Benzo[a]acridinylmethyl esters as pH sensitive fluorescent photoactive precursors: Synthesis, photophysical, photochemical and biological applications

Article abstract of DOI:10.1039/c3ob42600a

A newsworthy class of carboxylate esters based on the (benzo[a]acridin-12- yl)methyl (BAM) chromophore has been shown to perform dual functions as a "pH sensitive fluorescent probe" and a "phototrigger" for acids. The photophysical properties of all the BAM ester conjugates were investigated and found to be highly sensitive to solvent polarity, H-bonding capability and pH of the environment. On irradiation using UV light (≥410 nm), BAM ester conjugates underwent heterolytic cleavage of C-O bonds resulting in efficient release of carboxylic and amino acids. Interestingly, the newly synthesized BAM chromophore was also explored for the construction of a drug delivery system (DDS). In the current DDS, the BAM chromophore plays two important roles: (i) a "fluorophore" for cell imaging and (ii) a "phototrigger" for the drug release. In vitro biological studies revealed that the newly developed BAM based DDS has a good biocompatibility, cellular uptake properties and efficient photoregulated anticancer drug release ability. the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42600a

Organic &
Biomolecular Chemistry
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Benzo[a]acridinylmethyl esters as pH sensitive
fluorescent photoactive precursors: synthesis,
photophysical, photochemical and biological
applications†
Cite this: Org. Biomol. Chem., 2014,
12, 3459
Mohammed Ikbal,a Biswajit Saha,b Shrabani Barman,a Sanghamitra Atta,a
Deb Ranjan Banerjee,a Sudip Kumar Ghoshb and N. D. Pradeep Singh*a
A newsworthy class of carboxylate esters based on the (benzo[a]acridin-12-yl)methyl (BAM) chromophore
has been shown to perform dual functions as a “pH sensitive fluorescent probe” and a “phototrigger” for
acids. The photophysical properties of all the BAM ester conjugates were investigated and found to be
highly sensitive to solvent polarity, H-bonding capability and pH of the environment. On irradiation using
UV light (≥410 nm), BAM ester conjugates underwent heterolytic cleavage of C–O bonds resulting in
efficient release of carboxylic and amino acids. Interestingly, the newly synthesized BAM chromophore
was also explored for the construction of a drug delivery system (DDS). In the current DDS, the BAM
chromophore plays two important roles: (i) a “fluorophore” for cell imaging and (ii) a “phototrigger” for the
drug release. In vitro biological studies revealed that the newly developed BAM based DDS has a good
biocompatibility, cellular uptake properties and efficient photoregulated anticancer drug release ability.
Received 29th December 2013,
Accepted 24th March 2014
DOI: 10.1039/c3ob42600a
www.rsc.org/obc
ger whose fluorescence properties can respond rapidly and
sensitively to intracellular pH distributions and cellular pH
Introduction
Recently there has been a great demand for fluorescent photo- fluctuations, then we can have a single chromophore moiety
triggers particularly in the field of photoresponsive drug deli- which can act both as a “pH sensitive fluorescent probe” and
very systems (DDSs), because of their ability to perform dual as a “phototrigger for the drug release”.9
roles as a “fluorophore” for cell imaging and a “phototrigger”
Fluorescent pH probes10 are very useful tools for monitor-
for the drug release.1 To meet the growing need, chromo- ing pH changes in biological samples. However, most of the
phores based on polycyclic aromatic and heteroaromatic current pH probes respond to the pH changes by increasing or
compounds such as anthraquinone,2 anthracene,3 pyrene,4 decreasing the fluorescence intensity. The only intensity based
perylene,5 phenanthrene,2 coumarin6 and fluorescein7 have sensing is compromised by many other non-pH factors like
been targeted for developing fluorescent phototriggers. Among changes of probe concentration, solvent polarity and other
them coumarin8 based chromophores have been extensively environmental effects. An ideal pH responsive probe should
utilized for the synthesis of several fluorescent phototriggers exhibit shifted absorption or emission spectra upon pH
due to their strong fluorescence and faster photorelease changes, high fluorescence quantum yield for pH imaging in
ability. Though, coumarin based fluorescent phototriggers cells, good membrane permeability, low cytotoxicity and clear
exhibited strong fluorescence properties which is useful for cellular location.11
live cell imaging experiments. But to a larger extent their fluo-
The acridine chromophore has been well explored for the
rescent properties were found to be insensitive to the cellular development of pH sensitive probes suitable for in vivo
pH fluctuations. By any means, if we can develop a phototrig- imaging. The acridine moiety exhibits a difference in fluo-
rescence lifetime between the neutral and protonated states,
over the 6.0 to 8.0 pH range in solution. The neutral acridine
aDepartment of Chemistry, Indian Institute of Technology Kharagpur,
Kharagpur-721302, India. E-mail: ndpradeep@yahoo.co.in; Fax: (+) 91-3222-282252;
Tel: (+) 91-3222-282324
shows a lifetime of ∼10 ns, while the protonated form has a
lifetime of 31 ns.12 Thus acridine undergoes a pH-dependent
emission shift to longer wavelengths in acidic environments.
Despite their interesting pH sensitive fluorescent nature, only
recently a phototrigger, namely, acridin-9-methyl, has been
developed for the release of carboxylic and amino acids.13
bDepartment of Biotechnology, Indian Institute of Technology Kharagpur,
Kharagpur-721302, India
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob42600a
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Hence, we thought of designing a new pH sensitive fluorescent
phototrigger based on the acridine chromophore with
improved photophysical and photochemical properties com-
pared to acridin-9-methyl.
Results and discussion
Synthesis of BAM esters (5a–f and 7a–b)
Complying with Scheme 1, the esters (5a–f and 7a–b, Table 1)
Recent studies have shown that extended angular acridine of (benzo[a]acridin-12-yl)methyl were prepared. A Friedel–
derivatives exhibit improved photophysical properties com- Crafts reaction of N-phenyl-2-napthyl amine (1) with AcOH, in
pared to the parent acridine moiety such as (i) a stronger the presence of ZnCl2, fetched 12-methylbenzo[a]acridine
molar absorption coefficient, (ii) a much more red shifted (2),15 which on treatment with N-bromosuccinimide (NBS) in
absorption band, (iii) stronger fluorescence and (iv) the pres- the presence of benzoyl peroxide in carbon tetrachloride (CCl4)
ence of an intramolecular electron-transfer state of high under refluxing conditions gave 12-(bromomethyl)benzo[a]-
energy and long lifetime. The above improved photophysical acridine (3).16 Finally, treatment of 3 with carboxylic acids
properties of extended angular acridine derivatives are mainly (4a–f) and N-protected amino acids (6a–b) in the presence of
attributed to their strong skeletal rigidity and larger π-conju- potassium carbonate (K2CO3) in dry DMF at 50 °C resulted in
gation system.14 Further, it has been also shown that extended esters (5a–f) and (7a–b) respectively, with moderate to good
angular acridine derivatives (four fused six-membered rings) yields (70–82%, Table 1). The BAM esters were purified and
with a crescent shape and a larger surface area intercalate characterized by 1H, 13C NMR, IR and mass spectral analysis.
