Design, Synthesis and Antibacterial Activity of Coumarin-1,2,3-triazole Hybrids Obtained from Natural Furocoumarin Peucedanin

Synthesis of 1,2,3-triazole-substituted coumarins and also 1,2,3-triazolyl or 1,2,3-triazolylalk-1-inyl-linked coumarin-2,3-furocoumarin hybrids was performed by employing the cross-coupling and copper catalyzed azide-alkyne cycloaddition reaction approaches. The synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus, Bacillius subtilis, Actinomyces viscosus and Escherichia coli bacterial strains. Coumarin-benzoic acid hybrids 4с, 42с and 3-((4-acetylamino-3-(methoxycarbonyl)phenyl)ethynyl)coumarin (29) showed promising activity against S. aureus strains, and the 1,2,3-triazolyloct-1-inyl linked coumarin-2,3-furocoumarin hybrid 37c was endowed with high selectivity against B. subtilis and E. coli species. The in vitro antibacterial activity of 4с, 29, 37c and 42с can potentially be compared with that of a number of modern antibiotic drugs used in the clinic, suggesting promising prospects for further research. A detailed study of the molecular interactions with the targeted protein MurB was performed using docking simulations and the obtained results are quite promising.


Introduction
As stated in a WHO report, in the last few decades, the incidence of microbial infections has increased dramatically together with emergence of antimicrobial-resistant strains [1]. Increasing instances of antimicrobial drug resistance requires the design and synthesis of new small molecules with higher affinity and specificity for their potential targets to serve as antibiotics. Coumarins, naturally plant-derived compounds with a benzopyrone moiety, possess a wide variety of biological activities. Series of coumarin derivatives are being extensively studied due to their broad array of biological activities, low toxicity, and lower drug resistance properties [2].
Various naturally-isolated coumarins, as well as their chemically modified analogs, are active against numerous bacterial strains, including those which have developed multidrug resistance [2]. Among these compounds of interest are the coumarin-1,2,3-triazole hybrids ( Figure 1) [3,4]. 1,2,3-Triazoles have been nuclei of choice in recent years because of their excellent favorable safety profile, latent ability to form hydrogen bonds, moderate dipole character, rigidity and stability under in vivo conditions and capability of interacting with biomolecular targets [5,6]. A set of coumarin hybrids A ( Figure 1) having a 1,2,3-triazole moiety in the C-7 position on the coumarin core was assessed for their in vitro antimicrobial activities against Gram-positive and Gram-negative pathogens [7]. The obtained results showed that all hybrids of type A displayed considerable activity against the tested strains, and SAR studies revealed that the substituent and the length of alkyl spacers in the 1 and 4 position of the triazole ring have profound effects on the antimicrobial potency. Morpholinylmethyl-and piperazinylmethyl (N-R 1 ,R 2 )-containing hybrids exhibited noticeable activity against various bacterial pathogens and were more potent than the hybrids incorporating a phthalimidomethyl moiety in the 4 position of the triazole ring. The 6,8-disubstituted coumarin-1,2,3-triazole conjugates B (Figure 1) exhibited significant in vitro antibacterial activity [8,9]. Investigation of the antimicrobial activity of this type of hybrid compounds led to the identification of several different structural frameworks.
The obtained results suggested that both the length of the spacer (n) and also the nature of the substituent (H, OH, OMe) in the 6 and 8 positions modified the lipophilicity of the hybrids, and this in turn affected the antibacterial activity. In these molecules, the triazole rings are linked with the coumarins by a methylene oxygen. The antibacterial activity was correlated with the length of the spacer (n), and the contribution order of substituents was the following: H > OMe > OH.
