properties as well as high
photostability is needed in order to create an effective and applicable theranostic compound.
The compound, dicyanomethylene-4H-pyran (DCM) is a model NIR fluorophore since it has
emission in the NIR region, a large Stokes shift and high photostability.25-27 DCM has been used
as a fluorescent sensing material 28,29, nonlinear optic material,30 logic gates 31,32, photovoltaic
sensitizers33 as well as in vivo and in vitro imaging. Because of the photostability and
penetration properties of the fluorophore DCM, we chose it as a NIR fluorophore to monitor
release of CA-4 upon GSH cleavage (Figure 1).
Figure 1: Theranostic prodrug of combretastatin A-4.
4

Herein we utilize the disulfide bond cleavage mechanism to deliver the drug CA-4 to
TNBC cells. We designed and synthesized a novel NIR cleavable CA-4 prodrug YK-5-252. We
validated disulfide bond cleavage using the DCM fluorophore in live cell imaging and FACS
analysis (Figure 2). Finally we show that YK-5-252 acts via the same mechanism as CA-4.
These results demonstrate the promise of this prodrug YK-5-252 as a new strategy in the
treatment of TNBC.
CA-4
Traceless linker
DCM-NH2
OCH3
OCH3
O
H
N
O
S
S
OCH3
O
O
O
O
OCH3
GSH
YK-5-252
NC
CN
OCH3
OCH3
H
N
O
SH
O
O
O
HS
OCH3
+
O
O
OCH3
NC
CN
O
O
S
S
O
O
NH2
OCH3
OCH3
O
OCH3
HO
NC
CN
DCM-NH2
(Near-IR Fluorophore)
OCH3
Combretastatin A-4
(Tubulin Polymerization Inhibitor)
Figure 2: Mechanism of YK-5-252 disulfide bond cleavage
2. Results and Discussion
5

2.1 Synthesis of YK-5-252
The GSH-activatable fluorescent prodrug, YK-5-252 was synthesized by combining a
NIR fluorescent agent with a GSH-activatable disulfide linker, which in turn was conjugated to
CA-4. Three distinct steps were followed: 1) 2-(2-methyl-4H-chromen-4-ylidene) malononitrile
(DCM) synthesis, which was accomplished according to established procedures;29 2) NIR
fluorophore DCM-NH2 synthesis;29 3) conjugation of DCM-NH2 with a GSH-cleavable disulfide
(S-S) linker;34 and 4) finally connection with the anticancer agent CA-4 (Figure 3).
Figure 3. Synthesis of YK-5-252
2.2 Fluorescent Properties of YK-5-252
Upon completion of the synthesis of YK-5-252, its fluorescence properties and ability to
be activated via GSH cleavage was evaluated. DCM-NH2 has NIR fluorescence emission at
650 nm with an excitation of 492 nm (Figure 4a). However when conjugated with CA-4, YK-5-
6

252 has no NIR emission (Figure 4b). This is due to the loss of electron donating ability of the
amine group when attached to the linker. To ascertain the stability of YK-5-252 toward GSH,
fluorescence was monitored in the presence of GSH. As expected, treatment with GSH (250
mM; pH 8) resulted in emission at 650 nm (Figure 4b).
Figure 4: a) Emission spectra of the DCM (1 mM) b) Emission spectra of prodrug YK-5-252 (1
mM) alone, YK-5-252 (1 mM) in the presence of GSH (250 mM; pH 8) in DMSO/PBS solution
(50/50, v/v).
Further HPLC analysis confirms that CA-4 release is concurrent with the release of
DCM-NH2 in a traceless manner (Figure 5). The retention time for YK-5-252 was found to be
10.5 minutes (Figure 5a). After treatment with GSH (Figure 5b), the YK-5-252 peak at 10.5
minutes disappeared while two new peaks at 6.8 and 8.6 minutes appears which corresponded
to the release of CA-4 and DCM-NH2, respectively (Figure 5c and 5d).
7

