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

Article abstract of DOI:10.3390/molecules24112126

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.

Full text of DOI:10.3390/molecules24112126

molecules
Article
Design, Synthesis and Antibacterial Activity
of Coumarin-1,2,3-triazole Hybrids Obtained from
Natural Furocoumarin Peucedanin
Alla V. Lipeeva 1, Danila O. Zakharov 1, Liubov G. Burova 2, Tatyana S. Frolova 3,4
,
Dmitry S. Baev 1 , Ilia V. Shirokikh 2, Alexander N. Evstropov 2, Olga I. Sinitsyna 3,4
,
Tatyana G. Tolsikova 1 and Elvira E. Shults 1,4,
*
1
Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences,
Lavrentyev Ave. 9, 630090 Novosibirsk, Russia; mond_05@list.ru (A.V.L.); zakharov@nioch.nsc.ru (D.O.Z.);
mitja2001@gmail.com (D.S.B.); TG_tolstikova@mail.ru (T.G.T.)
2
3
Department of Microbiology, Immunology and Virology, Novosibirsk State Medical University,
Krasny Prospect 52, 630091 Novosibirsk, Russia; mic.bio.lgb@mail.ru (L.G.B.); ishirok@gmail.com (I.V.S.);
mic.bio.ave@mail.ru (A.N.E.)
The Federal Research Center Institute of Cytology and Genetics, Acad. Lavrentyev Ave. 10,
630090 Novosibirsk, Russia; frolova@bionet.nsc.ru (T.S.F.); oisinits@bionet.nsc.ru (O.I.S.)
Novosibirsk State University, Pirogova Str. 1, 630090 Novosibirsk, Russia
4
*
Correspondence: schultz@nioch.nsc.ru; Tel.: +7-(383)-330-8533
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 12 May 2019; Accepted: 31 May 2019; Published: 5 June 2019
Abstract:
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.
Keywords: coumarin; furocoumarin; Sonogashira coupling; CuAAC reaction; antibacterial activity
1. 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
Molecules 2019, 24, 2126; doi:10.3390/molecules24112126
www.mdpi.com/journal/molecules

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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 [
(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 [ ]. The obtained
results showed that all hybrids of type displayed considerable activity against the tested strains,
5,6]. A set of coumarin hybrids A
7
A
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 [ ]. Investigation of the antimicrobial activity of
8,9
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.
Figure 1. Chemical structures of coumarin-1,2,3-triazole hybrids of types A–D.
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
–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-C30 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

Molecules 2019, 24, 2126
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synthesis. The antimicrobial activity of the triazolyl-substituted coumarins against a panel of clinically
relevant bacterial strains were also investigated and discussed.
Scheme 1. Synthesis 6-(1-(carboxyphenyl)-1H-1,2,3-triazol-4-ylcarbamoyl)umbelliferones 4a–c
.
Reagents and Conditions: (
a
) 20% eq. NaOH, dioxane, reflux, 20 min; (
b
) DCC, HOBt, Et3N, DMF, rt,
.
48 h; (c) CuSO4 5H2O (5 mol%), sodium ascorbate (15 mol%), H2O-CH2Cl2, 20→40 ◦C.
2. Results and Discussion
2.1. Chemical Synthesis
The umbelliferon carboxylic acid
coumarins 4a (Scheme 1), was conveniently obtained by treatment of peuruthenicin (
dioxane [19]. The peptide coupling reaction of with propargyl amine using DCC as a coupling reagent
gave the corresponding 6-(prop-2-ynylcarbamoyl)umbelliferone ( ) in 67% yield. We have chosen
3
, the starting compound for the synthesis of triazolyl substituted
–
c
2) with NaOH in
3
5
6
the most simple and well-characterized variant, namely, carrying out the reaction in a CH2Cl2-water
mixture, with catalysis of the Cu(I) ions generated in situ from CuSO4 and sodium ascorbate [12].
CuAAC-reaction of ethynyl coumarin
6-[1-(carboxyphenyl)-1,2,3-triazol-4-yl]carbamoyl)- umbelliferones 4a–c in 50–62% yields.
The synthetic route followed for the synthesis of the desired novel coumarins substituted with
a triazole ring in the C-3 position 10 is outlined in Scheme 2. Bromination of peuruthenicin
) with dioxane dibromide (2.2 eq) in CH2Cl2 proceeds selectively and led to the formation of
6 with three positional azidobenzoic acid isomers 7a–с led to the
8–
(2
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 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.

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Scheme 2. Synthesis of 3-(triazolyl)-substituted coumarins
dibromide, CH2Cl2, rt, 16 h; (
): NaN3, DMF, 40 ◦C, 14 h; (
(1:1), rt, 3 h, then 40 ◦C, 1 h; (d): CuI, Et3N, MeCN, rt, 10 h.
8
–
10. Reagents and Conditions: (
a): dioxane
b
c) CuSO4, sodium ascorbate, eq. CH2Cl2
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 or 10 in high yield by performing this reaction in the presence of
9
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%.
Thus we obtained 3-(1H-1,2,3-triazol-1-yl)peuruthenicin derivatives 8–10. It was also important
for us to identify the influence of the position of coumarin as the substituent in the C-4 or C-1 of the
triazole ring. To obtain 3-(1H-1,2,3-triazol-4-yl)peuruthenicins we were interested in the synthesis of
3-(ethynyl)peuruthenicin (16, Scheme 3).

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Scheme 3.
Synthesis 3-arylethynylcoumarins 17
,
24
–
a
29
): Pd(PPh3)2Cl2, CuI, Et3N, benzol,
c) Pd(PPh3)2Cl2, CuI, Et3N, toluene, MAOS,
,
3-(alkynyl)coumarins 31
,
33,
and 3-(ethynyl)peuruthenicin (34). Reagents and Conditions: (
110 ◦C, 12 h; (
) Pd(PPh3)2Cl2, CuI, Et3N, Bu4NBr, DMF; (
100 ◦C, 2 h; (d) CsF, MeOH, TEBA, rt, 10 h.
b
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
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
–24 proceeds with the formation of
–
(
(
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

Molecules 2019, 24, 2126
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operation, increased reaction rates, and improved reaction yields. Performing the microwave-assisted
reaction of 3-bromopeuruthenicin (11) and trimethylsilylacetylene (33) using 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 CH2Cl2-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).
(a)
(b)
Scheme 4. Synthesis of coumarin-2,3-dihydrofurocoumarin hybrids linked with 1H-1,2,3-triazole
.
rings. Reagents and Conditions: (
a
): CuSO4 5H2O (5 mol%), sodium ascorbate (15 mol%), H2O-CH2Cl2,
.
20
→
40 ◦C; (
b
): CuSO4 5H2O, sodium ascorbate, H2O-CH2Cl2, 40 ◦C, 5 h; (
c) Pd(PPh3)2Cl2, CuI, Et3N,
benzene, 80 ◦C, 14 h.
We also performed the synthesis of coumarin-2,3-dihydrofurocoumarin hybrids 37a
containing a triazolylmethylene-1-inyl linker group (Scheme 4). The synthetic route involved
the Sonogashira’s reaction of new 2-ethynylalkyltriazolyl substituted furocoumarins 38a with
3-bromopeuruthenicin (11, Scheme 4). Compounds 38a (yield 62–74%) were prepared by the CuAAC
reaction of 2-azidooreoselone (35), with 1,6-heptadiyne (39), 1,7-octadiyne (40) or 1,9-decadiyne (41).
Reaction of 3-bromopeuruthenicin (16) with alkynes 38a in the Sonogashira reaction conditions
–c,
–
c
–c
–
с
afforded the coumarin-2,3-dihydrofurocoumarin hybrids linked through 1H-1,2,3-triazolylalk-1-inyl
groups 37a–c (52–57% yields).
2.2. Biological Screening
The new coumarins 4a
7-triazolylsubstituted coumarins 42a
paper [23] were screened for their in vitro antibacterial activity against Gram positive bacterial strain
–c,
11
,
29
,
30, coumarin-dihydrofurocoumarin hybrids 36
, 37a–c, and the
–
c (Figure 2), the synthesis of which was described in our previous

Molecules 2019, 24, 2126
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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
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 g/mL against
µg/mL and less.
µ
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).
Figure 2.
Chemical structures of antibacterial coumarin-benzoic acid hybrids and
coumarin-furocoumarin hybrids with a 1,2,3-triazolyl linker.
Table 1. The in vitro antibacterial activity of compounds 4a c, 11, 26, 29, 30, 37a–c, 42a–c and starting
–
compounds 1–3 against bacterial strain S. aureus 209p ATCC6538P.
S. aureus 209p
S. aureus 209p
MIC, µg/mL
Compound
Compound
MIC, µg/mL
800 ± 28.87
>1000
1
2
30
162.5 ± 23.94
250 ± 20.41
425 ± 47.87
51.25 ± 3.15
>1000
37a
3
933.3 ± 66.67
>1000
37b
37c
4a
4b
4c
11
26
29
416.7 ± 60.09
0.16 ± 0.08
92.5 ± 4.79
412.5 ± 42.7
0.41 ± 0.13
42a
42b
>1000
42c
0.31 ± 0.23
0.97 ± 0.02
1.89 ± 0.08
Ceftriaxonum
Streptomycin
In the series of coumarin-benzoic acid hybrids 4a
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 and 42a , respectively, so the carboxylic acid
–c, 42a–c in view of the position of the carboxylic
,
,
b
,b
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

