Synthesis and evaluation of an imidazole derivative-fluorescein conjugate

Article abstract of DOI:10.1016/j.bmc.2012.11.026

The murine double minute (MDM2) oncogene a negative regulator of protein 53 (p53) tumor suppressor, is found overexpressed in many different types of cancer and the interaction between MDM2 and p53 has become the target of intensive research. MDM2 inhibitors represent a promising class of p53 activating compounds that may be effective in cancer treatment and diagnostic imaging. Nutlins, a family of cis-imidazoline analogues and small-molecule MDM2 antagonists, have the potential use in cancer therapies. We have synthesized an imidazole derivative (Nutlin-Glycine) conjugated to the commonly used fluorophore, 6-carboxyfluorescein (FAM) and evaluated its possible use as an imaging agent. Cellular uptake studies demonstrated that the fluorescence intensity in human osteosarcoma (SJSA-1) and colon carcinoma (HCT116) cells were significantly increased with the treatment of Nutlin-Glycine-FAM when compared with FAM (control). Blocking studies also confirmed that our imidazole-fluorescein conjugate may be a good candidate for imaging tumors, suggesting the need for further in vivo evaluation by positron emission tomography.

Full text of DOI:10.1016/j.bmc.2012.11.026

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of an imidazole derivative–fluorescein conjugate
Pradip Ghosh a, , Jiawei Zhang b, Zheng-Zheng Shi c, King Li d,
⇑
⇑
a The University of Texas Health Science Center at Houston, The Brown Foundation Institute of Molecular Medicine, Center for Molecular Imaging, Houston, TX 77030, USA
b Cancer Institute, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
c Department of Radiology, The Methodist Hospital Research Institute, Houston, TX 77030, USA
d Department of Radiology, Wake Forest School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The murine double minute (MDM2) oncogene a negative regulator of protein 53 (p53) tumor suppressor,
is found overexpressed in many different types of cancer and the interaction between MDM2 and p53 has
become the target of intensive research. MDM2 inhibitors represent a promising class of p53 activating
compounds that may be effective in cancer treatment and diagnostic imaging. Nutlins, a family of cis-imi-
dazoline analogues and small-molecule MDM2 antagonists, have the potential use in cancer therapies.
We have synthesized an imidazole derivative (Nutlin–Glycine) conjugated to the commonly used fluoro-
phore, 6-carboxyfluorescein (FAM) and evaluated its possible use as an imaging agent. Cellular uptake
studies demonstrated that the fluorescence intensity in human osteosarcoma (SJSA-1) and colon
carcinoma (HCT116) cells were significantly increased with the treatment of Nutlin–Glycine–FAM when
compared with FAM (control). Blocking studies also confirmed that our imidazole–fluorescein conjugate
may be a good candidate for imaging tumors, suggesting the need for further in vivo evaluation by pos-
itron emission tomography.
Received 24 August 2012
Revised 7 November 2012
Accepted 15 November 2012
Available online xxxx
Keywords:
MDM2
p53
Fluorescence imaging
FAM
Nutlin-2
Glycine
PET
Published by Elsevier Ltd.
SJSA-1
HCT116
1. Introduction
strategy.14,15 Several studies have shown that disruption of the
p53–MDM2 interaction by different macromolecular approaches
The development of molecular imaging in the clinical setting has
only just begun and could yield tremendous patient benefit in the
form of earlier lesion detection, treatment response monitoring,
and a truly individualized approach to treatment of cancer. In this
early phase of the molecular-targeted patient-friendly cancer che-
motherapy, there is a need for novel viable anticancer molecular
targets. The MDM2 oncoprotein has been recently validated as a po-
tential target for cancer drug development. MDM2 is a zinc finger
oncoprotein1–4 that has been well characterized as the principal
negative regulator of the p53 tumor suppressor protein.5 The tu-
mor-suppressor p53 is a short-lived protein that is maintained at
low, often undetectable levels in normal cells. Stabilization of the
protein in response to an activating signal, such as DNA damages,
results in a rapid rise in p53 level with subsequent inhibition of cell
growth. MDM2 binds the p53 tumor-suppressor with high affinity
and negatively and modulates its transcriptional activity and stabil-
ity.6,7 The MDM2 gene has been found amplified or overexpressed
in many human malignancies with impaired p53 function.8–13
Therefore, activation of the p53 pathway through inhibition of
MDM2 has been proposed as an attractive therapeutic
or by the suppression of MDM2 expression can lead to the
activation of p53 and tumor growth inhibition.16,17 Inhibition with
small molecules is a more attractive proposal due to the pharmaco-
logical advantages of this class of drugs in stability and oral
bioavailability.