Figure 5: HPLC traces of a) YK-5-252 (200 µM) b) YK-5-252 (200 µM) + GSH (50 mM), and c)
CA-4 (200 µM) d) DCM-NH2 (200 µM).
2.3 Live cell imaging
In order to test the ability of YK-5-252 to provide selective release of CA-4 in cancer
cells, we monitored the presence of DCM-NH2 through confocal microscopy. After 1 hour of
treatment, the fluorescent was observed in higher quantities in the TNBC cells (MDA-MB-231;
Figure 6 a-c) versus normal breast cells (MCF10A; Figure 6d-f). The result demonstrates that
YK-5-252 is rapidly cleaved by TNBC cells, which produce higher concentrations of GSH. Also
this experiment confirms the utility of YK-5-252 to monitor drug delivery and release.
Figure 6 a) Confocal microscopy of MDA-MB-231 cells (TNBC) treated with YK-5-252 (1
micromolar) for 1 hr. b) Nuclear staining with Hoescht dye c) Merged image of YK-5-252 and
8

nuclear stain d) Confocal microscopy of MCF10A (normal breast cells) treated with YK-5-252 (1
micromolar) for 1 hr. e) Nuclear stained with Hoescht dye f) Merged image of YK-5-252 and
nuclear stain.
2.4 Flow cytometry
To further investigate the cellular activation of YK-5-252, we used fluorescence-activated
cell sorting (FACS). Cells were treated with YK-5-252 for 48 hours. After treatment FACS
analysis was conducted to determine how much of the cell population has the fluorophore DCM-
NH2 present. FACS analysis showed that out of the entire cell population treated with YK-5-252
(approcimately 106 cells), 72% of the TNBC cells (MDA-MB-231) contained DCM-NH2 (Figure
7a) whereas only about 5% of normal cells (MCF10A) (Figure 7b) contained the fluorophore
DCM-NH2. This experiment along with the imaging results demonstrates that the TNBC cells
release more DCM-NH2 than the normal cells.
Figure 7: Flow cytometry analysis of cellular uptake of YK-5-252 (1 ꢀM) in a) MDA-MB-231 and
b) normal breast cancer cells (MCF10A) after 48 hours of treatment.
The difference in prodrug cleavage in cancer cells versus normal cells is most likely due
to the higher levels of GSH produced in cancer cells. We have confirmed that GSH levels in
9

MDA-MB-231 cancer cells were significantly higher than levels in MCF10A normal cells (Figure
8). Using a colorimetric assay, the GSH concentration in TNBC cells was found to be 20 times
higher than the concentration in normal cells.
Figure 8: Concentration of GSH produced by TNBC cells (MDA-MB-231) and normal breast
cells (MCF10A).
2.5 Tubulin polymerization
To determine the biological effects of YK-5-252, we investigated its mechanism of action
using immunofluorescence staining. Since YK-5-252 is a prodrug of CA-4, we expected it to be
cleaved in cells and cause tubulin polymerization in the same manner as CA-4. Treatment of
TNBC cells with YK-5-252 (50 nM) did not cause microtubule disruption after 1 hour of
treatment (Figure 9a) whereas CA-4 (50 nM) completely inhibited tubulin polymerization within
1 hour (Figure 9b & d). However after 6 hours of treatment YK-5-252 began to disrupt cellular
microtubules (Figure 9c).
10

Figure 9: Immunofluorescence staining of tubulin in TNBC cells (MDA-MB-231) a) treated with
YK-5-252 (50nM; 1hr), b) cells treated with CA-4 (50nM; 1hr), c) cells treated with YK-5-252
(50nM; 6hr) and d) cells treated with CA-4 (50nM; 6hr)
2.6 Cell Proliferation
To evaluate the toxicity of YK-5-252 in cells, we measured the inhibition of cell
proliferation in normal cells (MCF10A) and TNBC cells (MDA-MB-231). In normal cells, YK-5-
252 has a GI50 of approximately 19.2 nM whereas CA-4 has a GI50 less than 1 pM. In TNBC
cells, YK-5-252 has a GI50 of 40.3 nM whereas CA-4 had a GI50 of 28.1 nM. The lower GI50 of
CA-4 in normal and TNBC cells indicates that YK-5-252 reduces the toxicity of CA-4. However
both CA-4 and YK-5-252 are highly potent in normal cells (Table 1). This lack of selectivity is
likely due to the toxicity of YK-5-252 rather than a failure of the cleavage mechanism.
11

