Synthesis and Properties of 6-Aryl-4-azidocinnolines and 6-Aryl-4-(1,2,3-1H-triazol-1-yl)cinnolines

An efficient approach towards the synthesis of 6-aryl-4-azidocinnolines was developed with the aim of exploring the photophysical properties of 6-aryl-4-azidocinnolines and their click reaction products with alkynes, 6-aryl-4-(1,2,3-1H-triazol-1-yl)cinnolines. The synthetic route is based on the Richter-type cyclization of 2-ethynyl-4-aryltriazenes with the formation of 4-bromo-6-arylcinnolines and nucleophilic substitution of a bromine atom with an azide functional group. The developed synthetic approach is tolerant to variations of functional groups on the aryl moiety. The resulting azidocinnolines were found to be reactive in both CuAAC with terminal alkynes and SPAAC with diazacyclononyne, yielding 4-triazolylcinnolines. It was found that 4-azido-6-arylcinnolines possess weak fluorescent properties, while conversion of the azido function into a triazole ring led to complete fluorescence quenching. The lack of fluorescence in triazoles could be explained by the non-planar structure of triazolylcinnolines and a possible photoinduced electron transfer (PET) mechanism. Among the series of 4-triazolylcinnoline derivatives a compound bearing hydroxyalkyl substituent at triazole ring was found to be cytotoxic to HeLa cells.


Introduction
During last decade azido-substituted heterocycles have been broadly studied in several areas. The first one is photoaffinity labeling [1] which is an important biological tool for the investigation of different biological processes by specific modification of proteins using their interaction with singlet nitrenes generated from azidoheterocyles under UV irradiation ( Figure 1A) [2][3][4]. The second approach involves use of azidoheterocycles in the synthesis of triazolylheterocycles, mainly by Cu-catalyzed alkyne-azide cycloaddition (CuAAC) [5,6]. This methodology allowed a great variety of biologically active triazolyl substituted heterocycles to be synthesized and tested in numerous biological assays ( Figure 1B) [7][8][9][10]. The third main field where azidoheterocycles are in great demand is as azide-based fluorophore tags for biological imaging ( Figure 1C) [11][12][13][14][15][16].

Synthesis of 6-aryl-4-azidocinnolines
Several routes have been established for introduction of an azido group into a heterocyclic ring [41]. The most common techniques are azidodediazoniation of arenediazonium salts, synthesis of azides from the corresponding heteroarylboronic acids and nucleophilic substitution of activated halogens. In most cases involving 4-azidoquinolines nucleophilic substitution of a chlorine atom at the C4 position of a quinoline ring has been employed [42][43][44][45]. A few examples are known of bromine substitution [46,47] along with azidodediazoniation [48] and conversion of the corresponding quinolones to azidoquinolines employing diphenylphospharylazide [49].

Synthesis of 6-aryl-4-azidocinnolines
Several routes have been established for introduction of an azido group into a heterocyclic ring [41]. The most common techniques are azidodediazoniation of arenediazonium salts, synthesis of azides from the corresponding heteroarylboronic acids and nucleophilic substitution of activated halogens. In most cases involving 4-azidoquinolines nucleophilic substitution of a chlorine atom at the C4 position of a quinoline ring has been employed [42][43][44][45]. A few examples are known of bromine substitution [46,47] along with azidodediazoniation [48] and conversion of the corresponding quinolones to azidoquinolines employing diphenylphospharylazide [49].
To reach the target 4-azido-6-arylcinnolines compounds we decided to use 4-bromocinnolines, which are synthetically accessible by the Richter cyclization in higher yields compared to corresponding chloro derivatives [54], while the bromine atom at the C4 position should be still reactive towards nucleophilic substitution. Firstly, two synthetic routes (A and B) have been proposed for target molecules (Scheme 1).

