Synthesis and cytotoxicity study of novel 3-(triazolyl)coumarins based fluorescent scaffolds

Article abstract of DOI:10.1016/j.bmcl.2016.09.078

Recently a choice of fluorescent bioimaging probes have been developed as medical diagnostic tools. Herein, we have introduced a series of coumarin-based target specific probes for cancer theranostic application which play a dual role in the field of both diagnosis and therapy. A fluorogenic version of 1,3-dipolar cycloaddition between azides and alkynes (DBCO) has been introduced to develop the triazolylcoumarin based fluorescent scaffolds. These scaffolds were screened for their anticancer activity against breast cancer (MCF7) and human epitheloid cervix carcinoma (HeLa) cell line. It was established that triazolylcoumarins (5c and 5d) are having electronegative substitution in the benzene ring displayed most effective anticancer profile in both the cell lines. Compounds 5a and 5d exhibited maximum quantum yield and strong cellular uptake in the MCF-7 cell line.

Full text of DOI:10.1016/j.bmcl.2016.09.078

Accepted Manuscript
Synthesis and cytotoxicity study of novel 3-(triazolyl)coumarins based fluores-
cent scaffolds
Sohini Sinha, P.K. Anuja, Debasish Mishra, Priyankar Paira
PII:
S0960-894X(16)31027-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.078
BMCL 24302
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 February 2016
17 September 2016
30 September 2016
Please cite this article as: Sinha, S., Anuja, P.K., Mishra, D., Paira, P., Synthesis and cytotoxicity study of novel 3-
(triazolyl)coumarins based fluorescent scaffolds, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.09.078
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Synthesis and cytotoxicity study of novel 3-
(triazolyl)coumarins based fluorescent scaffolds
Sohini Sinha,† Anuja PK,† Debasish Mishra, Priyankar Paira*
aDepartment of Chemistry, School of advanced sciences, VIT University,
Vellore-632014
bSchool of Bioscience & Technology, VIT University, Vellore-632014, Tamilnadu, India
Email: priyankar.paira@vit.ac.in
ABSTRACT: Recently a choice of fluorescent bioimaging probes have been developed as medical diagnostic tools.
Herein, we have introduced a series of coumarin-based target specific probes for cancer theranostic application
which play a dual role in the field of both diagnosis and therapy. A fluorogenic version of 1, 3-dipolar cycloaddition
between azides and alkynes (DBCO) has been introduced to develop the triazolylcoumarin based fluorescent
scaffolds. These scaffolds were screened for their anticancer activity against breast cancer (MCF7) & human
Epitheloid Cervix Carcinoma (HeLa) cell line. It was established that traizolylcoumarins (5c & 5d) are having
electronegative substitution in the benzene ring displayed most effective anticancer profile in both the cell lines.
Compound 5a & 5d exhibited maximum quantum yield and strong cellular uptake in the MCF-7 cell line.
Keywords: triazolylcoumarin, anticancer activity, theranostic application, fluorescence study,
Fluorescence imaging
The term "theranostic" signifies a specific combination of the diagnostic and therapeutic
capabilities into a single agent (called "companion diagnostic"). This includes the use of
diagnostic tests to identify a particular disease, select a treatment regimen for it and monitor the
patient compliance. The diagnostic tests are used to identify a biomarker which allows the use of
the new drug.1 Theranostic promises significantly in the area of personalized medicine which
customizes healthcare to individual patients and for better patient compliance. The efficient
process of theranostic development is quite complex as it requires a stepwise phasing from
development to remuneration. All the stages of the system should be harmonized to process
simultaneous availability and accessibility of the drugs. In spite of this, it is essential to simplify
the rules and regulations to create a better modification of theranostic towards the robustness of
invention.2 The execution of theranostic is challenging since it possess considerable economic
disputes for industrial organisations as well as the process and timelines for drug and test
development are different from one another.
Corresponding author. Tel.: +91-416-2243091; fax: 91-416-2243092; e-mail: priyankar.paira@vit.ac.in
1