YK-5-252
(micromolar)
CA4
(micromolar)
<10-6
MCF10A
0.01918
(CI95= 0.004844 to 0.07596)
MDA-MB-231*
0.04033
(CI95= 0.0168 to 0.0968)
0.002806
(CI95= 0.000641 to 0.01229)
Table 1. Inhibition of cell proliferation in normal cells (MCF10A) and TNBC cells (MDA-
MB-231). *Values determined after 72hr treatment.
3. Conclusion
YK-5-252 is a prodrug, which uses a disulfide bond linkage to reduce the toxicity of CA-4
and connect a NIR fluorophore to monitor drug delivery. We have shown that the NIR
fluorophore, DCM-NH2, can be activated through cleavage of the disulfide bond linkage by GSH
and can be monitored through live cell imaging. We also show that the toxicity of CA-4 toward
normal and cancerous cells was decreased by this prodrug strategy. Immunofluorescence
staining shows that YK-5-252 causes a delay in inhibition of tubulin polymerization compared to
CA-4, which is likely to contribute to its reduced toxicity. Therefore YK-5-252 is a proof-of-
concept prodrug, which uses a disulfide bond cleavage mechanism and is a theranostic tool for
drug monitoring. Further optimization will be conducted to improve the selectivity of this drug.
4. Experimental Section
4.1 Chemistry
All reagents and solvents were purchased from commercial suppliers and used as
received unless noted otherwise. Flash column chromatography separations were done on a
Biotage SP1 system monitoring at 254 and 310 nm. NMR spectra were recorded on a Varian
12

1
13
400 spectrometer at 25 °C, operating at 400 MHz for H and 100 MHz for C NMR. The
chemical shifts are expressed in ppm downfield from TMS as an internal standard. Reactions
were monitored by thin-layer chromatography (TLC) on silica gel 60 glass slides. The structure
of the synthesized compounds follows unequivocally from the mode of synthesis and the m/z
values found in their low-and high-resolution mass spectra, TLC and NMR spectroscopy verified
the purity.
4.1.1. Synthesis of 1-(2-hydroxyphenyl) butane-1,3-dione (1)
1-(2-hydroxyphenyl)ethanone (5.0g, 36.2 mmol) was dissolved in 100 mL ethyl acetate,
and then sodium (4.0 g, 0.17 mmol) was added into the solution. The resulting mixture was
stirred at room temperature for 3 hrs. The solid was filtered off and dissolved in 50 mL water,
and adjusted the pH value to neutral by 6 N HCl. Extracted with ethyl acetate and the organic
layers were dried over Na2SO4. The solvent was removed under vacuo to produce a brown solid
3.6 g (54%), which was directly used for the next step without further purification.
4.1.2. Synthesis of 2-methyl-4H-chromen-4-one (2)
To a solution of 2 (3.6 g, 20.2 mmol) in acetic acid (35 ml) was added dropwise sulfuric
acid 3.0 ml. The mixture was refluxed for 30 min and then poured into ice water. The aqueous
solution was neutralized with sat. Na2CO3, extracted with ethyl acetate. The organic layers were
dried over Na2SO4, filtered and concentrated to yield crude red solid. The crude product was
purified by silica column chromatography with Hexane/ethyl acetate as eluent to afford a pale
1
yellow solid 2.3 g (74%). H NMR (CDCl3, 400 MHz) ꢁ (ppm): 8.00 (m, 1H), 7.46 (m, 1H), 8.23
13
(m, 2H), 7.20 (m, 2H), 6.00(s, 1H), 2.21 (s, 3H); C NMR (100 MHz) ꢁ 177.92, 166.12, 156.26,
133.29, 125.33, 124.73, 123.34, 117.67, 110.32, 20.40.
4.1.3. Synthesis of 2-(2-methyl-4H-chromen-4-ylidene) malononitrile (3, DCM)
13