In 2004, Vassilev et al. (Hoffman-La Roche Inc., Nutley, New
Jersey) first described a class of antagonists that inhibit the
MDM2–p53 complex. These antagonists are a group of cis-imidaz-
oline analogues designated as the Nutlins (for Nutley inhibitor).18
These cis-imidazoline derivatives bind tightly into the p53 pocket
of MDM2 and displace p53 from its complexes with MDM2
in vitro with IC50 in the 100–300 nM range. Historically, it has been
difficult to develop small-molecule inhibitors of nonenzyme pro-
tein–protein interactions because of their large and shallow inter-
faces. Through X-ray crystallography, the MDM2–p53 complex
showed a well-defined hydrophobic cleft which represented the
binding site for p53. These inhibitors could disrupt the MDM2–
p53 interaction by binding specifically in this cleft and liberating
functional p53.19–21 These imidazole analogues (Nutlin-1 and 2)
are not commercially available except Nutlin-3, which is currently
under clinical investigation. Nutlin-3 inhibits p53–MDM2
interaction with an IC50 of 0.09 l
M.18 It induces the expression of
p53-regulated genes and exhibits potent antiproliferative activity
in cells with functional mutated p53.
⇑
Corresponding authors.
E-mail address: pghosh38@yahoo.com (P. Ghosh).
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.11.026
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

2
P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Several imidazole derivatives have been developed as diagnos-
ative Nutlin-2, which is not commercially available, and prepared
Nutlin–Glycine derivative according to the reported procedure27–
tic agents for positron emission tomography (PET) or optical
imaging.22–26 We prepared an imidazole derivative (Nutlin-2) with
glycine moiety, ‘Nutlin–Glycine’, according to the reported proce-
dures with some modifications by our lab.27–29 Using this
compound as a starting material for synthesizing MDM2 selective
ligands, we obtained a Nutlin analog that targets to intracellular
MDM2 and renders biological activities in tumor cells with wild
type p53 but not cells with a mutant p53. In this report we will also
describe using this analog conjugated to the commonly used fluo-
rophore, 6-carboxyfluorescein, to serve as an imaging moiety in
cultured cells.
29
with some modifications to achieve high yield. The modifica-
tions we developed utilized the emerging microwave technologies
and techniques to accelerate the synthesis process. The synthetic
scheme for Nutlin–Glycine–FAM conjugate is illustrated in Figure
1. The compound 8, Nutlin–Glycine was purified by high perfor-
mance liquid chromatography (HPLC) and isolated in 82% yield.
The final conjugate ‘Nutlin–Glycine–FAM’ was prepared by the
reaction of Nutlin–Glycine, 8 with a known fluorophore, 6-car-
boxyfluorescein N-hydroxysuccinimide ester (6-FAM-NHS) in
DMF in the presence of triethylamine at ambient temperature for
overnight. The crude conjugate 9 was purified by semi preparative
reversed-phase HPLC and isolated in 71% yield. Nutlin–Glycine–
2. Results and discussions
FAM was dissolved in DMSO and filter sterilized using a 0.2
mini-syringe filter prior to use.
l
The MDM2 oncoprotein has been validated as a potential target
for cancer drug development. MDM2 amplification and/or overex-
pression occur in a wide variety of human cancers, several of which
can be treated experimentally with MDM2 antagonists. Small
molecules, including synthetic chalcones, norbornane derivatives,
cis-imidazoline derivatives (Nutlins), a pyrazolidinedione sulfon-
amide and 1,4-benzodiazepine-2,5-diones, etc., have been reported
to disrupt the p53–MDM2 binding, thereby enhancing p53 activity
to elicit anticancer effects.30 We have selected the imidazole deriv-
To determine the effect of Nutlin-2 on cell growth and viability,
nine cancer cell lines, five with wild-type p53 status and four with
mutant p53 status, were incubated with a serial concentration of
Nutlin-2 for 5 days, followed with the MTS [3-(4,5-dimethylthia-
zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-
razolium, inner salt)] cell viability assay. As shown in Figure 2,
there were dose-dependent antiproliferative and cytotoxic effects
in cells with wild-type p53, however, the cells with mutant p53
Br
CN
CHO
OMe
C
OEt
OEt
HN
H
OEt
OEt
a
b
e
N
N
OCH3
4
EtO
OMe
1
OMe
2
Br
5
Br
Br
Br
Br
Br
NH2
NH2
d
c
N
NH
f
O
CHO
Br
Br
4
3
Boc
NH
O
O
OH
NH2
Br
Br
N
Br
N
Br
N
O
O
O
N
O
O
N
N
N
N
g
h
N
OCH3
OCH3
OCH3
N
N
N
EtO
6
EtO
8
EtO
7
Br
Br
N
O
O
i
O
OH
N
N
Br
Br
O
H
O
O
O
N
OCH3
9
HO
N
EtO
Figure 1. Synthetic scheme of Nutlin–Glycine–FAM conjugate. Reagents and conditions: (a) 30% NH4OH, IBA, rt, 4 h, 81%; (b) AcCl2, EtOH, rt, overnight, 63%; (c) NH4OAc,
microwave, 90 °C, 2 h, 67%; (d) (i) H2SO4, microwave, 80 °C, 3 h, (ii) 10(M)NaOH, rt, 2 h, 66%; (e) EtOH, Et3N, DMAP, 4, microwave, 80 °C, 4 h, 75%; (f) Phosgene, DMAP, Et3N,
DCM, rt, 1 h, piperzinyl ethanol, 73%; (g) Glycine-Boc, DCM, DMAP, DCC, rt, overnight, 73% ; (h) TFA, DCM, rt, 1 h, HPLC, 82%; (i) FAM-NHS, DMF, Et3N, rt, overnight, HOAc,
HPLC, 71%.
