Synthesis of Natural and Biologically Active Quinoxaline Analogs

Article abstract of DOI:10.1007/s10600-019-02728-1

Reactions of quinoxalines and quinoxalin-2-ones with C-nucleophiles under acid-catalysis conditions gave products from nucleophilic substitution of hydrogen. Substitution of F atoms in the aromatic core of quinoxalines was studied. Antibacterial and fungistatic activity of the synthesized compounds was studied.

Full text of DOI:10.1007/s10600-019-02728-1

DOI 10.1007/s10600-019-02728-1
Chemistry of Natural Compounds, Vol. 55, No. 3, May, 2019
SYNTHESIS OF NATURAL AND BIOLOGICALLY
ACTIVE QUINOXALINE ANALOGS
Yu. A. Azev,1* O. S. Koptyaeva, A. N. Tsmokalyuk,
1
1
1
2
2
T. A. Pospelova, N. A. Gerasimova, N. P. Evstigneeva,
N. V. Zil′berberg, N. V. Kungurov, and O. N. Chupakhin
2
2
1,3
Reactions of quinoxalines and quinoxalin-2-ones with C-nucleophiles under acid-catalysis conditions gave
products from nucleophilic substitution of hydrogen. Substitution of F atoms in the aromatic core of
quinoxalines was studied. Antibacterial and fungistatic activity of the synthesized compounds was studied.
Keywords: quinoxalines, 6,7-difluoroquinoxalines, C-, N-, O-nucleophiles, antibacterial and fungistatic activity.
The pyrazine heterocycle appears in various biologically active and natural compounds. Benzoannelated pyrazine
(
quinoxaline) occurs in vitamin B and the antibiotics echinomycin (natural peptide antibiotic) and actinoleutin, which inhibit
2
growth of Gram-positive bacteria and are active against various tumors [1, 2]. Currently, preparations of quinoxidine and
dioxidine, which incorporate quinoxaline into their structures [3], are used in medical practice for serious microbial infections.
In the present work, quinoxalines were functionalized on the heterocyclic core using environmentally and chemically
benign nucleophilic substitution of hydrogen [4].
Electron paramagnetic resonance (EPR) studies of the reactivity of quinoxaline in the presence of electron donors or
acceptors found two different types of radicals. Previously, quinoxalines were reported to form cation-radicals in a couple of
instances [5, 6].
Quinoxaline cation-radical A was detected in an MeCN (or C H ) solution of 1a (Scheme 1, Fig. 1a). The triplet EPR
6
6
1
4
spectrum with intensity distribution 1:1:1 (Fig. 1a) was characteristic of splitting by one N nucleus, which confirmed the
structure was quinoxaline cation-radical A. The coupling constant of the unpaired electron with the N nucleus was
1
4
αN = 1.58 mT in the hyperfine structure of the spectrum. An EPR study of the reaction mixture from 1a or 1b and a nucleophile
(
dimedone) in DMSO in the presence of acid detected stable cation-radical C of a diprotic quinoxalinium salt (Fig. 1b and 1c) [7].
NH
The coupling constants of the unpaired electron with the two equivalent N nuclei (α
= 0.67 mT) and the protons
N
NH
bonded to them (αH = 0.37 mT) were similar for a solution of 6,7-difluoroquinoxaline 1b and dimedone in the presence of
HCl (Fig. 1c).
-
-
2
Cl
Cl
H
N
H
N
+
+
_
N
+
N
-
R
R
N
N
R
R
R
R
-
a
e
b
+e (Nu)
.
.
+
N
N
H
H
A
R = H (a)
1a,b
R = H (a), F (b)
B
C
a. CH CN/C H (+TBAF); b. HCl, DMSO
3
6 6
Scheme 1
1
) Ural Federal University named after the first President of Russia B. N. Yeltsin, 19 Mira St., Ekaterinburg, 620002
Russia, e-mail: azural@yandex.ru; 2) Ural Research Institute for Dermatology, Venereology and Immunopathology, Ministry
of Health, Sverdlovsk Region, Ekaterinburg, 620076 Russia; 3) I. Ya. Postovsky Institute of Organic Synthesis, Ural Branch,
Russian Academy of Sciences, Ekaterinburg, 620990 Russia. Translated from Khimiya Prirodnykh Soedinenii, No. 3,
May–June, 2019, pp. 441–447. Original article submitted October 1, 2018.
0
009-3130/19/5503-0513 ©2019 Springer Science+Business Media, LLC
513