This step was performed with modified method as in the literature.29 Malononitrile (1.0 g,
15.0 mmol) and 3 (2.0 g, 12.5 mmol) were dissolved in 15 ml acetic anhydride. The solution was
refluxed for 5 hrs and 300 mg more malononitrile was added and refluxed for extra 4 hrs. The
solvent was evaporated in vacuo to dryness, water (40 ml) was added and the mixture was
refluxed for 0.5 h. The water was removed to dryness and the crude was applied to silica
column with hexane/ethyl acetate as eluent to yield compound 3 (DCM) as an orange solid
1
(0.78 g, 30 %). H NMR (CDCl3, 400 MHz) ꢁ (ppm): 8.76 (d, 1H, J = 8.4 Hz), 7.64 (dd, 1H, J =
13
7.2, 7.6 Hz), 7.34 (m, 2H), 6.59(s, 1H), 2.36 (s, 2H); C NMR (100 MHz) ꢁ 162.00, 153.27,
152.96, 134.75, 126.09, 125.70, 118.82, 117.52, 116.67, 115.51, 105.48, 62.14, 20.60.
4.1.4. Synthesis of (E)-2-(2-(4-amnostyryl)-4H-chromen-4-ylidene) malononitrile (4, DCM-
NH2)
To a solution of DCM (252 mg, 1.20 mmol) and 4-acetamido benzaldehyde (147 mg, 1.2
mmol) in toluene (40 mL) was added piperidine (0.6 mL) and acetic acid (0.6 mL), the reaction
mixture was refluxed for 3 h. The orange solid was filtered off, and con. HCl/ethanol (45 mL,
2:1) was added to the filtrate, followed by further reflux for another 2 h. The mixture was
neutralized with Sat. Na2CO3 to pH 8, then extracted with ethyl acetate. The ethyl acetate layers
were combined and washed with brine, dried over Na2SO4. The organic solvent was removed in
vacuo, the residue was purified by silica chromatography with hexane/ethyl acetate as elute to
1
afford a deep red solid 5 (146 mg, 39 %). H NMR (DMSO, 400 MHz): ꢁ 8.72 (dd, 1H, J = 1.2,
1.2 Hz), 7.89 (m, 1H), 7.77 (m, 1H), 7.64 (d, 1H, J = 15.6 Hz), 7.58 (m, 1H), 7.49 (d, 2H, J = 8.4
Hz), 7.08 (d, 1H, J = 16 Hz), 6.87 (s, 1H), 6.61 (d, 2H, J = 8.8 Hz), 6.00 (s, 2H).
4.1.5. Synthesis of (E)-2-((2-hydroxyethyl)disulfanyl)ethyl 4-(2-(4-(dicyanomethylene)-4H-
chromen-2-yl)vinyl)phenylcarbamate (5, DCM-S)
14

To a mixture of DCM-NH2 (20 mg, 0.064 mmol), triphosgene (76 mg, 0.26 mmol) in dry
toluene (10 mL) was added DIEA (132.3 mg, 1.0 mmol) dropwise at room temperature. The
mixture was reflux for 3 h. Remove the unreacted phosgene by flushing the N2 gas, then added
2, 2’-dithiodiethanol (98.7 mg, 0.64 mmol) in CH2Cl2/THF (1:1, 4 mL), the mixture was stirred at
room temperature overnight. The solvent was removed and purified by silica column
1
chromatography to afford an orange-yellow solid DCM-S (15 mg, 48 %). H NMR (DMSO, 400
MHz) ꢁ (ppm): 8.92 (dd, 1H, J = 1.6, 1.6 Hz), 7.73 (m, 1H), 7.53 (m, 8H), 7.06 (s, 1H), 6.85 (s,
1H), 6.73 (d, 1H, J = 15.6 Hz), 4.47 (t, 2H, J = 6.4 Hz), 3.93 (t, 2H, J = 5.6, 6.0 Hz), 3.03 (t, 2H,
–
J = 6.4 Hz), 2.94 (t, 2H, J = 6.4, 6.0 Hz); HRMS [M-H] calcd for C25H20N3O4S2, 490.0895; found,
490.0901.
4.1.6. Synthesis of 2-((2-((2-methoxy-5-(3,4,5-trimethoxystyryl) phenoxy) carbonyloxy)
ethyl)
disulfanyl)
ethyl
4-((E)-2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)
phenylcarbamate (YK-5-252)
A mixture of DCM-S (20 mg, 0.04 mmoL), triphosgene (5 mg, 0.016 mmoL) and DMAP
(6 mg, 0.049 mmoL) in chloroform/THF (1:1, 10 mL) was stirred at r.t for 3 hr. Then the solvent
was evaporated in vacuo. The residue was dissolved in THF (5 mL), then a chilled solution of
CA-4 (13 mg, 0.04 mmol) in 0.09 mL of 0.5 M NaOH and 1 mL THF was added. The reaction
mixture was stirred at r.t overnight. Neutralized the mixture with Sat. NaHCO3 to pH = 6-7,
concentrated and purified by silica chromatography (20% Toluene-EtOAc) to afford a dark red
1
solid (8 mg, 24 %). H NMR (DMSO, 400 MHz) ꢁ (ppm): 8.92 (d, 1H, J = 8.4 Hz), 7.74 (m, 1H),
7.56 (m, 2H), 7.42 (m, 6H), 7.17 (m, 2H), 6.87 (m, 2H), 6.70 (d, 1H, J = 15.6 Hz), 6.50 (s, 2H),
6.47 (d, 2H, J = 3.6), 4.53 (t, 2H, J = 7.2, 7.6 Hz), 4.46 (t, 2H, J = 6.0 Hz), 3.83 (s, 3H), 3.82 (s,
_
3H), 3.68 (s, 6H), 3.01 (m, 4H); HRMS [M+Na]: calcd for C44H39N3O10S2 Na+, 856.1975; found,
856.1979.
15