"Click chemistry" is a selective, quantitative, cost effective and pH-insensitive green
approach,3,4 and hence this approach is useful for the synthesis of a variety of bio conjugates
including peptides,5 proteins,6 polysaccharides,7 and even entire viruses8 and cells.9 However, the
conventional "Click Chemistry" requires Cu(I) catalyst for 1,3-dipolar cycloaddition between
azides and terminal alkynes which is toxic to most organisms10 and thus, prevents its use in many
biological systems. The novel Copper-free Click Chemistry is based on the reaction of a
cyclooctyne (DBCO) moiety with an azide-labelled reaction partner, known as strain-promoted
alkyne azide cycloaddition (SPAAC).11 SPAAC has several benefits over the conventional click
technique as it is extremely fast at room temperature, biocompatible, bioorthogonal along with
highly specific and stable in nature.12
3-Azido coumarin, a profluorophore, reacts with terminal alkynes to generate highly
fluorescent triazole compounds which are very useful for biological applications, especially for
in vivo labelling.13 Furthermore, substitutions at the 3- and 7-positions of coumarin dyes are
known to have a strong impact on their fluorescence properties.14 Coumarin derivatives have also
been developed as an anti-tumour agents and their metabolite 7-hydroxycoumarin was tested in
several human tumour cell lines.15 However, the biological actions of coumarins are greatly
enhanced by attaching the triazole rings with these moieties. These unique templates have been
associated with anti-viral, anti-fungal, anti-tumour, anti-bacterial, anti-inflammatory and also
CNS activities.16 These scaffolds were also reported as anti-microbial, anti-inflammatory, anti-
tubercular, anti-HIV, anti-malarial, cardiovascular and diuretic agents.17-20 In light of these
research works going on, we have successfully developed a series of azido coumarins which on
further treatment with DBCO provide a class of fluorescent triazoles for theranostic application.
Our approach was to develop a new class of coumarin-based fluorogenic probes having
cytotoxicity against various cancer cell lines by the application of 1,3-dipolar cycloaddition of
azides and alkynes. Initially, 3-acetamido coumarin analogues (1a-e) were synthesized by using
a choice of substituted salicylaldehyde and N-acetyl glycine in presence of acetic anhydride
under microwave conditions. Microwave is an efficient tools for the construction of carbon-
carbon building blocks with high reaction rate and yield.21 These coumarins were then refluxed
with HCl/Ethanol (1:1) mixture for 2 h and further treated with sodium nitrite followed by
sodium azide to get the desired 3-azido coumarin derivatives (2a-e) (scheme 1).22 Subsequently,
Compound 2a was treated with dibromoethane for the formation of desired
2

bromoalkoxyazidocoumarin (3f). Briefly, compound 2a was dissolved in acetone, K2CO3 (1:10)
was added to the reaction mixture and the resulting solution was refluxed for 30 min at 80°C.
Thereafter, dibromoethane (1:12) was added to it and the mixture was refluxed for 5 hours.
Progress of the reaction was monitored by TLC in hexane/ethyl acetate (3:1) solvent system.
After complete conversion of the starting materials, the solution was filtered and the filtrate was
evaporated to dryness to get the desired product (3f). The structure of compound 3f was
confirmed by 1H-NMR spectroscopy and ESI-MS. The characteristic peaks of two CH2 exhibited
at σ 3.65 & 4.35 as two distinct triplets. Subsequently, DBCO (4) was treated with 3-
azidocoumarin analogues (2a-e, 3f) in 0.5 ml of DMSO-d6 at ambient temperature for 30 min.
The progress of the reaction was monitored by TLC. The reaction resulted in the formation of
two distinct regioisomers (1,4-triazole and 1,5-triazole regioisomer), which is a well known
phenomenon for copper free click reaction. But in all the cases we could not isolate the
regioisomer even after HPLC purification.23 The structure of triazolylcoumarins (5a) was further
confirmed by 1H NMR, 13C NMR and Mass spectroscopy. The characteristic peaks of three CH2
and one NH2 were observed in the range of σ 1.86-1.88, 2.62, 3.18 and 5.04-5.07 ppm
respectively. In the 13C NMR spectra characteristic peaks of tertiary amide & ketone was
observed at σ 169.8 and 159.0 ppm respectively. Likewise, three CH2 peaks were appeared in the
range of σ 33-54 ppm. The quantitative formation of all the 3-(triazolyl)coumarins derivatives
(5a-f) were supported by NMR & Mass spectroscopy.24
3

