key: cord-0719676-4f8ynh0w
authors: Pršir, Kristina; Horak, Ema; Kralj, Marijeta; Uzelac, Lidija; Liekens, Sandra; Steinberg, Ivana Murković; Krištafor, Svjetlana
title: Design, Synthesis, Spectroscopic Characterisation and In Vitro Cytostatic Evaluation of Novel Bis(coumarin-1,2,3-triazolyl)benzenes and Hybrid Coumarin-1,2,3-triazolyl-aryl Derivatives
date: 2022-01-19
journal: Molecules
DOI: 10.3390/molecules27030637
sha: e5d73bc362200cc735c7cd306c18743238a40b6b
doc_id: 719676
cord_uid: 4f8ynh0w

In this work, a series of novel 1,2,3-triazolyl-coumarin hybrid systems were designed as potential antitumour agents. The structural modification of the coumarin ring was carried out by Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition of 7-azido-4-methylcoumarin and terminal aromatic alkynes to obtain 1,4-disubstituted 1,2,3-triazolyl-coumarin conjugates 2a–g, bis(1,2,3-triazolyl-coumarin)benzenes 2h–i and coumarin-1,2,3-triazolyl-benzazole hybrids 4a–b. The newly synthesised hybrid molecules were investigated for in vitro antitumour activity against five human cancer cell lines, colon carcinoma HCT116, breast carcinoma MCF-7, lung carcinoma H 460, human T-lymphocyte cells CEM, cervix carcinoma cells HeLa, as well as human dermal microvascular endothelial cells (HMEC-1). Most of these compounds showed moderate to pronounced cytotoxic activity, especially towards MCF-7 cell lines with IC(50) = 0.3–32 μM. In addition, compounds 2a–i and 4a–b were studied by UV-Vis absorption and fluorescence spectroscopy and their basic photophysical parameters were determined.

Coumarin (2H-chromen-2-one) has become a privileged heterocyclic moiety in biomedical chemistry and novel drug discovery. The chemistry of coumarin has been profoundly examined as a result of its wide distribution in nature, and the extensive biological activity of its derivatives, including anticancer [1, 2] , antioxidant [3] , antiviral [4] , antibacterial and antitubercular properties [5] . Due to its lipophilic, aromatic and planar structure, the coumarin ring can easily bind different biological targets by π-π, hydrogen or dipole-dipole interactions and therefore is incorporated in the structure of various important bioactive compounds [6] .

In addition, the therapeutic potential of coumarins strongly depends on their basic structure [7] as seen in simple, bis-and fused polycyclic coumarins [8] [9] [10] [11] [12] . Furthermore, the nature and position of substituents on the benzopyrone core can affect the pharmacological properties and biological activities of the resulting molecule. Design and synthesis of coumarin-based hybrid molecules by introducing one or more heterocyclic moieties in the coumarin core is a promising approach in the development of novel therapeutic agents with improved and diverse activity [13] .

In the context of easy structural modification, the concept of click chemistry has received great attention. The Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition is considered to be the typical click reaction in which 1,4-disupstituted 1,2,3-triazole is formed [14] [15] [16] . Over the past decade, the 1,2,3-triazole ring has emerged as one of the most important building blocks in the synthesis of molecular hybrids, and a vast number of molecules have been developed on the basis of this structure [17] . Besides the synthetic simplicity and modular nature of the reaction, aromatic 1,2,3-triazoles obtained in this way have an important effect on the pharmacological profile of the resulting molecule. Triazole rings mostly act as linkers between various heterocyclic motifs in molecular hybrids, but can also directly participate in the binding of the target analyte. Because of the large dipole moment, triazoles can act as hydrogen bond donors, mimicking amide functional group. More importantly, they are resistant to metabolic degradation and hydrolysis, and are stable under acidic, basic and redox conditions in living biological systems.

In recent years, derivatives conjugated with 1,2,3-triazole at the C-3, C-4 and C-7 positions of the coumarin ring have been evaluated as promising antitumour (I) [18, 19] , antiinflammatory (II) [20] , neuroprotective (III) [21] and antitubercular agents (IV-V) [8, 22] , and representatives are shown in Figure 1 .

of coumarin-based hybrid molecules by introducing one or more heterocyclic moieties in the coumarin core is a promising approach in the development of novel therapeutic agents with improved and diverse activity [13] .