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The only mentioned example of a 4-azidocinnoline molecule was also obtained from the corresponding chlorocinnoline by nucleophilic substitution with sodium azide in ethanol [29].
To reach the target 4-azido-6-arylcinnolines compounds we decided to use 4-bromocinnolines, which are synthetically accessible by the Richter cyclization in higher yields compared to corresponding chloro derivatives [54], while the bromine atom at the C4 position should be still reactive towards nucleophilic substitution. Firstly, two synthetic routes (A and B) have been proposed for target molecules (Scheme 1). Scheme 1. Proposed synthetic "Routes A" and "B" towards 6-aryl-4-azidicinnolines.
The first step for both routes is the chemoselective Sonogashira coupling of the C-I group of 4-bromo-2-iodophenyltriazene with hept-1-yne (Scheme 1), which has been used previously for the synthesis of poly(arylene ethynylene)s [40].
"Route A" seems to be more divergent and rational than "Route B", because it allows for variations of the aromatic substituent during the last synthetic step. Therefore we chose "Route A" to start with. The Richter-type cyclization followed by the regioselective substitution of bromine atom with sodium azide in absolute DMF at the activated C4 position proceeded smoothly and did not affect the bromine atom at C6. Hence 4-azido-6-bromocinnoline 5 was obtained in good yield (Scheme 2). Next we tried to carry out the Suzuki coupling. Despite the fact that this transformation of substrates bearing aromatic azido groups is known, yields usually are low, because the whole process is accompanied by several side reactions that are common for both Suzuki coupling (homocoupling, reductive dehalogenation) and for azides (denitrogenative decomposition through nitrene intermediates and through the Staudinger reaction if phosphine ligands are present in the reaction mixture) [65]. Two types of conditions tested differed in the base (Na2CO3 or K3PO4) and solvents (toluene/dioxane/H2O or only dioxane, respectively) used (Scheme 2). The formation of the desired 4-azido-6-phenylcinnoline 7a as the main product was observed in both cases. However the isolated yields of the product 7a were found to be between low (conditions d, Scheme 2) to moderate (conditions e, Scheme 2). Moreover the Suzuki coupling proceeded with the formation of many byproducts that required time-consuming purification of the target azide by column chromatography. Therefore we turned to "Route B". Scheme 1. Proposed synthetic "Routes A" and "B" towards 6-aryl-4-azidicinnolines.
The first step for both routes is the chemoselective Sonogashira coupling of the C-I group of 4-bromo-2-iodophenyltriazene with hept-1-yne (Scheme 1), which has been used previously for the synthesis of poly(arylene ethynylene)s [40].
"Route A" seems to be more divergent and rational than "Route B", because it allows for variations of the aromatic substituent during the last synthetic step. Therefore we chose "Route A" to start with. The Richter-type cyclization followed by the regioselective substitution of bromine atom with sodium azide in absolute DMF at the activated C4 position proceeded smoothly and did not affect the bromine atom at C6. Hence 4-azido-6-bromocinnoline 5 was obtained in good yield (Scheme 2). Next we tried to carry out the Suzuki coupling. Despite the fact that this transformation of substrates bearing aromatic azido groups is known, yields usually are low, because the whole process is accompanied by several side reactions that are common for both Suzuki coupling (homocoupling, reductive dehalogenation) and for azides (denitrogenative decomposition through nitrene intermediates and through the Staudinger reaction if phosphine ligands are present in the reaction mixture) [65]. Two types of conditions tested differed in the base (Na 2 CO 3 or K 3 PO 4 ) and solvents (toluene/dioxane/H 2 O or only dioxane, respectively) used (Scheme 2). The formation of the desired 4-azido-6-phenylcinnoline 7a as the main product was observed in both cases. However the isolated yields of the product 7a were found to be between low (conditions d, Scheme 2) to moderate (conditions e, Scheme 2). Moreover the Suzuki coupling proceeded with the formation of many byproducts that required time-consuming purification of the target azide by column chromatography. Therefore we turned to "Route B".
The scope of "Route B" was then checked with different aryl-and heteroarylboronic acids 6b-g. "Conditions a" worked well for the CF3 (b), MeO (c) and PhO (d) series. However, these Suzuki coupling conditions failed when applied to other boronic acids bearing NHBoc (6e), CN (6f) and 3-thienyl (6g) groups. Thus, either complete or partial decomposition of the starting triazenes occurred when a mixture of organic solvent with water in the presence of sodium carbonate as a base was used. Excluding water from the reaction mixture and changing the base to potassium phosphate allowed all three triazenes 8e−g to be obtained in high yields. The Richter cyclization and the subsequent nucleophilic substitution of bromine by an azido group for all compounds The Suzuki coupling of aromatic halotriazenes has been employed recently in the initial steps of the synthesis of hexadehydrotribenzo [12]annulene [66]. Similar conditions (Na 2 CO 3 , toluene/dioxane/H 2 O, 100 • C) for our substrates gave 4-phenyltriazene 8a in 72% yield. Applying next the Richter-type cyclization and nucleophilic substitution of bromine with sodium azide under the same conditions as for "Route A" enabled us to produce the final 4-azido-6-phenylcinnoline 7a in better overall yield (42%, "Route B") (Scheme 3) compared to "Route A" (24%) (Scheme 2). Same routes? The Suzuki coupling of aromatic halotriazenes has been employed recently in the initial steps of the synthesis of hexadehydrotribenzo [12]annulene [66]. Similar conditions (Na2CO3, toluene/dioxane/H2O, 100 °C) for our substrates gave 4-phenyltriazene 8a in 72% yield. Applying next the Richter-type cyclization and nucleophilic substitution of bromine with sodium azide under the same conditions as for "Route A" enabled us to produce the final 4-azido-6-phenylcinnoline 7a in better overall yield (42%, "Route B") (Scheme 3) compared to "Route A" (24%) (Scheme 2). Same routes? The scope of "Route B" was then checked with different aryl-and heteroarylboronic acids 6b-g. "Conditions a" worked well for the CF3 (b), MeO (c) and PhO (d) series. However, these Suzuki coupling conditions failed when applied to other boronic acids bearing NHBoc (6e), CN (6f) and 3-thienyl (6g) groups. Thus, either complete or partial decomposition of the starting triazenes occurred when a mixture of organic solvent with water in the presence of sodium carbonate as a base was used. Excluding water from the reaction mixture and changing the base to potassium phosphate allowed all three triazenes 8e−g to be obtained in high yields. The Richter cyclization and the subsequent nucleophilic substitution of bromine by an azido group for all compounds The scope of "Route B" was then checked with different aryl-and heteroarylboronic acids 6b-g. "Conditions a" worked well for the CF 3 (b), MeO (c) and PhO (d) series. However, these Suzuki coupling conditions failed when applied to other boronic acids bearing NHBoc (6e), CN (6f) and 3-thienyl (6g) groups. Thus, either complete or partial decomposition of the starting triazenes occurred when a mixture of organic solvent with water in the presence of sodium carbonate as a base was used. Excluding water from the reaction mixture and changing the base to potassium phosphate allowed all three triazenes 8e-g to be obtained in high yields. The Richter cyclization and the subsequent nucleophilic substitution of bromine by an azido group for all compounds proceeded smoothly under the same conditions as for the unsubstituted phenyltriazene 8a, providing a series of key 6-aryl(heteroaryl)-4-azidocinnolines 7 in high yields (Scheme 3).

