Click-on fluorescent triazolyl coumarin peptidomimetics as inhibitors of human breast cancer cell line MCF-7

Article abstract of DOI:10.1039/c7nj02712e

The development of a series of multipurpose triazolyl coumarin peptidomimetics that can also work as click-on fluorescent inhibitors of human breast cancer cell line MCF-7 is described. The quantum mechanical calculations indicate that the click-on fluore

Full text of DOI:10.1039/c7nj02712e

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Received 00th
January 20xx,
Click-on Fluorescent Triazolyl Coumarin Peptidomimetics as
Inhibitors of Human Breast Cancer Cell line MCF-7
P. Thasnima and D. Bahulayana*
The development of a series of multipurpose triazolyl coumarin peptidomimetics that can also work as click-on fluorescent
inhibitors of human breast cancer cell lineMCF-7 is described. The quantum mechanical calculations indicate that the click-
on fluorescence observed when the azidomethyl cumarin precursors up on click cycloaddition reactions with various
alkynes could be due to the decrease in electron density and increase in the gap between the HOMO energy levels of the
excited coumarin unit and the methyl group upon triazole formation which in turn reduce the process of acceptor
photoinduced electron transfer (a-PeT). The tunable and intense click-on fluorescence observed when the cumarin alkynes
replaced with bioisosteric open chain ketoamidespoint to the possibility for developing large number of multipurpose
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
probs/anticancer
agents
throgh
systamatic
structure
alterations
in
the
system.
background contrast. Recently, various click chemistry
methods such as Cu(I) catalyzed azide-alkyne cycloaddition
reactions,9 enamine10 and enolate11 mediated organocatalytic
Introduction
Coumarin framework is a privileged scaffold in medicinal
chemistry and are found in many natural products and
therapeutics.1Several coumarin derivatives have anti-bacterial,
anti-fungal,anti-coagulant and anti-HIV activities.2 The
pharmacological, biochemical and therapeutic properties of
coumarin derivatives have strong dependence on the nature
as well as positioning of its structural substituents.3For
example, 3-phenyl coumarin derivatives containing carbamate
groups are efficient for inhibiting the cell cycle arrest activity of
the HIV-1Vpr.4 Similarly, coumarin derivatives bearing an
electron donating heteroatom at the 7-position are widely
used as fluorophores since the presence of an electron
donating group at 7-position strongly enhances their
fluorescence.5 Fluorogenic substrates based on 4-
methylumbelliferones (4-methyl-7-hydroxycoumarin) have
been widely used for the detection of phosphatase and
glycosidase activities which are directly related to human
breast and colon cancer.6However, thesesubstrates require an
alkaline pH to obtain maximum fluorescence.7In order to
overcome such issues, a wide variety of fluorescent probes
have been developed over the years for imaging and
quantification of biological entities.8 However, a majority of
cycloaddition
reactions,strain
promoted
azide-alkyne
cycloaddition reactions12 and sulfur(VI) fluoride exchange
(SuFEx) click reactions13etc. have been emerged as viable tools
for the synthesis of multifunctional hybrid molecules including
various fluorescent probes. Click-on fluorescent probes are of
interest because these probes have low background emission
due to the masking of the fluorophore with donor azide or
acceptor alkyne functionality present in these scaffolds before
the click reaction. The ligation of such functionalized scaffolds
through click reaction produces a linker triazole which can
effectively delocalize the local electrons to reinstate the
fluorescence. Successful examples of such click-on probes
include scaffolds based on coumarin14, anthracene15 and
naphthalimide16 etc. Motivated with these developments and
also in continuation of our research on the development of
multifunctional small molecules based on click chemistry, here
we report a rational design and experimental validation of a
series of click-on blue emitting triazole functionalized
coumarin scaffolds (TC1-4&ATC1-6) which are active against
human breast cancer cell line MCF-7.