Molecules 2019, 24, 2126
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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
and 2 exhibited a lack of activity (Table 1).
1
The results of study of promising substances 4с
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-
, 29, 37c and 42с on other strains of Staphylococcus
,
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.
Table 2. The in vitro antibacterial activity results of compounds 4c
,
29
,
37c and 42c against S. aureus
and A. viscosus U-18 strains.
S. aureus C-18
MIC, µg/mL
S. aureus “Viotko”
A. viscosus U-18
MIC, µg/mL
Compound
MIC, µg/mL
3.75 ± 1.73
2.0 ± 1.01
68.8 ± 18.75
0.21 ± 0.08
1.03 ± 0.32
4c
29
0.65 ± 0.22
1.78 ± 1.10
68.75 ± 11.97
6.28 ± 2.37
6.50 ± 1.29
15.1 ± 1.56
75.2 ± 14.43
Not Tested
166.7 ± 44.1
933.3 ± 33.33
37c
42c
Ceftriaxone
Next set of experiments was dedicated to the analysis of the antibacterial potential
of coumarin-2,3-dihydrofurocoumarin hybrids 36 37a (Table 3). The in vitro antibacterial
,
–c
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).
It can be observed that from the
series of coumarin-2,3-dihydrofurocoumarin hybrids only dimeric compound 37c with the
1,2,3-triazolyloct-1-inyl linker group displayed promising activity against B. subtilis and also E. coli
(JM 109) bacterial strains. On the contrary to this, variously 1,2,3-triazolyl or 1,2,3-triazolylhex-1-inyl
or 1,2,3-triazolylpent-1-inyl linked hybrids 36, 37a,b were deprived of anti-bacterial activities (MICs
were >1000 mg/mL).

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Table 3. Antimicrobial activities of coumarin-furocoumarin hybrids 36, 37a–c and parent compounds
1, 2 against B. subtilis and E. coli strains (* P < 0.05 relative to the control).
MIC ± SEM, µg/mL
Compound
Bacillius subtilis
Escherichia coli (JM 109)
1
>1000
>1000
2
36
>1000
>1000
>1000
>1000
37a
>1000
>1000
37b
>1000
>1000
37c
0.02 ± 0.003 *
10.8 ± 2.50 *
2.0 ± 0.68
0.15 ± 0.04 *
12.79 ± 1.88 *
1.0 ± 0.35
NQO
Streptomycin
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
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
–c, 8, 9, and 42a–c and also coumarin-furocoumarin
,
–
−
−
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
in their structure a large number of polar groups and -systems (Figure 3). The presence of the carboxy
function in compounds 4c 42c allowed the formation of hydrogen bond with amino acid ARG225
, 37c and 42c successfully combine
π
,
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.

Molecules 2019, 24, 2126
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Table 4. Values of the minimum binding energy for new compounds to MurB protein.
Ligand
FAD
42c
37c
42a
4c
Binding Energy *, kcal/mol
Ligand
Binding Energy *, kcal/mol
−12.081
−8.983
−8.977
−8.898
−8.473
−8.416
−8.171
2
37b
42b
8
−7.884
−7.844
−7.441
−6.578
−6.509
−6.300
−5.399
1
4b
4a
36
9
* Value is not genuine binding energy but estimated docking score.
(A)
(B)
Figure 3. Cont.

Molecules 2019, 24, 2126
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(C)
(D)
Figure 3. Noncovalent interactions of compounds (3A
dotted lines: green—hydrogen bonds, purple—stacking interactions, orange—electrostatic interactions.
Hydrophobic interactions and nonpolar hydrogens are omitted. ( ) 3D interactions of ligand 4c at the
FAD binding site of MurB Protein. ( ) 3D interactions of ligand 42c at the FAD binding site of MurB
Protein. ( ) 3D interactions of ligand 37c at the FAD binding site of MurB Protein. ( ) 3D interactions
of FAD molecule at the binding site of MurB Protein.
—4c, 3B—42c, 3C—37c, 3D—FAD) are shown by
A
B
C
D
3. 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
29 and 30 with bromine, and aryl(hetaryl)substituents in the 3 position revealed significant MIC values
(between 0.41 to 2.0 g/mL for compound 29), indicating that 3-substituted coumarin analogues show
µg/mL and 0.21–6.28 µg/mL respectively. Most of the compounds of series 11,
µ
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

Molecules 2019, 24, 2126
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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.
4. Experimental Section
4.1. General Information
NMR spectra were acquired on Bruker AV-400 (1H: 400.13 MHz, 13C: 100.78 MHz) or Bruker AV-600
(1H: 600.30 MHz, 13C: 150.95 MHz) instruments (Bruker BioSpin GmbH, Rheinstetten, Germany),
using tetramethylsilane (TMS) as an internal standart. In the description of the 1H- and 13C-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 [
polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA), and expressed in (deg
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
α
]
D were determined on a PolAAr 3005
·
mL)/(g dm),
·
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 (CHCl3-EtOH, 9:1; detection under UV light or by treatment with iodine vapor).
The chemicals used: dioxane dibromide, trifluoromethanesulfonic anhydride, NaN3, CuSO4,
sodium ascorbate, CuSO4, CuI, N,N-dicyclohexylcarbodiimide (DCC), propargyl amine hydrochloride
(
(
5
), propargyl bromide (80% in toluene solution), aryl acetylenes 17
31), trimethylsilylacetylene (33), 1,6-heptadiyne (39), 1,7-octadiyne (40) and 1,9-decadiyne (41
Louis, MO, USA) or Alfa Aesar (GmbH, Karlsruhe,
Germany). Dichlorobis(triphenylphosphine)palladium(II) was obtained as described in [30].
Peucedanin ( ) was isolated from Peucedanum morisonii according to [31]. Peuruthenicin ( ) [32],
umbelliferone carboxylic acid 19], azidobenzoic acids 7a 33], methyl ether of 9-aminopelargonic
, 19–2, 24, 2-methylbut-3-yn-2-ol
)
were purchased from Aldrich (St.
1
2
3
[
–с [
acid hydrochloride [34], D,L-2-aminobutyric acid and L-phenylalanine hydrochlorides [35],
methyl 2-(prop-2-ynylamino)butanoate (14), and (S)-methyl 3-phenyl-2-(prop-2-ynylamino)propanoate
(15) [36], methyl ethynylantranilate (23) [37] and 2-azidooreoselone (35) [14] were synthesized according
to published procedures. Solvents (CH2Cl2, CHCl3, MeCN, DMF, THF, benzene, dioxane) and Et3N
were purified by standard methods and distilled under a stream of argon just before use. Copies of
NMR spectra (1H & 13C) are given in Supplementary Materials.
4.2. Syntheses and Spectral Data
7-Hydroxy-2-oxo-N-(prop-2-ynyl)-2H-chromene-6-carboxamide
umbelliferone-6-carboxylic acid ( , 500 mg, 2 mmol) and hydroxybenzotriazole (135 mg,
1 mmol) in DMF (5 mL) was added propargylamine hydrochloride ( , 120 mg, 2.1 mmol) and Et3N
(
6)
To
a
stirred solution of
3
5
(0.27 mL, 2 mmol) and the reaction mixture was left for 5 min. Then diisopropylcarbodiimide (300 mg,
2.4 mmol) was added and the mixture was stirred for 2 days. The reaction mixture was poured into
a Petri dish to evaporate in air, the residue was dissolved in CH2Cl2 (10 mL) and acetone was added