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
carcinoma cells (wild-type p53) showed increased G0/G1 phase
fraction and sharply decreased S-phase compartment after incuba-
tion with Nutlin-2. But there was no significant change in MDA-
MB-435S cells (mutant p53). Therefore, Nutlin-2 could cause
growth inhibition and cell cycle arrest specifically in cancer cells
with wild-type p53.
To test the effect of Nutlin–Glycine on cell growth and viability,
six cancer cell lines were used for this experiment; three cell lines
with wild-type p53 and three with mutant p53. Each cell line was
incubated with serial dilutions of Nutlin–Glycine, Nutlin-2, or
Nutlin-3 (positive controls) for 5 days, and followed by cell viabil-
ity analysis. As shown in Figure 4a, there was a dose-dependent
antiproliferative effect observed in cells with wild-type p53;
however, the cells with mutant p53 did not exhibit any growth
inhibition even when incubated with the highest dose (10 lM) of
the agents (Fig. 4b). The addition of the glycine to Nutlin-2,
Nutlin–Glycine, did not diminish biological activity when com-
pared with Nutlin-2 (native) or Nutlin-3.
The Nutlin–Glycine–FAM conjugate was tested for cellular
uptake in SJSA-1 cells (osteosarcoma) and HCT116 cells (colon
carcinoma). As shown in Figure 5, cellular uptake of Nutlin–Gly-
Figure 2. Antiproliferative and cytotoxic activity of Nutlin-2. Exponentially grow-
ing cultured cancer cells with wild-type p53 (A549, HCT116, HepG2, RKO, and SJSA-
1) or mutant p53 (Hela, MDA-MB-435S, PC-3 and SW480) were incubated with
Nutlin-2 for 5 days and the cell viability were determined by the MTS assay. The
curves represent percentages of viable cells relative to untreated controls
(mean SD, n = 4).
cine–FAM was significantly increased at 1 and 5 lM in SJSA-1
and HCT116 cells. The fluorophore by itself, FAM, was used as a
negative control at the same concentrations. The cellular uptake
of FAM was minimal and non-specific in both cell lines.
In blocking studies we used SJSA-1 and HCT116 cells. In this
experiment, we used 10-fold excess of Nutlin–Glycine derivative
as a blocking agent. As shown in Figure 6, Nutlin–Glycine–FAM
did not exhibit any impairment even incubated with the highest
dose of Nutlin-2.
To investigate the effect of Nutlin-2 on cell cycle, three cancer
cell lines, two with wild-type p53 and one with mutant p53, were
incubated with Nutlin-2 for 48 h, and then the cells were stained
with propidium iodide (PI) for cell cycle analysis by flow cytometry
( Fig. 3). SJSA-1 human osteosarcoma and A549 human lung
was significantly blocked at a concentration of 5 lM when com-
Figure 3. Cell cycle analysis with Nutlin-2. Cultured MDA-MB-435S, A549 and SJSA-1 cells were incubated with Nutlin-2 or an equivalent amount of solvent for 48 h,
followed by PI staining and flow cytometry analysis. The percentages of cells in each cell cycle phase are shown in the inset above the histogram.
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

4
P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
pared with uptake alone (no block) in both cell lines (71.71% for
SJSA-1 and 77.34% for HCT116). FAM or DMSO was used as
series pump (Agilent, Germany), with UV detector operated at
254 or 494 nm, using a semi-preparative and analytical C-18 re-
negative controls at the same concentration (5
minary findings warrant further investigation into the possible
use of Nutlin–Glycine–FAM conjugate as an imaging agent.
l
M). These preli-
verse phase column, Luna SCX 100A (5
(5
m, 150 ꢀ 4.6 mm).
lm, 250 ꢀ 10 mm) or
l
4.2. Chemistry
3. Conclusion
4.2.1. 2-Ethoxy-4-methoxybenzonitrile, 1
We report herein an efficient synthesis and evaluation of an
imidazole–fluorescein conjugate for imaging tumors by detecting
MDM2 oncoprotein. The preliminary cellular uptake and also
blocking studies demonstrated that Nutlin–Glycine–FAM conju-
gate might be useful as a tumor imaging agent and also help to
identify a potential probe for further investigations for MDM2 in
the future, but needs further in vivo evaluation, for example, by
labeling the Nutlin–Glycine with a positron nuclide followed by
positron emission tomography.
2-Hydroxy-4-anisaldehyde (2.0 g, 11.10 mmol) was dissolved in
30% ammonium hydroxide (15 mL) and 5 mL of acetonitrile (3:1),
which resulted in the formation of a turbid solution. To this turbid
solution, 2-iodoxybenzoic acid (6.22 g, 22.20 mmol) was added
slowly with constant stirring at room temperature for 4 h. The
yellowish-brown solution becomes colorless which indicates com-
pletion of the reaction (TLC). The reaction mixture was filtered and
evaporated under high vacuum, and the residue was dissolved in
dichloromethane (100 mL). The solution was washed with water
(3 ꢀ 100 mL). The organic phase was dried (MgSO4), evaporated
to dryness and purified on a silica gel column using 2% methanol
in dichloromethane. The pure compound 1 (1.6 g) was obtained
in 81% yield. 1H NMR 1 (CDCl3) d: 7.46 (1H, d, J = 8.4 Hz, ArH),
6.53–6.43 (2H, m, ArH), 4.11 (2H, q, CH2), 3.85 (3H, s, CH3), 1.51
(3H, t, J = 7.1 Hz, CH3). MS: (M+1) calculated 178.08, found 178.11.