In the context of easy structural modification, the concept of click chemistry has received great attention. The Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition is considered to be the typical click reaction in which 1,4-disupstituted 1,2,3-triazole is formed [14] [15] [16] . Over the past decade, the 1,2,3-triazole ring has emerged as one of the most important building blocks in the synthesis of molecular hybrids, and a vast number of molecules have been developed on the basis of this structure [17] . Besides the synthetic simplicity and modular nature of the reaction, aromatic 1,2,3-triazoles obtained in this way have an important effect on the pharmacological profile of the resulting molecule. Triazole rings mostly act as linkers between various heterocyclic motifs in molecular hybrids, but can also directly participate in the binding of the target analyte. Because of the large dipole moment, triazoles can act as hydrogen bond donors, mimicking amide functional group. More importantly, they are resistant to metabolic degradation and hydrolysis, and are stable under acidic, basic and redox conditions in living biological systems.

In recent years, derivatives conjugated with 1,2,3-triazole at the C-3, C-4 and C-7 positions of the coumarin ring have been evaluated as promising antitumour (I) [18, 19] , antiinflammatory (II) [20] , neuroprotective (III) [21] and antitubercular agents (IV-V) [8, 22] , and representatives are shown in Figure 1 . Another essential class of nitrogen containing heterocycles in novel drug development is benzofused azoles. Benzimidazole and benzothiazole represent important pharmacophores in medicinal chemistry, primarily because of their structural similarity with naturally occurring molecules. Among the various benzazole-based compounds, 2-thio- Another essential class of nitrogen containing heterocycles in novel drug development is benzofused azoles. Benzimidazole and benzothiazole represent important pharmacophores in medicinal chemistry, primarily because of their structural similarity with naturally occurring molecules. Among the various benzazole-based compounds, 2thiobenzazoles have attracted considerable attention in medicinal research and their deriva- tives have been reported as potential antitubercular (VI) [23] , antiproliferative (VII) [24] , antiviral (VIII) [25] and anti-inflammatory agents (IX) [26] (Figure 2 ). benzazoles have attracted considerable attention in medicinal research and their derivatives have been reported as potential antitubercular (VI) [23] , antiproliferative (VII) [24] , antiviral (VIII) [25] and anti-inflammatory agents (IX) [26] (Figure 2 ).

Despite the large number of known bioactive coumarin-based molecules, there is a need for novel therapeutic agents with improved activity, especially those substituted at C-7 position of the coumarin ring. Taking into account the expressed activity of these three classes of heterocycles, their combination in one target molecule may lead to enhanced bioactivity when compared to individual entities. This type of molecular hybridisation is considered an effective and powerful tool in drug discovery for development of novel hybrids or conjugates with improved and diverse pharmacological properties [13, 17] . Therefore, we have employed a molecular hybridisation strategy in order to design new hybrids that incorporate coumarin, 1,2,3-triazole and/or benzofused benzazole moieties in their scaffold. Here, we report the synthesis of hybrids containing coumarin and 1,2,3triazole moieties in one scaffold, via a Cu(I)-catalysed 1,3-dipolar cycloaddition reaction of 7-azidocoumarin and terminal alkynes. The compounds consist of 7-(1,2,3-triazolyl)coumarin as a central backbone and aryl-substituted subunit linked to the 4-position of the triazole ring. Moreover, planar bis-(coumarin-1,2,3-triazolyl)benzenes were designed by connecting two 7-(1,2,3-triazolyl)coumarin nuclei to enhance the activity allowing additional interactions. Further modification of the triazolyl-coumarin core was carried out by introducing benzofused heterocycles through a thiomethylene linker. In addition to the compounds being potentially biologically active, fluorescence was observed with some of them, making them potentially suitable for application in fluorescence bioimaging. Coumarins are well-known fluorophores in labelling of biomolecules [27] because of the relatively large Stokes shift and high quantum yields, while in combination with 1,2,3-triazoles, new opportunities are opening up to investigate their potential as fluorescent probes for detecting biologically and environmentally important metal cations [28, 29] , anions [30] and biomolecules [31] . Therefore, the antiproliferative activity and basic photophysical properties of these newly prepared hybrids has been examined.