Molecules 2019, 24, 2126
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(5 mL). The precipitate was filtered, the solution was evaporated and residue was dried. The product
was isolated by fractional precipitation from acetone. Compound was obtained in 67% yield (320 mg).
Colorless powder. М.р. 188 ◦C (dec.). IR (KBr, , cm−1): 3388, 3286, 3243, 3059, 2921, 2852, 2100, 1737,
6
ν
1708, 1649, 1620, 1598, 1570, 1500, 1392, 1344, 1310, 1234, 1213, 1144, 911, 854, 837, 735, 688, 639, 628.
1
UV (EtOH)
λmax, (lg
ε
): 222(3.22), 240 (3.35), 329 (2.89), 372 (2.88) nm. H-NMR (CDCl3 + CD3OD,
400 MHz, δH): 3.16 (s, 1Н,
≡
CН), 4.20 (s, 2Н, CН2), 6.26 (d, J = 9.6 Hz, 1Н, Н-4), 6.83 (s, 1Н, Н-8), 7.76
(d, J = 9.6 Hz, 1Н, Н-3), 8.10 (s, 1Н, Н-5). 13C-NMR (CDCl3+CD3OD, 100 MHz, δC): 45.8, 71.0, 78.8,
103.9, 111.4, 112.6, 113.8, 129.3, 144.0, 157.1, 161.2, 162.4, 167.1 (C=O). Anal. calcd for C13H9NO4: C,
64.20; H, 3.73; N, 5.76; found: C 64.33; Н3.77; N 5.65.
4.2.1. General Method for the Synthesis of Compounds 4a–c
A solution of the appropriate azidobenzoic acid 7a–с (35 mg, 0.14 mmol) in CH2Cl2 (10 mL) was
mixed with a solution of sodium ascorbate (42 mg, 15 mol%, 0.021 mmol) and CuSO4
·
5H2O (1.2 mg,
5 mol%, 0.007 mmol) in Н2О(5 mL) and 6-(N-propynyl)carboxamidocoumarin ( , 20 mg, 0.14 mmol)
6
was added while stirring. The reaction mixture was stirred for 3 h at room temperature and additional
◦
for 1 h at 40 C. After the completion of the reaction, the mixture was diluted with Н2О(10 mL),
the product was extracted with CH2Cl2 (4
MgSO4, and the solvent was removed at reduced pressure. The product was purified by column
chromatography (eluent CHCl3).
×
10 mL), the combined extracts were dried over anhydrous
2-(4-((7-Hydroxy-2-oxo-2H-chromene-6-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid
Colorless powder, m.p. 201–202 ◦C (ethanol). Yield 50%. IR (KBr, , cm−1): 3367, 3257, 3066, 2922, 2852,
2666, 1740, 1691, 1596, 1575, 1485, 1446, 1392, 1304, 1265, 1148, 1078, 1020, 908, 829, 796, 752, 687, 640.
UV (EtOH) max, (lg
): 246 (3.45), 301 (2.76), 331 (3.51), 357 (3.23) nm. 1H-NMR (CDCl3+CD3OD,
(4a).
ν
λ
ε
400 MHz, δH): 4.93 (s, 2Н, CН2), 6.22 (d, J = 9.8 Hz, 1Н, Н-3), 6.82 (s, 1Н, Н-8), 7.13 (dd, J = 7.6, 7.2 Hz,
1Н, Н-400), 7.18 (1Н, d, J = 8.0 Hz, 1Н, Н-600), 7.48 (dd, J = 8.0, 7.2 Hz, 1Н, Н-500), 7.63 (d, J = 9.8 Hz, 1Н,
Н-4), 7.78 (s, 1Н, Н-5), 7.88 (d, J = 7.6 Hz, 1Н, Н-300), 7.98 (s, 1Н, Н-50). 13C-NMR (CDCl3+CD3OD,
100 MHz, δC): 44.5, 104.3, 111.8, 113.4, 119.8, 121.3, 123.0, 124.1, 125.9, 129.5, 130.7, 132.1, 135.9, 142.3,
143.8, 158.5, 160.5, 163.8, 167.6, 169.2. Anal. calcd for C20H14N4O6: C, 59.12; H, 3.47; N, 13.79; found: C,
58.88; H, 3.77; N, 14.14.
3-(4-((7-Hydroxy-2-oxo-2H-chromene-6-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid
Yellowish powder, m.p. 186–187 ◦C (hexane). Yield 64%. IR (KBr, , cm−1): 3427, 3095, 3062, 2923, 2852,
2665, 1738, 1687, 1631, 1600, 1581, 1456, 1305, 1236, 1153, 1113, 1097, 1056, 1008, 931, 912, 893, 800, 795,
(4b).
ν
754, 698, 673. UV (EtOH)
λ
max, (lg
ε
): 225 (3.66), 246 (3.53), 301 (3.23), 336 (3.22), 347 (3.18), 357 (3.23)
1
nm. H-NMR (CDCl3, 400 MHz, δH): 4.97 (s, 2Н, CН2), 6.30 (d, J = 9.8 Hz, 1Н, Н-3), 6.87 (s, 1Н, Н-8),
7.45 (d, J = 8.2 Hz, 1Н, Н-600), 7.47 (dd, J = 8.2, 7.6 Hz, 1Н, Н500), 7.62 (d, J = 9.8 Hz, 1Н, Н-4), 7.77 (s,
1Н, Н-5), 7.88 (d, J = 7.6 Hz, 1Н, Н-400), 8.01 (s, 2Н, Н-50, 200), 11.19 (br.s, 3H, OH, NH). 13C-NMR
(CDCl3, 100 MHz, δC): 44.2, 104.9, 111.9, 114.2, 120.4, 121.7, 123.4, 124.3, 126.9, 127.1, 130.0, 130.7, 135.7,
140.8, 143.1, 158.9, 160.2, 161.0 (C-7), 164.2, 170.8. Anal. calcd for C20H14N4O6: C, 59.12; H, 3.47; N,
13.79; found: C, 59.55; H, 3.49; N, 13.43.
4-(4-((7-Hydroxy-2-oxo-2H-chromene-6-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid
Grayish powder, m.p. 203–204 ◦C (hexane). Yield 60%. IR (KBr, , cm−1): 3433, 3062, 2958, 2924, 2852,
2673, 1734, 1676, 1630, 1600, 1579, 1448, 1427, 1392, 1288, 1236, 1215, 1143, 1113, 1076, 946, 904, 858, 827,
(4c).
ν
796, 768, 732. UV (EtOH)
λ
max, (lg
ε
): 225 (3.66), 246 (3.78), 270 (3.65), 293 (3.53), 324 (3.36), 351 (3.21)
1
nm. H-NMR (CDCl3, 400 MHz, δH): 4.95 (s, 2Н, CН2), 6.28 (d, J = 9.8 Hz, 1Н, Н-3), 6.87 (s, 1Н, Н-8),
7.10 (d, J = 8.4 Hz, 2Н, Н-200,600), 7.61 (d, J = 9.8 Hz, 1Н, Н-4), 7.98 (s, 1Н, Н-5), 8.02 (s, 1Н, Н-50), 8.03
(br.s, 1Н, NН), 8.10 (d, J = 8.4 Hz, 2Н, Н-300,500), 11.19 (br.s, 2Н, ОН). 13C-NMR (CDCl3, 100 MHz, δC):
42.5, 104.9, 111.9, 114.1, 118.9 (2C), 121.8, 125.3, 125.7, 127.6, 130.7, 132.1 (2C), 143.0, 145.7, 158.9, 160.1,
164.2, 169.5, 170.7. Anal. calcd for C20H14N4O6: C, 59.12; H, 3.47; N, 13.79; found: C, 59.16; H, 3.14;
N, 13.42.

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3-Bromopeuruthenicin (11). A solution of 1.0 g (4.5 mmol) of peuruthenicin (
2
) and 2.48 g (9 mmol) of
dioxane dibromide in CH2Cl2 (10 mL) was stirred at rt for 16 h. Then the reaction mixture was filtered,
and the filtrate was evaporated. 3-Bromopeuruthenicin (11, yield 86%, 1.14 g) was finally purified by
crystallization from chloroform. White solid; m.p. 187–188 ◦C (ether). IR (KBr, , cm−1): 3480, 3350,
ν
3116, 3050, 2956, 2853, 1720, 1675, 1621, 1616, 1598, 1440, 1311, 1293, 1231, 1163, 1124, 1089, 1030, 989,
964, 918, 845, 783, 750, 729, 719. UV (EtOH) max, (lg ): 244 (4.19), 314 (4.02), 334 (4.10), 400 (3.39) nm.
λ
ε
1H-NMR (600 MHz, CDCl3, δH): 3.99 (s, 3H, OCH3), 6.85 (s, 1H, H-8), 7.97 (s, 2H, H-4,5), 11.21 (s, 1H,
OH). 13C-NMR (150 MHz, CDCl3, δC): 52.9, 104.8, 108.8, 110.5, 112.5, 129.9, 143.8, 156.4, 157.9, 164.3,
169.2. HRMS (ESI), m/z (Irel, %): 298(66), 269(15.9), 240(24), 220(18), 188(31), 160(16), 131(17), 103(19),
79(12); calcd for C11H7BrO5 [M]: 297.9471; found: 297.9468; Anal. Calcd. for C11H7BrO5: C, 44.18; Н,
2.36; Br, 26.72. Found: C, 44.08; Н, 2.16; Br, 26.70.
3-Azidopeuruthenicin (12). To a stirred solution of 25 mg (0.84 mmol) of 3-bromopeuruthenicin (11
)
in DMF at room temperature was added NaN3 (82 mg, 1.26 mmol). The reaction mixture was
heated at 40 ◦C for 14h. After consumption of the bromocoumarin, the mixture was cooled to room
temperature, quenched with eq. NaCl (10 mL), and the products and were extracted with CH2Cl2
(5
×
5 mL). The combined organic phases were dried over anhydrous MgSO4, after filtration, the
solvent was removed under reduced pressure and was further dried in a freeze drier at oil pump
vacuum. (P = 1 mm). The product (0.15 g, 68%) was obtained as a yellow solid, m.p. 161–162 ◦C.
IR (KBr,
ν
, cm−1): 3455, 3390, 3357, 3299, 3004, 2971, 2923, 2858, 2121, 1727, 1621, 1467, 1434, 1375, 1309,
1
1290, 1263, 1193, 975, 869, 728. UV (EtOH)
λmax, (lg
ε
): 223(4.51), 243(4.4), 329(4.2), 373(3.97). H-NMR
(CDCl3, 400 MHz, δH): 3.99 (s, 3H, OCH3), 6.83 (s, 1H, H-8), 7.58 (s, 1Н, H-4), 8.00 (s, 1H, H-5), 11.19 (s,
1H, OH). 13C- NMR (CDCl3, 100 MHz, δC): 52.7, 104.8, 109.9, 111.9, 114.0, 120.6, 142.9, 158.8, 159.9,
164.2, 169.4. Anal. Calcd for C11H7N3O5: C, 50.58; Н, 2.70; N, 16.09; found C, 51.04; Н, 3.12; N, 15.74.
4.2.2. 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 CH2Cl2 (30 mL). The organic
phase was separated and the aqueous phase was extracted with CH2Cl2 (3
were combined, dried (MgSO4), filtered and the solvent evaporated under reduced pressure. The residue
×
10 mL). The organic phases
was purified by column chromatography to give secondary amines 13, 14, 15.
Methyl 9-(prop-2-ynylamino)nonanoate (13). Yellowish oil. 1H-NMR (CDCl3, 400 MHz, δH): 1.22 (m, 4H,
CH2-110,120), 1.39 (2H, CH2-130), 1.52 (2H, CH2-100), 2.12 (4H, CH2-90,140), 2.21 (m, 2H, CH2-150),
2.58 (2H, CH2-80), 3.32 (s, 2H, CH2-60), 3.57 (s, 3H, OCH3).13C-NMR (CDCl3, 100 MHz, δC): 25.2, 27.4,
29.3, 29.5, 30.0, 34.32, 38.40, 48.90 (
C13H23NO2: C, 69.29; Н, 10.29; N, 6.22; found C, 68.74; Н, 10.10; N, 5.94.
C
H2-120,90,50), 51.62 (OCH3), 71.38, 73.02 (C
CH). Anal. calcd for
≡
Methyl 7-hydroxy-3-(4-((9-methoxy-9-oxononylamino)methyl)-1H-1,2,3-triazol-1-yl)-2-oxo-2H-chromene-
6-carboxylate ( ). A solution of 3-azidopeuruthenicin (12, 50 mg, 0.19 mmol) in methylene chloride
(10 mL) and a solution of sodium ascorbate (15 mol%, 0.255 mmol) and CuSO4 5H2O (5 mol%,
8
×
0.085 mmol) in water (10 mL) were mixed and the terminal alkyne 13 (63 mg, 0.28 mmol) was added
with stirring. The reaction mixture was stirred for 3 h at room temperature and then for 1 h at 40 ◦C.
The cooled mixture was diluted with water (10 mL) and the product was extracted with methylene
chloride (4
vacuo. The residue was purified by column chromatography to give 73 mg (67%) of compound
a yellow oil. IR (KBr,
, cm−1): 3465, 3396, 3118, 3047, 2954, 2923, 2852, 1751, 1735, 1673, 1623, 1602,
×
10 mL). The combined extracts were dried over MgSO4, and the solvent was removed in
8
as
ν
1573, 1469, 1438, 1367, 1288, 1230, 1162, 1124, 1089, 1031, 962, 943, 865, 792. UV (EtOH)
242 (4.49), 299 (4.17), 313 (4.26), 334 (4.34), 390 (3.39) nm. H-NMR (CDCl3, 400 MHz, δH): 1.25 (m,
4H, CH2-110,120), 1.66 (2H, CH2-130), 2.18 (2H, CH2-100), 2.40 (4H, CH2-90,140), 2.47 (m, 2H, CH2-150),
λ
max, (lg
ε
):
1