Molecules 2019, 24, x FOR PEER REVIEW 2 of 22 1,2,3-Triazoles have been nuclei of choice in recent years because of their excellent favorable safety profile, latent ability to form hydrogen bonds, moderate dipole character, rigidity and stability under in vivo conditions and capability of interacting with biomolecular targets [5,6]. A set of coumarin hybrids A ( Figure 1) having a 1,2,3-triazole moiety in the C-7 position on the coumarin core was assessed for their in vitro antimicrobial activities against Gram-positive and Gram-negative pathogens [7]. The obtained results showed that all hybrids of type A displayed considerable activity against the tested strains, and SAR studies revealed that the substituent and the length of alkyl spacers in the 1 and 4 position of the triazole ring have profound effects on the antimicrobial potency. Morpholinylmethyl-and piperazinylmethyl (N-R1,R2)-containing hybrids exhibited noticeable activity against various bacterial pathogens and were more potent than the hybrids incorporating a phthalimidomethyl moiety in the 4 position of the triazole ring. The 6,8-disubstituted coumarin-1,2,3-triazole conjugates B (Figure 1) exhibited significant in vitro antibacterial activity [8,9]. Investigation of the antimicrobial activity of this type of hybrid compounds led to the identification of several different structural frameworks. The obtained results suggested that both the length of the spacer (n) and also the nature of the substituent (H, OH, OMe) in the 6 and 8 positions modified the lipophilicity of the hybrids, and this in turn affected the antibacterial activity. In these molecules, the triazole rings are linked with the coumarins by a methylene oxygen. The antibacterial activity was correlated with the length of the spacer (n), and the contribution order of substituents was the following: H > OMe > OH. The substituted coumarins with 1,2,3-triazole substituent in the 3 position (compounds C) exhibited activity which was dependent on the nature of the substituent on the triazole ring (alkylamide, alkylpeptide) [10].
All coumarin-1,2,3-triazole hybrids of the type D ( Figure 1) displayed promising antibacterial and antifungal properties, which were comparable with the reference drug griseofulvin [11]. The SAR revealed that the substituent at C-5, C-6, and/or C-8 position of coumarin moiety have a great influence on the antimicrobial activity, and 6-Br was favorable for activity against the bacterial strain E. coli and the fungus A. niger, while a 6-Me benefitted the activity against B. subtilis; monohalo hybrids were more potent than their bis-halo counterparts (in the 6 and 8 positions) against all strains.
Thus, the design and synthesis of novel coumarinotriazole derivatives offers a prospective route for accessing new molecules with improved antibacterial activity profiles. Our research interests include the design of convenient ways to access polysubstituted natural furocoumarins [12][13][14][15][16] and coumarins [17,18] and the assessment of their potential as bioactive agents. Herein we report the synthesis of a range of coumarins containing a triazole substituent in the 3 or 6 position of the coumarin core. Our attention was concentrated on the synthesis of compounds bearing both a previously identified pharmacophoric moiety and a substituted triazole ring. Thus, we describe the synthesis of a range of mixed compounds having coumarin-2,3-dihydrofurocoumarin hybrid structures linked through an 1H-1,2,3-triazole ring or alkyne-methylene-triazolyl bridge at the C2-C3 atoms. As starting compounds we used the natural linear furocoumarin peucedanin (1) and the coumarin peuruthenicin (officinalin, 7-hydroxy-6-(methoxycarbonyl)coumarin, 2, Scheme 1). The Pd-catalyzed coupling methods and Cu-catalyzed azide-alkyne cycloaddition (CuAAC reaction) were the main routes of synthesis. The antimicrobial activity of the triazolyl-substituted The substituted coumarins with 1,2,3-triazole substituent in the 3 position (compounds C) exhibited activity which was dependent on the nature of the substituent on the triazole ring (alkylamide, alkylpeptide) [10].
All coumarin-1,2,3-triazole hybrids of the type D ( Figure 1) displayed promising antibacterial and antifungal properties, which were comparable with the reference drug griseofulvin [11]. The SAR revealed that the substituent at C-5, C-6, and/or C-8 position of coumarin moiety have a great influence on the antimicrobial activity, and 6-Br was favorable for activity against the bacterial strain E. coli and the fungus A. niger, while a 6-Me benefitted the activity against B. subtilis; monohalo hybrids were more potent than their bis-halo counterparts (in the 6 and 8 positions) against all strains.