The synthesis of 1,2,3-triazolyl-coumarin hybrids 2a-i is outlined in Scheme 1. Azido coumarin 1, as a key precursor, was synthesised by two methods using commercially available 7-amino-4-methylcoumarin as starting material. Transformation of amino group Despite the large number of known bioactive coumarin-based molecules, there is a need for novel therapeutic agents with improved activity, especially those substituted at C-7 position of the coumarin ring. Taking into account the expressed activity of these three classes of heterocycles, their combination in one target molecule may lead to enhanced bioactivity when compared to individual entities. This type of molecular hybridisation is considered an effective and powerful tool in drug discovery for development of novel hybrids or conjugates with improved and diverse pharmacological properties [13, 17] . Therefore, we have employed a molecular hybridisation strategy in order to design new hybrids that incorporate coumarin, 1,2,3-triazole and/or benzofused benzazole moieties in their scaffold. Here, we report the synthesis of hybrids containing coumarin and 1,2,3-triazole moieties in one scaffold, via a Cu(I)-catalysed 1,3-dipolar cycloaddition reaction of 7-azidocoumarin and terminal alkynes. The compounds consist of 7-(1,2,3-triazolyl)coumarin as a central backbone and aryl-substituted subunit linked to the 4-position of the triazole ring. Moreover, planar bis-(coumarin-1,2,3-triazolyl)benzenes were designed by connecting two 7-(1,2,3-triazolyl)coumarin nuclei to enhance the activity allowing additional interactions. Further modification of the triazolyl-coumarin core was carried out by introducing benzofused heterocycles through a thiomethylene linker. In addition to the compounds being potentially biologically active, fluorescence was observed with some of them, making them potentially suitable for application in fluorescence bioimaging. Coumarins are well-known fluorophores in labelling of biomolecules [27] because of the relatively large Stokes shift and high quantum yields, while in combination with 1,2,3-triazoles, new opportunities are opening up to investigate their potential as fluorescent probes for detecting biologically and environmentally important metal cations [28, 29] , anions [30] and biomolecules [31] . Therefore, the antiproliferative activity and basic photophysical properties of these newly prepared hybrids has been examined.

The synthesis of 1,2,3-triazolyl-coumarin hybrids 2a-i is outlined in Scheme 1. Azido coumarin 1, as a key precursor, was synthesised by two methods using commercially available 7-amino-4-methylcoumarin as starting material. Transformation of amino group to azido substituent in 1 comprised of diazotization and nucleophilic substitution of traditional diazonium salt with azidotrimethylsilane, TMSN 3 (Method A) [32] or diazotization and tosylation followed by nucleophilic substitution of corresponding coumarinyldiazonium tosylate (ArN 2 + TsO − ) with NaN 3 as azide source (Method B) [33] . In the simple and practical Method A, tert-butyl nitrite (t-BuONO) was used as diazotizing agent for 7-amino-4-methylcoumarin followed by reaction of arenediazonium salt thus obtained with TMSN 3 . Alternatively, Method B involved a one-pot procedure to azidocoumarin 1 via the non-usual diazonium tosylate as an intermediate. Conversion of 7-amino-4methylcoumarin to the corresponding azide 1 started by diazotization with NaNO 2 in water in the presence of p-toluenesulfonic acid, PTSA, followed by azide substitution with NaN 3 . Neither method required purification and afforded the pure azidocoumarin 1 without isolation of diazonium intermediates, but Method B provided 1 in better yield (68% and 40%, respectively). Azidocoumarin 1 was then subjected to copper-catalysed Huisgen 1,3-dipolar cycloaddition with various commercially available or synthesised aromatic terminal alkynes and target 1,4-disubstituted 1,2,3-triazolyl-coumarin conjugates 2a-i were prepared. to azido substituent in 1 comprised of diazotization and nucleophilic substitution of traditional diazonium salt with azidotrimethylsilane, TMSN3 (Method A) [32] or diazotization and tosylation followed by nucleophilic substitution of corresponding coumarinyldiazonium tosylate (ArN2 + TsO − ) with NaN3 as azide source (Method B) [33] . In the simple and practical Method A, tert-butyl nitrite (t-BuONO) was used as diazotizing agent for 7amino-4-methylcoumarin followed by reaction of arenediazonium salt thus obtained with TMSN3. Alternatively, Method B involved a one-pot procedure to azidocoumarin 1 via the non-usual diazonium tosylate as an intermediate. Conversion of 7-amino-4-methylcoumarin to the corresponding azide 1 started by diazotization with NaNO2 in water in the presence of p-toluenesulfonic acid, PTSA, followed by azide substitution with NaN3. Neither method required purification and afforded the pure azidocoumarin 1 without isolation of diazonium intermediates, but Method B provided 1 in better yield (68% and 40%, respectively). Azidocoumarin 1 was then subjected to copper-catalysed Huisgen 1,3-dipolar cycloaddition with various commercially available or synthesised aromatic terminal alkynes and target 1,4-disubstituted 1,2,3-triazolyl-coumarin conjugates 2a-i were prepared. The Cu(I) catalyst was generated in situ from Cu(II) salt using sodium ascorbate as reducing agent. To enhance the catalytic effect of Cu(I) in the azide-alkyne click reaction, polytriazolylamine ligand, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TBTA [34] was also used and compounds 2a-i were obtained in low to moderate yields (11% to 59%). Propargylated benzazole derivatives 3a and 3b were prepared via nucleophilic addition of propargyl bromide with 2-mercaptobenzthiazole or 2-mercaptobenzmidazole in the presence of K2CO3 as a base (Scheme 2). Finally, the Cu-catalysed Huisgen cycloaddition reaction of azidocoumarin 1 and propargyl benzazoles 3a and 3b yielded the desired coumarin-triazole-benzazole hybrids 4a and 4b, respectively. The Cu(I) catalyst was generated in situ from Cu(II) salt using sodium ascorbate as reducing agent. To enhance the catalytic effect of Cu(I) in the azide-alkyne click reaction, polytriazolylamine ligand, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TBTA [34] was also used and compounds 2a-i were obtained in low to moderate yields (11% to 59%). Propargylated benzazole derivatives 3a and 3b were prepared via nucleophilic addition of propargyl bromide with 2-mercaptobenzthiazole or 2-mercaptobenzmidazole in the presence of K 2 CO 3 as a base (Scheme 2). Finally, the Cu-catalysed Huisgen cycloaddition reaction of azidocoumarin 1 and propargyl benzazoles 3a and 3b yielded the desired coumarin-triazole-benzazole hybrids 4a and 4b, respectively. 