Molecules 2019, 24, 2126
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2.72 (2H, CH2-80), 3.65 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 4.02 (s, 2H, CH2-60), 6.87 (s, 1H, H-8), 7.97 (s,
1Н, H-4), 7.98 (s, 1H, H-5), 8.01 (s, 1H, H-50), 11.25 (br.s, 2H, OH, NH). 13C-NMR (CDCl3, 100 MHz, δC):
29.7, 30.8, 31.9, 35.5, 39.6, 40.1, 40.5, 45.1, 52.9, 61.9, 104.7, 108.7, 110.5, 112.5, 121.4, 124.9, 142.2, 143.8,
156.4, 157.8, 164.3, 169.2, 174.4. Anal. calcd. for C24H30N4O7: C, 59.25; H, 6.22; N, 11.52; found: C,
59.02; H, 6.12; N, 11.27.
4.2.3. Preparation of 3-(Triazolyl)coumarins 9, 10
A stirred solution of 3-azidopeuruthenicin (12, 90 mg, 0.35 mmol) in MeCN (15 mL) was
successively treated with alkyne 14 or 15 (1.2 equiv.), CuI (0.1 equiv.) and Et3N (1.1 equiv.) and the
reaction mixture was stirred at room temperature until complete consumption of the starting material.
Then, water (5 mL) was added and the products were extracted with CH2Cl2 (3
organic phases were dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by
column chromatography, eluting with chloroform, chloroform:ethanol 100:1.
×
10 mL). The combined
Methyl 7-hydroxy-3-(4-((1-methoxy-1-oxobutan-2-ylamino)methyl)-1H-1,2,3-triazol-1-yl)-2-oxo-2H-chromene-
6-carboxylate (9). Yield 56%. Yellow oil. IR (KBr, ν
, cm−1): 3463, 3398, 3307, 3050, 2954, 2923, 2852,
1733, 1677, 1625, 1575, 1465, 1440, 1367, 1309, 1292, 1232, 1220, 1164, 1124, 1089, 1012, 968, 865, 794, 948.
1
UV (EtOH)
λ
max, (lg
ε
): 207 (4.69), 244 (4.29), 300 (3.95), 315 (4.02), 336 (4.1), 387 (3.41) nm. H-NMR
(CDCl3+CD3OD, 400 MHz, δH): 0.81 (t, 3H, J = 7.0 Нz, CH3-100), 31.55 (m, 2H, CH2-90), 3.27 (m, 1H,
H-80), 3.62 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 3.90 (m, 2H, H-60), 6.76 (s, 1H, H-8), 7.90 (s, 1Н, H-4),
7.91 (s, 1H, H-5), 8.09 (s, 1H, H-50). 13C-NMR (CDCl3, 100 MHz, δC): 9.5, 25.8, 36.3, 51.6, 51.8, 60.7,
104.4, 108.2, 110.3, 112.3, 121.3, 124.8, 141.0, 144.1, 156.5, 157.5, 164.0, 168.9, 174.8. Anal. calcd. for
C19H20N4O7: C, 54.81; H, 4.84; N, 13.46; found, %: C, 55.13; H, 5.02; N, 13.59.
(S)-Methyl 7-hydroxy-3-(4-((1-methoxy-1-oxo-3-phenylpropan-2-ylamino)methyl)-1H-1,2,3-triazol-1-yl)-2
-oxo-2H-chromene-6-carboxylate (10). Yield 62% (by method for snthesis of compound 8–34%). Yellow oil.
[α] +12.04 (с 1.00, CHCl3). IR (ν
, cm−1): 3459, 3395, 3290, 3049, 2954, 2925, 2854, 1737, 1673, 1623,
D
1602, 1573, 1537, 1490, 1438, 1367, 1288, 1232, 1162, 1124, 1087, 962, 943, 865, 792. UV (EtOH)
λ
,
max
1
(lgε
): 244 (4.22), 312 (3.94), 334 (4.02), 399 (3.26) nm. H-NMR (CDCl3, 400 MHz, δH): 2.90-3.03 (m,
2H, H-90), 3.36 (dd, J = 16.3, 1.8 Hz, 2H, H-60), 3.65 s (3H, OCH3), 3.73 (dd, J = 6.4, 7.0 Hz, 1H, H-80),
3.98 (s, 3H, OCH3), 6.84 s (1H, H8), 7.16-7.18 (m, 2H, o-Ph), 7.19-7.22 (m, 1H, p-Ph), 7.26-7.32 (m, 2H,
m-Ph), 7.93 s (1Н, H-4), 7.95 (s, 1H, H-5), 8.08 (s, 1H, H-50), 11.13 (br s, 2H, OH, NH). 13C-NMR (CDCl3,
100 MHz, δC): 36.7, 39.3, 51.8, 52.8, 61.0, 104.9, 108.4, 110.3, 112.4, 120.5 (C-4), 125.1, 126.9, 129.1 (2C),
129.5 (2C), 132.5, 141.3, 143.8, 156.1, 157.7, 164.2, 169.2, 174.2. Anal. calcd. for C24H22N4O7: C, 60.25; H,
4.63; N, 11.71; found: C, 60.16; H, 4.96; N, 11.55.
4.2.4. General Procedure for the Sonogashira Cross-coupling Reactions of 3-Bromopeuruthenicin 11
with Aryl Alkynes 17, 19–24 or 2-methylbut-3-yn-2-ol 31
To a solution of 3-bromopeurutenicin (11, 100 mg, 0.34 mmol) and terminal alkynes 17, 19–24 or
31 (0.54 mmol) in benzene (5 mL) was added CuI (6 mg, 10 mol%), Pd(PPh3)2Cl2 (12 mg, 5 mol%),
and Et3N (0.076 mL, 0.44 mmol; 1.3 (eq. to 11) under argon. The reaction mixture was stirred at
80 ◦C for 12–14 h (TLC). The mixture was cooled and 5 mL of water was added. The separated water
layer was extracted with CН2Cl2 (5
×
4 mL). The combined organic extracts was washed with water,
dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was subjected to
column chromatography on silica gel. Eluting with chloroform and crystallization from ether afforded
the corresponding 3-(R)-alkynylcoumarins 18, 25–30, 32.
Methyl 7-hydroxy-2-oxo-3-(phenylethynyl)-2H-chromene-6-carboxylate (18). Yield 65% (70 mg). Yellow oil.
IR (
ν
, cm−1): 3430, 3350, 3052, 2954, 2925, 2852, 2112 (C
≡
C), 1736, 1682, 1616, 1571, 1442, 1367, 1290, 1224,
ε
1160, 1128, 1091, 966, 873, 792, 750. UV (EtOH)
335 (3.83) nm. H-NMR (CDCl3, 400 MHz, δH): 3.85 (s, 3H, OCH3), 6.79 (s, 1H, H-8), 7.48–7.56 (m, 5H,
H-Ph), 7.92 s(1H, H-4), 7.93 (s, 1H, H5), 11.21 (1H, OH). 13C-NMR (CDCl3, 100 MHz, δC): 52.1, 83.3,
λmax, (lg ): 237 (4.14), 272 (3.86), 280 (3.88), 315 (3.78),
1