Thus, the design and synthesis of novel coumarinotriazole derivatives offers a prospective route for accessing new molecules with improved antibacterial activity profiles. Our research interests include the design of convenient ways to access polysubstituted natural furocoumarins [12][13][14][15][16] and coumarins [17,18] and the assessment of their potential as bioactive agents. Herein we report the synthesis of a range of coumarins containing a triazole substituent in the 3 or 6 position of the coumarin core. Our attention was concentrated on the synthesis of compounds bearing both a previously identified pharmacophoric moiety and a substituted triazole ring. Thus, we describe the synthesis of a range of mixed compounds having coumarin-2,3-dihydrofurocoumarin hybrid structures linked through an 1H-1,2,3-triazole ring or alkyne-methylene-triazolyl bridge at the C2-C3 atoms. As starting compounds we used the natural linear furocoumarin peucedanin (1) and the coumarin peuruthenicin (officinalin, 7-hydroxy-6-(methoxycarbonyl)coumarin, 2, Scheme 1). The Pd-catalyzed coupling methods and Cu-catalyzed azide-alkyne cycloaddition (CuAAC reaction) were the main routes of synthesis. The antimicrobial activity of the triazolyl-substituted coumarins against a panel of clinically relevant bacterial strains were also investigated and discussed.
The synthetic route followed for the synthesis of the desired novel coumarins substituted with a triazole ring in the C-3 position 8-10 is outlined in Scheme 2. Bromination of peuruthenicin (2) with dioxane dibromide (2.2 eq) in CH2Cl2 proceeds selectively and led to the formation of 3-bromopeuruthenicin (11) as the sole product in 88% isolated yield. 3-Аzidocoumarin 12 was conveniently obtained in 68% yield, by reacting the corresponding 3-bromopeuruthenicin (11) with sodium azide in DMF. The second alkyne building block was obtained from amino acids in two steps. At the first step, 9-aminopelargonic acid, D,L-2-aminobutyric acid or L-phenylalanine hydrochloride were transformed to subsequent methyl ethers of the amino acid hydrochloride in nearly quantitative yield requiring no further purification. Reaction of amino acids methyl esters hydrochloride with propargyl bromide in the presence of K2CO3 in dry DMF gave alkynes 13-15, which were purified through the column chromatography on silica gel using a CHCl3/EtOH mixture as an eluent.
The synthetic route followed for the synthesis of the desired novel coumarins substituted with a triazole ring in the C-3 position 8-10 is outlined in Scheme 2. Bromination of peuruthenicin (2) with dioxane dibromide (2.2 eq) in CH 2 Cl 2 proceeds selectively and led to the formation of 3-bromopeuruthenicin (11) as the sole product in 88% isolated yield. 3-Azidocoumarin 12 was conveniently obtained in 68% yield, by reacting the corresponding 3-bromopeuruthenicin (11) with sodium azide in DMF. The second alkyne building block was obtained from amino acids in two steps. At the first step, 9-aminopelargonic acid, d,l-2-aminobutyric acid or L-phenylalanine hydrochloride were transformed to subsequent methyl ethers of the amino acid hydrochloride in nearly quantitative yield requiring no further purification. Reaction of amino acids methyl esters hydrochloride with propargyl bromide in the presence of K 2 CO 3 in dry DMF gave alkynes 13-15, which were purified through the column chromatography on silica gel using a CHCl 3 /EtOH mixture as an eluent.