Study of 6-aryl-4-azidocinnoline's Reactivity in the Sythesis of 6-aryl-4-triazolylcinnolines
All azidocinnolines were found to be stable crystalline compounds when stored at −18 • C and slowly decomposed in solution. Thus complete decomposition of azidocinnoline 7a in acetone-d 6 was detected after a week. Despite this fact, all azides were stable under conditions used for CuAAC. Thus carrying out CuAAC of azidocinnolines 7a-g both with terminal aromatic alkynes bearing EWG, EDG and aliphatic alkynes in the mixture of THF/H 2 O using the copper (II) sulfate/sodium ascorbate catalytic system afforded the corresponding 4-triazolylcinnolines mostly in good yields (Scheme 4). proceeded smoothly under the same conditions as for the unsubstituted phenyltriazene 8a, providing a series of key 6-aryl(heteroaryl)-4-azidocinnolines 7 in high yields (Scheme 3).

Study of 6-aryl-4-azidocinnoline's Reactivity in the Sythesis of 6-aryl-4-triazolylcinnolines
All azidocinnolines were found to be stable crystalline compounds when stored at −18 °C and slowly decomposed in solution. Thus complete decomposition of azidocinnoline 7a in acetone-d6 was detected after a week. Despite this fact, all azides were stable under conditions used for CuAAC. Thus carrying out CuAAC of azidocinnolines 7a−g both with terminal aromatic alkynes bearing EWG, EDG and aliphatic alkynes in the mixture of THF/H2O using the copper (II) sulfate/sodium ascorbate catalytic system afforded the corresponding 4-triazolylcinnolines mostly in good yields (Scheme 4). One modification of the well-studied CuAAC is to carry out the reaction in the presence of tris(triazolyl) ligands, which improves the yields, shortens the reaction time and improves the synthetic accessibility of triazole derivatives that are not available when the common Cu(II)/ ascorbate catalytic system is used [67]. Surprisingly, when the CuAAC of azidocinnoline 7c with 3,4-dimethoxyphenylacetylene was run in the presence of the TBTA ligand (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine), the reaction proceeded very slowly and full conversion was not achieved even after two days. Therefore triazolylcinnoline 11c was isolated in only 35 % yield (the yield of 11c without TBTA ligand was 76%).
Next we checked the reactivity of azidocinnolines synthesized in reactions other than CuAAC used for the synthesis of 1H-1,2,3-triazole derivatives, like the enol-mediated organocatalytic synthesis of triazoles and strain-promoted alkyne-azide cycloaddition (SPAAC). Unfortunately, enol-mediated reaction [68] of azide 7b with propionic aldehyde catalysed by 1,8-diazabicyclo- [5.4.0]undec-7-ene (DBU) did not go as expected. The only isolated product was 4-aminocinnoline, which was presumably formed from the corresponding nitrene as an intermediate of azide decomposition (Scheme 5). One modification of the well-studied CuAAC is to carry out the reaction in the presence of tris(triazolyl) ligands, which improves the yields, shortens the reaction time and improves the synthetic accessibility of triazole derivatives that are not available when the common Cu(II)/ascorbate catalytic system is used [67]. Surprisingly, when the CuAAC of azidocinnoline 7c with 3,4-dimethoxyphenylacetylene was run in the presence of the TBTA ligand (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine), the reaction proceeded very slowly and full conversion was not achieved even after two days. Therefore triazolylcinnoline 11c was isolated in only 35 % yield (the yield of 11c without TBTA ligand was 76%).