these dyes shows continuous fluorescence before and afterthe Experimental
imaging process and the presence of anyunreacted dyecould
also contribute to this, resulting in a reduced signal to
Starting materials were purchased from Aldrich and Merck. Alkynes
and azides were synthesized by following literature procedure. High
resolution mass spectra were measured with a Thermo Exactive
Orbitrap with MeOH as solvent. FT-IR spectra were recorded on a
JASCO-FTIR-4100 spectrometer. Absorption spectra of the
compounds were recorded on
a
JASCO V-550 UV/Vis
Spectrophotometer and fluorescence measurements were carried
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out with a Perkin Elmer LS55 Fluorescence Spectrometer. The Hydroxycoumarin (1mmol) and K2CO3 (414 mg, 3 mmol) in DMF
fluorescence quantum yield (Φf) was computed using the below were kept at 50 °C for half an hour and then cooled to room
equation,
temperature. To this, propargyl bromide (0.119 mL, I mmol) was
added and stirred for 4 hours. The mixture was then poured into ice
water. The precipitate formed was filtered and washed with water
2
2
Φf = ΦfRFSARɳS / FRASɳR
where FS and FR are the integrated fluorescence intensities of the to afford pure propargylatedcoumarin1-3.
sample and reference, AS and AR are the absorbance of the sample
and reference at excitation wavelength, ɳS and ɳR are the refractive Synthesis of type coumarin alkyne 4
indexes of the solvents used for the sample and reference.
Anthracene in ethanol solution was used as the reference (Φfl
=
A solution of coumarin-3-carboxylic acid (1.9 g, 0.01 mol), propargyl
0.27) as standard. Fluorescence decay curves were recorded with a alcohol (0.56 mL, 0.01 mol), N,N-dicyclohexylcabodiimide (2.3 g,
Horiba Fluorolog Fluorescence Spectrometer with TCSPC and 011 mol), and 4-dimethylaminopyridine (0.122 g, 0.001 mol) in
lifetime τ was calculated using the below equation using three dichloromethane were stirred at room temperature for 6 h. After 6
exponentials where T stands for Time and B stands for relative h, the precipitated N,N-dicyclohexylurea was filtered off and the
amplitude.
filtrate was washed with water, 5% acetic acid, and again with
water. The filtrate was then dried over magnesium sulphate and the
solvent was removed under vacuum. The residue obtained was
washed with petroleum ether to afford the alkyne 4(prop-2-yn-1-yl
2-oxo-2Hchromene-3-carboxylate) (2.4 g, 96%).
τ = T1B1+T2B2+T3B3…/100
Synthesis of 4-Bromomethyl umbeliferone BMU
Placed a solution of 4-methylumbeliferone (176 mg, 10 mmol) in20
mL glacial acetic acid in a 500 mL flask. To this, 1.598 g (0.4982 mL, Synthesis ketoamide alkynes 5-10
10 mmol) of bromine in 10 mL glacial acetic acid was added from a
dropping funnel and the mixture was stirred vigorously during the A round bottom flask was charged with propargyl derivative of
addition of bromine while maintaining the temperature between 0- hydroxybenzaldehyde (2 mmol), ketone (2 mmol), acetyl chloride (2
5 °C. After the addition of bromine, the mixture was kept at room mL), acetonitrile (1 mL) and catalytic amount of BF3.Et2O and the
temperature for another 30 minutes and then poured into excess mixture was kept under stirfor 3h at room temperature.The
water and stirred well. The precipitate obtained was filtered, progress of the reaction was monitored by TLC at regular intervals.
washed with cold water and crystallized from dilute ethanol to After 3h, the mixture was quenched with cold water. The solid
afford pure BMU (white powder). 4-(bromomethyl)-7-hydroxy-2H- residue separated was collected and purified by passing it through a
chromen-2-one. FT-IR (KBr) νmax ; 1698, 1620, 1600, 1548, 1511, silica gel column. Elution of the column with petroleum ether (60-
1441, 1375, 1352, 1263, 1232, 1204, 1141, 1075, 1000, 945, 848, 80 ºC) / ethyl acetate (9: 1 v/v) afforded 5-10.
836, 814, 750, 739, 725, 692, 633, 618, 556, 516,99, 492, 471 ,458,
453, 444, 434, 429, 421, 413, 406, 401 cm-1 1H-NMRδ: 2.490 (s, General procedure for the Cu (I) catalyzed (3+2) azide-alkyne 1, 3-
;
2H), 6.972-6.994 (d, 2H), 7.682-7.703 (d, 2H), 8.232 (s, 1H) ppm; dipolar cycloaddition reactions for the synthesis of TC1-4 and ATC
HRMS (EI) m/z calcd for C10H7BrO3[M+H]+: 254.9579 , found: 1-6
[M+H]+: 255. 0365.