The interaction of the new azide 12 with methyl 9-(prop-2-ynylamino)nonanoate 13 under above catalytic conditions was carried out at 20 °C for 4 h (monitoring by TLC) and led to the target compound 8 in the isolated yield 67%. The reaction of azide 12 with terminal alkynes 14, 15, proceeds with the formation of substituted coumarins 9 or 10 in high yield by performing this reaction in the presence of CuI and Et3N (method d). By using of CuSO4 and sodium ascorbate as the catalysts in the reaction of 12 with 15, the isolated yield of coumarin 10 was decreased to 34%. For this purpose we studied the activity of 3-bromopeuruthenicin (11) in the Sonogashira cross-coupling reaction. Besides providing data about the reactivity of 3-bromopeuruthenicin (11) in the cross-coupling reaction with various terminal alkynes, this study served to make available a larger panel of peuruthenicin derivatives for testing their antimicrobial activity. Therefore, the reaction between 3-bromopeurutenicine (11) with phenyl acetylene (17) was optimized first, attempting to obtain the cross-coupling derivative 18 under different conditions. After considerable experimentation, we found that the cross-coupling proceeds in a benzene solution in the presence of a catalytic amounts of trans-dichlorobis(triphenylphosphine)palladium(II), copper(I) iodide, and Et3N as a base (conditions a, Scheme 3) and led to the corresponding 3-(phenylethynyl)coumarin (18) in 65%yield after column chromatography on silica gel. Using Bu4NBr as an additive, DMF as a solvent and Et3N as a base did not improve the yield of compound 18 (conditions b). On the other hand, using K2CO3 as a base in benzene or DMF at 80-100 °C also met with failure, and the reaction afforded yields of 45% or 42%, respectively, and a yield of 20% was recorded when the transformation was performed using (iPr)2NH as a base. With the set of optimum conditions in hands, we found that the reaction of coumarin 11 with aryl(hetaryl)acetylenes 19-24 proceeds with the formation of compounds 25-30 (52-68% yields. A perspective route for synthesis of terminal The interaction of the new azide 12 with methyl 9-(prop-2-ynylamino)nonanoate 13 under above catalytic conditions was carried out at 20 • C for 4 h (monitoring by TLC) and led to the target compound 8 in the isolated yield 67%. The reaction of azide 12 with terminal alkynes 14, 15, proceeds with the formation of substituted coumarins 9 or 10 in high yield by performing this reaction in the presence of CuI and Et 3 N (method d). By using of CuSO 4 and sodium ascorbate as the catalysts in the reaction of 12 with 15, the isolated yield of coumarin 10 was decreased to 34%.
Pd(PPh3)2Cl2, CuI as catalysts and Et3N as a base in toluene solution under microwave irradiation at 100 °C for 2 h give the cross-coupling product 34 in 64% isolated yield. Thus, the use of microwaves was found to significantly improve the reaction yield and shorten the reaction time. Desilylation of compound 34 using CsF in MeOH in the presence of benzyltriethylammonium chloride (TEBA) afforded 3-ethynylpeuruthenicin (16, 85% yield) which was purified by column chromatography on silica gel using CHCl3 as an eluent. At the next step of our study, the acetylene building block 16 was reacted with 2-azidooreozelone (35) [14]. Carrying out the reaction in a СH2Сl2-water mixture, with catalysis of the Cu(I) ions generated in situ from CuSO4 and sodium ascorbate we obtained the coumarin-2,3-dihydrofurocoumarin hybrid linked through an 1H-1,2,3-triazole ring 36 isolated in 70% yield (Scheme 4).
We also performed the synthesis of coumarin-2,3-dihydrofurocoumarin hybrids 37a-c, containing a triazolylmethylene-1-inyl linker group (Scheme 4). The synthetic route involved the For this purpose we studied the activity of 3-bromopeuruthenicin (11) in the Sonogashira cross-coupling reaction. Besides providing data about the reactivity of 3-bromopeuruthenicin (11) in the cross-coupling reaction with various terminal alkynes, this study served to make available a larger panel of peuruthenicin derivatives for testing their antimicrobial activity. Therefore, the reaction between 3-bromopeurutenicine (11) with phenyl acetylene (17) was optimized first, attempting to obtain the cross-coupling derivative 18 under different conditions. After considerable experimentation, we found that the cross-coupling proceeds in a benzene solution in the presence of a catalytic amounts of trans-dichlorobis(triphenylphosphine)palladium(II), copper(I) iodide, and Et 3 N as a base (conditions a, Scheme 3) and led to the corresponding 3-(phenylethynyl)coumarin (18) in 65% yield after column chromatography on silica gel. Using Bu 4 NBr as an additive, DMF as a solvent and Et 3 N as a base did not improve the yield of compound 18 (conditions b). On the other hand, using K 2 CO 3 as a base in benzene or DMF at 80-100 • C also met with failure, and the reaction afforded yields of 45% or 42%, respectively, and a yield of 20% was recorded when the transformation was performed using (iPr) 