4.2. Cell Culture
Human breast normal cell line MCF-10A was grown in DMEM/F12 medium (Life
Technology) supplemented with 5% horse serum, hydrocortisone, insulin and pen/strep at 37 °C
(5% CO2). MDA-MB231 was grown in DMEM supplemented with 10% fetal bovine serum at 37
°C (5% CO2).
4.2.1. Cellular Imaging
Cells were plated onto fluorodishes in 1 mL of culture medium for 24 h. Cells were
exposed to YK-5-252 (1 M) or YK-5-252 (1 M) plus GSH (250 M) for 1-3hrs. After the
desired time, the cell cultures were washed with PBS (1X) and fresh medium was added.
Images were taken using a confocal laser scanning microscope (Zeiss LSM 510). YK-5-252 was
observed using a 488 nm laser, and the emission wavelength was read from 570 to 620 nm and
expressed as red.
4.2.2. Fluorescence-activated cellular sorting (FACS) analysis
Cells were treated with YK-5-252 for the indicated time period in 6-well plate. After
treatment cells were trypsinized and fixed with cold ethanol. Cells were subsequently analyzed
with a FACSort system (Becton Dickinson).
4.2.3, Immunofluorescence Chemistry
Cells were treated with YK-5-252 for the indicated time. Cells were then fixed with cold
methanol for 5 minutes and blocked with 10% normal calf serum in PBC for 20 minutes. Cells
were then stained with monoclonal ꢂ-tubulin antibody (T-4026; Sigma) in 10% normal calf
serum in PBS for 60 minutes. After a series of washes cells were incubated with FITC-
conjugated sheep antimouse IgG (F3008; Sigma) for 60 minutes. After a series of washes, cells
16

were stained with NucBlue and examined on a confocal laser scanning microscope (Zeiss LSM
510). Images were compiled using the J-image software.
4.2.4. In Vitro Cytotoxicity Assay
The cytotoxicity of YK-5-252 and Combretastatin 4A to cancer cell lines was determined
by MTT assay. Briefly, cells (4000-5000 cells per well) were evenly plated into 96-well plates
and incubated overnight. Then cells were exposed to serial dilutions of drugs and further
incubated for 48 h. Subsequently, the medium in each well was replaced with fresh culture
medium containing 0.5 mg/mL MTT. The absorbance in each individual well was determined at
570 nm with a microplate spectrophotometer (Wallace Victor2 1420 Multilabel Counter). Each
drug concentration was tested in triplicate and in three independent experiments. GI50 values
were calculated by nonlinear regression analysis using GraphPad Prism 4.0.
Acknowledgements
The authors would like to thank the financial support of the Georgetown University
Center for Drug Discovery. A patent application has been filed by Georgetown University on the
behalf of the inventors that are listed as authors in this article.
17

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20

Dual action prodrug, YK-5-252, reduces the toxicity of combretastatin A-4 (CA-4) and provides a
theranostic imaging tool
Graphical abstract
21