An equimolar amount of 4-Azidomethyl umbelliferone4-AMU (1
Synthesis of 4-Azidomethyl umbeliferone 4-AMU
mmol) and any one of the alkynes 1-4 or 5-10 (1 mmol) are
dissolved in minimum amount of DMSO. To this, 2 mL of t-BuOH, 1
BMU (254 mg, 1 mmol), K2CO3 (414 mg, 3 mmol) and sodium azide mL of water, CuSO4-5H2O (200 mg) and sodium ascorbate (150 mg)
(65 mg, 1 mmol) were stirred at room temperature in DMF. After are added and stirred at room temperature for 12 h, and then
completion of the reaction, the mixture was poured into ice cold poured in to cold water. The precipitated click product was filtered,
water. The solid product obtained was filtered and dried under washed with water and dried under vacuum to afford the
vacuum to afford 4-(azidomethyl)-7-hydroxy-2H-chromen-2-one(4- respective TC or ATC in pure form. Typical spectral data for 7-
AMU) in pure form (Brown powder). FT-IR (KBr) νmax; 3392, 2920, hydroxy-4-((4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)-1H-1,2,3-
2851, 2122, 1701, 1608, 1550, 1507, 1445, 1377, 1340, 1315, 1261, triazol-1-yl)methyl)-2H-chromen-2-one (TC1- white powder): FT-IR,
1201, 1161, 1146, 1105, 810, 754, 725, 631, 580, 552, 529, 456 cm- KBr, νmax: 3439 , 2922, 2852, 1715, 1619, 1572, 1509, 1467, 1389,
1
;
1H-NMR, δ: 2.142 (2, 2H), 6.669 (s, 1H), 6. 732-6. 762 (d, 1H), 1349, 1326, 1295, 1225, 1206, 1151, 1063, 1019, 984, 941, 844,
6.820-6.841 (d, 1H), 7.361-7.383 (d, 1H), 7.668-7.690 (d, 1H), 822, 765, 686 cm-1; 1H NMR (400 MHz, DMSO-d6) δ; 2.297-2.321 (d,
8.229(s,1H)ppm;13C NMR (100 MHz, DMSO-d6): δ167.01, 163.01, 3H), 4.022-4.070 (q, 2H), 5.023 (s, 1H), 5.464-5.468 (d, 2H), 5.998 (s,
156.67, 154.65, 129.31, 127.95, 125.01, 113.65, 112.75, 102.13, 2H), 6.165 (s, 1H), 7.314-7.438 (m, 3H), 7.554 (s, 1H), 7.570-80121
56.30, 40.12, 39.91, 39.70, 39.08, 38.07 ppm; HRMS (EI) m/z calcd (m, 4H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 168.20, 160.80,
for C10H7N3O3 [M]+:217.0487, found: [M]+: 217.0118.
160.27, 156.05, 141.43, 135.53, 135.07, 131.75, 130.04, 127.80,
127.47, 127.27, 114.52, 114.33, 87.47, 66.48, 60.66, 48.33,45.33,
40.12, 39.31, 33.24, 33.07,30.87, 30.66 ppm ; HRMS (EI) m/z calcd
for C22H15N3O6 [M+H]+: 418.0961 , found: [M+H]+: 418.1029.
General procedure for the synthesis of coumarin alkynes 1-3
2 | J. Name., 2012, 00, 1-3
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Results and discussion
Scheme 3.List of TriazolylCoumarins (TC1-4) synthesized.
Our study began with the synthesis of an azidomethylcoumarin
4-AMU) from 4-methyl umbeliferone. 4-AMU was obtained in
(
We then performed the Cu (I) catalyzed azide -alkyne [3+2]
cycloaddition reaction20 between the coumarinazide 4- AMU
and coumarin alkynes 1-4 to study the fluorogenicity after the
triazole linker formation between the two non-fluorescent
a two-stepprocess starting with the bromination of 4-methyl
umbeliferone (4-MU) using Br2/CH3COOH.17 The structure of
this compound was confirmed by FT-IR (750 cm-1, C-Br
stretching), 1H NMR (3.97 ppm, 2H, s), 13C NMR (43 ppm, C
atom of –CH2Br) and HRMS analysis. The 4-bromomethyl
coumarin scaffolds. Various triazole products, namely TC1-4
,
were obtained as shown in scheme 3 and were characterized
by by spectroscopic analysis.
umbeliferone thus obtained
(4-BMU) showed a weak
fluorescence at 438 nm, due to the less electron donating
effect of bromomethyl group at the 4- position of the
coumarin ring. In the subsequent step, 4-BMU was treated
with NaN3 in DMF at 60-65 °C to obtain the azidoderivative, 4-
AMU 18
. 4-AMU also showed a very weak fluorescence at 413
nm due to the quenching effect imparted by the electron-rich
nitrogen atoms of the azido group. The overall synthetic
procedure for obtaining 4-AMU is shown in Scheme 1.