2 NH as a base. With the set of optimum conditions in hands, we found that the reaction of coumarin 11 with aryl(hetaryl)acetylenes 19-24 proceeds with the formation of compounds 25-30 (52-68% yields. A perspective route for synthesis of terminal alkynes (for example, 3-ethynylpeuruthenicin (16) was the Sonogashira coupling of 11 with accessible 2-methylbut-3-yn-2-ol (31) and subsequent elimination of acetone from the 3-alkynylcoumarin 32. We found that the reaction of 11 with alkyne 31 (conditions a) led to the formation of 3-(3-hydroxy-3-methylbut-1-ynyl)coumarin (32, (66% yield). However, the transformation of the alcohol 32 into the terminal alkyne using the known conditions [20] was unsuccessful. The copper and palladium-catalyzed Sonogashira coupling of 3-bromopeuruthenicin 11 with trimethylsilylacetylene (33) (using conditions b) rqwuired a long reaction time and proceeds with the formation of traces of 3-(trimethylsilyl-ethynyl)peuruthenicin (34). Those results forced us improve this reaction. Currently, microwave-assisted organic synthesis (MAOS) is receiving increasing attention as a valuable alternative to the conventional heating to speed up chemical reactions, and microwave irradiation has been applied to Sonogashira coupling reactions [21,22]. It should be noted that MAOS has significant advantages that include simplicity in operation, increased reaction rates, and improved reaction yields. Performing the microwave-assisted reaction of 3-bromopeuruthenicin (11) and trimethylsilylacetylene (33) using Pd(PPh 3 ) 2 Cl 2 , CuI as catalysts and Et 3 N as a base in toluene solution under microwave irradiation at 100 • C for 2 h give the cross-coupling product 34 in 64% isolated yield. Thus, the use of microwaves was found to significantly improve the reaction yield and shorten the reaction time. Desilylation of compound 34 using CsF in MeOH in the presence of benzyltriethylammonium chloride (TEBA) afforded 3-ethynylpeuruthenicin (16, 85% yield) which was purified by column chromatography on silica gel using CHCl 3 as an eluent.
At the next step of our study, the acetylene building block 16 was reacted with 2-azidooreozelone (35) [14]. Carrying out the reaction in a CH 2 Cl 2 -water mixture, with catalysis of the Cu(I) ions generated in situ from CuSO 4 and sodium ascorbate we obtained the coumarin-2,3-dihydrofurocoumarin hybrid linked through an 1H-1,2,3-triazole ring 36 isolated in 70% yield (Scheme 4).

Biological Screening
The new coumarins 4a-c, 11, 29, 30, coumarin-dihydrofurocoumarin hybrids 36, 37a-c, and the 7-triazolylsubstituted coumarins 42a-c (Figure 2), the synthesis of which was described in our previous paper [23] were screened for their in vitro antibacterial activity against Gram positive bacterial strain Staphylococcus aureus 209 ATCC 6538P. The antibacterial activity was studied by serial dilution in a liquid nutrient medium [24]. The minimum inhibition concentrations (MIC) of the test cultures were determined. The most promising substances were those with indicators of MIC = 100 µg/mL and less. Table 1 reveals that coumarino-1,2,3-triazole derivatives 4c and 42c and 3-arylethynylpeuruthenicin 29 showed excellent antibacterial activity, with MIC values ranging from 0.16-0.41 µg/mL against the tested microorganism and they prove to be better than that of the reference drugs ceftriaxone (0.97 µg/mL) and streptomycin (1.89 µg/mL).  In the series of coumarin-benzoic acid hybrids 4a-c, 42a-c in view of the position of the carboxylic group in the benzoic acid moiety, we considered that compounds containing this substituent in the 4 position 4c, 42c were more active than compounds 4a,b and 42a,b, respectively, so the carboxylic acid group is an excellent microbial ligand which can bind more effectively at the active site of receptor. The difference in antibacterial activity profile of 3-bromocoumarin 11 and 3-(arylethynyl) coumarins 26, 29, 30 indicates the importance of the nature of the functional substituent on the triple bond in the C-3 position of coumarin. Thus, compound 29 containing an anthranilic acid methyl ester fragment on the 3-ethynylcoumarin nucleus showed greater activity than compounds with 4-tolyl-or pyridine substituents at the triple bond in 3-ethynylcoumarin. It should be noted that the biological effect of anthranilic acid derivatives is based on their ability to act as modulators of the nuclear peroxisome proliferator activated receptors (PRAR) and the farnesoid X (FXR) receptors, which fulfill crucial roles in metabolic balance [25,26]. In this direction the antibacterial activity of compounds 37a-c, having a methylene-triazolyl-furocoumarin substituent on the triple bond of 3-ethynylcoumarin was dependent on the length of the C-methylene linker. The activity of the most active hybrid compound 37c is due the presence of a hexamethylene-1-inyl linker group. Coumarin-2,3-dihydrofurocoumarin hybrid 37c was found to exhibit good potency at 51.25 mg/mL of MIC, while the parent compounds 1 and 2 exhibited a lack of activity ( Table 1).