4. Materials and methods
4.1. Reagents and instrumentations
All reagents and solvents were purchased from Aldrich Chemi-
cal Co. (Milwaukee, WI), and used without further purification.
Nutlin-3 was purchased from Cayman Chemical (Ann Arbor, MI),
and stored frozen as a 1 mM stock solution in DMSO. Thin layer
chromatography (TLC) was performed on pre-coated Kieselgel 60
F254 (Merck) glass plates. Proton and Carbon NMR spectra were
recorded on a Brucker 300 or 500 MHz spectrometer using tetra-
methylsilane as an internal reference and hexafluorobenzene as
an external reference, respectively, at The University of Texas M.
D. Anderson Cancer Center. The mass spectra were obtained on a
LCQ Fleet mass spectrometer using electrospray ionization (ESI)
technique. Microwave synthesis was performed on a Biotage Initi-
ator Eight microwave synthesizer (Uppsala, Sweden). High perfor-
mance liquid chromatography (HPLC) was performed on a 1200
4.2.2. Ethyl 2-ethoxy-4-methoxybenzimidate, 2
Compound 1 (1.0 g, 5.64 mmol) was dissolved in 5 mL of anhy-
drous ethanol in a dry flask. Acetyl chloride (3.2 mL, 45.15 mmol)
was added slowly with stirring at 0 °C for 1 h and continued at
room temperature for overnight. The reaction mixture was
quenched with 1(N) sodium bicarbonate solution (0.5 mL). The
crude product was extracted into dichloromethane (100 mL) and
the organic solution was washed with water (3 ꢀ 100 mL) and
brine. The residue was purified on a silica gel column using 3%
methanol in dichloromethane. The pure compound 2 (0.9 g) was
obtained in 63% yield. 1H NMR 2 (CDCl3) d: 8.69 (1H, br s, NH),
7.78 (1H, d, J = 7.8 Hz, ArH), 6.51–6.48 (2H, m, ArH), 4.29 (2H, q,
Figure 4a. Antiproliferative and cytotoxic activity of Nutlin analogs. Exponentially growing cultured cancer cells with mutant p53 (Hela, MDA-MB-435S, SW480) were
incubated with Nutlin-2, Nutlin-3 and Nutlin–Glycine derivative for 5 days and the cell viability were determined by the MTS assay. The curves represent percentages of
viable cells relative to untreated controls (mean SD, n = 4).
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Figure 4b. Antiproliferative and cytotoxic activity of Nutlin analogs: Cultured cancer cells with wild-type p53 (HCT116, RKO, and SJSA-1) were incubated with Nutlin-2,
Nutlin-3 and Nutlin–Glycine derivative for 5 days and the cell viability were determined by the MTS assay. The curves represent percentages of viable cells relative to
untreated controls (mean SD, n = 4).
1800
SJSA-1
HCT116
1500
1200
900
600
300
0
MEDIA
DMSO
FAM 1uM
FAM 5uM
Nut-Gly-FAM Nut-Gly-FAM
1uM 5uM
Figure 5. Cell uptake studies in cultured human osteosarcoma and colon cancer cells. Cultured SJSA-1 human osteosarcoma and HCT116 colon cancer cells were grown at
37 °C for in a tissue culture incubator. DMSO, FAM and Nutlin–Glycine–FAM were added to the separate culture media and allowed to incubate for 1 h at 37 °C, followed by
cell harvest and fluorescence measurements. The culture media itself was incubated with both cell lines (no drug, no DMSO). Relative fluorescence values were plotted for the
vehicle (Media or DMSO), FAM and Nutlin–Glycine–FAM at two final concentrations (1 and 5 lM). Error bars indicate standard error of the mean for 3 runs.
CH2), 4.13 (2H, q, CH2), 3.82 (3H, s, CH3), 1.50 (3H, t, J = 7.2 Hz,
CH3), 1.39 (3H, t, J = 7.2 Hz, CH3). MS: (M+1) calculated 224.26,
found 224.31.