Molecules 2019, 24, 2126
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95.2, 108.1, 112.0, 113.1, 114.7, 129.8 (2C), 130.0, 130.8, 132.8, 133.6 (2C), 144.4, 157.6, 158.4, 164.6, 169.7.
Anal. Calcd for C19H12O5: 320.0745; found: 320.0743.
Methyl 3-((4-fluorophenyl)ethynyl)-7-hydroxy-2-oxo-2H-chromene-6-carboxylate (25). Yield 68% (78 mg),
yellowish powder, m.p. 167–168 ◦C (ether). IR (KBr, , cm−1): 3425, 2933, 2836, 2145, 1733, 1673, 1608,
ν
1517, 1463, 1450, 1373, 1346, 1257, 1228, 1124, 1012, 939, 873, 852, 840, 792, 753. UV (EtOH)
λ
max, (lg
ε
):
1
218 (4.56), 225 (4.54), 245 (4.64), 305 (4.3), 329 (4.41) nm. H-NMR (CDCl3, 400 MHz, δH): 3.91 (s, 3H,
OCH3), 6.77 (s, 1H, H-8), 6.93 (dd, J = 8.2, 7.2 Нz, 2H, H-30,50), 7.40 (dd, J = 8.2, 3.4 Нz, 2H, H-20,60),
7.74 (s, 1H, H-4), 7.92 (s, 1H, H-5), 11.26 (1H, OH). 13C-NMR (CDCl3, 100 MHz, δC): 53.3, 82.0, 95.7,
104.7, 110.7, 111.2, 112.1, 118.9 (2C), 128.6, 130.5, 131.8 (2C), 143.4, 158.0, 160.1(C-40, JC-F 259.1 Hz),
161.3, 164.4, 169.4. Anal. calcd for C19H11FO5: C, 67.46; Н, 3.28; F, 5.62; found: C, 68.08; Н, 3.50; F, 5.12.
Methyl 7-hydroxy-2-oxo-3-(p-tolylethynyl)-2H-chromene-6-carboxylate (26). Yield 61% (70 mg). brownish
powder, m.p. 132–133 ◦C (ether). IR (KBr, , cm−1): 3430, 3299, 3259, 3049, 2956, 2852, 2121, 1753, 1735,
ν
1697, 1677, 1594, 1516, 1436, 1367, 1321, 1294, 1234, 1203, 1162, 1087, 960, 848, 794, 748, 734. UV (EtOH)
max, (lg
): 237 (4.41), 251 (4.21), 272 (4.13), 281 (4.16), 314 (4.04), 338 (4.11) nm. 1H-NMR (CDCl3,
λ
ε
400 MHz, δH) 2.36 (s, 3H, CH3), 4.00 (s, 3H, OCH3), 6.88 (s, 1H, H-8), 7.15 (d, J = 8.2 Нz, 2H, H30,50),
7.45 (d, J = 8.2 Нz, 2H, H-20,60), 7.81 (s, 1H, H-4), 8.00 (s, 1H, H-5), 11.24 (1H, OH). 13C-NMR (CDCl3,
100 MHz, δC): 21.7, 52.9, 82.4, 95.9, 104.8, 110.5, 111.2, 112.1, 119.00, 129.2 (2C), 130.5, 131.8 (2C), 139.3,
143.9, 158.0, 158.8, 164.4, 169.5. Anal. calcd for C20H14O5: C 71.85, Н4.22, found: C 71.36, Н3.94.
Methyl 3-((4-ethylphenyl)ethynyl)-7-hydroxy-2-oxo-2H-chromene-6- carboxylate (27). Yield 52% (61 mg)
brownish powder, m.p. 130–131 ◦C (ether). IR (KBr, , cm−1): 3380, 3139, 3049, 2929, 2856, 2185, 1736,
ν
1673, 1625, 1579, 1481, 1439, 1389, 1351, 1288, 1230, 1162, 1126, 1091, 1037, 962, 946, 865, 825, 750, 736.
1
UV (EtOH)
λmax, (lg
ε
): 225 (4.1), 249 (3.67), 259 (3.59), 335 (4.18) nm. H-NMR (CDCl3, 400 MHz, δH):
1.21 (t, J = 6.8 Нz, 3H, Et), 2.62 (q, J = 6.8 Нz, 2H, Et), 3.97 (s, 3H, OCH3), 6.85 (s, 1H, H-8), 7.13 (dd,
J = 8.4 Нz, 2H, H-30,50), 7.40 (d, J = 8.4 Нz, 2H, H-20,60), 7.96 (s, 1H, H4), 7.97 (s, 1H, H5), 11.21 (1H,
OH). 13C-NMR (CDCl3, 100 MHz, δC): 17.3, 28.2, 53.5, 81.9, 97.3, 105.6, 112.1, 113.5, 114.8, 122.9, 129.2,
129.9 (2C), 132.6 (2C), 144.2, 144.5, 158.8, 159.5, 165.2, 170.2. Anal. calcd for C21H16O5: C, 72.41; Н, 4.63;
found: C, 72.18; Н, 4.90.
Methyl 7-hydroxy-3-((4-methoxyphenyl)ethynyl)-2-oxo-2H-chromene-6-carboxylate (28). Yield 58% (70 mg)
brownish powder, m.p. 134–135 ◦C (ether). IR (KBr, , cm−1): 3430, 3303, 3216, 3049, 3014, 2956, 2129,
ν
1732, 1677, 1594, 1516, 1491, 1436, 1367, 1321, 1294, 1267, 1234, 1203, 1163, 1124, 1088, 960, 865, 849,
794, 748, 734. UV (EtOH)
λ
max, (lg
ε
): 237 (4.18), 265 (3.78), 277 (3.91), 280 (3.92), 324 (3.84), 335 (3.88)
1
nm. H-NMR (CDCl3, 400 MHz, δH): 3.91 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.86 (s, 1H, H-8), 7.13 (d,
J = 8.6 Нz, 2H, H-30,50), 7.41 (d, J = 8.6 Нz, 2H, H-20,60), 7.97 (s, 1H, H-4), 7.98 (s, 1H, H-5), 11.18 (1H,
OH). 13C-NMR (CDCl3, 100 MHz, δC): 53.2, 53.6, 81.6, 95.4, 105.1, 111.1, 111.7, 112.8, 118.7, 119.4 (2C),
129.5, 133.3 (2C), 144.1, 157.9, 158.7, 161.6, 164.7, 169.8. Anal. calcd for C20H14O6: C, 68.57; Н, 4.03;
found: C, 68.88; Н, 3.90.
Methyl 3-((4-acetylamino-3-(methoxycarbonyl)phenyl)ethynyl)-7-hydroxy-2-oxo-2H-chromene-6- carboxylate
(
29). Yield 45% (66 mg) yellowish powder, m.p. 154–155 ◦C (ethanol). IR (KBr, , cm−1): 3430, 3299,
ν
3259, 3216, 3049, 2956, 2925, 2852, 2208, 1753, 1735, 1697, 1677, 1620, 1594, 1515, 1491, 1437, 1367, 1321,
1294, 1234, 1203, 1162, 1124, 1088, 960, 920, 866, 849, 795, 748, 735. UV (EtOH)
272 (4.00), 280 (4.01), 316 (3.92), 335 (3.96) nm. H-NMR (CDCl3, 400 MHz, δH): 2.19 (s, 3H,
λ
max, (lg
ε
): 237 (4.28),
H3-CO),
1
C
3.88 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.82 (s, 1H, H-8), 7.55 (dd, J = 8.8, 2.0 Нz, 1H, H-60), 7.94 (s, 1H,
H-4), 7.95 (s, 1H, H-5), 8.09 (d, J = 2.0 Нz, 1H, H-20), 8.58 (d, J = 8.8 Нz, 1H, H-50), 11.16 (br.s, 2H, NH,
OH). 13C-NMR (CDCl3+CD3OD, 100 MHz, δC): 25.2, 52.5, 52.8, 71.3, 82.2, 104.6, 108.5, 112.4, 116.2,
120.0, 130.0, 130.5, 133.7, 134.6, 137.7, 141.2, 143.9, 156.5, 157.7, 166.4, 167.9, 169.1, 169.3. Anal. calcd for
C23H17NO8: C, 63.45; Н, 3.94; N, 3.22; found: C, 63.18; Н, 3.92; N, 3.12.
Methyl 7-hydroxy-2-oxo-3-(pyridin-2-ylethynyl)-2H-chromene-6-carboxylate (30). Yield 55% (60 mg) brown
oil. IR (ν
, cm−1): 3444, 2971, 2929, 2856, 2133, 1735, 1674, 1626, 1579, 1481, 1438, 1388, 1352, 1300, 1230,