Photophysical Properties of 6-aryl-4-azidocinnolines and 6-aryl-4-triazolylcinnolines
Having both azidocinnolines and triazolylcinnolines in hand, we next studied their photophysical properties. Fluorogenic azides represent a class of fluorescent dyes which are only weakly fluorescent without conversion into triazoles. However, when a triazole moiety is introduced into the molecule, it imparts fluorescent properties to the entire molecular scaffold. Fluorogenic azides are of special interest for bioimaging, because they allow avoiding a series of steps to wash the excess of dye from the labeled materials and thus they eliminate the problem of background fluorescence. Syntheses and applications of various fluorogenic heterocyclic azides have been reported recently and discussed in the Introduction section [11] (Figure 1). Taking into account that the aryl substituent, azide group and azo-group of a cinnoline core are conjugated, we decided to investigate whether 6-aryl-4-azidocinnolines could be used as fluorogenic azide dyes.
Firstly, the absorption and emission spectra of azidocinnoline solutions in THF were measured (Figures 3). The obtained data revealed that both EWG and EDG groups at the para-position of an aryl ring attached to C6 atom provided an increase in fluorescence brightness. To quantify the observed fluorescence, the absolute quantum yields of fluorescence (QY) of azidocinnolines 7 were measured (Table 1).

Photophysical Properties of 6-aryl-4-azidocinnolines and 6-aryl-4-triazolylcinnolines
Having both azidocinnolines and triazolylcinnolines in hand, we next studied their photophysical properties. Fluorogenic azides represent a class of fluorescent dyes which are only weakly fluorescent without conversion into triazoles. However, when a triazole moiety is introduced into the molecule, it imparts fluorescent properties to the entire molecular scaffold. Fluorogenic azides are of special interest for bioimaging, because they allow avoiding a series of steps to wash the excess of dye from the labeled materials and thus they eliminate the problem of background fluorescence. Syntheses and applications of various fluorogenic heterocyclic azides have been reported recently and discussed in the Introduction section [11] (Figure 1). Taking into account that the aryl substituent, azide group and azo-group of a cinnoline core are conjugated, we decided to investigate whether 6-aryl-4-azidocinnolines could be used as fluorogenic azide dyes.
Firstly, the absorption and emission spectra of azidocinnoline solutions in THF were measured (Figures 3). The obtained data revealed that both EWG and EDG groups at the para-position of an aryl ring attached to C6 atom provided an increase in fluorescence brightness. To quantify the observed fluorescence, the absolute quantum yields of fluorescence (QY) of azidocinnolines 7 were measured (Table 1). Scheme 6. Reactivity of 4-azidocinnoline 7g in SPAAC.