Scheme 1: Plausible pathway for the synthesis of 3-(triazolyl)coumarin (5a-f)
The fluorescence spectra of compounds 5a-f were determined at concentrations of 3x10-
6mol L-1 in water. Emission spectra of compounds 5a-f are presented in Figure 1. The
fluorescence excitation wavelengths (λex/nm) at 315, 296, 315, 325, 325 & 300 nm were used
for compounds 5a, 5b, 5c, 5d, 5e and 5f respectively. It was also observed that all the
compounds are significantly fluorescent in water (Figure 1, Table 1). The characteristic
fluorescence of compound 5d in DMSO-d6 (reaction solvent) was depicted in Figure 2.
We have calculated the emission of quantum yield (Φ) of the fluorescence active
compounds in water (Table 1). The ΦR was calculated by using below formula, Φ = (ΦR * IS *
ODR * ղs) / ( IR * ODS * ղR) Where ΦR = emission of quantum yield of reference, IS and IR
= integral area of reference and sample respectively, ODS and ODR = excited absorbance of
sample and reference respectively, ղS and ղR = refractive index of sample solvent and reference
solvent respectively. Here, we have used Quinine Sulphate as a standard for calculating emission
of quantum yield (Table 1). We have used 0.5 M H2SO4 & water as solvent for the reference and
synthesized compounds respectively. From Table 1 we found that compound 5a & 5d exhibited
best quantum yields (~ 0.20) among all the synthesized scaffolds.
5f
5b
5c
3000
2500
2000
1500
1000
500
5d
5e
5a
0
360
390
420
450
480
Wavelength (nm)
(a)
(b)
(c)
Figure 1. UV & Fluorescence emission spectra of 3-(triazolyl)coumarin (5a-f) in water
4

4
3d
5d
Figure 2 Fluorescence images of Starrting material (4, 3d) & product (5d) in DMSO-d6 under
UV chamber (λ )
Table 1. Spectroscopic data for 3-(triazolyl)coumarin (5a-f) at 298 K in water.
a
b
Entry
λmax
λmax
Stokes
ODc
Id
Φe
(abs,nm) (em,nm)
Shift (nm)
315
296
315
325
325
300
350
382
382
382
378
384
378
452
67
86
67
53
59
0.12
2.4
2.46
0.24
1.68
1.3
79195
46928
68596
182975
7867
0.16
0.00478
0.006
0.20
0.0011
0.04
5a
5b
5c
5d
5e
78
102
168450
63905
5f
Quinine
Sulphate
0.029
0.57
aabs = absorbance; bem = emission; cOD = excited absorbance; dI = integral area; eΦ = emission of quantum yield
The cytotoxicity of all the synthesized compounds (2a–e & 5a-f) (Table 2) was evaluated
using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) bioassay
beside a panel of cell lines such as human epithelioid cervix carcinoma (HeLa) and human breast
carcinoma cell line (MCF-7) in triplicates. These cells were incubated with compounds (2a–e &
5a-f) along with Cisplatin and Doxorubicin as standard drugs at concentration 1–100 µM for 24
5