The results of study of promising substances 4с, 29, 37c and 42с on other strains of Staphylococcus aureus and Actinomyces viscosus U-18 are presented in Table 2. Further study on the S. aureus strain confirmed the high activity of compounds 4с, 29 and 42c. Compound 4c (carboxamidotriazolyl-benzoic acid substitution at the C-6 position of the coumarin core) showed good activity against A. viscosus compared with compound 42c which showed moderate activity against A. viscosus. Characteristically, that compound 42c with a triazolylbenzoic acid substituent in the C-7 position possessed the highest activity against S. aureus "Viotko" bacterial strains. Of interest was also the high antibacterial activity of 3-ethynylcoumarin with methylanthranilate substituent 29 on the all tested S. aureus strains. This compound will be further used as the scaffold for structural optimization to develop more potent and selective antibacterial agents. Chemical structures of antibacterial coumarin-benzoic acid hybrids and coumarin-furocoumarin hybrids with a 1,2,3-triazolyl linker. In the series of coumarin-benzoic acid hybrids 4a-c, 42a-c in view of the position of the carboxylic group in the benzoic acid moiety, we considered that compounds containing this substituent in the 4 position 4c, 42c were more active than compounds 4a,b and 42a,b, respectively, so the carboxylic acid group is an excellent microbial ligand which can bind more effectively at the active site of receptor. The difference in antibacterial activity profile of 3-bromocoumarin 11 and 3-(arylethynyl) coumarins 26, 29, 30 indicates the importance of the nature of the functional substituent on the triple bond in the C-3 position of coumarin. Thus, compound 29 containing an anthranilic acid methyl ester fragment on the 3-ethynylcoumarin nucleus showed greater activity than compounds with 4-tolyl-or pyridine substituents at the triple bond in 3-ethynylcoumarin. It should be noted that the biological effect of anthranilic acid derivatives is based on their ability to act as modulators of the nuclear peroxisome proliferator activated receptors (PRAR) and the farnesoid X (FXR) receptors, which fulfill crucial roles in metabolic balance [25,26]. In this direction the antibacterial activity of compounds 37a-c, having a methylene-triazolyl-furocoumarin substituent on the triple bond of 3-ethynylcoumarin was dependent on the length of the C-methylene linker. The activity of the most active hybrid compound 37c is due the presence of a hexamethylene-1-inyl linker group. Coumarin-2,3-dihydrofurocoumarin hybrid 37c was found to exhibit good potency at 51.25 mg/mL of MIC, while the parent compounds 1 and 2 exhibited a lack of activity ( Table 1).
The results of study of promising substances 4с, 29, 37c and 42с on other strains of Staphylococcus aureus and Actinomyces viscosus U-18 are presented in Table 2. Further study on the S. aureus strain confirmed the high activity of compounds 4с, 29 and 42c. Compound 4c (carboxamidotriazolylbenzoic acid substitution at the C-6 position of the coumarin core) showed good activity against A. viscosus compared with compound 42c which showed moderate activity against A. viscosus. Characteristically, that compound 42c with a triazolylbenzoic acid substituent in the C-7 position possessed the highest activity against S. aureus "Viotko" bacterial strains. Of interest was also the high antibacterial activity of 3-ethynylcoumarin with methylanthranilate substituent 29 on the all tested S. aureus strains. This compound will be further used as the scaffold for structural optimization to develop more potent and selective antibacterial agents. Next set of experiments was dedicated to the analysis of the antibacterial potential of coumarin-2,3-dihydrofurocoumarin hybrids 36, 37a-c ( Table 3). The in vitro antibacterial activity of those compounds was additionally tested against Bacillius subtilis and Escherichia coli (JM 109) bacterial strains. The obtained results were compared with the known tumorogenic compound 4-nitroquinolin-1-oxide (NQO) and presented as an average concentration of inhibitory 50% bacterial proliferation (incubation time 20 h).