more strongly with DNA and RNA duplexes than the tricyclic
Photophysical properties of (benzo[a]acridin-12-yl)methyl
(BAM) esters
planar derivatives (e.g. the acridines and phenanthridines),
which will be really beneficial in delivering the drug inside the
nucleus of the cell. The above interesting photophysical pro-
perties and strong intercalating ability of extended angular
Absorption and emission properties of BAM esters. The UV-
vis absorption spectra of a degassed 2 × 10−5 (M) solution of
acridine derivatives prompted us to develop a pH sensitive the BAM esters (5a–f and 7a–b) in methanol (MeOH) were
fluorescent phototrigger based on the benzo[a]acridine moiety.
recorded. Fig. 1 shows the normalized absorption and emis-
In this paper, we report a new pH sensitive fluorescent
sion spectra of the representative ester 5d in methanol. The
phototrigger based on (benzo[a]acridin-12-yl)methyl (BAM) for absorption spectrum of 5d shows an intense band centered at
374 nm with ε = 50 × 103 mol−1 L cm−1, while in the emission
carboxylic acids and amino acids. The protection of carboxylic
acids and amino acids by coupling with the phototrigger BAM
was discussed. The pH sensitive fluorescence behavior of ester All the carboxylate esters exhibited similar kinds of absorption
spectrum, the emission maxima were red shifted to 430 nm.
conjugates of BAM was investigated. The photorelease ability
of BAM was studied by irradiating the ester conjugates using
and emission spectra, independent of the substituent attached
to the carboxylic group (see ESI Fig. S1 & S2, page-S12†). The
UV light above 410 nm. Further, we also constructed a molar absorptivities, fluorescence quantum yield, and absorp-
DDS using the BAM chromophore and demonstrated its ability
to perform dual functions as a fluorescent imaging agent
tion and emission maxima of the BAM esters are summarized
in Table 1. The fluorescence quantum yield was calculated
and as a phototrigger for delivery of the anticancer drug using 9,10-diphenylanthracene as a standard (Φ = 0.95 in
ethanol).17
chlorambucil.
Scheme 1 Synthesis of BAM esters of carboxylic (5a–f) and amino acids (7a–b).
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Table 1 Synthetic yield, UV-vis absorption and fluorescence data of
BAM esters (5a–f and 7a–b) in methanol
UV-vis
absorption
Fluorescence
Stokes’
Synthetic
yielda (%)
λmax
λmax
shifte
(nm)
b
d
log εc
(nm)
Φ f
f
Ester
(nm)
5a
5b
5c
5d
5e
5f
75
80
82
78
75
79
70
79
372
374
377
374
374
375
372
371
3.82
3.82
3.91
3.92
3.93
3.89
3.42
3.44
429
430
429
430
430
431
431
432
57
56
52
56
56
56
57
58
0.12
0.27
0.25
0.27
0.25
0.14
0.16
0.17
7a
7b
a Based on isolated yield. b Maximum absorption wavelength. c Molar
absorption coefficient at the maximum absorption wavelength.
d Maximum emission wavelength. e Difference between maximum
absorption wavelength and maximum emission wavelength.
f Fluorescence quantum yield (excitation wavelength 374 nm, error
limit within 5%).
Fig. 2 Fluorescence spectra of ester 5d in different solvents (2.0 × 10−5 M).
Fluorescence properties of the BAM esters in aqueous binary
mixtures. We also investigated the fluorescence properties of
BAM ester in an aqueous binary solvent system to understand
the effect of hydrogen bonding interaction. We recorded the
emission spectra (Fig. 3) of 5c in MeOH–H2O binary mixtures.
We observed that upon increasing the amount of water in the
MeOH–H2O mixture, the fluorescence intensity of 5c gradually
increased. The above fact can be attributed to solute–solvent
interactions through the formation of hydrogen-bonds
between the solvent and the nitrogen atom of the benzoacri-
dine moiety.19
pH dependent fluorescence properties of BAM esters. To
investigate the pH responsive behaviour of the BAM esters,
ester 5b was taken for the model study. The emission spectra
(Fig. 4a) of 5b were recorded in EtOH–HEPES buffer (20 : 80)
solution covering a broad spectrum of pH values ranging from
3.13 to 10.51. The fluorescence spectra of 5b showed greater
sensitivity to pH change. Further we also noticed fluorescent
colour change (blue to green) of ester 5b as we move from
basic pH to acidic pH (Fig. 4b).
Fig. 1 Normalized UV-vis absorption spectrum (black line) and emis-
sion spectrum (red line) of ester 5c in MeOH.
Interestingly, we noted an isosbestic point in the emission
spectra at around 475 nm, indicating the presence of two dis-
Solvent dependent fluorescence properties of BAM esters. tinct species in the equilibrium. This is because at lower pH,
By fluorescence emission spectroscopy, the excited state pro- protonation is favoured and hence protonated 5b is the pre-
perties of BAM esters were investigated. Like other nitrogen dominant species. Thus the fluorescence spectrum at lower pH
heterocycles (e.g. acridine, isoquinoline) the fluorescence pro- is mainly observed due to protonated 5b in the excited state. At
perties of the BAM esters were also found to be strongly depen- neutral pH, the fluorescence spectrum obtained corresponds
dent on solvent polarity.18
to 5b in the excited state. The fluorescence maximum of 5b in
As a representative example, the emission spectra (Fig. 2) of higher pH is around 420 nm, which is red shifted to ≈520 nm
5d in solvents of increasing polarity are shown; this displays a at lower pH.
strong solvent dependence. Further, the molar absorptivities,
The photophysical studies showed that all the BAM
fluorescence quantum yield, and absorption and emission esters exhibited strong fluorescence, large Stokes’ shift,
maxima of the BAM ester 5d in different solvents were moderate fluorescence quantum yield, and more interestingly
recorded and are summarized in Table 2. The above fluo- their fluorescence properties were found to be highly sensi-
rescence solvatochromism of the 5d can be attributed to its tive to the pH of the medium which implies that the BAM
closely lying 1π–π* and 1n–π* excited states, similar to its esters can be targeted as a potential pH sensitive fluorescent
parent acridine moiety.19
probe.