Scheme 1.Synthesis of 4-Azidomethyl umbeliferone (4-AMU).
Figure 1.Enhancement of fluorescence upon Click reaction of 4-AMU
and alkyne 1.
Next, we synthesized 4 different coumarin alkynes (1-4) by the
propargylation of various coumarin derivatives.19 These
molecules also didn’t show any fluorescence.
The photophysical properties of 4-AMU and its click ligation
products TC1-4 were measured in DMSO at pH 6.8 and are
shown in Table 1. As mentioned before, 4-AMU showed weak
fluorescence at 413 nm, however, after the click reaction, the
molecule become turn-on or click-on fluorescent and the
intensity of the emission was significantly increased with a
bathochromic shift in wavelength to the range of 432-459 nm
in accordance with the presence of various functional groups
in them (Figure 1).To evaluate fluorescence enhancement
upon triazole formation, fluorescence quantum yields of 4-
AMU and TC1-4 were measured using anthracene in ethanol
(Φfl= 0.27) as standard. Before the click reaction, 4-AMU had a
quantum yield of 0.14 and after the click reaction, this values
increased to the range of 0.74 to 0.87 with the highest value of
O
O
O
Br
O
K2CO3,
R1
R1
R3
R3
DMF, 55 °C
R2
R2
1
1-4
O
R1 = H
R2 = OH
R3 = H
R1 = H
R2 = H
R3 = OH
R1 = H
O
O
O
O
2
3
4
O
1 (82 %)
2 (86 %)
O
O
O
R2 = CH3
R3 = OH
R1 = COOH
R2 = H
O
O
O
O
3 (79 %)
R3 = H
4 (80 %)
Scheme 2.Synthesis of coumarin alkynes 1-4
.
0.87 for TC1
.
Table 1. Photophysical properties of click products and their
O
O
precursors
CH2N3
O
O
CuSO4 / Na Ascorbate
3o Butanol:H2O:DMSO
(4:2:1)
OH
λmax
(abs)
(nm) [a]
340
λmax
(emi)
(nm) [a]
413
Stoke’s
shift
(nm) [b]
73
O
O
N
N
CH2
[c]
ɸfl/Ɛmax
ɸflxƐmax
N
Compound
HO
O
O
O
O
1-4
4-AMU
O
TC1-4
O
4-AMU
TC1
0.14/0.0078
0.87/7.21
0.78/6.93
0.80/7.82
0.74/7.34
0.50/6.06
0.58/6.32
0.60/7.56
0.62/8.13
0.55/6.88
0.52/6.98
0.0011
6.27
5.40
6.26
5.43
3.03
3.66
4.53
5.04
3.78
3.63
315
459
144
O
O
TC2
TC3
TC4
ATC1
ATC2
ATC3
ATC4
ATC5
ATC6
322
326
337
334
325
340
335
342
432
452
446
474
468
458
473
469
110
126
109
140
143
118
138
127
O
O
O
O
N
N
N
N
O
N
N
O
TC1 (85 %)
Abs/Em(nm)
3 315/459
OH
TC2 (79 %)
Abs/Em(nm)
322/432
OH
CH
O
O
O
O
O
O
N
N
OH
N
OH
O
N
O
N
O
N
TC3 (82 %)
Abs/Em(nm)
326/452
O
O
TC4 (75 %)
Abs/Em(nm)
337/446
310
450
140
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[a] Absorption and emission maxima were measured in DMSO and are given in
nm. [b] Given in nm. [c] Quantum yields were determined by using anthracene in
ethanol (Φfl = 0.27) as standard, extinction coefficients Ɛmaxare in 103 M-1cm-1.
Figure 3: Fluorescence decays measured by time-correlated single photon
counting.
The Stokes shift of 4-AMU and TC1-4 were calculated and are
listed along with absorption and emission maxima in Table 1.
All the four click ligation products showed high Stokes shift
values with a maximum of 144 nm for TC1. The high Stokes
shift values observed for these compounds revealed that these
click-on dyes have high signal to background contrast. The
maximum absorption (336 nm) and least Stokes shift (109 nm)
observed forTC4 could be due to the presence of an ester
group on the alkyne part. The Stokes shift values observed for
these new click-on dyes are superior to the same reported for
In order to understand the fluorescence enhancement upon click
reaction, we measured the fluorescence lifetime of 4-AMU and
TC1and the decay curves are shown in Figure 3. 4-AMU has a
fluorescence lifetime of 0.97 ns, whereas TC1 has a fluorescence
lifetime of 2.04 ns. The high fluorescence lifetime observed for
these molecules also supports the high quantum yield obtained for
these dyes.