Molecular Docking
Molecular docking protocol is an essential tool to mimic the natural course of interaction of the ligand with active sites of receptors through lowest binding energies in which two molecules fit together in 3D space. Here, we discuss the mechanism of interaction between coumarinotriazole derivatives 3, 4 and 5 with the MurB protein (PDB ID: 1HSK). MurB is an attractive target protein in bacterial infection diseases and it is an essential enzyme which takes part in amino sugar metabolism which reduces the enolpyruvyluridine diphosphate N-acetyl glucosamine (EP-UNAG) as an intermediate in the assembly of the UNAM-pentapeptide (m-A2pm) protein to uridine diphosphate N-acetylMuamic acid (UNAM) of cell wall precursor [27]. For the calculations, the XRD model of S. aureus N-acetylenolpyruvylglucosamine reductase (MurB) with PDB ID 1HSK was chosen (resolution 2.3 Å) [28]. To model a possible mechanism of MurB inhibition, molecular docking of new coumarins was performed at the binding site of flavin adenine dinucleotide (FAD) in the Glide application [29]. We have screened the coumarinotriazoles 4a-c, 8, 9, and 42a-c and also coumarin-furocoumarin hybrids 36, 37b, and 37c. The molecules 4a-c, 37c and 42a,c strongly approach the MurB protein receptor as shown by their minimum binding energies −8.416-−8.983 Kcal/mol ( Table 4). The docking results were found to be in good agreement with in vitro antibacterial experimental MIC values (Tables 1-3). The FAD binding site is saturated with polar amino acids. This facilitates formation of a large number of hydrogen bonds due to the large number of polar groups in the FAD molecule. Additional stabilization in the binding site provided stacking interactions of aromatic cycles of adenine. Inspection of the binding mode demonstrated, that compounds 4c, 37c and 42c successfully combine in their structure a large number of polar groups and π-systems ( Figure 3). The presence of the carboxy function in compounds 4c, 42c allowed the formation of hydrogen bond with amino acid ARG225 residue ( Figure 3A-C). As shown in Figure 3C, the inhibitor 37c formed addition hydrogen bonding interactions with the active site residues: the furan ring C=O with SER 238 (2.92 Å) and the coumarin ring C=O with TYR 155. All those interactions contribute to a spatial orientation close to FAD and the formation of hydrogen bonds with the same amino acid residues as FAD ( Figure 3D). We found that the MurB protein receptor amino acids ASN80, SER82, SER238, GLY81 are the most active sites responsible for interactions with the ligand. Important interaction centers at the binding site are arginines 225 and 310, protein chain section 79-83, glycines 145, 146 and 153, isoleucine 140 and proline 141, valine 199. New coumarins interact with almost all these centers, except for 140-141, and also form bonds characteristic only for these molecules. The formation of interactions with aromatic amino acid residues increases the stability of the conformations of new coumarins at the binding site. with the same amino acid residues as FAD ( Figure 3D). We found that the MurB protein receptor amino acids ASN80, SER82, SER238, GLY81 are the most active sites responsible for interactions with the ligand. Important interaction centers at the binding site are arginines 225 and 310, protein chain section 79-83, glycines 145, 146 and 153, isoleucine 140 and proline 141, valine 199. New coumarins interact with almost all these centers, except for 140-141, and also form bonds characteristic only for these molecules. The formation of interactions with aromatic amino acid residues increases the stability of the conformations of new coumarins at the binding site.