Compounds 2a-i, 4a and 4b were evaluated for their in vitro cytostatic activities against a panel of human malignant tumour cell lines, as follows: colon carcinoma (HCT116), breast adenocarcinoma (MCF-7), lung carcinoma (H 460), human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa). In addition, we included one nontumour cell line, human dermal microvascular endothelial cells (HMEC-1), to evaluate the selectivity of the newly prepared compounds. The biological data are presented in Table  1 and Figure 3 . Amongst all coumarin-1,2,3-triazole-aryl hybrids 2a-g, compound 2b bearing an aminophenyl moiety showed the most pronounced cell cytotoxicity and selectivity against the MCF-7 cell line with an IC50 = 0.6 μM. Besides, compound 2b also showed significant cytotoxic activity against HCT116 cell line with IC50 = 3 μM. Interestingly, its N,Ndimethylated analogue 2c indicated loss of activity and retained only moderate inhibitory effect against MCF-7 with IC50 = 21 μM. Furthermore, compound 2a with an unsubstituted phenyl moiety at 1,2,3-triazole ring did not show any specific cytostatic activity. Moreover, introduction of methyl (2d), methoxy (2f) or formyl substituents (2g) at the phenyl moiety of the molecular structure did not have any favourable effect on the cytostatic activity of the conjugates, and only moderate activity against HCT116 cell line was observed (IC50 = 36, 22 and 77 μM, respectively). In addition, the bromo substituent on the phenyl ring in 2e had mild influence on cytotoxicity against tested cell lines.

Bis(coumarin-1,2,3-triazolyl)benzenes 2h and 2i were generally the most cytotoxic compounds, especially 1,3-bis(coumarin-triazolyl) derivative 2h with strong inhibitory activity against breast (MCF-7: IC50 = 0.3 μM) and colon carcinoma (HCT116: IC50 = 9 μM). Its 1,4-disubstituted structural isomer 2i displayed slightly reduced cytotoxicity with IC50 = 1 and 7 μM against MCF-7 and HCT116 cells, respectively.

Two coumarin-1,2,3-triazole-benzazole hybrids 4a and 4b were also evaluated and demonstrated almost the same moderate activity against all cell lines. Additionally, benzothiazole derivative 4a showed more pronounced selectivity than its benzimidazole congener 4b against MCF-7 cell lines, with IC50 = 3 μM.

Interestingly, MCF-7 cell line was especially sensitive towards almost all synthesised compounds with IC50 = 0.3-32 μM except for 2d, f and g, which were devoid of activity against this cancer cell line. The HMEC-1 cell line was almost completely insensitive to all tested compounds. 

Compounds 2a-i, 4a and 4b were evaluated for their in vitro cytostatic activities against a panel of human malignant tumour cell lines, as follows: colon carcinoma (HCT116), breast adenocarcinoma (MCF-7), lung carcinoma (H 460), human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa). In addition, we included one nontumour cell line, human dermal microvascular endothelial cells (HMEC-1), to evaluate the selectivity of the newly prepared compounds. The biological data are presented in Table 1 and Figure 3 . Amongst all coumarin-1,2,3-triazole-aryl hybrids 2a-g, compound 2b bearing an aminophenyl moiety showed the most pronounced cell cytotoxicity and selectivity against the MCF-7 cell line with an IC 50 = 0.6 µM. Besides, compound 2b also showed significant cytotoxic activity against HCT116 cell line with IC 50 = 3 µM. Interestingly, its N,N-dimethylated analogue 2c indicated loss of activity and retained only moderate inhibitory effect against MCF-7 with IC 50 = 21 µM. Furthermore, compound 2a with an unsubstituted phenyl moiety at 1,2,3-triazole ring did not show any specific cytostatic activity. Moreover, introduction of methyl (2d), methoxy (2f) or formyl substituents (2g) at the phenyl moiety of the molecular structure did not have any favourable effect on the cytostatic activity of the conjugates, and only moderate activity against HCT116 cell line was observed (IC 50 = 36, 22 and 77 µM, respectively). In addition, the bromo substituent on the phenyl ring in 2e had mild influence on cytotoxicity against tested cell lines. 