h. Most of these triazolocoumarin (5a-f) scaffolds exhibited comparable anticancer activity with
cisplatin. However, these compounds showed less potency than doxorubicin. Likewise,
azidocoumarin analogues (2a-e) & DBCO (4) were not showing any potency in both the cell
lines. DMSO was used as control where it was also not showing any inhibition of cell. In MCF-7
cells, the IC50 values for triazolylcoumarin derivatives were in the range of 9–21µM (Table 2).
Similarly, in HeLa cells, the IC50 values were found in the range of 17–26 µM. In the literature, it
was found that substitution of strong electronegative atom such as chloro/bromo at the para
position of the aromatic ring increases the lipophilicity of molecules which is accountable for
enhanced cytotoxicity in MTT model.25 In our present work, it was observed that compounds 5c
& 5d having halogen substitution in the benzene ring shows very significant cytotoxicity in both
the cells whereas corresponding azidocoumarin derivatives (2c & 2d) did not show any potency.
Moreover, starting DBCO moiety was also not showing any potency in both the cancer cell lines.
Therefore, the cyclooctyne fused triazole moiety might be the key template for high potency in
cancer cell lines. The structure–activity relationship studies revealed that the introduction of
electronegative chlorine & bromine atom in the benzene ring (compound 5c & 5d) enhanced the
cytotoxicity. While, the anticancer efficacy is being highly reduced once introduction of electron
releasing groups (OH, OMe, alkyl) in the triazolylcoumarin rings (5a, 5b, 5f). Herein, we have
used normal human fetal lung fibroblast (MRC-5) as a model of healthy cell to estimate the
selective potency of these scaffolds in cancer cell lines with respect to normal cell line. It was
observed that most of the 3-(triazolyl)coumarins are 3-11 fold more selective in cancer cell line
than normal fibroblast. Among all these scaffolds, compound 5d displayed best potency &
selectivity profile in all the reported cancer cell lines. While, cisplatin was not exhibited any
selectivity in those cancer cell lines.
6

Table: 2 Preliminary MTT cytotoxicity screening of synthesized triazolylcoumarin derivatives
(5a-f), azidocoumarins (2a-2e) and DBCO (4) at 24 h of drug exposure
Compounds
IC50 (µM)a
SFb
Cell lines
HeLa
MCF-7
MRC-5
90.2±0.87
92.8±1.54
100.2±1.88
185.22±1.65
110±1.43
HeLa
4.27
3.54
5.49
10.58
5.55
6.19
1.50
1.61
2.48
2.43
2.37
1.88
-
MCF-7
4.90
4.24
9.40
18.84
8.68
6.52
1.63
1.85
2.97
3.60
2.63
2.15
-
21.12±0.91 18.4±0.87
26.21±0.64 21.87±1.67
18.24±1.15 10.65±0.68
17.5±1.22 9.83±0.69
19.8±0.88 12.66±1.11
5a
5b
5c
5d
5e
5f
2a
2b
19.5±0.66 18.51±0.90 120.87±1.88
41.62±0.81 38.4±0.97
36.41±0.54 31.54±2.17
28.34±1.15 23.65±0.68
27.6±1.22 18.63±1.69
29.6±1.88 26.66±1.11
62.7±1.44
58.66±1.89
70.4±1.66
67.2±0.88
70.2±1.11
60.4±1.11
-
2c
2d
2e
32.06±1.22
-
28±1.22
-
4
DMSO
CisPlatin
Doxorubicin
18±0.5
5.2±0.92
10±0.6
3.83±0.49
20.2±1.2
70.6±1.55
1.12
13.57
2.02
18.43
aIC50 is the concentration at which 50% of cells were undergoing cytotoxic cell death due to
synthesized compound treatment. cSF(selectivity factor) = ratio of IC50 for MRC-5/IC50 for all
the cancer cell lines. MRC-5 fibroblasts are typically chosen as models for health cells to
evaluate the selectivity of chemotherapeutic drugs.
We performed cellular imaging experiment using MCF-7 cell line. MCF-7 cells were
incubated for 2 hour at 37° C with compound 5a & 5d (20 μM). We observed strong green
fluorescence in live cells using excitation and absorbance filters 320 and 400 nm, respectively
(Figure 3). In Figure 3, a strong cellular uptake of compound 5a & 5d was observed. Hence, we
concluded that compound 5a & 5d might be applicable as cancer theranostic for its high
quantum yield as well as high potency & selectivity in cancer cell lines.
7