Photophysical Properties of 6-aryl-4-azidocinnolines and 6-aryl-4-triazolylcinnolines
Having both azidocinnolines and triazolylcinnolines in hand, we next studied their photophysical properties. Fluorogenic azides represent a class of fluorescent dyes which are only weakly fluorescent without conversion into triazoles. However, when a triazole moiety is introduced into the molecule, it imparts fluorescent properties to the entire molecular scaffold. Fluorogenic azides are of special interest for bioimaging, because they allow avoiding a series of steps to wash the excess of dye from the labeled materials and thus they eliminate the problem of background fluorescence. Syntheses and applications of various fluorogenic heterocyclic azides have been reported recently and discussed in the Introduction section [11] (Figure 1). Taking into account that the aryl substituent, azide group and azo-group of a cinnoline core are conjugated, we decided to investigate whether 6-aryl-4-azidocinnolines could be used as fluorogenic azide dyes.
Firstly, the absorption and emission spectra of azidocinnoline solutions in THF were measured ( Figure 3). The obtained data revealed that both EWG and EDG groups at the para-position of an aryl ring attached to C6 atom provided an increase in fluorescence brightness. To quantify the observed fluorescence, the absolute quantum yields of fluorescence (QY) of azidocinnolines 7 were measured ( Table 1).
The obtained data revealed that all 6-aryl-4-azidocinnolines 7 possess weak fluorescence with quantum yield values of less than 1%, with the highest QY being observed for 4-azido-6-(4-methoxyphenyl)cinnoline. Hoping to observe fluorescent properties in triazole derivatives 11, 16 the absorption and emission spectra for the series of triazolylcinnolines 11a−c,e−g, 16   Unfortunately, all triazoles were found to be devoid of any fluorescence. Emission spectra of azide/triazole pairs with MeO group 7c/11c and 3-thienyl group 7f/11f are presented on Figure 5 (for the spectra of the whole series see Appendix A- Figures A1, A2).
The obtained data revealed that all 6-aryl-4-azidocinnolines 7 possess weak fluorescence with quantum yield values of less than 1%, with the highest QY being observed for 4-azido-6-(4methoxyphenyl)cinnoline. Hoping to observe fluorescent properties in triazole derivatives 11, 16 the absorption and emission spectra for the series of triazolylcinnolines 11a−c,e−g, 16 The obtained data revealed that all 6-aryl-4-azidocinnolines 7 possess weak fluorescence with quantum yield values of less than 1%, with the highest QY being observed for 4-azido-6-(4-methoxyphenyl)cinnoline. Hoping to observe fluorescent properties in triazole derivatives 11, 16 the absorption and emission spectra for the series of triazolylcinnolines 11a−c,e−g, 16   Unfortunately, all triazoles were found to be devoid of any fluorescence. Emission spectra of azide/triazole pairs with MeO group 7c/11c and 3-thienyl group 7f/11f are presented on Figure 5 (for the spectra of the whole series see Appendix A- Figures A1, A2). Unfortunately, all triazoles were found to be devoid of any fluorescence. Emission spectra of azide/triazole pairs with MeO group 7c/11c and 3-thienyl group 7f/11f are presented on Figure 5 (for the spectra of the whole series see Appendix A- Figures A1 and A2). To explain the observed loss of fluorescence for triazoles, quantum chemical calculations for S 0 states of the azide/triazoles pairs 7c/11c and 7f/11f were carried out. Geometry optimization was achieved using DFT calculations (B3LYP 6-311 + G(2d,2p)). The obtained data revealed that triazoles 11c and 11f are non-planar molecules: the triazole rings of both compounds lay out of the corresponding cinnoline plane ( Table 2). X-Ray studies confirmed the non-planar geometry of triazole 11f in the solid state ( Figure 6). The dihedral angle between the triazole and cinnoline rings was found to be 64.8°. Therefore, despite the extension of the conjugated chain, the non-planar geometry of 4-triazolylcinnolines could be one of reasons for the observed absence of fluorescence. Analysis of the frontier molecular orbitals