The antiviral activity of synthesised compounds (2a-c, 2h-i and 4a-b) was tested against Herpes simplex virus 1 and 2, vaccinia virus, adeno virus-2, human coronavirus, vesicular stomatitis virus, respiratory syncytial virus, reovirus-1, Sindbis virus, Coxsackie Bis(coumarin-1,2,3-triazolyl)benzenes 2h and 2i were generally the most cytotoxic compounds, especially 1,3-bis(coumarin-triazolyl) derivative 2h with strong inhibitory activity against breast (MCF-7: IC 50 = 0.3 µM) and colon carcinoma (HCT116: IC 50 = 9 µM). Its 1,4-disubstituted structural isomer 2i displayed slightly reduced cytotoxicity with IC 50 = 1 and 7 µM against MCF-7 and HCT116 cells, respectively.

Two coumarin-1,2,3-triazole-benzazole hybrids 4a and 4b were also evaluated and demonstrated almost the same moderate activity against all cell lines. Additionally, benzothiazole derivative 4a showed more pronounced selectivity than its benzimidazole congener 4b against MCF-7 cell lines, with IC 50 = 3 µM.

Interestingly, MCF-7 cell line was especially sensitive towards almost all synthesised compounds with IC 50 = 0.3-32 µM except for 2d, f and g, which were devoid of activity against this cancer cell line. The HMEC-1 cell line was almost completely insensitive to all tested compounds.

The antiviral activity of synthesised compounds (2a-c, 2h-i and 4a-b) None of the compounds showed any significant antiviral activity (data not shown).

The structures of all compounds were determined by NMR analysis (based on inspecting H-H coupling constants and chemical shifts). Generally, 1 H NMR spectra of all 1,2,3-triazolyl-coumarin derivatives showed characteristic protons at C5 of triazole at 8.95-9.66 ppm, and proton at C2 of coumarin moiety at approximately 6.5 ppm. 1 H and 13 C NMR and HRMS spectral data for compounds 2a-i and 4a-b are summarised in Table S1 .

Coumarin derivatives are widely used as fluorophores in fluorescent probes and fluorescent tags for biological molecules. Conventional coumarin dyes typically absorb at and are excited by UV light, with emission in the blue spectral region [35] . However, excitation at short wavelengths is not desirable for bioimaging applications because of possible autofluorescence and photo-induced damage of tissue. With an appropriate substitution in the 3-and/or 7-position of the coumarin ring, the derivatives obtained can emit strong fluorescence in the blue-green region (400-550 nm). For example, introduction of an 1,2,3-triazole ring at 3-or 7-position of coumarin has been shown to strongly affect their fluorescent properties [36, 37] . Therefore, spectroscopic characterisation of newly synthesised coumarin derivatives 2a-g and 4a-b containing different substituents conjugated via 1,2,3-triazole linker, as well as bis(triazolyl-coumarin)benzenes 2h-i, was conducted. UV-Vis absorption and fluorescence emission spectra were measured in dimethyl sulfoxide (DMSO) at the compound concentration of 1.0 × 10 −5 mol dm −3 and are presented in Figure 4 .

an emission band with a maxima at 406 nm, while substitution at phenyl ring in compounds 2b-g resulted in bathochromic shifts of emission maxima (5 to 111 nm), with the exception of bromo-substituted derivative 2e. As shown in Figure 4b , p-tolyl substituted derivative 2d showed the most pronounced fluorescence intensity at 445 nm, while 2c did not fluoresce at all in DMSO. Despite emitting at 517 nm and showing the largest Stokes shift of all compounds (187 nm), 2f displayed quite low fluorescence in comparison to other conjugates. It should be noted that all the derivatives 2 exhibit large Stokes shifts (64-187 nm) and therefore have the potential for application as molecular probes and light-emitting markers in chemistry and biology. Additionally, it can be noticed that introduction of strong electron-donating substituents on the phenyl ring results in lowering of quantum yields. As the electron-donating strength of substituents on the phenyl ring decreases in order 2c (NMe2), 2b (NH2), 2f (OMe), the respective quantum yields increase in the same order 0.004, 0.035, 0.058 reaching the highest value of 0.445 for weakly electron-donating methyl group in 2d. The respective molar absorption coefficients follow the same trend and increase with increasing electron-donating ability of the substituent ranging from 15,100 for 2d to 41,000 M −1 cm −1 for 2c.