(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 2. Fluorescence microscopy images and phase photos of live cells: (a) Phase photo of MCF-7 cells
without the drug (b) Fluorescent image of MCF-7 with compound 5d (20 µM in PBS buffer); incubation
time 2 h (c) Fluorescent image of MCF-7 with compound 5d (20 µM in PBS buffer); incubation time 2 h,
zoom scan (d) Phase photo of MCF-7 cells with compound 5d; 8 h incubation (e) Fluorescent image of
MCF-7 with compound 5d (20 µM in PBS buffer); incubation time 8 h (f) Fluorescent image of MCF-7
with compound 5a (20 µM in PBS buffer); incubation time 2 h (g) Fluorescent image of MCF-7 with
compound 5a (20 µM in PBS buffer); incubation time 2 h, zoom scan
8

Conclusion
In review, a class of 3-(triazolyl)coumarins derivatives was synthesized using a simplest
& effective technique. This technique allows a great compact of synthetic flexibility and offers
the opportunity of synthesizing newer 3-(triazolyl)coumarins analogues for anticancer screening.
Interestingly, 3-(triazolyl)coumarins (5d) is having two electronegative chlorine substitutions in
the benzene ring shows most potency in both the cancer cell lines. Compound 5d might be
utilized for cancer theranostic because of its high quantum yield, high potency and strong
cellular uptake.
Acknowledgement:
We are thanking to the VIT University-India for financial support in our project. We
acknowledge to DST, New Delhi for VIT-DST-FIST project. We also acknowledge to DST-
SERB, India for young scientist grant.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/....
†Equal contribution
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24. Preparation of dibenzo triazolo[4,5-d] azocinyl chromen-2-one (5a-f): 3-Azido coumarin derivatives
(2a-e, 3f) were shaken with DBCO (4) (1:1 mole ratio) in 0.5 ml of DMSO-d6 at room temperature
for 30 minutes at 1000 rpm. The reaction was completed within 30 minutes which was confirmed by
TLC. Consequently, NMR was performed with the solution. The Solvent has been evaporated and a
mixture of regioisomers (1:1) was formed with high yield.
3-(8-(3-aminopropanoyl)-8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-3-yl)-7-hydroxy-
1
2H-chromen-2-one (5a): Yield: 95%; mp: 128-130°C; Rf (2% MeOH in EA): 0.36; H NMR (400
MHz, DMSO-d6): σ 1.86-1.88 (m, 2H, CH2), 2.62 (brs, 2H, CH2), 3.18 (s, 2H, CH2), 5.04-5.07 (m,
2H, NH2), 7.23-7.40 (m, 7H, ArH), 7.48-7.51 (m, 3H, ArH), 7.63-7.65 (m, 2H, ArH); 13C NMR (125
MHz, DMSO-d6): σ 33.4 (CH2), 35.8 (CH2), 54.9 (CH2), 101.8 (CH), 107. 9 (CH), 109.0 (CH), 113.0
(CH), 114.3 (CH), 121.5 (CH), 122.3 (CH), 125.2 (CH), 125.7 (C), 126.9 (CH), 127.8 (C), 128.1
(CH), 128.4 (C), 128.8 (C), 129.1 (CH), 129.6 (CH), 130.3 (C), 132.4 (CH), 148.2 (C), 149.3 (C),
150.9 (C), 156.2 (-COH), 159.0 (-OCO), 169.8 (-NCO); ESI-MS (MeOH): 480 [M+H]+.
3-(8-(3-aminopropanoyl)-8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-3-yl)-6-bromo-2H-
1
chromen-2-one (5c): Yield: 97%; mp: 138-140°C; Rf (2% MeOH in EA): 0.48; H NMR (400 MHz,
DMSO-d6): σ 1.84-1.88 (m, 2H, CH2), 2.61-2.66 (m, 2H, CH2), 3.63 (s, 2H, CH2), 5.0-5.05 (m, 2H,
NH2), 7.03 (d, 1H, J = 8.8 Hz, ArH), 7.25 (brs, 1H, ArH), 7.28-7.30 (m, 1H, ArH), 7.32-7.38 (m, 3H,
ArH), 7.44-7.49 (m, 4H, ArH), 7.61-7.63 (m, 2H, ArH); 13C NMR (125 MHz, DMSO-d6): σ 35.9
(CH2), 36.9 (CH2), 54.8 (CH2), 103.7 (CH), 108.1 (CH), 114.2 (CH), 115.7 (CH), 116.6 (C), 121.4
(C), 122.3 (C), 122.4 (C), 123.8 (C), 125.2 (CH), 126.7 (CH), 127.7 (CH), 128.1 (CH), 128.2 (CH),
10