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Table 2 Photophysical data of ester 5d with increasing solvent polarity and H-bonding capability
d
δH hydrogen
εmax
Stokes’
εa (25 °C)
bondingb
λmax c (nm)
(M−1 m−1
)
λem e (nm)
shiftf (nm)
Φf
g
Solvent
Hexane
1.88
2.38
37.50
20.70
9.10
24.55
33.00
0
372
374
372
372
374
374
374
6750
4295
4875
7080
5915
4620
7610
421
425
426
426
427
430
430
49
51
54
54
53
56
56
0.39
0.51
0.48
0.52
0.51
0.34
0.24
Toluene
Acetonitrile
Acetone
DCM
Ethanol
Methanol
2.0
6.1
7.1
7.1
19.4
22.3
a Dielectric constant at 25 °C. b Hydrogen bonding capability. c Maximum absorption wavelength. d Molar absorption coefficient at the maximum
absorption wavelength. e Maximum emission wavelength. f Difference between maximum absorption wavelength and maximum emission
wavelength. g Fluorescence quantum yield (excitation wavelength 374 nm, error limit within 5%).
Fig. 3 Fluorescence spectra of the ester 5c in MeOH and in increasing
percentage of water in MeOH (2.0 × 10−5 M).
Phototrigger ability of (benzo[a]acridin-12-yl)methyl (BAM)
esters
The effect of solvent on photorelease of BAM esters. To
select the best solvent system for the photorelease of acids by
BAM esters, we irradiated BAM ester 5b in different solvents
Fig. 4 (a) Fluorescence spectra of the ester 5b (1 × 10−4 M) at different
pH. (b) Pictorial representation of fluorescent colour changes of 5b at
different pH.
systems, and results are summarized in Table 3. We found that
ester 5b generated benzoic acid in relatively high chemical and
quantum yield in ACN–H2O (50 : 50) compared to other sol-
vents used for the study.
Photolysis of BAM esters (5a–f and 7a–b). Based on the
above solvent studies, we carried out the photolysis of all the
The course of the photocleavage reaction was monitored by
BAM esters (5 mL of 1.0 × 10−4 M solution) individually, in UV (see ESI page-12, Fig. S3†), reverse phase HPLC and 1H
acetonitrile–H2O (50 : 50 v/v) using a 125 W medium pressure NMR spectroscopy. As a representative example, we have pro-
1
Hg lamp as the light source (≥410 nm) and 1 N NaNO2 as the vided the H NMR spectra (Fig. 5) of ester 5d at regular inter-
UV cut-off filter (Scheme 2). We observed that all the BAM vals of irradiation.
esters released their corresponding carboxylic (4a–f) and
In each case, the photolysis was stopped when conversion
amino acids (6a–b) as a significant photoproduct with good reached at least 95% (as indicated by HPLC). For all the BAM
chemical (80–92%) and quantum (0.127–0.083) yields (Фp) as esters, the photolysis products (corresponding acids and the
summarised in Table 4. The quantum yield (Фp) for the release photoproduct 8) were confirmed by isolating and matching
of carboxylic acids was calculated using potassium ferrioxalate their 1H NMR spectra with their corresponding authentic
as an actinometer.20
samples.
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Table 3 Photolytic data of ester 5b on irradiation by visible light
(≥410 nm) in different solvents
Photolytic data of carboxylate ester 5b
% of 5b
% of 4b
Quantum
Solvent
depleteda
releasedb
yieldc (Φp)
CH3CN
10
40
67
90
65
85
8
30
65
85
60
75
0.012
0.047
0.102
0.111
0.079
0.098
CH3CN–H2O (90 : 10)
CH3CN–H2O (70 : 30)
CH3CN–H2O (50 : 50)
CH3OH
CH3OH–H2O (50 : 50)
a % of 5b depleted as determined by HPLC. b % of 4b released as
determined by HPLC. c Photochemical quantum yield for the release of
4b (error limit within 5%).
Fig. 5 1H NMR spectral changes of ester 5d (2 × 10−4 M) in ACN–H2O
(50 : 50 v/v) at regular intervals of irradiation (0–360 min).
Scheme 2 Photorelease of carboxylic and amino acids by BAM esters.
Table 4 Photolytic data of BAM esters (5a–f and 7a–b) on irradiation
(≥410 nm) in acetonitrile–H2O (50 : 50 v/v)
Photolytic data of ester (5a–f and 7a–b)
Scheme 3 Mechanism for the photorelease of carboxylic and amino
acids.
Time of
Carboxylic and
Ester amino acid
photolysis % of acid
(min)
Quantum
generateda yieldb (Φp)
carboxylate anion react with solvent molecules to yield (benzo-
[a]acridin-12-yl)methanol (8) and acids respectively (Scheme 3).
After the successful demonstration of the BAM chromo-
phore as a fluorescent phototrigger, we were interested in
exploring the applicability of the BAM chromophore in the
construction of a photoresponsive drug delivery system (DDS).
5a
5b
5c
5d
5e
5f
C6H5CH2CO2H
C6H5CO2H
p-CH3C6H4CO2H
p-CH3OC6H4CO2H
p-(CH2vCH)C6H4CO2H 430
p-NO2C6H4CO2H
400
357
360
340
90
85
90
92
83
80
83
85
0.106
0.112
0.117
0.127
0.090
0.083
0.085
0.086
450
460
465
7a
7b
NH(Boc)(CH2)3CO2H
CH3CH(NHBoc)CO2H
a
A photoresponsive DDS based on (benzo[a]acridin-12-yl)-
%
of carboxylic acid released as determined by HPLC.
b Photochemical quantum yield for the generated carboxylic acids methyl (BAM) for regulated release of an anticancer drug
(error limit within 5%).
Synthesis of the (benzo[a]acridin-12-yl)methyl–chlorambucil
(BAM–Cbl) conjugate. We have synthesized the (benzo[a]-
acridin-12-yl)methyl–chlorambucil (BAM–Cbl) conjugate as
shown in Scheme 4. Treatment of 12-(bromomethyl)benzo[a]-
Possible mechanism for the photorelease of acids. Based on
literature reports3,6 and our solvent studies, we suggest the
acridine (3) with the anticancer drug chlorambucil (9) in the
possible mechanism for photorelease of carboxylic and amino
acids. The initial photochemical step involves excitation of the
BAM-ester to its singlet excited state, which then undergoes
facile singlet–triplet intersystem crossing. Subsequently, from
the triplet excited state it undergoes either homolytic C–O
bond cleavage followed by an electron transfer to produce the
(benzo[a]acridin-12-yl)methyl cation and the carboxylate anion
or it may undergo heterolytic C–O bond cleavage to produce
the above mentioned ion pair. After ion-pair separation in
Scheme 4 Synthesis of the (benzo[a]acridin-12-yl)methyl–chlorambu-
an aqueous polar solvent, the methylenic carbocation and the cil (BAM–Cbl) conjugate.