a
b
c
3
465 3485
3505
Field [G]
3525 3545
3485
3505
3525
3545
3485
3505
3525
3545
Field [G]
Field [G]
Fig. 1. EPR spectra of quinoxaline radicals: cation-radical 1a in MeCN or C H (a); quinoxaline 1a in DMSO with added HCl
6
6
(
b); quinoxaline 1b in DMSO with added HCl (c).
Obviously, the tendency of quinoxalines 1a,b to form radicals in the presence of weak electron acceptors (C H ,
6
6
MeCN) indicated that the compounds had pronounced electron-donating properties (reactions with electrophiles, e.g., acids).
However, the formation of protonated quinoxaline salt radicals under acid catalysis conditions was indicative of pronounced
electron-accepting properties (reactions with nucleophiles).
Quinoxaline reacted smoothly under mild conditions with C-nucleophiles in our previous work [8, 9]. Amore detailed
study of these reactions detected quinoxaline dimers 7 and 8 in 3–5% yields. The present work showed that purging air
through the mixture of quinoxaline (1a or 1b) and nucleophile (Scheme 2) increased significantly (by 10–15%) the yield of
target product whereas the yields of dimers 7 and 8 were 2–5%. The yields of dimers 7 and 8 increased to 12–20% and the
yields of target compounds 2a,b–6a,b decreased by 7–10% if the reaction was performed under N2.
R
N
R
N
R
N
a
+
i, ii
N
N
R
R
Nu
R
N
R
N
R
N
1
a,b
2a,b - 6a,b
7,8
i: 45-61%
ii: 28-44%
i: 2-5%
ii: 12-20%
O
N
O
OH
O
Nu:
CH
CH
O
3
3
N
CH
3
N
O
O
O
OH
5a,b
N
Ph
CH
3
O
CH
3
2
a,b
3a,b
4a,b
6a,b
a. HNu, HCl, DMSO, 20°C, 2 h, (i) air; (ii) N2
Scheme 2
Obviously, products from monosubstitution of hydrogen during the reaction under N (or with an O deficiency)
2
2
2
were formed by addition of nucleophiles to quinoxaline C and oxidation of the resulting σ-adducts by starting quinoxalines
1
a,b or their protonated salts. In turn, the intermediates were oxidized and reduced the starting protonated quinoxaline salts to
cation-radicals. The reaction of cation-radicals with the starting quinoxalines was a typical hetarylation reaction that produced
dimers 7 and 8.
Thus, the dimerization products confirmed that radicals formed in the reaction mixture and underwent nucleophilic
substitution of hydrogen.
H
N
H
N
O
O
a
N
N
Nu
1
c
9-11, 43-54%
OH
OCH
3
CH
3
Nu:
N
O
N
OH
3
H CO
OCH
3
Ph
9
10
11
a. HNu, AcOH, Δ, 40 h
Scheme 3
514