of white crystals (67% yields). 1H NMR 3 (CDCl3) d: 8.61 (1H, s, CH),
8.01 (1H, br s, NH), 7.88 (6H, d, J = 8.2 Hz, ArH), 7.69 (2H, d,
J = 7.8 Hz, ArH), 7.58 (2H, d, J = 7.1 Hz, ArH), 7.47 (2H, d,
J = 7.8 Hz, ArH), 7.15 (4H, d, J = 6.5 Hz, ArH), 5.33 (1H, d,
J = 7.1 Hz, CH), 5.18 (1H, d, J = 7.5 Hz, CH). MS: (M+1) calculated
721.08, found 721.90
4.2.3. 4-Bromo-N-(2-(4-bromobenzylideneamino)-1,2-(4-
bromophenyl)ethyl)benzamide, 3
4-Bromobenzaldehyde (1.0 g, 5.41 mmol) and ammonium
acetate (2.08 g, 27.05 mmol) in water (5 mL) was heated with
microwaves in a sealed tube at 90 °C for 2 h. The reaction mixture
was cooled and precipitate was filtered off. The white precipitate
was washed with water and evaporated to dryness. The crude
product is crystallized from ethyl acetate–methylcyclohexane
solvent mixture to give 2.6 g of the expected product 3 in the form
4.2.4. 1,2-Bis(4-bromophenyl)ethane-1,2-diamine, 4
Compound 3 (2.5 g, 3.47 mmol) was taken in 3 mL of sulfuric
acid and 10 mL of water in a sealed tube and heated with
microwaves at 80 °C for 3 h. The residue was cooled and precipi-
tate was filtered off. The crude precipitate was dissolved in 10
(M) sodium hydroxide solutions and stirred for further 2 h at room
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

6
P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
2400
2100
1800
1500
1200
900
SJSA-1
HCT116
600
300
0
DMSO
FAM 5uM
Nut-Gly-FAM 5uM
(Block)
Nut-Gly-FAM 5uM
Figure 6. Cell blocking studies in cultured human osteosarcoma and colon cancer cells. Cultured SJSA-1 and HCT116 cancer cells were grown at 37 °C for in a tissue culture
incubator. Nutlin–Glycine was added to the separate culture media and allowed to incubate for 1 h at 37 °C prior treatment with Nutlin–Glycine–FAM and further allowed to
incubate for 1 h, followed by cell harvest and fluorescence measurements. FAM and DMSO were incubated with both cell lines for 1 h as negative controls. Relative
fluorescence values were plotted for the vehicle (DMSO), FAM and Nutlin–Glycine–FAM at a final concentration (5
runs.
lM). Error bars indicate standard error of the mean for 3
temperature. After that the residue was dissolved in dichlorometh-
ane (150 mL) and washed with water (3 ꢀ 150 mL). The organic
phase was dried, evaporated to dryness and purified on a silica
gel column using 3% isopropanol in ethyl acetate as eluent. The
mixture of epimers 4, 0.85 g was obtained in 66% yield. 1H NMR
4 (CDCl3) d: 7.46 (4H, d, J = 8.1 Hz, ArH), 7.20 (4H, d, J = 8.1 Hz,
ArH), 3.98 (4H, s, NH2), 1.38 (2H, s, CH). MS: (M+1) calculated
371.08, found 371.10.
2-piperazin-1-yl-ethanol (1.8 g, 14.1 mmol) in anhydrous dichlo-
romethane (5 mL) at 0 °C over 10–20 min. The reaction mixture
was stirred at 0 °C for 1 h and warmed to room temperature for
30 min. The reaction mixture was worked up with 0.3 mL of water,
extracted with dichloromethane (100 mL) and washed with water
(3 ꢀ 100 mL) and brine. The organic extract was dried and purified
on a silica gel column and using 5% methanol in dichloromethane
as eluent to isolate 6, Nutlin-2 (0.71 g) in 73% yield. 1H NMR 6
(CDCl3) d: 7.55 (1H, d, J = 8.4 Hz, ArH), 7.22 (4H, dd, J = 8.1 Hz,
ArH), 6.85 (4H, dd, J = 8.4 Hz, ArH), 6.56 (1H, d, J = 8.7 Hz, ArH),
6.51 (1H, s, ArH), 5.61 (1H, d, J = 9.6 Hz, CH), 5.43 (1H, d,
J = 9.6 Hz, CH), 4.28–3.91 (2H, m, CH2), 3.88 (3H, s, CH3), 3.59
(2H, t, J = 5.4 Hz, CH2), 3.18 (4H, br s, CH2), 2.44 (2H, t, J = 5.1 Hz,
CH2), 2.16 (4H, br s, CH2), 1.45 (3H, t, J = 6.9 Hz, CH3). 13C NMR 6
(CDCl3) d: 16.6, 43.2, 44.4, 56.6, 56.9, 58.6, 60.1, 60.9, 62.6, 68.4,
69.5, 109.2, 111.3, 112.1, 124.4, 125.3, 128.4, 128.7, 130.2, 130.6,
131.5 (2), 1.33.4, 135.1 (2), 141.5, 145.3, 156.2, 159.1, 164.9 and
169.3. High resolution MS: M+1, calculated 687.4341, found
687.4342.
4.2.5. 4,5-Bis(4-bromophenyl)-2-(2-ethoxy-4-methoxyphenyl)
dihydroimidazole, 5
Compound
2 (0.45 g, 2.01 mmol), triethylamine (0.28 mL,
2.01 mmol), 4-dimethylamino pyridine (0.12 g, 1.0 mmol) and
N-(4-Bromobenzoyl)-meso-1,2-di(4-bromophenyl)-1,2-diaminoe-
thane, 4 (0.75 g, 2.01 mmol) was taken in 6 mL of anhydrous etha-
nol in a sealed tube and heated with microwaves at 80 °C for 4 h.