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Fig. 6 Docked poses of DNA–BAM–Cbl caged conjugate: (a) surface
representation showing the minor groove binding of 10; (b) stereoview
of the docked conformations of 10 showing that the interactions are
mainly hydrophobic and van der Waals type.
Fig. 7 Time course for the photorelease of chlorambucil from the
BAM–Cbl conjugate under visible light irradiation (≥410 nm).
under UV light (≥410 nm) by a 125 W medium pressure Hg
lamp using a suitable filter (1 N NaNO2 solution) and the
course of the photorelease was followed by reverse phase
HPLC.
A known amount of aqueous suspension of the photolysate
(0.1 mL) was taken at regular intervals of time and subjected
to reverse phase HPLC. The course of the photorelease of the
anticancer drug was then quantified by plotting the HPLC
peak area obtained for chlorambucil (Fig. 7). The reaction was
followed until the consumption of the BAM–Cbl conjugate is
less than 5% of the initial area.
presence of K2CO3 in dry DMF yielded the final photoproduct
10 in good yield. The conjugate was purified and characterized
by 1H, 13C NMR, IR and mass spectral analysis.
Intercalation and preferred binding mode of the benzo[a]-
acridin-12-yl)methyl–chlorambucil (BAM–Cbl) conjugate with
DNA. To substantiate the intercalation and preferred binding
mode of the prodrug BAM–Cbl conjugate with DNA, we per-
formed molecular docking studies. The lowest energy docked
conformation is used in analysis. Due to the helical shape of
the benzo[a]acridine moiety, it binds at the helical turning
close to the major helix. The interactions are mainly hydro-
phobic and van der Waals type. No hydrogen bonding is
observed. T-shaped pi stacking interactions have been found
(Fig. 6) between the benzo[a]acridine moiety and DA9 of chain-
A and DT10 of chain-B. The binding energy of this docked con-
In vitro cellular imaging studies using the BAM–Cbl conjugate
To investigate real-time cellular uptake properties of the BAM–
Cbl conjugate, we carried out cell imaging studies using the
HeLa cell line. A cellular uptake study after 4 h of incubation
reveals that the BAM–Cbl conjugate was internalized by the
cell membrane (Fig. 8a and b). To study the localization of the
BAM–Cbl conjugate inside the HeLa cell, we stained the cell
nuclei with propidium iodide. After exposing the HeLa cells to
the BAM–Cbl conjugate for 4 h, followed by propidium iodide
formation is −4.08 kcal mol−1
.
The docking study between the benzoacridine–chlorambu-
cil conjugate and the DNA duplex reveals that there are good
interactions between the benzo[a]acridine moiety and the DNA
duplex. Hence, this theoretical study suggests that the benzo[a]-
acridine moiety may act as a good carrier to deliver the anti-
cancer drug chlorambucil inside the nucleus of the cell.
Photoinduced release of the anticancer drug chlorambucil by
the BAM–Cbl conjugate
A suspension of the BAM–Cbl conjugate in the ACN–H2O
(50 : 50) solvent system (1 × 10−4 M) was irradiated (Scheme 5)
Fig. 8 Confocal images of HeLa cells: (a) bright field image, (b, c) cells
were incubated with 20 µM BAM–Cbl conjugate for 4 h followed by
fixation of cells and nuclear staining by PI, (b) the uptake of the BAM–
Cbl conjugate (λex = 410 nm), (c) the uptake of PI (25 nM, λex = 535 nm),
(d) fluorescent overlay images of (b) and (c) showing that both the BAM–
Cbl conjugate and PI are located at the cell nuclei. The blue fluor-
escence is from the BAM–Cbl conjugate and the red fluorescence is
from PI used to stain the nuclei.
Scheme 5 Photorelease of the anticancer drug.
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(PI), we noted that conjugates were readily internalized and
found to be located mostly in the cell nucleus as detected by
confocal microscopy (Fig. 8c and d).
Cell cytotoxicity assay of the BAM–Cbl conjugate before and
after irradiation
After a successful demonstration of the pronounced accumu-
lation of the BAM–Cbl conjugate within the nucleus of HeLa
cells, we evaluated the cytotoxicity of the BAM–Cbl conjugate,
chlorambucil and BAM–OH (8) in vitro using the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a
yellow tetrazole) assay in the HeLa cell line. The cytotoxic
effect of each treatment was expressed as percentage of cell via-
bility relative to the untreated control cells. The percentage of
cell viability was plotted versus the concentration. It was
observed that cell viability remains above 80% at different con-
centrations of the BAM–Cbl conjugate and BAM–OH, whereas
increasing cytotoxicity was observed on addition of increasing
amounts of chlorambucil (Fig. 9).
For the light exposure experiment, cells incubated with
different concentrations of the BAM–Cbl conjugate on
irradiation for 35 min under UV light (≥410 nm) resulted in
the release of the anticancer drug chlorambucil, thereby
causing cytotoxicity in cancerous HeLa cell lines as validated
by the MTT toxicity data (Fig. 10).
On the other hand, there was no significant cell death
observed when the cells were irradiated in the presence of
BAM–OH, indicating that the cytotoxicity was likely caused by
the released drug, chlorambucil, upon light irradiation
(Fig. 10). On comparison with the same concentration of chlor-
ambucil as that of the BAM–Cbl conjugate, the BAM–Cbl con-
jugate showed much lower cytotoxicity compared to
chlorambucil. But upon irradiation, the BAM–Cbl conjugate
showed an enhanced cytotoxicity to cancer cells in comparison
to chlorambucil, because of the efficient photorelease of chlor-
ambucil inside the cell. Thus the BAM–Cbl conjugate serves as
a versatile photoresponsive DDS for the release of the anti-
cancer drug inside the cell nucleus.
Fig. 10 Cell viability test of (1) BAM–OH, (2) BAM–Cbl and (3) chloram-
bucil against the HeLa cell line in different concentrations after
irradiation (values are presented as mean standard deviation).
Conclusion
In summary, we have demonstrated that caged esters based on
the (benzo[a]acridin-12-yl)methyl (BAM) chromophore can
perform dual functions as a pH sensitive fluorescent probe and
a fluorescent phototrigger. Photophysical studies revealed that
the BAM esters are highly fluorescent in nature and more
importantly their fluorescence properties were found to be
highly sensitive to pH of the medium. On the other hand, BAM
ester conjugates on photolysis efficiently released carboxylic
and amino acids. Finally, we have developed an excellent bio-
compatible fluorescent photoresponsive DDS using the BAM
chromophore for both in vitro cellular imaging application and
regulated delivery of the anticancer drug inside the cell nucleus.