Molecules 2019, 24, 2126
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1163, 1126, 1092, 1038, 962, 947, 865, 825, 750, 736, 723. UV (EtOH)
λ
max, (lg
ε
): 227 (4.25), 238 (4.15),
1
294 (3.93), 306 (3.92), 351 (4.13) nm. H-NMR (CDCl3, 400 MHz, δH): 3.76 (s, 3H, OCH3), 6.72 (s, 1H,
H-8), 7.18 (ddd, J = 7.4, 5.2, 1.4 Нz, 1H, H-40), 7.31 (dd, J = 6.6, 1.4 Нz, 1H, H-60), 7.54 (ddd, J = 7.4, 6.6,
1.8 Нz, 1H, H-50), 7.69 (s, 1H, H-4), 7.77 (s, 1H, H-5), 8.49 (ddd, J = 7.2, 1.8 Нz, 1H, H-30), 11.20 (1H,
OH). 13C-NMR (CDCl3, 100 MHz, δC): 52.9, 82.6, 94.8, 105.0, 112.8, 115.1, 116.6, 123.5, 129.4, 130.4,
134.1, 138.1, 141.6, 144.4, 156.9, 158.1, 164.4, 169.7. Anal. calcd for C18H11NO5: C, 67.29; Н, 3.45; N, 4.36;
found: C, 67.08; Н, 3.38; N, 4.22.
Methyl 7-hydroxy-3-(3-hydroxy-3-methylbut-1-ynyl)-2-oxo-2H-chromene-6-carboxylate (32
). Yield 66%
(68 mg), m.p.114–115 ◦C (ethanol). IR (KBr, , cm−1): 3282, 3221, 2981, 2932, 2868, 2250, 1741, 1679,
ν
1612, 1600, 1584, 1462, 1446, 1363, 1296, 1269, 1210, 1170, 1120, 1096, 1058, 998, 964, 954, 900, 889, 842,
1
792, 735, 723. UV (EtOH)
λmax, (lg
ε
): 223 (4.04), 246 (3.84), 338 (3.67), 365 (3.57) nm. H-NMR (CDCl3,
400 MHz, δH): 1.34 (s, 6H, 2
1H, H-5). C-NMR (CDCl3, 100 MHz,
×
CH3), 3.95 (s, 3H, OCH3), 6.78 (s, 1H, H-8), 7.76 (br. s, 1H, H-4), 7.93 (s,
c): 30.9, 31.0, 52.7, 65.3, 66.2, 84.1, 104.8, 109.9, 110.5, 114.0,
3
δ
130.6, 143.1, 157.8, 160.0, 164.1, 169.4. Anal. calcd for C16H14O6: C, 63.57; Н, 4.67; found: C, 63.22; Н,
4.44. HRMS, m/z calcd.: 302.0785; found 302.0783 [M].
Methyl 7-hydroxy-2-oxo-3-((trimethylsilyl)ethynyl)-2H-chromene-6-carboxylate (34). A sealed 10 mL glass
tube containing 3-bromopeuruthenicin (11, 100 mg (0.34 mmol), toluene (3 mL), trimethylsilylacetylene
(
33, 0.1 mL, 0.68 mmol), CuI (3 mg, 5 mol %), Pd(PPh3)2Cl2 (23 mg, 10 mol. %), and Et3N (0.061 mL, 1.3
eq.) was placed in the cavity of a microwave reactor and irradiated for 2 h at 100 ◦C and power 50 W
in an Anton Paar Microwave 50 reactor. After cooling to 25 ◦C, the tube was removed from the reactor.
Then the mixture was cooled and 5 mL of water was added. The separated water layer was extracted
with CН2Cl2 (5
filtered, and concentrated under reduced pressure. The residue was crystallized from ether, yield 64%
(68 mg), m.p. 104–107 ◦C. IR (KBr, , cm−1): 3437, 2924, 2852, 2223, 1735, 1672, 1314, 1597, 1508, 1425,
×
4 mL). The combined organic extracts was washed with water, dried over MgSO4,
ν
1388, 1328, 1236, 1222, 1159, 1107, 908, 839, 752, 694, 680. UV (EtOH)
λ
max, (lg
ε
): 325 (3.97), 304 (3.85),
1
241 (4.01) nm. H-NMR (CDCl3, 400 MHz, δH) 0.20 (s, 9H, SiMe3), 3.93 (s, 3H, OCH3), 6.76 (s, 1H, H-8),
7.74 (s, 1H, H-4), 7.91 (s, 1H, H-5), 10.97 (s, 1H, OH). 13C-NMR (CDCl3, 100 MHz, δC): 8.6, 52.7, 73.8,
81.5, 101.6, 110.2, 110.4, 111.7, 128.4, 145.1, 158.0, 158.3, 164.45, 169.2. Anal. calcd for C16H16O5Si: C,
60.74; Н, 5.10; Si, 8.88; found: C, 60.55; Н, 5.18; Si, 8.54.
Methyl 3-ethynyl-7-hydroxy-2-oxo-2H-chromene-6-carboxylate (16). To a solution of compound 34 (60 mg,
0.00019 mol) in methanol (3 mL) were added CsF (14 mg, 0.00095 mol) and TEBA (5 mg). The mixture
was stirred at rt for 10 h in an argon flow (TLC). Then 10 mL of water was added and the mixture was
extracted with methylene chloride (5
×
4 mL). The combined extract was washed with water, dried
over MgSO4, filtered, and concentrated under reduced pressure. The residue was subjected to column
chromatography on silica gel. Eluting with chloroform and crystallization from ether gave compound
ν
16, yield 85%, m.p. 84- 88 oC. IR (KBr, , cm−1): 3463, 2956, 2923, 2854, 2102, 1741, 1677, 1621, 1581,
1494, 1444, 1416, 1367, 1295, 1234, 1186, 1157, 1120, 1099, 1024, 952, 914, 856, 721. UV (EtOH)
λ
,
max
1
(lgε
): 225 (4.1), 246 (3.96), 321 (3.84), 344 (3.92). H-NMR (CDCl3, 400 MHz) 2.16 (s, 1H, CH), 3.99 (s,
3H, OCH3), 6.86 (s, 1H, H-8), 7.83 (s, 1H, H-4), 8.00 (s, 1H, H-5), 11.29 (s, 1H, OH). 13C- NMR (CDCl3,
100 MHz, δC): 52.3, 75.4, 82.2, 105.1, 112.6, 114.0, 114.8, 128.5, 143.3, 159.4, 159.7, 164.7, 168.9. Anal.
calcd for C13H8O5 C 63.94, Н3.30 found: C 63.65, Н3.84.
Methyl 7-hydroxy-3-[1-(2-isopropyl-3,7-dioxo-2,3-dihydro-7H-furo[3,2-g]chromen-2-yl)-1H-1,2,3-triazol-4
-yl]-2-oxo-2H-chromene-6-carboxylate (36). A solution of 2-azidooreoselone (35, 100 mg, 0.35 mmol) and
3-ethynylpeurutenicin (16, 85 mg, 0.35 mmol) in CH2Cl2 (10 mL) was mixed with a solution of sodium
ascorbate (10 mg, 15 mol%) and CuSO4
stirred for 3 h at rt and additional for 1 h at 40 ◦C. After the completion of the reaction, the mixture
was diluted with Н2О(10 mL), the product was extracted with CH2Cl2 (4 10 mL), the combined
extracts were dried over anhydrous MgSO4, filtered, and the solvent was removed at reduced pressure.
·
5H2O (4 mg, 5 mol%) in Н2О(5 mL). The reaction mixture was
×