The reaction mixture was quenched with 1(N) sodium bicarbonate
solution (0.3 mL). The crude product was extracted into dichloro-
methane (100 mL) and the organic solution was washed with
water (3 ꢀ 100 mL) and brine. The organic phase was dried
(MgSO4), evaporated to dryness and the crude residue was purified
on a silica gel column and using 2% methanol in dichloromethane
as eluent. This afforded product 5 as yellowish color powder (0.8 g,
75% yield). 1H NMR 5 (CDCl3) d: 8.26 (1H, d, J = 8.7 Hz, NH), 7.21
(4H, d, J = 8.1 Hz, ArH), 6.86 (4H, d, J = 8.4 Hz, ArH), 6.64 (2H, d,
J = 6.6 Hz, ArH), 6.34 (1H, d, J = 6.1 Hz, ArH), 5.36 (2H, br s, CH),
4.16 (2H, q, CH2), 3.88 (3H, s, CH3), 1.42 (3H, t, J = 6.9 Hz, CH3).
MS: (M+1) calculated 531.26, found 531.28.
4.2.7. 2-(4,5-Bis(4-bromophenyl)-2-(2-ethoxy-4-
methoxyphenyl)dihydroimidazole-carbonyl)piperazinyl)ethyl
2-(tert-butoxycarbonylamino) acetate, 7
Compound
6
(0.5 g, 0.73 mmol) and N-(tert-butoxycar-
bonyl)glycine (0.19 g, 1.09 mmol) were dissolved in anhydrous
dichloromethane (10 mL) in a dry flask under argon atmosphere
and N,N0-dicyclohexylcarbodiimide (0.45 g, 2.19 mmol) was added
followed by the addition of 4-dimethylamino pyridine (89 mg,
0.73 mmol) at room temperature. The reaction mixture was stirred
for overnight when TLC showed no starting material remained.
Solvent was evaporated and crude residue was purified on a silica
gel column using 3% methanol in dichloromethane to isolate 7
(0.45 g) in 73% yield. 1H NMR 7 (CDCl3) d: 8.11 (1H, br s, NH),
7.90 (1H, d, J = 8.1 Hz, ArH), 7.65 (4H, d, J = 8.3 Hz, ArH), 7.01 (4H,
dd, J = 8.1 Hz, ArH), 6.54 (1H, d, J = 8.0 Hz, ArH), 6.52 (1H, s, ArH),
5.41 (1H, d, J = 6.4 Hz, CH), 5.28 (1H, d, J = 7.1 Hz, CH), 4.22 (2H,
t, J = 7.3 Hz, CH2), 4.18 (2H, br s, CH2), 4.03 (2H, q, CH2), 3.79 (3H,
s, CH3), 3.41 (4H, t, J = 6.1 Hz, CH2), 3.02 (2H, t, J = 6.3 Hz, CH2),
2.46 (4H, t, J = 6.0 Hz, CH2), 1.36 (3H, s, CH3), 1.33 (3H, t,
J = 7.1 Hz, CH3). MS: M+1, calculated 844.60, found 844.61.
4.2.6. 4,5-Bis(4-bromophenyl)-2-(2-ethoxy-4-methoxyphenyl)
dihydroimidazolyl)(4-(2-hydroxyethyl)piperazinyl)methanone,
6
Compound 5 (0.75 g, 1.41 mmol) was dissolved in anhydrous
dichloromethane (6 mL) in a dry flask under argon atmosphere
and triethylamine (0.2 mL, 1.41 mmol) was added followed by
the addition of 4-dimethylamino pyridine (86 mg, 0.71 mmol) at
room temperature. Phosgene (0.29 g, 2.82 mmol, 20% in toluene)
was added dropwise into the mixture and stirred for 1 h at room
temperature and evaporated. The residue was further dried under
high vacuum for 1–2 h. The mixture was dissolved in 3 mL of
anhydrous dichloromethane and added dropwise to a solution of
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
4.2.8. 2-(4,5-Bis(4-bromophenyl)-2-(2-ethoxy-4-
methoxyphenyl)dihydroimidazole-carbonyl)piperazinyl)ethyl-
2-aminoacetate, 8
executed with the compounds indicated in Section 2. After the
treatment, cells were harvested, washed twice by PBS, fixed with
70% ethanol, and incubated overnight at 4 °C. After thawing, cells
Compound 7 (0.3 g, 0.36 mmol) was dissolved in dichlorometh-
ane (5 mL) under argon atmosphere. Trifluroacetic acid (0.5 mL)
was added dropwise in to the mixture and stirred for 30 min at
0 °C and 1 h at room temperature when TLC showed no starting
material remained. Solvent was evaporated and crude residue
was purified on a semi preparative high-performance liquid chro-
matography (HPLC). Purification was performed on a Luna SCX
were washed twice in cold PBS and suspended in PI (50 lg/mL),
0.1 mg/mL RNase A, 0.05% Tritin X-100 solution, followed with
an incubation at 37 °C for 45 min. Finally, 2 mL of PBS was added,
the cells were pelleted at 1000 rpm for 5 min and re-suspended in
500 lL of PBS for flow cytometer analysis with a BD LSR II
cytometer.