Experimental section
Chemicals and starting materials
All starting materials, reagents, and solvents were obtained
from commercial sources and used without further purifi-
cation unless otherwise stated. All anhydrous reactions were
performed under a dry nitrogen atmosphere.
Methods and techniques
1H NMR (200 MHz) spectra were recorded on a BRUKER-AC
200 MHz spectrometer. Chemical shifts are reported in ppm
from tetramethylsilane with the solvent resonance as the
internal standard (deuterochloroform: 7.26 ppm). Data are
reported as follows: chemical shifts, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, dd = doublet of doublet,
m = multiplet), and coupling constant (Hz). 13C NMR (50 MHz)
spectra were recorded on a BRUKER-AC 200 MHz Spectrometer
with complete proton decoupling. Chemical shifts are reported
in ppm from tetramethylsilane with the solvent resonance as
the internal standard (deuterochloroform: 77.0 ppm). Chroma-
tographic purification was done with 60–120 mesh silica gels
(Merck). For reaction monitoring, precoated silica gel 60 F254
TLC sheets (Merck) were used. UV-vis absorption spectra were
Fig. 9 Cell viability test of (1) BAM–Cbl, (2) BAM–OH and (3) chloram-
bucil against the HeLa cell line in different concentrations before
irradiation (values are presented as mean standard deviation).
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recorded on a Shimadzu UV-2450 UV/vis spectrophotometer. pletion of the reaction (as indicated by TLC) cold distilled
FT-IR spectra were recorded on a Perkin Elmer RXI spectro- water was poured into the reaction mixture and diluted with
meter. High-resolution mass spectra (HRMS) were recorded DCM. The organic layer was separated, dried over Na2SO4 and
using an LCT micro mass spectrometer. HPLC was performed the solvent was removed by rotary evaporation under reduced
using Shimadzu Prominence (LC 20 AT) liquid chromato- pressure, and the crude residue was purified by column
graphy on a C18 column (4.5 mm × 250 mm) with a UV/vis chromatography using ethylacetate (EtOAc) in pet ether.
detector. Photolysis of all the esters was carried out using a
(Benzo[a]acridin-12-yl)methyl 2-phenylacetate (5a). The
125 W medium pressure mercury lamp supplied by SAIC (India). crude product was purified by column chromatography (20%
ethyl acetate–hexane) to give the title compound 5a (130 mg,
75%) as a brown solid, Rf (30% ethyl acetate–hexane) 0.45; m.p.
Synthesis of 12-methylbenzo[a]acridine (2)
A mixture of N-phenyl 2-naphthyle amine (3.0 g, 13.6 mmol), 118–120 °C; FTIR (KBr) νmax (cm−1): 1734; 3150, 1H NMR
acetic acid (3.0 g, 50 mmol) and zinc chloride (13 g, (CDCl3, 200 MHz) δ: 3.86 (s, 2H), 6.0 (s, 2H), 7.33–7.42 (m, 6H),
95.6 mmol) was heated up to 180 °C with continuous stirring. 7.60–7.67 (m, 2H), 7.84–7.89 (m, 4H), 8.10–8.19 (m, 1H), 8.26
Then excess acetic acid was removed from the reaction mixture (d, 1H, J = 8.6 Hz), 8.3 (d, 1H, J = 8.0 Hz); 13C NMR (CDCl3,
by distillation and the reaction mixture was heated at 250 °C 50 MHz) δ: 41.8, 62.5, 124.0, 125.3, 126.9, 127.0, 127.1, 127.5,
for an additional 5 h followed by the addition of an aqueous 128.0, 128.1 (2C), 128.6, 128.8 (2C), 128.9, 129.1, 129.4, 129.6,
ammonia solution. The resulting yellow precipitates were col- 129.7, 132.5, 132.8, 133.7, 135.8, 147.2, 149.9, 171.4; HRMS
lected by filtration. The residue was dissolved in chloroform (ES+) calcd for C26H20NO2 [M + H]+ 378.1488, found 378.1480.
and neutralized by washing with aqueous NaHCO3 and dried
(Benzo[a]acridin-12-yl)methyl benzoate (5b). The brown
over anhydrous Na2SO4. The organic solvent was then removed solid compound 5b (133 mg, 80%) was obtained by purifi-
under reduced pressure to give a crude product which was cation of the crude product using column chromatography
further purified by column chromatography.
(25% ethyl acetate–hexane), Rf (30% ethyl acetate–hexane)
1
12-Methylbenzo[a]acridine (2). Brown solid (2.67 g, 80%), Rf 0.40; m.p. 120–125 °C; FTIR (KBr) νmax (cm−1): 1712, 3250; H
(20% ethyl acetate–hexane) 0.45; m.p. 128–130 °C; FTIR (KBr) NMR (CDCl3, 200 MHz) δ: 6.41 (s, 2H), 7.50–7.57 (m, 2H),
1
νmax (cm−1): 3110, 1571; H NMR (CDCl3, 200 MHz) δ: 3.43 (s, 7.59–7.80 (m, 4H), 7.89–8.21 (m, 4H), 8.22–8.36 (m, 2H),
3H), 7.60–7.66 (m, 3H), 7.74–7.89 (m, 4H), 8.21–8.33 (m, 2H), 8.41–8.60 (m, 2H), 8.61–8.64 (m, 1H); 13C NMR (CDCl3,
8.54 (m, 1H); 13C NMR (CDCl3, 50 MHz) δ: 19.9, 123.8, 124.4, 100 MHz) δ: 62.5, 124.1, 124.5, 125.4, 127.1, 127.4, 128.3,
125.7, 125.9, 126.9, 127.1, 128.5, 128.9, 129.2 (2C), 129.4, 128.5, 128.7 (3C), 129.0, 129.1, 129.2, 129.4, 129.5, 130.0, 130.2
130.3, 132.1, 132.6, 141.7, 146.6, 149.6; HRMS (ES+) calcd for (2C), 132.8, 133.0, 133.3, 133.7, 149.5, 166.3; HRMS (ES+) calcd
C18H14N [M + H]+ 244.1120, found 244.1123.
for C25H18NO2 [M + H]+ 364.1332, found 364.1340.
Preparation of 12-(bromomethyl)benzo[a]acridine (3). 500 mg
(Benzo[a]acridin-12-yl)methyl 4-methylbenzoate (5c). Purifi-
(2.05 mmol) of 12-methylbenzo[a]acridine was dissolved in cation of crude carboxylate by column chromatography (20%
20 ml of CCl4. N-Bromosuccinimide (2.0 mmol) was added in ethyl acetate–hexane) gave the title compound 5c (142 mg,
the presence of benzoyl peroxide (0.82 mmol) and the reaction 82%) as a light yellow solid, Rf (30% ethyl acetate–hexane)
1
mixture was heated under refluxing conditions for 2 h. After 0.45; m.p. 115–118 °C; FTIR (KBr) νmax (cm−1): 1717; 3161, H
completion of the reaction, it was diluted with DCM. The NMR (CDCl3, 200 MHz) δ: 2.38 (s, 3H), 6.25 (s, 2H), 7.24 (d,
organic layer was separated, dried over Na2SO4 and the solvent 2H, J = 8.0 Hz), 7.56–7.68 (m, 3H), 7.84–7.90 (m, 4H), 8.06 (d,
was removed under reduced pressure to yield 12-(bromo- 2H, J = 8.0 Hz), 8.27–8.33 (m, 2H), 8.51–8.55 (m, 1H); 13C NMR
methyl)benzo[a]acridine.