128.8 (C), 128.9 (CH), 129.3 (C), 129.6 (CH), 132.4 (CH), 144.6 (C), 148.3 (C), 151.4 (C), 154.0 (-
OCO), 170.5 (-NCO); ESI-MS (MeOH): 542 [M+H]+.
3-(8-(3-aminopropanoyl)-8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-3-yl)-6,8-dichloro-
1
2H-chromen-2-one (5d): Yield: 90%; mp: 129-131°C; Rf (2% MeOH in EA): 0.44; H NMR (400
MHz, DMSO-d6): σ 1.86-1.93 (m, 2H, CH2), 2.68-2.76 (m, 2H, CH2), 3.20 (s, 2H, CH2), 5.01-5.06
(m, 2H, NH2), 6.99 (s, 1H, ArH), 7.28-7.30 (m, 1H, ArH), 7.32-7.39 (m, 3H, ArH), 7.45-7.49 (m, 3H,
ArH), 7.57 (s, 1H, ArH), 7.61-7.62 (m, 2H, ArH); 13C NMR (125 MHz, DMSO-d6): σ 31.6 (CH2),
35.1 (CH2), 54.8 (CH2), 107.7 (CH), 112.7 (CH), 114.3 (CH), 120.1 (C), 121.5 (C), 122.3 (C), 124.1
(C), 125.3 (CH), 126.2 (C), 126.8 (C), 126.9 (CH), 127.8 (CH), 127.9 (C), 128.2 (CH), 128.4 (CH),
128.6 (CH), 129.2 (C), 129.4 (CH), 132.3 (CH), 143.0 (C), 148.0 (C), 150.1 (C), 157.4 (-OCO),
169.4 (-NCO); ESI-MS (MeOH): 532 [M+H]+
3-(8-(3-aminopropanoyl)-8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-3-yl)-2H-
1
chromen-2-one (5e) Yield: 94%; mp: 108-110°C; Rf (2% MeOH in EA): 0.40; H NMR (400 MHz,
DMSO-d6): σ 1.83-1.90 (m, 2H, CH2), 3.05-3.10 (m, 2H, CH2), 3.66-3.68 (m, 2H, CH2), 5.05-5.08
(m, 2H, NH2), 6.68-6.74 (m, 2H, ArH), 6.97 (t, 1H, J = 6.0 Hz), 7.10-7.36 (m, 2H, ArH), 7.38-7.46
(m, 5H, ArH), 7.47-7.51 (m, 2H, ArH), 7.64 (t, 1H, J = 7.2 Hz, ArH); ESI-MS (MeOH): 464 [M+H]+
25. Hari, H. P.; Umashankar, D.; Wilson, J. Q.; Masami, K.; Hiroshi, S.; Jonathan, R.D. Eur. J. Med.
Chem.2008, 43, 1.
11

Graphical Abstract
Synthesis and cytotoxicity study of novel 3-(triazolyl)
coumarin based fluorescent scaffolds
Sohini Sinha,a† Anuja PK,a† Debasish Mishra,b Priyankar Pairaa*
aDepartment of Chemistry, School of advanced sciences, VIT University, Vellore-632014, Tamilnadu, India
bSchool of Bioscience & Technology, VIT University, Vellore-632014, Tamilnadu, India