Bis(coumarin-triazolyl)benzenes showed rather different emission spectra. 1,3-bis derivative 2h exhibited an emission maximum at the lowest wavelength of all compounds, emitting at 394 nm. However, 1,4-disubstitution on benzene ring resulted in a bathocromic shift, and the observed emission maximum of 2i was at 415 nm ( Figure 4d) .

Interestingly, triazolyl-coumarins with benzazole moiety 4a and 4b showed dual emission in DMSO, with maxima at 350 and 416 nm (4a), and 357 and 418 nm (4b) ( Figure  3c ). Organic fluorophores with dual emission properties are especially interesting for application in white light electroluminescence and luminescent bioimaging [38] . Dual emission most commonly occurs as a result of photoinduced structural rearrangement and charge re-distribution in fluorophore molecules, so further studies are underway to investigate this. The basic photophysical properties of 2a-i and 4a-b in DMSO are summarised in Table 2 . •

The coumarin oxygen atom has sp 2 hybridization and is a part of the π-system of the molecule, so the spectrum may show an n→π* electronic transition in addition to a number of π→π* transitions. Compound 2a, containing an unsubstituted phenyl ring exhibited spectra with the absorption maximum at 328 nm. Compounds 2a-g displayed absorption spectra of similar general shape as 2a, with the main absorption band in the range of 325-331 nm and generally lower molar absorption coefficient depending on substituents on the phenyl ring (ε = 13,500-23,200 M -1 cm -1 ) (Figure 4a ). However, aminophenyl and N,N-dimethylaminophenyl substituted derivatives 2b and 2c show greatly enhanced absorption in the range 284-296 nm with high extinction coefficients (ε > 39,000 M −1 cm −1 ) accompanied by a shoulder peak at around 325 nm. Methoxyphenyl-substituted 2f showed slight bathochromic shift (4 to 6 nm) compared to compounds 2a-e and 2g. The observed absorption bands are assigned to the π→π* electronic transitions characteristic for the whole coumarin ring system, while the expected n→π* transition may be submerged under the stronger π→π* transition. It should be noted that all the derivatives 2 exhibit large Stokes shifts (64-187 nm) and therefore have the potential for application as molecular probes and light-emitting markers in chemistry and biology. Additionally, it can be noticed that introduction of strong electron-donating substituents on the phenyl ring results in lowering of quantum yields. As the electron-donating strength of substituents on the phenyl ring decreases in order 2c (NMe 2 ), 2b (NH 2 ), 2f (OMe), the respective quantum yields increase in the same order 0.004, 0.035, 0.058 reaching the highest value of 0.445 for weakly electron-donating methyl group in 2d. The respective molar absorption coefficients follow the same trend and increase with increasing electron-donating ability of the substituent ranging from 15,100 for 2d to 41,000 M −1 cm −1 for 2c.

Bis(coumarin-triazolyl)benzenes showed rather different emission spectra. 1,3-bis derivative 2h exhibited an emission maximum at the lowest wavelength of all compounds, emitting at 394 nm. However, 1,4-disubstitution on benzene ring resulted in a bathocromic shift, and the observed emission maximum of 2i was at 415 nm ( Figure 4d) .

Interestingly, triazolyl-coumarins with benzazole moiety 4a and 4b showed dual emission in DMSO, with maxima at 350 and 416 nm (4a), and 357 and 418 nm (4b) (Figure 3c ). Organic fluorophores with dual emission properties are especially interesting for application in white light electroluminescence and luminescent bioimaging [38] . Dual emission most commonly occurs as a result of photoinduced structural rearrangement and charge re-distribution in fluorophore molecules, so further studies are underway to investigate this. The basic photophysical properties of 2a-i and 4a-b in DMSO are summarised in Table 2 . Absorption and fluorescence spectra were recorded at compound concentration c = 1 × 10 −5 M, λ exc = λ abs,max , slit width = 5-5. Stock solutions of compounds were prepared in DMSO.