(CDCl3, 50 MHz) δ: 21.7, 62.3, 124.1, 125.1, 126.8, 127.1, 128.0
12-(Bromomethyl)benzo[a]acridine (3). Deep brown solid (3C), 128.6, 128.7 (2C), 129.3, 129.6 (2C), 129.8, 130.0 (3C),
(561 mg, 85%), Rf (20% ethyl acetate–hexane) 0.40; m.p. 132.4, 132.8, 136.2, 144.3, 147.2, 149.9, 166.3; HRMS (ES+)
98–101 °C; FTIR(KBr) νmax (cm−1): 3113, 1569; 1H NMR (CDCl3, calcd for C26H20NO2 [M + H]+ 378.1488, found 378.1490.
200 MHz) δ: 5.54 (s, 2H), 7.52–7.59 (m, 2H), 7.80–7.87 (m, 2H),
(Benzo[a]acridin-12-yl)methyl 4-methoxybenzoate (5d). The
7.92–7.96 (m, 2H), 8.16 (d, 1H, J = 7.0 Hz), 8.41 (d, 1H, J = compound 5d (141 mg, 78%) was obtained as a yellow solid on
8.2 Hz), 8.58 (d, 1H, J = 8.2 Hz), 9.04 (d, 1H, J = 7.6 Hz); purification of the crude product through column chromato-
13C NMR (CDCl3, 50 MHz) δ: 31.2, 123.2, 124.0, 124.8, 125.4, graphy (20% ethyl acetate–hexane), Rf (30% ethyl acetate–
126.9, 127.2, 127.9, 128.2, 129.1, 129.2, 130.0, 132.0, 132.8, hexane) 0.45; m.p. 130–135 °C; FTIR(KBr) νmax (cm−1): 1715,
1
133.0, 138.4, 146.7, 149.7; HRMS (ES+) calcd for C18H13BrN 3158, H NMR (CDCl3, 200 MHz) δ: 3.86 (s, 3H), 6.27 (s, 2H),
[M + H]+ 322.0225, found 322.0230.
6.93 (d, 2H, J = 9.0 Hz), 6.36–7.71 (m, 3H), 7.81–7.94 (m, 4H),
8.12 (d, 2H, J = 9.0 Hz), 8.32 (d, 2H, J = 8.2 Hz), 8.56 (d, 1H, J =
8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ: 55.5, 62.3, 113.9 (2C),
121.9, 124.2, 125.2, 127.1 (3C), 128.1, 128.7 (2C), 129.3, 129.7
General procedure for the synthesis of carboxylate esters
(5a–f and 7a–b)
12-(Bromomethyl)benzo[a]acridine (150 mg, 0.46 mmol) was (3C), 132.1 (3C), 132.5, 132.9, 136.3, 147.3, 150.0, 166.1; HRMS
dissolved in 15 mL of dry DMF, and then potassium carbonate (ES+) calcd for C26H20NO3 [M + H]+ 394.1438, found 393.1443.
(0.05 mmol) and carboxylic acid (0.65 mmol) were added. The
(Benzo[a]acridin-12-yl)methyl 4-vinylbenzoate (5e). The
reaction mixture was stirred at 50–55 °C for 10–12 h. After com- dark yellow crude solid on purification by column chromato-
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graphy (30% ethyl acetate–hexane) gave the title compound 5e spectra were recorded using a fluorescence spectrophotometer.
(134 mg, 75%) as an orange solid/light yellow solid; Rf (40% The fluorescence quantum yield of the caged compounds was
ethyl acetate–hexane) 0.35; m.p. 115–118 °C; FTIR (KBr) νmax calculated using eqn (1).
(cm−1): 1714, 3156; 1H NMR (CDCl3, 200 MHz) δ: 5.33 (d, 1H, J
2
ðGradCGÞ ðηCG
Þ
Þ
= 10.8 Hz), 5.80 (d, 1H, J = 17.4 Hz), 6.23 (s, 2H), 6.68 (dd, 1H,
J1 = 11.0, J2 = 17.6), 7.39–7.43 (m, 2H), 7.56–7.65 (m, 4H),
7.82–7.87 (m, 3H), 8.05 (d, 2H, J = 8.2 Hz), 8.21–8.28 (m, 2H),
8.48 (d, 1H, J = 7.4 Hz); 13C NMR (CDCl3, 100 MHz) δ: 62.5,
117.0, 124.1, 125.1, 126.4 (2C), 127.0, 127.1 (2C), 128.1, 128.6
(2C), 128.8, 129.2, 129.6, 129.7 (2C), 130.3 (2C), 132.5, 132.8,
135.8, 136.1, 142.6, 147.2, 149.9, 166.2; HRMS (ES+) calcd for
C27H20NO2 [M + H]+ 390.1488, found 390.1495.
ðΦf ÞCG ¼ ðΦf ÞST
ð1Þ
2
ðGradSTÞ ðηST
where the subscripts CG and ST denote caged compound and
standard respectively. 9,10-Diphenylanthracene in ethanol was
taken as the standard. Φf is the fluorescence quantum yield;
Grad is the gradient from the plot of integrated fluorescence
intensity vs. absorbance, and η is the refractive index of the
solvent.
(Benzo[a]acridin-12-yl)methyl 4-nitrobenzoate (5f). Purifi-
cation of crude carboxylate by column chromatography (25%
ethyl acetate–hexane) gave the title compound 5f (148 mg,
79%) as a deep yellow solid, Rf (40% ethyl acetate–hexane)
Since fluorescence for both the caged compounds and the
standard was recorded in the same solvent, eqn (1a) was used
for the calculation of the fluorescence quantum yield.