of triazolylcinnolines 11c and 11f showed that the HOMO is associated with the donor part of the molecule (triazole) whereas the LUMO is concentrated in the acceptor cinnoline part. On the other hand the frontier molecular orbitals of azidocinnolines 7c and 7f are evenly distributed over the entire molecule (Table 2). Therefore, another reason for the lack of fluorescence of triazolylcinnolines compared to azidocinnolines could be intramolecular photoinduced electron transfer (PET) that is known to be responsible for fluorescence quenching [70]. For example, 1,4-bis(2-hydrohyphenyl)-1,2,3-triazole with a similar frontier molecular orbital separation between both 2-hydrohyphenyl substituents exhibited very weak fluorescence [71]. To explain the observed loss of fluorescence for triazoles, quantum chemical calculations for S 0 states of the azide/triazoles pairs 7c/11c and 7f/11f were carried out. Geometry optimization was achieved using DFT calculations (B3LYP 6-311 + G(2d,2p)). The obtained data revealed that triazoles 11c and 11f are non-planar molecules: the triazole rings of both compounds lay out of the corresponding cinnoline plane ( Table 2). X-Ray studies confirmed the non-planar geometry of triazole 11f in the solid state ( Figure 6). The dihedral angle between the triazole and cinnoline rings was found to be 64.8 • . Therefore, despite the extension of the conjugated chain, the non-planar geometry of 4-triazolylcinnolines could be one of reasons for the observed absence of fluorescence. To explain the observed loss of fluorescence for triazoles, quantum chemical calculations for S 0 states of the azide/triazoles pairs 7c/11c and 7f/11f were carried out. Geometry optimization was achieved using DFT calculations (B3LYP 6-311 + G(2d,2p)). The obtained data revealed that triazoles 11c and 11f are non-planar molecules: the triazole rings of both compounds lay out of the corresponding cinnoline plane ( Table 2). X-Ray studies confirmed the non-planar geometry of triazole 11f in the solid state ( Figure 6). The dihedral angle between the triazole and cinnoline rings was found to be 64.8°. Therefore, despite the extension of the conjugated chain, the non-planar geometry of 4-triazolylcinnolines could be one of reasons for the observed absence of fluorescence. Analysis of the frontier molecular orbitals of triazolylcinnolines 11c and 11f showed that the HOMO is associated with the donor part of the molecule (triazole) whereas the LUMO is concentrated in the acceptor cinnoline part. On the other hand the frontier molecular orbitals of azidocinnolines 7c and 7f are evenly distributed over the entire molecule (Table 2). Therefore, another reason for the lack of fluorescence of triazolylcinnolines compared to azidocinnolines could be intramolecular photoinduced electron transfer (PET) that is known to be responsible for fluorescence quenching [70]. For example, 1,4-bis(2-hydrohyphenyl)-1,2,3-triazole with a similar frontier molecular orbital separation between both 2-hydrohyphenyl substituents exhibited very weak fluorescence [71]. Analysis of the frontier molecular orbitals of triazolylcinnolines 11c and 11f showed that the HOMO is associated with the donor part of the molecule (triazole) whereas the LUMO is concentrated in the acceptor cinnoline part. On the other hand the frontier molecular orbitals of azidocinnolines 7c and 7f are evenly distributed over the entire molecule (Table 2). Therefore, another reason for the lack of fluorescence of triazolylcinnolines compared to azidocinnolines could be intramolecular photoinduced electron transfer (PET) that is known to be responsible for fluorescence quenching [70]. For example, 1,4-bis(2-hydrohyphenyl)-1,2,3-triazole with a similar frontier molecular orbital separation between both 2-hydrohyphenyl substituents exhibited very weak fluorescence [71].   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.   1 Geometry optimization (B3LYP 6-311 + G(2d,2p)); 2 Torsion angle between triazole and cinnoline rings in optimized strucures.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 μM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.