100A column (5
l
m, 250 ꢀ 10 mm). The flow was 4 mL/min, with
4.5. In vitro uptake studies
the mobile phase starting from 90% solvent A (0.1% trifluoroacetic
acid in water) and 10% solvent B (0.1% trifluoroacetic acid in aceto-
nitrile) to 10% solvent A and 90% solvent B at 20 min. The fraction
containing Nutlin–Glycine, 8 was collected and dried in 82% yield
(0.22 g). 1H NMR 8 (CDCl3) d: 7.96 (1H, d, J = 8.4 Hz, ArH), 7.61
(4H, d, J = 8.1 Hz, ArH), 7.13 (4H, dd, J = 8.4 Hz, ArH), 6.64 (1H, d,
J = 8.0 Hz, ArH), 6.50 (1H, s, ArH), 5.45 (1H, d, J = 6.1 Hz, CH), 5.33
(1H, d, J = 6.4 Hz, CH), 4.31 (2H, t, J = 7.1 Hz, CH2), 4.26 (2H, s,
CH2), 4.06 (2H, q, CH2), 3.81 (3H, s, CH3), 3.40 (4H, t, J = 7.2 Hz,
CH2), 2.91 (2H, t, J = 6.1 Hz, CH2), 2.56 (4H, t, J = 6.5 Hz, CH2), 1.59
(2H, br s, NH2), 1.34 (3H, t, J = 6.9 Hz, CH3). 13C NMR 8 (CDCl3) d:
16.4, 42.3, 45.2, 52.0, 54.5, 55.6, 56.4 (2), 60.2, 63.3, 66.1, 74.7,
108.6, 113.0, 116.1, 122.5, 123.1, 127.1, 127.7, 131.4, 131.9,
135.5, 136.2 (2), 139.8, 141.3, 145.5, 151.1, 158.9, 164.2, 165.7,
166.8, and 171.4. High resolution MS: M+1, calculated 744.4934,
found 744.4937.
Wild type p53 human osteosarcoma cells (SJSA-1) and colon
cancer cells (HCT116) were grown in a 24-well plate at a density
of 400,000 cells/well. They were allowed to attach for 24 h prior
to treatment. FAM and Nutlin–Glycine–FAM conjugate were
directly added to the separate culture media at a concentration
of 1 and 5 lM and incubated for 1 h at 37 °C. DMSO (5 lM) or cul-
ture media itself was also incubated for 1 h at 37 °C. Cells were
then washed three times with pre-warmed PBS (37 °C), harvested
by scraping, and counted and diluted to 100,000 cells/mL. Approx-
imately 10,000 cells per well were seeded in 96-well black-wall
plates, and the fluorescence intensity in cells was measured in a
fluorometric plate reader (FLUOstar OPTIMA, Durham, NC) with a
485/520 nm filter at an optical magnification of 20ꢀ. For each sam-
ple tested a total of 3 runs were assayed.
4.6. Cell blocking studies
4.2.9. Nutlin–Glycine–FAM conjugate, 9
A solution of compound 8 (50 mg, 0.067 mmol) and triethyl-
amine (11 lL, 0.077 mmol) in anhydrous N,N-dimethylformamide
(0.3 mL) was cooled to 0 °C for 10–20 min under argon filled bal-
loon. 6-carboxyfluorescein N-hydroxysuccinimide ester (48 mg,
0.10 mmol) in anhydrous N,N-dimethylformamide (0.1 mL) was
added dropwise and stirred in the dark at ambient temperature
for overnight. The reaction mixture was quenched by adding
In this experiment, wild type p53 human osteosarcoma cells
(SJSA-1) and colon cancer cells (HCT116) were used at a density
of 600,000 cells/well. They were allowed to attach for 24 h prior
to treatment. Nutlin–Glycine derivative (10-fold excess) was added
to a separate culture media at a concentration of 5
ment with Nutlin–Glycine–FAM for 1 h at 37 °C. Nutlin–Glycine–
FAM (5 M) was added into this media and further incubated for
1 h at 37 °C. FAM and DMSO were also incubated for 1 h at a con-
centration of 5 M as negative controls. Cells were then washed,
lM prior treat-
l
100 lL of 5% acetic acid in water. The solvent was evaporated to
dryness under high vacuum. The purification of the crude product
was carried out on a semi preparative high-performance liquid
chromatography (HPLC) and above solvent system at 4 mL/min
flow rate. The fraction containing Nutlin–Glycine–FAM conjugate
was collected, dried and stored in the dark at ꢁ20 °C until use.
The pure conjugate 9, 52 mg was obtained in 71% yield and was
characterized by high resolution mass spectroscopy (HRMS). HRMS
(M+1): calculated 1100.1675, found 1100.1678.
l
harvested and approximately 10,000 cells per well were seeded
in 96-well black-wall plates, and the fluorescence intensity in cells
was measured in a fluorometric plate reader with a 485/520 nm fil-
ter. For each sample tested a total of 3 runs were assayed.
Acknowledgments
This project was supported by The Methodist Hospital Research
Institute, the M.D. Anderson Foundation, and the Vivian L. Smith
Foundation.