1
0.40; m.p. 158–162 °C; FTIR (KBr) νmax (cm−1): 1722, 3162; H
ðGradCG
Þ
ðΦfÞCG ¼ ðΦfÞST
ð1aÞ
ðGradST
Þ
NMR (CDCl3, 200 MHz) δ: 6.37 (2H), 7.60–7.71 (m, 4H),
7.80–7.97 (m, 4H), 8.24–8.34 (m, 2H), 8.40–8.47 (m, 3H), 8.93
(s, 1H); 13C NMR (CDCl3, 50 MHz) δ: 63.4, 123.7, 124.9, 125.2,
126.8, 127.1, 127.3, 127.9, 128.2, 128.4, 128.7, 129.0, 129.1,
129.8, 129.9 (2C), 131.3, 132.5, 133.0, 135.2, 135.4, 147.3,
148.4, 150.0, 164.3; HRMS (ES+) calcd for C25H17N2O4 [M + H]+
409.1182, found 409.1176.
(Benzo[a]acridin-12-yl)methyl 4-(tert-butoxycarbonylamino)-
butyrate (7a). The crude product was purified by column
chromatography (20% ethyl acetate–hexane) to give the title
compound 7a (143 mg, 70%) as a light yellow solid; Rf (30%
ethyl acetate–hexane) 0.45; m.p. 111–114 °C; FTIR (KBr) νmax
(cm−1): 1683, 1729, 3340; 1H NMR (CDCl3, 200 MHz) δ: 1.43
(brs, 9H), 2.04–1.90 (m, 2H), 2.62 (t, 2H, J = 14.8), 3.26 (dd, 2H,
J1 = 6.4 Hz, J2 = 12.8 Hz), 4.66 (brs, 1H), 6.10 (s, 2H), 7.74–7.67
(m, 3H), 7.95–7.81 (m, 4H), 8.22 (d, 1H, J = 8.6 Hz), 8.32 (d,
1H, J = 8.6 Hz), 8.41 (m, 1H); 13C NMR (CDCl3, 100 MHz) δ:
25.5, 28.3 (3C), 29.6, 31.5, 39.7, 62.0, 123.8, 125.0, 126.7, 127.0,
128.0, 128.5, 128.7, 129.0 (2C), 129.5, 129.6, 132.5, 132.7 (2C),
136.0, 147.0, 149.8, 155.9, 172.9; HRMS (ES+) calcd for
C27H29N2O4 [M + H]+ 445.2121, found 445.2119.
Photolysis of esters (5a–f and 7a–b)
A solution of 2 × 10−4 M of the carboxylates (5a–f and 7a–b)
was prepared in acetonitrile individually. Nitrogen was passed
through the solution for 30 min and irradiated using a 125 W
medium pressure Hg lamp as a light source (≥410 nm) and
1 N NaNO2 solution as a UV cut-off filter. At regular intervals of
time, 20 μl of the aliquots was taken and analyzed by RP-HPLC
using mobile phase methanol, at a flow rate of 1 ml min−1
(detection: UV 254 nm). Peak areas were determined by
RP-HPLC, which indicated a gradual decrease of the caged
compound with time, and the average of three runs. The reac-
tion was followed until the consumption of the ester is less
than 5% of the initial area. Based on HPLC data for each
caged compound, we plotted normalized [a] (HPLC peak area)
versus irradiation time. We observed an exponential correlation
for the disappearance of the caged compounds which
suggested a first order reaction. Further, the quantum yield
for the photolysis of caged compounds was calculated using
eqn (2)
(R)-Benzo[a]acridin-12-ylmethyl 2-(tert-butoxycarbonylamino)-
propanoate (7b). Purification of the crude product by column
chromatography (25% ethyl acetate–hexane) gave the title com-
pound 7b (156 mg, 79%) as a light yellow solid, Rf (30% ethyl
acetate–hexane) 0.45; m.p. 115–118 °C; FTIR (KBr) νmax (cm−1):
1683, 1732, 3352; 1H NMR (CDCl3, 200 MHz) δ: 1.25 (s, 3H),
1.45 (s, 9H), 4.52 (t, 1H, J = 6.8 Hz), 5.08 (brs, 1H), 6.13 (s, 2H),
7.74–7.67 (m, 3H), 7.93–7.81 (m, 5H), 8.22 (d, 1H, J = 8.6 Hz),
8.48–8.35 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ: 18.6, 28.5
(3C), 49.5, 62.6, 80.2, 123.9, 127.4 (2C), 128.4, 129.0 (2C), 132.1
(2C), 132.6 (2C), 133.1 (2C), 133.4, 133.7, 139.0, 148.4, 153.0,
ðkpÞCG
ðΦpÞCG
¼
ð2Þ
I0 ðFCG
Þ
where the subscript ‘CG’ denotes caged compound. Φp is the
photolysis quantum yield, kp is the photolysis rate constant, I0
is the incident photon flux and F is the fraction of light
absorbed. Potassium ferrioxalate was used as an actinometer.
Determination of the incident photon flux (I0) of the UV lamp
by potassium ferrioxalate actinometry
154.4, 176.4; HRMS (ES+) calcd for C26H27N2O4 [M + H]+ Potassium ferrioxalate actinometry19,20 was used for the deter-
431.1965, found 431.1967.
mination of the incident photon flux (I0) of the UV lamp used
for irradiation. Solutions of potassium ferrioxalate, 1,10-phe-
nanthroline and the buffer solution were prepared following
Photophysical properties of carboxylates (5a–f and 7a–b)
The UV-vis absorption spectra of a degassed 2 × 10−5 M solu- the literature procedure.19
tion of esters (5a–f and 7a–b) in methanol were recorded on a
0.006 M solution of potassium ferrioxalate was irradiated
UV-vis spectrophotometer and the fluorescence emission using a 125 W medium pressure Hg lamp as a visible light
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source (≥410 nm) and 1 N NaNO2 solution as a UV cut-off
(Benzo[a]acridin-12-yl)methyl 4-(4-(bis(2-chloroethyl)amino)-
filter. At regular intervals of time (3 min), 1 ml of the aliquots phenyl)butanoate (10). The crude product on purification by
was taken out and to it 3 ml of 1,10-phenanthroline solution column chromatography (20% ethyl acetate–hexane) yielded
and 2 ml of the buffer solution were added and the whole solu- the title ester 10 (253 mg, 75%) as a light yellow solid; Rf (30%
tion was kept in the dark for 30 minutes. The absorbance of ethyl acetate–hexane) 0.45; FTIR (KBr) νmax (cm−1): 1718, 3152;
the red phenanthroline–ferrous complex formed was then 1H NMR (CDCl3, 200 MHz) δ: 2.07 (t, 2H, J = 7.4 Hz), 2.56–2.70
measured spectrophotometrically at 510 nm. The amount of (m, 4H), 3.58–3.69 (m, 8H), 6.09 (s, 2H), 6.61 (d, 2H, J = 8.8
Fe2+ ions was determined from the calibration graph. The cali- Hz), 7.07 (d, 2H, J = 8.6 Hz), 7.65–7.71 (m, 4H), 7.87–7.94 (m,
bration graph was plotted by measuring the absorbance of the 3H), 8.23 (d, 1H, J = 8.4 Hz), 8.35 (d, 1H, J = 8.6 Hz), 8.42–8.46
phenanthroline–ferrous complex at several known concen- (m, 1H); 13C NMR (CDCl3, 100 MHz) δ: 27.1, 34.0, 34.2, 40.7
trations of Fe2+ ions in the dark. From the slope of the graph (2C), 53.7 (2C), 62.1, 112.3 (2C), 125.3, 127.0, 127.2 (2C), 127.4,
the molar absorptivity of the phenanthroline–ferrous complex 128.4, 128.5 (2C), 128.8, 129.1 (2C), 129.3, 129.6 (3C), 129.9,
was calculated to be 1.10 × 104 M−1 cm−1 at 510 nm which is 130.1, 130.4, 133.0, 136.7, 144.6, 173.5; HRMS (ES+) calcd for
found to be similar to the reported value. Using the known C32H31Cl2N2O2 [M + H]+ 545.1757, found 545.1761.