Biological Studies of 6-aryl-4-triazolylcinnolines
Next we turned to biological activity screening tests. Both the triazole fragment and cinnoline moiety can be found in variety of compounds with antibacterial, antifungal and anticancer properties. Therefore we studied the antibacterial and antifungal activities of triazoles 11a,c,e−g, 16 against Escherichia coli and Saccharomyces cerevisiae, respectively. The cytotoxicity of triazolylcinnolines and their activity as DNA-cleavage agents were also tested.
The obtained data revealed that the studied triazolylcinnolines 11a,c,e−g, 16 are inactive towards the Gram-negative bacterium Escherichia coli and fungus Saccharomyces cerevisiae. On the other hand the MTT test for screening of cytotoxicity allowed identifying triazolylcinnoline 11a as being active. Despite the fact that other triazolylcinnolines did not display reduced HeLA cell viability, the IC 50 for compound 11a bearing hydroxyalkyl substituent was found to be 56.5 µM (Figure 7). Triazolylcinnoline 11a possess a flat heteroaromatic moiety−6-arylcinnoline, which might have DNA intercalating activity. Therefore we tested the ability of triazolylcinnolines to cleave DNA. None of the compounds affected the DNA plasmid pBR322, indicating a different mechanism of cytotoxicity.

General Information
Solvents and reagents used for reactions were purchased from commercial suppliers. Catalyst Pd(PPh3)4 was purchased from Sigma-Aldrich (München, Germany). Solvents were dried under standard conditions; chemicals were used without further purification. 4-Bromo-2-iodoaniline [72], 1-(4-bromo-2-iodophenyl)-3-ethyl-3-phenyltriaz-1-ene (1) [40], Co2(CO)6-complex of diazacyclononyne 15 [69] and TBTA [67] were synthesized by known procedures without any modifications. Evaporation of solvents and concentration of reaction mixtures were performed in vacuum at 35 °C on a rotary evaporator. Thin-layer chromatography (TLC) was carried out on silica gel plates (Silica gel 60, F254, Merck (Darmstadt, Germany) with detection by UV or staining with a basic aqueous solution of KMnO4. Melting points (mp) determined are uncorrected. 1 H and 13 CNMR spectra were recorded at 400 and 100 MHz, respectively, at 25 °C in acetone-d6 without the internal standard using a 400 МHz Avance spectrometer (Bruker, Billerica, MA, USA). The 1 H-NMR data are reported as chemical shifts (δ), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), coupling constants (J, given in Hz), and number of protons. The 13 C NMR data are reported as the chemical shifts (δ) with coupling constant J(C−F) for F-containing compounds. Chemical shifts for 1 H and 13 C are reported as δ values (ppm) and referenced to residual solvent (δ = 2.05 ppm for 1 H; δ = 29.84 for 13 C-for spectra in acetone-d6, δ = 7.26 ppm for 1 H; δ = 77.16 ppm for 13 C-for spectra in CDCl3 and δ = 2.50 ppm for 1 H; δ = 39.52 ppm for 13 C-for spectra in DMSO-d6). High resolution mass spectra (HRMS) were determined using electrospray ionization (ESI) in the mode of positive ion registration with a Bruker microTOF mass analyzer (Billerica, MA, USA). UV-vis spectra for solutions of all compounds in THF were recorded on a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) at 20 °C. Fluorescence spectra for the same solutions were recorded on a FluoroMax-4 spectrofluorometer (Horiba Scientific, Glasgow, Scotland) at 20 °C. IR spectra were measured using a Nicolet 8700 spectrometer (Thermo Scientific, Madison, WI, USA) equipped with a Thermo Scientific Smart iTR™ as an Attenuated Total Reflectance (ATR) sampling accessory. Data for 11f were collected using an XtaLAB SuperNova diffractometer (Rigaku Oxford Diffraction, Tokio, Japan) equipped with an HyPix3000 CCD area detector operated with monochromated microfocused CuKα radiation (λ[CuKα] = 1.54184 Å). All the data were integrated and corrected for background, Lorentz, and polarization effects by means of the CrysAlisPro (Tokyo, Japan) [73] program complex. Absorption correction was applied using the empirical spherical model within the CrysAlisPro program complex using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. The unit-cell parameters were refined by the least-squares techniques. The structures were solved by

General Information
Solvents and reagents used for reactions were purchased from commercial suppliers. Catalyst Pd(PPh 3 ) 4 was purchased from Sigma-Aldrich (München, Germany). Solvents were dried under standard conditions; chemicals were used without further purification. 4-Bromo-2-iodoaniline [72], 1-(4-bromo-2-iodophenyl)-3-ethyl-3-phenyltriaz-1-ene (1) [40], Co 2 (CO) 6 -complex of diazacyclononyne 15 [69] and TBTA [67] were synthesized by known procedures without any modifications. Evaporation of solvents and concentration of reaction mixtures were performed in vacuum at 35 • C on a rotary evaporator. Thin-layer chromatography (TLC) was carried out on silica gel plates (Silica gel 60, F254, Merck (Darmstadt, Germany) with detection by UV or staining with a basic aqueous solution of KMnO 4 . Melting points (mp) determined are uncorrected. 1 H and 13 CNMR spectra were recorded at 400 and 100 MHz, respectively, at 25 • C in acetone-d 6 without the internal standard using a 400 МHz Avance spectrometer (Bruker, Billerica, MA, USA). The 1 H-NMR data are reported as chemical shifts (δ), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), coupling constants (J, given in Hz), and number of protons. The 13 C NMR data are reported as the chemical shifts (δ) with coupling constant J(C−F) for F-containing compounds. Chemical shifts for 1 H and 13 C are reported as δ values (ppm) and referenced to residual solvent (δ = 2.05 ppm for 1 H; δ = 29.84 for 13 C-for spectra in acetone-d 6 , δ = 7.26 ppm for 1 H; δ = 77.16 ppm for 13 C-for spectra in CDCl 3 and δ = 2.50 ppm for 1 H; δ = 39.52 ppm for 13 C-for spectra in DMSO-d 6 ). High resolution mass spectra (HRMS) were determined using electrospray ionization (ESI) in the mode of positive ion registration with a Bruker microTOF mass analyzer (Billerica, MA, USA). UV-vis spectra for solutions of all compounds in THF were recorded on a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) at 20 • C. Fluorescence spectra for the same solutions were recorded on a FluoroMax-4 spectrofluorometer (Horiba Scientific, Glasgow, Scotland) at 20 • C. IR spectra were measured using a Nicolet 8700 spectrometer (Thermo Scientific, Madison, WI, USA) equipped with a Thermo Scientific Smart iTR™ as an Attenuated Total Reflectance (ATR) sampling accessory. Data for 11f were collected using an XtaLAB SuperNova diffractometer (Rigaku Oxford Diffraction, Tokio, Japan) equipped with an HyPix3000 CCD area detector operated with monochromated microfocused CuKα radiation (λ[CuKα] = 1.54184 Å). All the data were integrated and corrected for background, Lorentz, and polarization effects by means of the CrysAlisPro (Tokyo, Japan) [73] program complex. Absorption correction was applied using the empirical spherical model within the CrysAlisPro program complex using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. The unit-cell parameters were refined by the least-squares techniques. The structures were solved by direct methods and refined using the SHELX [74] program incorporated in the OLEX2 [75] program package.