All chemicals and solvents were purchased from commercial suppliers Acros (Antwerp, Belgium), Sigma Aldrich (St. Louis, MO, USA) or Fluka (Buchs, Switzerland) and used as received without further purification. 1 H and 13 C NMR spectra were recorded on a Bruker Avance 300 (Bruker, Billerica, MA, USA) or Bruker Avance 600 (Bruker, Billerica, MA, USA) at 300, 600, and 150 and 75 MHz, respectively. All NMR spectra were measured in DMSO-d 6 solutions, while chemical shifts are reported in ppm (δ) relative to TMS as the internal standard. Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances. Melting points were recorded on SMP10 Bibby apparatus (Barloworld Scientific, Staffordshire, UK). The electronic absorption spectra were recorded on a Varian Cary 50 spectrometer (Varian, Inc., Palo Alto, California, USA) using a quartz cuvette (1 cm). Mass spectra were recorded on Bruker Microflex MALDI/TOF mass spectrometer (Bruker, Billerica, MA, USA) using positive ionization. All compounds were routinely checked by TLC using Merck silica gel 60F-254 glass plates. The spectral data can be found in Supplementary Materials in Table S1 and Figures S1-S25.

Method A: 7-Amino-4-methylcoumarin (300.0 mg, 1.71 mmol) was dissolved in anhydrous acetonitrile (10 mL) and cooled to 0 • C in an ice bath. Then, t-BuONO (0.34 mL, 2.86 mmol) and TMSN 3 (0.28 mL, 2.11 mmol) were added dropwise and the reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and crude orange/brown compound 1 (136.0 mg, 40%) was isolated.

Method B: To a solution of 7-amino-4-methylcoumarin (100.0 mg, 0.57 mmol) and p-toluenesulphonic acid monohydrate (977.4 mg, 5.14 mmol) in 5.0 mL of H 2 O, NaNO 2 (354.4 mg, 5.14 mmol) was gradually added. After stirring of the reaction mixture for 1 h at room temperature, NaN 3 (59.4 mg, 0.91 mmol) was added and immediate emission of N 2 was observed. The resulting mixture was stirred overnight, filtered, washed with water and pure compound 1 (78.0 mg, 68%) as orange/brown powder was isolated; m.p. 163-172 • C.

1 H NMR (600 MHz, DMSO-d 6 ): δ/ppm = 7.77 (d, 1H, J = 8.5 Hz), 7.14-7.10 (m, 2H), 6.32 (s, 1H), 2.40 (s, 3H). 13 mixture was stirred for 15 min at room temperature. Afterwards, propargyl bromide (1.0 eq) was added and mixture was heated to reflux till its completion. After 4h the solvent was evaporated under reduced pressure and crude residue was purified by recrystallisation from ethanol (5 mL). 13 13 Compound 4a was synthesised according to above procedure using azidocoumarin 1 and propargylated benzothiazole derivative 3a (1 eq). After stirring of reaction mixture at 60 • C for 24 h and purification of raw material, crude dark yellow compound 4a (50.0 mg, 55%) was isolated; m.p. 186-192 • C.

cence measurements were carried out on a Varian Cary Eclipse fluorescence spectrophotometer at 25 • C using 1 cm path quartz cells. Excitation wavelengths were determined from absorbance maxima. Emission spectra were recorded from 200 to 700 nm. Relative fluorescence quantum yields were determined according to Miller using Equation (1):

where Φ is the emission quantum yield, A is the absorbance at the excitation wavelength, D is the area under the corrected emission curve and n is the refractive index of the solvents used. The subscripts s and x refer to the standard and to the unknown, respectively. The standard employed was quinine sulphate with a published fluorescence quantum yield of 0.54 [39] .

The experiments were carried out on several human cell lines, as follows: HCT116 (colon carcinoma), H 460 (lung carcinoma) and MCF-7 (breast carcinoma), HeLa (cervical carcinoma), human T-lymphocyte cells (CEM) and HMEC-1 (dermal microvascular endothelial cell) according to the previously published experimental procedure [40, 41] . Briefly, the cells were grown in DMEM medium with the addition of 10% foetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, and cultured as monolayers at 37 • C in a humidified atmosphere with 5% CO 2 . HCT116, H 460 and MCF-7 cells were seeded at 2 × 10 3 cells/well in a standard 96-well microtiter plates and left to attach for 24 h. Next day, test compound was added in five serial 10-fold dilutions. The cell growth rate was evaluated after 72 h of incubation, using MTT assay. Obtained results were expressed as IC 50 value which stands for the concentration of the compound necessary for 50% of growth inhibition. The IC 50 values were calculated from concentration-response curve using linear regression analysis by fitting the test concentrations that give percentage of growth (PG) values above and below the reference value (i.e., 50%). However, if for a given cell line all of the tested concentrations produce PGs exceeding the respective reference level of effect (e.g., PG value of 50), then the highest tested concentration is assigned as the default value, which is preceded by a ">" sign. Each test was performed in quadruplicate in at least two individual experiments.