quantum yield for the potassium ferrioxalate actinometer at
406.7 nm, the number of Fe2+ ions formed during photolysis
and the fraction of light absorbed by the actinometer, the inci-
Molecular docking study
dent intensity (I0) of the 125 W Hg lamp was determined to be The 18-mer DNA duplex PDB file was borrowed from RCSB
5.767 × 1017 quanta S−1
.
PDB bank (PDB ID: 3MKY). The ligand file has been prepared
using Accelrys Discovery Studio client 3.1. Prior to docking,
energy minimization of the ligand file has been carried out
using CFF force field. A docking study was carried out using
Autodock4.2. The DNA file has been prepared in Autodock
Tools by adding hydrogens and calculating gasteiger charges.
A whole macro molecule has been selected for grid calculation
to perform blind docking. Default parameters have been used
for grid calculation and docking studies. After docking,
images of the lowest energy docked conformation have been
prepared using the PyMol visualizer.
Preparative photolysis
A
solution of caged compounds (5a–f) and (7a–b)
(0.05 mmol) in acetonitrile–H2O (50 : 50) individually was
irradiated using the procedure described under deprotection
photolysis. The irradiation was monitored by TLC at regular
intervals. After completion of photolysis, the solvent was
removed under vacuum and the photoproducts (benzo[a]-
acridin-12-yl)methanol, and the corresponding carboxylic
acid were isolated by column chromatography using EtOAc in
hexane as an eluant.
Cytotoxicity of the BAM–Cbl conjugate on the HeLa cell line
(Benzo[a]acridin-12-yl)methanol (8)
Cytotoxicity before photolysis. The cytotoxicity in vitro was
measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, a yellow tetrazole) assay on the
HeLa cell line. Briefly, cells growing in the log phase were
The yellow solid compound 8 (11 mg, 90%) was obtained by
purification of the crude reaction mixture using column
chromatography (10% ethyl acetate–hexane); yellow solid, Rf
(40% ethyl acetate–hexane) 0.20; m.p. 134–136 °C; FTIR (KBr)
seeded into a 96-well cell-culture plate at 1 × 104 cells mL−1
.
1
νmax (cm−1): 3354; H NMR (CDCl3, 200 MHz) δ: 5.66 (s, 2H),
Different concentrations of BAM–Cbl, BAM–OH and chloram-
bucil were added into the wells with an equal volume of PBS in
the control wells. The cells were then incubated for 72 h at
37 °C in 5% CO2. Thereafter, fresh medium containing
0.40 mg ml−1 MTT was added to the 95 well plates and incu-
bated for 4 h at 37 °C in 5% CO2. Formazan crystals thus
formed were dissolved in DMSO after decanting the earlier
media and the absorbance was recorded at 595 nm.
7.23–7.25 (m, 1H) 7.66–7.73 (m, 4H), 7.77–7.90 (m, 3H), 8.27
(d, 1H, J = 8.4 Hz), 8.50 (d, 1H, J = 8.2 Hz), 8.95–8.99 (m, 1H);
13C NMR (CDCl3, 50 MHz) δ: 60.3, 122.8, 124.1, 124.5, 126.6,
126.8, 127.1, 127.9, 128.6, 129.4 (2C), 129.6, 130.4, 132.5 (2C),
140.1, 147.8, 149.9. HRMS (ES+) calcd for C18H14NO [M + H]+
260.1069, found 260.1065.
Procedure for the synthesis of the benzo[a]acridin-12-yl)-
methyl–chlorambucil (BAM–Cbl) conjugate (10)
Cytotoxicity after photolysis. HeLa cells maintained in
minimum essential medium (in a 96-well cell-culture plate at a
12-(Bromomethyl)benzo[a]acridine (200 mg, 0.46 mmol) was concentration of 1 × 104 cells mL−1) containing different con-
dissolved in 15 mL of dry DMF, and then potassium carbonate centrations (0.5, 1, 5, 10, 15 and 20 μM) of BAM–Cbl, BAM–OH
(0.05 mmol) and chlorambucil (0.74 mmol) were added. The and chlorambucil were incubated for 4 h at 37 °C and 5% CO2.
reaction mixture was stirred at 50–55 °C for 10–12 h. After com- Then the cells were irradiated (keeping the cell-culture plate
pletion of the reaction (as indicated by TLC) cold distilled 5 cm apart from the light source) under UV light (≥410 nm) by
water was poured into the reaction mixture and diluted with a 125 W medium pressure Hg lamp using a suitable filter (1 N
DCM. The organic layer was separated, dried over Na2SO4 and NaNO2 solution). After irradiation the cells were again incu-
the solvent was removed by rotary evaporation under reduced bated for 72 h. Then cytotoxicity was measured using the MTT
pressure.
assay as described above.
3468 | Org. Biomol. Chem., 2014, 12, 3459–3469
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9 H. Lee, W. Akers, K. Bhushan, S. Bloch, G. Sudlow,
R. Tang and S. Achilefu, Bioconjugate Chem., 2011, 22, 777–
784.
Acknowledgements
We thank DST (SERB) for financial support and DST-FIST for
400 MHz NMR. M. Ikbal and D. Banerjee are thankful to CSIR 10 (a) W. F. Jager, T. S. Hammink, O. v. d. Berg and
(India), S. Atta to UGC (India), and B. Saha and S. Barman to
IIT KGP for their research fellowships.
F. C. Grozema, J. Org. Chem., 2010, 75, 2169–2178;
(b) M. Tian, X. Peng, J. Fan, J. Wang and S. Sun, Dyes Pigm.,
2012, 95, 112–115; (c) P. Song, X. Chen, Y. Xiang, L. Huang,
Z. Zhou, R. Wei and A. Tong, J. Mater. Chem., 2011, 21,
13470–13475.
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