Conclusions
In summary, we have synthesized new series of coumarinotriazole compounds using [2 + 3]-cycloaddition reactions or Sonogashira cross-coupling reactions as the main approaches. The synthesized coumarino-triazole type derivatives were screened for their in vitro antimicrobial activity . Compounds 4c and 42c

Conclusions
In summary, we have synthesized new series of coumarinotriazole compounds using [2 + 3]-cycloaddition reactions or Sonogashira cross-coupling reactions as the main approaches. The synthesized coumarino-triazole type derivatives were screened for their in vitro antimicrobial activity. Compounds 4c and 42c, having the 4-(carboxyphenyl)triazolyl substituent in the 6 or 7 position of the coumarin ring showed excellent antibacterial activity against S. aureus strains, with MIC values of 0.16-3.75 µg/mL and 0.21-6.28 µg/mL respectively. Most of the compounds of series 11, 29 and 30 with bromine, and aryl(hetaryl)substituents in the 3 position revealed significant MIC values (between 0.41 to 2.0 µg/mL for compound 29), indicating that 3-substituted coumarin analogues show promising antibacterial activity and are key compounds for further development as antibacterial agents. Coumarin-2,3-dihydrofurocoumarin hybrid compound 37c was found to be selective against Bacillius subtilis and E. coli, with MIC values of 0.02-0.15 µg/mL. A molecular docking study was performed for the most active compounds against the MurB protein. Molecular docking results were well corroborated with the in vitro antibacterial activity findings. Finally, the successful synthesis and antimicrobial evaluation along with docking study of new coumarino-triazole scaffolds obtained on the base of accessible plant coumarins peucedanin and peuruthenicin will provide further advantages to design and develop triazole derivatives with selective antibacterial activity.

General Information
NMR spectra were acquired on Bruker AV-400 ( 1 H: 400.13 MHz, 13 C: 100.78 MHz) or Bruker AV-600 ( 1 H: 600.30 MHz, 13 C: 150.95 MHz) instruments (Bruker BioSpin GmbH, Rheinstetten, Germany), using tetramethylsilane (TMS) as an internal standart. In the description of the 1 H-and 13 C-NMR spectra, the furocoumarin and coumarin skeleton atoms numeration system given in structures 1 and 2 was used. Chemical shifts are reported in parts per million (ppm). The IR spectra were recorded by means of the KBr pellet technique on a Bruker Vector-22 spectrometer. The UV spectrum were obtained on an HP 8453 UV Vis spectrometer (Hewlett-Packard, Waldbronn, Germany). HRMS spectra were recorded on a DFS mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), evaporator temperature 180-220 • C, EI ionization at 70 eV). The specific rotation values [α] D were determined on a PolAAr 3005 polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA), and expressed in (deg·mL)/(g·dm), while concentration was expressed in g per 100 mL of solution. Melting points were determined using Stuart SMP30 melting point apparatus (Bibby Scientific, Staffordshire, UK). The microwave irradiation reaction was performed in a Microwave 50 reactor (Anton Paar, Graz, Austria). Elemental analysis was carried out on an 1106 Elemental analysis instrument (Carlo-Erba, Milan, Italy). The molecular weights of compounds 37a-c, 38c were determined using a Knauer vapor pressure osmometer (Knauer, Berlin, Germany). Spectral and analytical investigations were carried out at Collective Chemical Service center of Siberian Branch of the Russian Academy of Sciences.
Reaction products were isolated by column chromatography on silica gel 60 (0.063-0.200 mm, Merck KGaA, Darmstadt, Germany) eluting with chloroform and chloroform-ethanol (100:1; to 25:l). The reaction progress and the purity of the obtained compounds were monitored by TLC on Silufol UV-254 plates (CHCl 3 -EtOH, 9:1; detection under UV light or by treatment with iodine vapor).

General Method for the N-Propargylation of Amino Acid Methyl Esters
Propargyl bromide (10.5 mmol, 80% in toluene solution) was added to a solution of the appropriate amino acid ether hydrochlorides (10 mmol) and potassium carbonate (21 mmol) in dry DMF at room temperature under argon. The mixture was stirred for 1-3 days (TLC control), the solvent was evaporated under reduced pressure and the residue partitioned between water (30 mL) and CH 2 Cl 2 (30 mL). The organic phase was separated and the aqueous phase was extracted with CH 2 Cl 2 (3 × 10 mL). The organic phases were combined, dried (MgSO 4 ), filtered and the solvent evaporated under reduced pressure. The residue was purified by column chromatography to give secondary amines 13, 14, 15.