Molecules 2019, 24, 2126
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Yield 70% (0.130 g), yellowish solid. IR (KBr,
ν
, cm−1): 3469, 3049, 2983, 2956, 2883, 2850, 1739, 1675,
1629, 1556, 1467, 1438, 1390, 1353, 1284, 1132, 1162, 1128, 1093, 1025, 964, 944, 869, 827, 736. UV (EtOH)
1
λ
max, (lg
ε
): 217 (4.57), 249 (4.59), 294 (4.26), 307 (4.27), 337 (4.27), 381 (3.76), 393 (3.36) nm. H-NMR
(CDCl3, 400 MHz, δH): 0.89 (d, J = 7.0 Нz, 3H, CH3), 1.11 (d, J = 7.0 Нz, 3H, CH3), 3.30 (m, 1H,
CH(Me)2), 3.99 (s, 3H, OCH3), 6.41 (d, J = 9.6 Нz, 1H, H-6), 6.88 (s, 1H, H-80), 7.24 (s, 1H, H-9), 7.71 (d,
J = 9.6 Нz, 1H, H-5), 7.87 (s, 1H, H-4), 7.97 (s, 1H, H-40), 7.98 (s, 1H, H-50), 8.13 (s, 1H, H-100), 11.29 (s,
1H, OH). 13C-NMR (CDCl3, 100 MHz, δC): 16.2, 16.3, 30.1, 53.3, 96.5, 101.4, 105.2, 110.7, 111.8, 112.9,
115.7, 117.3, 117.5, 125.7, 128.0, 130.3, 142.9, 143.5, 144.3, 158.5, 159.3, 162.1, 164.7, 165.8, 169.6, 172.4,
194.7. Anal. calcd for C27H19N3O9: C, 61.25; Н, 3.62; N, 7.94; found: C, 61.01; Н, 3.54; N, 7.62.
4.2.5. General Method for the Preparation of Monoalkynes 38a–c
A solution of 2-azidooreoselone (35, 1.7 mmol) in methylene chloride (10 mL) and a solution of
sodium ascorbate (15 mol%, 0.255 mmol) and CuSO4
·
5H2O (5 mol%, 0.085 mmol) in water (10 mL) were
mixed and the appropriate terminal alkyne 39
–41 (0.85 mmol) was added with stirring. The reaction
◦
mixture was stirred for 5 h at 40 C. The cooled mixture was diluted with water (10 mL) and the
product was extracted with methylene chloride (4
MgSO4, filtered, the solvent was removed to give the desired products 38a–с.
×
10 mL). The combined extracts were dried over
2-Isopropyl-2-(4-(pent-4-ynyl)-1H-1,2,3-triazol-1-yl)-2H-furo[3,2-g]chromene-3,7-dione (38a). Yield 66%
(0.420 g), brownish oil. IR (ν
, cm−1): 3120, 2981, 2932, 2867, 2140, 1741, 1678, 1600, 1584, 1461, 1446,
1381, 1363, 1295, 1269, 1210, 1169, 1120, 1058, 964, 953, 909, 889, 792, 749. UV (EtOH)
λ
max, (lg
ε
):
1
231 (3.91), 282 (3.58), 320 (3.52), 348 (3.43) nm. H-NMR (CDCl3, 400 MHz, δH): 0.89 (d, J = 7.0 Hz,
3Н, CН3), 0.95 (d, J = 7.0 Hz, 3Н, CН3), 1.40 (m, 2H, CH2-70), 1.85 (s, 1H, H-100), 2.06 (m, 2H, CH2-60),
2.55 (m, 2H, CH2-80), 3.06 (m, 1H, C
H(CH3)2), 6.28 (d, J = 9.6 Нz, 1H, H-6), 6.99 (s, 1H, H-9), 7.39 (s, 1H,
H-4), 7.67 (d, J = 9.6 Нz, 1H, H-5), 7.82 (s, 1H, H-50). 13C-NMR (CDCl3, 100 MHz, δC): 15.4, 15.8, 19.5,
28.1, 28.2, 33.7, 69.2, 82.3, 98.4, 100.4, 115.4, 115.8, 117.0, 126.7, 127.5, 137.4, 145.9, 161.2, 164.0, 173.6,
191.5. Anal. calcd for C21H19N3O4: C, 66.83; Н, 5.07; N, 11.13; found: C, 67.01; Н, 5.12; N, 10.88.
2-(4-(Hex-5-ynyl)-1H-1,2,3-triazol-1-yl)-2-isopropyl-2H-furo[3,2-g]chromene-3,7-dione (38b). Yield 62%
ν
(0.415 g). Yellowish powder, m.p. 111–112 ◦C (ether). IR (KBr, , cm−1): 2971, 2828, 2856, 2115, 1735,
1673, 1625, 1579, 1481, 1438, 1388, 1351, 1230, 1126, 1091, 1037, 997, 962, 946, 865, 825, 750. UV (EtOH)
max, (lg
): 221 (4.12), 255 (4.47), 292 (3.98), 308 (3.89), 351 (4.03) nm. 1H-NMR (CDCl3, 400 MHz,
δH): 0.89 (d, J = 7.0 Hz, 3Н, CН3), 1.03 (m, 2H, CH2-80), 1.12 (d, J = 7.0 Hz, 3Н, CН3), 1.24 (m, 2H,
CH2-70), 1.92 (s, 1H, H-110), 2.14 (m, 2H, CH2-60), 2.30 (m, 1H, C (CH3)2), 3.40 (m, 2H, CH2-90 ), 6.35 (d,
λ
ε
H
J = 9.6 Нz, 1H, H-6), 7.02 (s, 1H, H-9), 7.43 (s, 1H, H-4), 7.69 (d, J = 9.6 Нz, 1H, H-5), 7.84 (s, 1H, H-50).
13C- NMR (CDCl3, 100 MHz, δC): 14.9, 15.3, 17.8, 27.6, 27.7, 27.73, 33.2, 68.7, 81.8, 97.9, 100.6, 114.9,
115.3, 116.1, 125.4, 126.9, 136.8, 144.7, 162.4, 165.6, 173.1, 191.0. Anal. calcd for C22H21N3O4: C, 67.51;
Н, 5.41; N, 10.74; found: C, 67.17; Н, 5.21; N, 10.72.
2-Isopropyl-2-(4-(oct-7-ynyl)-1H-1,2,3-triazol-1-yl)-2H-furo[3,2-g]chromene-3,7-dione (38c). Yield 74% (0.527
g). Yellowish powder, m.p. 121–122 ◦C (ether). IR (KBr, , cm−1): 3143, 3084, 2976, 2935, 2858, 2113,
ν
1735, 1627, 1581, 1481, 1419, 1390, 1352, 1286, 1226, 1128, 1093, 1039, 997, 947, 866, 825, 739. UV (EtOH)
max, (lg
): 221 (4.16), 255 (4.5), 294 (4.00), 307 (3.94), 339 (4.05), 351 (4.06) nm. 1H-NMR (CDCl3,
λ
ε
400 MHz, δH): 0.94 (d, J = 7.0 Hz, 3Н, CН3), 0.97 (d, J = 7.0 Hz, 3Н, CН3), 1.27 (m, 2H, CH2-80), 1.34 (m,
2H, CH2-90),1.46 (m, 2H, CH2-100), 1.55 (m, 2H, (m, 2H, CH2-70), 1.89 (s, 1H, H-130), 2.12 (m, 2H,
CH2-60), 2.60 (m, 2H, CH2-110), 3.09 (m, 1H, CH(CH3)2), 6.32 (d, J = 9.6 Нz, 1H, H-6), 7.04 (s, 1H, H-9),
7.44 (s, 1H, H-4), 7.72 (d, J = 9.6 Нz, 1H, H-5), 7.87 (s, 1H, H-50). 13C-NMR (CDCl3, 100 MHz, δC): 15.1,
15.5, 18.1, 27.9, 28.0, 28.4, 28.67, 28.73, 33.5, 68.1, 84.4, 98.1, 100.8, 115.1, 115.5, 116.3, 125.7, 128.4, 137.7,
143.1, 158.5, 161.4, 171.4, 191.6. Anal. calcd for C24H25N3O4: C, 68.72; Н, 6.01; N, 10.02; [M] 419; found:
C, 69.08; Н, 6.06; N, 10.27; М= 419.

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4.2.6. Sonogashira Cross-Coupling Reactions of Bromide 11 with Alkynes 38a–c
To a solution of 3-bromopeurutenicin (11, 100 mg, 0.34 mmol) and a terminal alkyne 38a–c
(0.54 mmol) in benzene (5 mL) was added CuI (6 mg, 10 mol%), Pd(PPh3)2Cl2 (12 mg, 5 mol%),
and Et3N (0.076 mL, 0.44 mmol; 1.3 equiv) under argon. The reaction mixture was stirred at 80 ◦C for
14 h (TLC). The mixture was cooled and 5 mL of water was added. The separated water layer was
extracted with CН2Cl2 (5
×
4 mL). The combined organic extracts was washed with water, dried over
MgSO4, filtered, and concentrated under reduced pressure. The residue was subjected to column
chromatography on silica gel (eluent—chloroform).
Methyl 7-hydroxy-3-(5-(1-(2-isopropyl-3,7-dioxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)-1H-1,2,3-triazol-4-
yl)-pent-1-ynyl)-2-oxo-2H-chromene-6-carboxylate (37a). Yield 52% (0.105 g). Brownish oil. IR (
3350, 3200, 2981, 2932, 2867, 2254, 1741, 1678, 1620, 1600, 1580, 1461, 1446, 1381, 1363, 1295, 1269, 1210,
ν
, cm−1):
1169, 1120, 1096, 1058, 964, 953, 889, 792, 735, 723. UV (EtOH)
λ
max, (lg
ε
): 234 (4.41), 256 (4.44), 273
1
(4.31), 310 (4.09), 339 (4.14), 354 (4.08) nm. H-NMR (CDCl3, 400 MHz, δH): 0.99 (d, J = 7.0 Hz, 3Н,
CН3), 1.02 (d, J = 7.0 Hz, 3Н, CН3), 1.60 (m, 2H, CH2-70), 2.18 (m, 2H, CH2-60), 2.65 (m, 2H, CH2-80),
3.13 (m, 1H, CH
(CH3)2), 3.97 (s, 3H, OCH3), 6.37 (d, J = 9.8 Hz, 1H, H-6), 6.84 (s, 1H, H-800), 7.08 (s, 1H,
H-9), 7.48 (d, 1H, J = 9.8 Hz, H-5), 7.76 (s, 1H, H-4), 7.71, 7.74 (both s, 2H, H-400,500), 7.97 (br.s, 2H, H-50,
OH). 13C-NMR (CDCl3, 100 MHz, δC): 15.0, 15.4, 20.0, 23.5, 32.4, 34.7, 55.7, 70.0, 85.7, 98.1, 99.9, 104.3,
107.2, 110.2, 112.3, 115.1, 116.0, 116.3, 125.5, 128.2, 130.7, 139.7, 142.9, 143.8, 156.1, 157.5, 158.5, 161.4,
162.0, 169.0, 171.4, 192.5. Anal. calcd for C32H25N3O9: C, 64.54; Н, 4.23; N, 7.06; [M] 598; found: C,
64.58, Н, 4.36, N, 7.07; М= 595.
Methyl 7-hydroxy-3-(6-(1-(2-isopropyl-3,7-dioxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)-1H-1,2,3-triazol-1-
yl)-hex-1-ynyl)-2-oxo-2H-chromene-6-carboxylate (37b). Yield 57% (0.118 g). Yellowish powder, m.p.
126–127 ◦C (ether). IR (KBr, , cm−1): 3388, 3139, 3049, 2929, 2856, 2235, 1735, 1673, 1626, 1579, 1481,
ν
1439, 1388, 1352, 1288, 1230, 1163, 1126, 1091, 1037, 997, 962, 946, 865, 825, 750, 737. UV (EtOH)
λ
,
max
1
(lgε
): 234 (4.39), 256 (4.43), 273 (3.81), 309 (4.09), 339 (4.13), 352 (3.62) nm. H-NMR (CDCl3, 400 MHz,
δH): 0.98 (d, J = 7.0 Hz, 3Н, CН3), 1.04 (d, J = 7.0 Hz, 3Н, CН3), 1.44–1.53 (m, 4H, CH2-70,80), 2.14 (m,
2H, CH2-60), 2.64 (m, 2H, CH2-90), 3.13 (m, 1H, CH(CH3)2), 3.95 (s, 3H, OCH3), 6.35 (d, J = 9.8 Hz, 1H,
H-6), 6.92 (s, 1H, H-800), 7.07 (s, 1H, H-9), 7.64 (d, J = 9.8 Hz, 1H, H-5), 7.71, 7.74 (both s, 2H, H-400,500),
7.88 (s, 1H, H-4), 7.97 (s, 1H, H50). 13C-NMR (CDCl3, 100 MHz, δC): 15.6, 16.0, 19.4, 32.5, 33.0, 36.7,
25.7, 54.2, 70.6, 88.5, 98.7, 100.6, 104.9, 108.8, 110.8, 112.8, 115.7, 116.6, 116.9, 126.1, 128.8, 132.3, 141.1,
143.5, 144.4, 156.6, 158.1, 159.1, 162.0, 162.6, 169.5, 172.0, 192.4. Anal. calcd for C33H27N3O9: C, 65.02;
Н, 4.46; N, 6.89; [M] 609; found: C, 65.08; Н, 4.06; N, 6.37; M = 605.
Methyl 7-hydroxy-3-(8-(1-(2-isopropyl-3,7-dioxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)-1H-1,2◦,3-triazol-4
-yl)-oct-1-ynyl)-2-oxo-2H-chromene-6-carboxylate (37c). Brownish powder, m.p. 152–153 C (ether).
Yield 55% (0.119 g). IR (KBr, ν
, cm−1): 3440, 3141, 3049, 2971, 2929, 2856, 2213, 1735, 1673, 1625, 1579,
1481, 1388, 1351, 1288, 1230, 1126, 1091, 1037, 962, 946, 865, 825, 750. UV (EtOH)
λ
max, (lg
ε
): 234 (4.63),
1
251 (4.67), 256 (4.67), 309 (4.33), 339 (4.37), 350 (4.34) nm. H-NMR (CDCl3, 400 MHz, δH): 0.91 (d,
J = 7.0 Hz, 3Н, CН3), 0.94 (d, J = 7.0 Hz, 3Н, CН3), 1.23, 1.29 (both m, 4H, CH2-80,90), 1.42 (m, 2H,
CH2-100), 1.51 (m, 2H, CH2-70), 2.07 (m, 2H, CH2-60), 2.56 (m, 2H, CH2-110), 3.07 (m, 1H, C
H(CH3)2),
3.92 (s, 3H, OCH3), 6.31 (d, J = 9.8 Hz, 1H, H-6), 6.78 (s, 1H, H-800), 7.03 (s, 1H, H-9), 7.46 (s, 1H, H-4),
7.68 (d, J = 9.8 Hz, 1H, H-5), 7.83 (s, 1H, H-50). 7.89, 7.90 (both s, 2H, H-400,500). 13C-NMR (CDCl3,
100 MHz, δC): 15.2, 15.6, 19.0, 25.2, 29.9, 30.7, 31.2, 33.6, 36.3, 52.7, 70.2, 87.4, 98.2, 101.0, 104.5, 108.4,
110.4, 112.4, 115.2, 115.6, 116.5, 125.7, 128.2, 131.8, 141.3, 143.0, 144.0, 156.2, 157.7, 158.9, 161.6, 162.2,
169.2, 171.5, 191.8. Anal. calcd for C35H31N3O9: C, 65.93; Н, 4.90; N 6.59; [M] 637; found: C, 65.38; Н,
4.66; N, 6.87; М= 635.