HeLa and HMEC-1 cells were seeded in 48-well plates at 10,000 cells/well. After 24 h, different concentrations of the compounds were added. After 3 days of incubation, the cells were trypsinized and counted in a Coulter counter. Suspension cells (human T-cell leukaemia Cem cells) were seeded in 96-well plates at 60,000 cells/well in the presence of different concentrations of compounds, allowed to proliferate for 96 h, and counted in a Coulter counter. The 50% inhibitory concentration (IC 50 ) was defined as the compound concentration required reducing cell proliferation by 50%.

In this work we present the synthesis of coumarin-triazole conjugates using the copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition. The synthesised compounds were characterised by 1 H-NMR, 13 C-NMR and HRMS. The synthesised compounds were tested against several human cancer cell lines (HCT116, MCF-7, H 460, CEM and HeLa) and non-tumour HMEC-1 cell line. The antiproliferative assay demonstrated moderate to pronounced activity of the compounds against some cancer cells, particularly against breast adenocarcinoma (MCF-7). Amongst coumarin-triazole-aryl hybrids, compound 2b with an amino substituent at the phenyl moiety showed the most pronounced activity towards MCF-7 cancer cells in submicromolar range (IC 50 = 0.6 µM) and enhanced activity against HCT116 cells with IC 50 = 3 µM. 1,3-Bis(coumarin-1,2,3-trazolyl)benzene 2h strongly inhibited growth of MCF-7 cells (IC 50 = 0.3 µM). Its 1,4 structural congener showed somewhat reduced antiproliferative activity with IC 50 = 1 µM against the same cancer cell line. The two coumarin-triazole-benzazole derivatives 4a and 4b showed very similar moderate activity against all of the cancer cell lines, whereas the presence of a benzothiazole or benz-imidazole moiety had no influence on antiproliferative activity for 4a or 4b, respectively. Generally, the MCF-7 cell line showed the most evident sensitivity towards most of the tested compounds, while non-tumour human HMEC-1 cell line remained almost completely unaffected. Thus, further appropriate structural modification of the most effective molecular hybrids is prerequisite to deliver more potent new compounds with broad and improved biological properties.

The absorption and emission spectra of the synthesised compounds revealed some interesting optical properties. These include large Stokes shifts of the coumarin-triazole-aryl hybrids 2a-2g (78-187 nm) and the dual emission peaks of the benzazole derivatives 4a and 4b. These spectroscopic properties suggest that the molecules may also be suitable for use as molecular probes or in bioimaging. Further detailed photophysical studies of the synthesised compounds are underway.

Supplementary Materials: The following supporting information can be downloaded online. Table S1 : 1 H and 13 C NMR and HRMS spectral data and melting points for 1,2,3-triazolyl-coumarin derivatives 2a-i and 4a-b, Figures S1-S14: 1 H and 13 

46 (s, 1H), 4.82 (s, 2H), 2.47 (s, 3H). 13 C NMR (150 MHz, DMSO-d 6 ): δ/ppm = 156

600 MHz, DMSO-d 6 ): δ/ppm = 12.91 (s, 1H), 8.95 (s, 1H), 7.95-7.89 (m, 3H), 7.55 (bs, 1H), 7.44 (bs, 1H), 7.15-7.13 (m, 2H)

MALDI TOF/TOF) m/z calcd for C 20 H 15 N 5 O 2 SNa

General Procedure for the Preparation of Bis(coumarin-1,2,3-triazolyl)benzenes 2h-i 7-azido-4-methyl-2H-chromen-2-one 1 (30.0 mg, 0.15 mmol) and the corresponding

Na-ascorbate (33.7 mg, 0.17 mmol)

3-Phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(4-methyl-2H-chromen-2-one) (2h) Compound 2h was synthesised following described method from azidocoumarin 1

After stirring for 48 h at r.t. and purification

49 (s, 2H), 1.33 (s, 6H). 13 C NMR (75 MHz, DMSO-d 6 ): δ/ppm = 159

1,4-Phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(4-methyl-2H-chromen-2-one) (2i) Compound 2i was prepared from azidocoumarin 1 (1.0 eq) and

After stirring for 72 h at r.t. and column chromatography

50 (s, 2H), 1.23 (s, 6H). 13 C NMR (75 MHz, DMSO-d 6 ): δ/ppm = 159

Spectroscopic Characterisation Spectrophotometric absorbance and fluorescence measurements were carried out using

UV-Vis absorption spectra were recorded, against the solvent

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We gratefully acknowledge the University of Zagreb (grant numbers 121054 and 121083) and Croatian Science Foundation (grant number IP-2014-09-3386) for the financial support. The authors would also like to acknowledge Matthew Steinberg, PhD for English language and style proofreading and correction.

The authors declare no conflict of interest.Sample Availability: Samples of the compounds are available from the authors.