4.3. Cell viability/cytotoxicity assay
Human cancer cells containing a wild-type p53 (A549, HCT116,
HepG2, RKO, and SJSA-1) and a mutant p53 (Hela, MDA-MB-435S,
PC-3 and SW480) were grown in the recommended medium
supplemented with 10% fetal bovine serum (Invitrogen, San Diego,
CA) in a humidified environment with 5% CO2 at 37 °C. Drugs were
dissolved in DMSO and stored as 1 mmol/L stock solutions in small
aliquots at ꢁ20 °C. Cells were seeded into 96-well plates and trea-
ted with Nutlin-2, Nutlin-3 and Nutlin–Glycine derivative at serial
References and notes
1. Cahilly-Snyder, L.; Yang-Feng, T.; Francke, U.; George, D. L. Somatic Cell Mol.
Genet. 1987, 13, 235.
2. Fakharzadeh, S. S.; Trusko, S. P.; George, D. L. EMBO J. 1991, 10, 1565.
3. Oliner, J. D.; Kinzler, K. W.; Meltzer, P. S.; George, P. L.; Vogelstein, B. Nature
1992, 358, 80.
4. Momand, J.; Zambetti, G. P.; Olson, D. C.; George, D.; Levine, A. J. Cell 1992, 69,
1237.
concentrations of 0–10 lM, respectively. After 5 days treatment,
5. Finlay, C. A.; Hinds, P. W.; Levin, A. J. Cell 1989, 1083, 57.
6. Kubbutat, M. H.; Jones, S. N.; Vousden, K. H. Nature 1997, 387, 299.
7. Picksley, S. M.; Lane, D. P. BioEssays 1993, 15, 689.
8. Freedman, D. A.; Wu, L.; Levine, A. J. Cell Mol. Life Sci. 1999, 55, 96.
9. Momand, J.; Jung, D.; Wilczynski, S.; Niland, J. Nucleic Acids Res. 1998, 26, 3453.
10. Bond, G. L.; Hu, W.; Levine, A. J. Curr. Cancer Drug Targets 2005, 5, 3.
11. Vousden, K. H.; Prives, C. Cell 2005, 120, 7.
cell viability was assessed by the MTS-based Cell Titer 96 Aqueous
One Solution Reagent (Promega, Madison, WI) according to manu-
facturer’s protocol.
4.4. Cell cycle analysis
12. Montes de, O.; Luna, R.; Wagner, D. S.; Lozano, G. Nature 1995, 378, 203.
13. Zhao, R.; Gish, K.; Murphy, M.; Yin, Y.; Notterman, D.; Hoffman, W. H.; Tom, E.;
Mack, D. H.; Levine, A. J. Genes Dev. 2000, 14, 981.
14. Chene, P. Nat. Rev. 2003, 3, 102.
15. Lane, D. P. Br. J. Cancer 1999, 80, 1.
Cell cycle was determined by flow cytometry analysis with pro-
pidium iodide (PI) staining (BD Pharmigen). One million cells were
seeded in 75-cm2 flasks and the subsequently treated were
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026

8
P. Ghosh et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
16. Chen, L.; Agrawal, S.; Zhou, W.; Zhang, R.; Chen, J. Proc. Nat. Acad. Sci. U.S.A.
1998, 95, 195.
24. Yang, D. J.; Wallace, S.; Cherif, A.; Li, C.; Gretzer, M. B.; Kim, E. E.; Podoloff, D. A.
Radiology 1995, 194, 795.
17. Chene, P.; Fuchs, J.; Bohn, J.; Garcia-Echeverria, C.; Furet, P.; Fabbro, D. J. Mol.
Biol. 2000, 299, 245.
25. Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 12063.
18. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong,
N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303,
844.
19. Patel, S.; Player, M. R. Expert Opin. Investig. Drugs 2008, 17, 1865.
20. Hu, C. Q.; Hu, Y. Z. Curr. Med. Chem. 2008, 15, 1720.
21. Shangary, S.; Wang, S. Clin. Cancer Res. 2008, 14, 5318.
22. Zhihao, Z.; Lin, Z.; Yajing, L.; Fenghua, Du; Hongmei, Gan; Jinpin, Qiao; Kung,
Hank F Nucl. Med. Biol. 2011, 38, 501.
26. Daniel, A.; Harki, N. S.; David, B. S.; Michael, E. P.; Peter, B. D. PNAS 2008, 105,
13039.
27. Bandgar, B. P.; Makone, S. S. Synth. Commun. 2006, 36, 1347.
28. Veejendra, K. Y.; Ganesh, K. B. Eur. J. Org. Chem. 2005, 452.
29. Proskurnina, M. V.; Lozinskaya, N. A.; Tkachenko, S. E.; Zefirov, N. S. Russ. J. Org.
Chem. 2002, 38, 1149.
30. John, K. B.; James, A.; Shantaram, K.; Shivaputra, P.; Darius, M.; Marian, O. Curr.
Cancer Drug Targets 2005, 5, 57.
23. Rasey, J. S.; Hofstrand, P. D.; Chin, L. K.; Tewson, T. J. J. Nucl. Med. 1999, 1072, 40.
Please cite this article in press as: Ghosh, P.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.11.026