The Richter-Type Cyclization Protocol of Triazenes 3, 8
To a solution of triazene 3 or 8 (1 equiv) in acetone (C = 0.1 M) HBr (48% aqueous solution, 20 equiv) was quickly added dropwise while maintaining the reaction mixture temperature of 20 • C by cooling the reaction mixture with a water bath. The resulting mixture was stirred for 10 minutes. Upon completion of the reaction, the reaction mixture was diluted with an aqueous solution of triethylamine (21 equiv). The resulting mixture was extracted with ethyl acetate, the combined organic layers were washed with water, brine and dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuum and the crude 4-bromocinnoline was purified by column chromatography.

General Procure for the Suzuki Coupling (Method A)
To a solution of triazene 3 or azidocinnoline 5 (1 equiv) in a mixture of toluene/1,4-dioxane/water (1:2:2) (C = 0.1 M) in a vial was added arylboronic acid 6 (1.5 equiv), Pd(PPh 3 ) 4 (5 mol%) and Na 2 CO 3 (2 equiv). The vial was sealed; the reaction mixture was evacuated and flushed with Ar several times. The vial with the reaction mixture was placed in a preheated oil bath (80−100 • C) and stirred for 1−24 h (TLC control). After completion the reaction, the mixture was cooled and poured into a saturated aqueous solution of NH 4 Cl and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous solutions of NH 4 Cl and brine, dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to yield the crude product, which was purified by column chromatography on silica gel.

General Procure for the Suzuki Coupling (Method B)
Triazene 3 or azidocinnoline 5 (1 equiv), ArB(OH) 2 6 (1.5 equiv), K 3 PO 4 (2 equiv), and Pd(PPh 3 ) 4 (5 mol %) were placed in a vial. The vial was sealed, and the mixture was evacuated and flushed with Ar several times. 1,4-Dioxane (C = 0.1 M) was added, and the vial with the reaction mixture was placed in a preheated oil bath (80−100 • C) and stirred for 1−20 h (TLC control). After cooling to rt, the reaction mixture was filtered through a pad silica gel and washed with ethyl acetate. Solvents were removed under reduced pressure, and the crude product was purified by column chromatography on silica gel.

General Procure for the Nucleophilic Substitution
Sodium azide (2-5 equiv) was added to a solution of bromocinnoline (1 equiv) in absolute DMF (C = 0.1 M). The mixture was degassed and stirred under argon at 50 • C for 24 h (TLC control). Upon completion the reaction, the reaction mixture was poured into water, extracted with ethyl acetate; the combined organic layers were washed three times with water and two times with brine, dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuum to yield the crude product, which was purified by column chromatography on silica gel.

DFT Calculations
All computations were carried out at the DFT/HF hybrid level of theory using Becke's three-parameter hybrid exchange functional in combination with the gradient-corrected correlation functional of Lee, Yang, and Parr (B3LYP) by using the GAUSSIAN 2003 program packages [76]. The geometries optimization was performed using the 6-311+G(2d,2p) basis set (standard 6-311 basis set added with polarization (d, p) and diffuse functions). Optimizations were performed on all degrees of freedom, and optimized structures were verified as true minima with no imaginary frequencies.
The Hessian matrix was calculated analytically for the optimized structures in order to prove the location of correct minima and to estimate the thermodynamic parameters.

The Absolute Fluorescence Quantum Yield Measurements
The absolute fluorescence quantum yield was measured on a Horiba Fluorolog-3 spectrometer (Edison, NJ, USA) equipped with an integrating sphere. A xenon lamp coupled to a double monochromator was used as excitation light source. The sample (1 cm quartz cuvette cell with molecular solution in THF) or blank (pure THF) were directly illuminated in the center of the integrating sphere. The optical density of all investigated sample solutions in THF did not exceed 0.1 at the luminescence excitation wavelength. Under the same conditions (e.g., excitation wavelength, spectral resolution, temperature), the luminescence spectrum of the sample E c , the luminescence spectrum of the blank E a , the Rayleigh scattering spectrum of the sample L c , and the Rayleigh scattering spectrum of the solvent L a were measured. The absolute fluorescence quantum yield was determined according to the formula:

Appendix A
Magnetic Resonance Research Centre, Chemical Analysis and Materials Research Centre, Centre for Optical and Laser Materials Research, Centre for X-ray Diffraction Studies, Chemistry Educational Centre. The authors are grateful to Dr. N. Samusik (Stanford University) for the helpful comments and additions to the text.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.