We then decided to study the effect of structural changes on
click-on fluorescence by replacing the coumarin alkyne part in
TC with an open chain scaffold which is a physico-chemical
equivalent of the cyclic lactone coumarin ring. Since esters and
amides are bioisosteres,23 we decided to replace coumarin
alkyne with an open chain ketoamide as shown in scheme 4.
commercially available bio-imaging probes like AlexaFluors(R)
.
We have also studied the solvent effect on fluorescence
intensity of these new click-on dyes since a change in solvent is
accompanied by a change in polarity, dielectric constant and
polarizability of the surrounding medium (Figure 2).21
TC1showed weak fluorescence in solvents such as ethyl
acetate, dichloromethane, acetonitrile and strong fluorescence
in polar solvents like DMF and DMSO. Interestingly, the
emission of TC1 in methanol showed a bathochromic shift of
50 nm which corresponds to a large Stokes shift of 193 nm.
The high Stokes shift observed for TC1 in methanol indicate
that the excited state geometry of TC1 in methanol could be
significantly different from that of the ground state, resulting
in a large value of excited state dipole moment.22Studies in
other solvents did not show any significant shift in the
emission maxima.
Scheme 4. Isosteric substitution of coumarin alkyne part in TC with a
ketoamide scaffold.
The ketoamides 5-10 were prepared by following the protocol
showed in scheme 524 and were connected to 4-AMU via Cu(I)
catalyzed azide
- alkyne [3+2] cycloaddition to obtain
triazolylcoumarins ATC1-6
.
Figure 2.Absorption (green line) and emission spectra of TC1 in DMSO (black
line), DMF (red line), ethyl acetate (blue line), dichloromethane (dark yellow
line), acetonitrile (magenta line) and methanol (dark cyan line).
Scheme 5.Synthesis of acetamidoketone alkynes 5-10.
The fluorescence properties of these molecules were also
examined (Table 1). Before click reaction the molecules did not
show any fluorescence. However, after click reaction, ATC1-6
become click-on fluorescent and they were also showed an
increase in fluorescence intensitywith a red shift in emission
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group, which restricts the a-PeT process to the coumarin core and
hence fluorescence could be restored.
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supplementary Information for cytotoxicity evaluation details).
Conclusions
In conclusion, we have designed
activatablecoumarin fluorescent inhibitors and probes by the
triazole derivatization of azidomethylcoumarin4-AMU 4-AMU
a
series of CuAAC-
.
showed little fluorescence (Φfl= 0.14) before click reaction.
However, upon triazole formation with various alkynes, a strong
fluorescence is induced with excellent enhancement of
fluorescence quantum yield at a long excitation wavelength above
450 nm. Quantum mechanical calculations indicate that conversion
of azidomethane to the corresponding triazoles results in
a
significant decrease in the electron density and the increase of the
HOMO energy level of the triazole-substituted methyl moiety. This
suggests that the fluorescence enhancement upon CuAAC reaction
is due to the inhibition of an a-PeT process. The cytotoxicity
evaluation results are promising and point to possibilities of these
molecules as potential inhibitors of human breast cancer cell line
MCF-7.
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Acknowledgements
Wang,Angew Chem., 2014, 126, 14410–14414. d) D. B.
P. Thasnim wishes to thank Council of Scientific and Industrial
Research (CSIR) New Delhi for research fellowship. We are
grateful to CSIR-NIIST Trivandrum, IISER Trivandrum, NIT
Calicut and Mahatma Gandhi University Kottayam for
analytical support.
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Table of Contents
Click-on Fluorescent Triazolyl Coumarin Peptidomimetics as Inhibitors of Human
Breast Cancer Cell line MCF-7
P. Thasnim and D. Bahulayan
A fluorogenic click reaction which enables the formation of fluorescent and bioactive triazolylcoumarins via photo induced
electron transfer process is described.

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New Journal of Chemistry
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DOI: 10.1039/C7NJ02712E
A fluorogenic click reaction which enables the formation of fluorescent and bioactive
triazolylcoumarin via photo induced electron transfer process is described.