Molecules 2019, 24, 2126
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4.3. Antibacterial Activity Assay
Method 1. Compounds 4a
–c,
11,
26,
29,
30
,
37a
–
c,
42a
–c
and parent compounds
1,
2, 3 were tested for
their in vitro antibacterial activity against Staphylococcus aureus 209рATCC 6538-P and strains from
the collection of Department of Microbiology, Immunology and Virology, Novosibirsk State Medical
University: Staphylococcus aureus C-18 - clinical isolated strain, Staphylococcus aureus “Viotko” and
Actinomyces viscosus U-18. Cultivation of bacterial cultures was carried out on agar and broth media
Muller-Hinton in aerobic conditions at a temperature of 37 ◦C. Cultivation time for Staphylococcus
aureus was 1–2 days, for Actinomyces viscosus U – 2–3 days. Analysis of antibacterial activity was
performed using the method of serial macrodilutions in a liquid medium in the total volume of
1.0 mL [24
to the desired concentration with 0.9% sodium chloride solution (0.2 mL) and nutrient broth. To assess
antibacterial activity, a number of double dilutions of the studied substances starting from 1000 g/mL
were used. Doses of substances above 1000 g/mL were not considered because of their low solubility.
The introduced dose of daily cultures of bacteria was determined using the standard of turbidity
according to McFarland and was controlled by seeding on a dense nutrient medium. It gave (2.75 0.85)
,38]. All compounds were primarily dissolved in 0.05 mL of 96% ethyl alcohol and brought
µ
µ
±
103 colony-forming units (CFU). For the minimum inhibitory concentration (MIC), the smallest dose of
the substance was taken, completely suppressing the growth of bacteria. The absence of signs of growth
in the liquid medium was controlled by seeding to the surface of a agar medium with subsequent
incubation under standard conditions. As a negative control, the test culture was introduced into
1 mL of broth and cultivated under the same conditions, followed by sowing on an agar nutrient
medium and taking into account the growth of bacteria. Based on the results of triplicate repeated
experiments, the mean value of the MIC and the standard error of measurement (M
calculated. The primary data of antibacterial activity are given in Supplementary Materials.
Method 2. Compounds 36 37a–с, and were tested for their in vitro antibacterial activity against
±
SEM) were
,
1
2
Bacillus subtilis and Escherichia coli (JM109). This strain is most similar to wild type of E. coli in our bacterial
collection. The known tumorogenic compounds 4-nitroquinolin-1-oxide (NQO) was used as the
reference compound [39]. This procedure was maintained according to the standard broth microdilution
method as recommended in guidelines of Clinical and Laboratory Standards Institute [24,40,41] and
the minimum inhibitory concentration (MIC) of compounds was tested. In short, testing was
performed in U-bottomed 96-well sterile plastic microdilution trays (Falcon 3077, Becton Dickinson
and Co., Cockeysville, MD, USA) in cation (Ca2+ and Mg2+) adjusted Mueller-Hinton broth medium
(Becton Dickinson and Co., Cockeysville, MD, USA). The concentration range of test compounds
was started from 1000 µg/mL by using serial two fold dilution. Standardized initial inoculum was
prepared by the direct colony suspension method to the final inoculum to 5 × 105 CFU/mL, as described
(CLSI M7-A7). After inoculation of previously prepared microdilution trays with tested compounds,
trays were incubated at 35
±
2 ◦C overnight (18–20 h) in an ambient air incubator. E. coli JM109 served
as quality control of MIC determination procedure, as well. The MIC was determined as the lowest
concentration of tested compound that completely inhibits growth of the organism in the microdilution
wells as detected by the unaided eye and comparing the amount of growth in the wells containing the
tested agent with the amount of growth in the growth-control wells (no antimicrobial agent). All testing
were done in triplicate.
4.4. Molecular Docking
Molecular modeling was carried out in the Schrodinger Maestro visualization environment using
applications from the Schrodinger Small Molecule Drug Discovery Suite 2016-1 (Python, NY, USA) [42].
Three-dimensional structures of the derivatives were obtained empirically in the LigPrep application
using the OPLS3 force field [43]. All possible tautomeric forms of compounds, as well as various states
of polar protons of molecules in the pH range of 7.0
for docking was selected automatically, based on the size and physico-chemical properties of FAD.
The extra precision (XP) algorithm of docking was applied. Docking was performed in comparison
±
2.0 were taken into account. The search area

Molecules 2019, 24, 2126
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with FAD. The three-dimensional structures of FAD was obtained in the PubChem database and
prepared in the LigPrep application. Non-covalent interactions of compounds in the binding site were
visualized using Biovia Discovery Studio Visualizer (Biovia, San Diego, CA, USA) [44].
Supplementary Materials: Supplementary data associated with this article can be found in the online version,
at Supporting information included the copies of NMR spectra (1H & 13C) of new compounds and also the primary
data of antibacterial activity.
Author Contributions: Project administration, E.E.S., A.N.E., O.I.S., and T.G.T.; Supervision, E.E.S. and L.G.B.;
Writing—original draft, E.E.S., A.V.L. and L.G.B.; Methodology, E.E.S., D.S.B., T.S.F; investigation, A.V.L., D.O.Z.,
L.G.B., D.S.B., I.V.S. and T.S.F; Writing—review & editing, E.E.S and L.G.B.
Funding: This work was performed under financial support in part from the Russian Science Foundation
(Grants 17-73-10099 and 18-13-00361).
Acknowledgments: Authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS for
spectral and analytical measurements.
Conflicts of Interest: The authors declare that they have no conflict of interests.
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Sample Availability: Samples of the compounds 1, 4a–c, 11, 16, 4a–c, 28, 29, 30, 36, 37a–c, 42a–c are available
from the authors.
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