Quinoxalones, i.e., quinoxaline derivatives with a 2-oxo group, are more stable in nucleophilic substitution of hydrogen
and do not form radical species. So, the yields of target compounds in reactions of quinoxalones are greater than those of
quinoxalines. The products from reactions of quinoxalones with several nucleophiles were previously reported [10, 11].
New examples of reactions of quinoxalones with C-nucleophiles were obtained by us. The reactions were carried out
with heating in AcOH and formed hydrogen substitution products 9–11 in good yields (Scheme 3).
Additional structural modifications were made possible by introducing F atoms into the quinoxaline aromatic fragment.
The conditions for monosubstitution of F atoms were especially interesting for 6,7-difluorinated quinoxalines. It was found
earlier that substitution of F atoms in polyfluorinated quinoxaline derivatives can occur stepwise in reactions with sodium
methoxide and dimethylamine [12, 13].
Our results for F-substitution of 6,7-difluoroquinoxaline (1b) by propargyl alcohol indicated that the reaction occurred
stepwise at 40–50°C with substitution of one F atom to give 12. Both F atoms were replaced at 90–100°C to produce disubstituted
derivative 13 (Scheme 4).
F
N
F
N
a
c
O
N
O
N
F
F
N
N
1
2, 58%
14, 42%
O
O
N
O
N
b
d
1
b
N
O
N
1
3, 55%
15, 54%
a. CH≡C-CH OH, 40–50°C, 1 h; b. CH≡C-CH OH, 90–100°C, 1 h; c. EtOH, NaOH, 40–50°C, 1h; d. EtONa, Δ, 1 h
2
2
Scheme 4
Heating 1b in EtOH at 40–50°C in the presence of base gave the product from monosubstitution of F (14). Quinoxaline
b reacted with NaOEt in refluxing EtOH to form the product from disubstitution of F (15).
1
Selective monosubstitution of F in 6,7-difluoroquinoxalines by amines was especially significant because it allowed
pharmacophores analogous to those in fluoroquinolone antibiotics to be incorporated into the molecular structure [14].
Previously, reactions of 6,7-difluoroquinoxalines with N-methylpiperazine were shown to form products corresponding
monosubstitution of the 7-F [9, 15]. We expanded the series of used nucleophiles and produced previously unknown
quinoxalones 17–20 (Scheme 5). The reactions of the quinoxalones with amines occurred smoothly in good yields upon
heating in DMSO.
O
HN
H
N
H
N
H
N
N
F
O
F
F
O
HN
NH
N
F
O
HN
O
a
a
N
N
N
R
1
= R
2
= H
N
H
N
R
2
N
R
2
R
1
R
1
1
7, 82%
16a-c
6a,18: R = R = H; 16b,19: R = CH , R = H; 16c,20: R = H, R = CH
3
18-20, 53-69%
1
1
2
1
3
2
1
2
a. DMSO, 135°C, 2 h
Scheme 5
The obtained quinoxalines were screened for antimicrobial and fungistatic activity. The biological activity tests also
included quinoxalines 21–25 that were reported by us earlier [7, 15] (Table 1). The tested compounds were screened preliminarily
in vitro) for microbial activity against bacteria strains of Escherichia coli, Klebsiella pneumoniae, Citrobacter braakii,
(
Proteus vulgaris, Serratia marcescens, Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and
Streptococcus pyogenes. Spectinomycin was used as a control. Table 1 presents the compounds with the highest activity
parameters.
The development of new antimicrobial agents for treating diseases caused by Neisseria gonorrhoeae is critical because
the resistance of this strain to cephalosporins, the latest line of antibiotics [16], is increasing globally. Studied quinoxalines
that were active against N. gonorrhoeae included 4a, 18, 19, 21, and 25, which were comparable to spectinomycin.
Compounds 18, 19, and 23 were highly active against an antibiotic-resistant strain of MRSA (S. aureus).
515

TABLE 1. Antimicrobial Activity of Synthesized Quinoxalines (MIC,* μg/mL)
Escherichia
coli
Shigella
flexneri
Staphylococcus
aureus
Streptococcus
piogenes
Staphylococcus
aureus (MRSA)
Neisseria
gonorrhoeae
Compound
4
à
> 250
> 250
> 250
> 250
15.6
> 250
> 250
–
31.2
62.5
31.2
125
7.8
7.8
> 250
> 250
31.2
> 250
> 250
–
–
3.9
3.9
> 250
> 250
–
15.6
31.2
> 250
62.5
7.8
31.2
125
> 250
4
b
10
17
18
19
20
21
22
23
24
25
–
250
15.6
> 250
> 250
> 250
–
31.2
15.6
62.5
31.2
> 250
> 250
62.5
15.6
16–32
31.2
7.8
> 250
> 250
> 250
> 250
> 250
125
31.2
> 250
> 250
15.6
31.2
31.2
> 250
–
62.5
31.2
31.2
–
> 250
> 250
15.6
15.6
125
15.6
Spectinomycin
15.6–31.2
31.2–62.5
_
*
_____
MIC = minimum inhibitory concentration.
TABLE 2. Antifungal Activity of Synthesized Quinoxalines (MIC, μg/mL)
Trichophyton
rubrum
Trichophyton
violaceum
Trichophyton mentagrophytes
Epidermophyton
floccosum
Microsporum
canis
Compound
var. interdigitale
1
1
1
2
2
7
8
9
0
5
50
25
> 100
25
> 100
50
100
> 100
50
> 100
> 100
100
> 100
> 100
50
> 100
100
100
100
50
100
> 100
25
50
50
25
100
Fluconazole
_____
6.25
3.12
100
_
*
MIC = minimum inhibitory concentration.
O
HN
N
H
H
H
N
R
R
N
N
N
O
N
F
N
O
O
OH
CH
N
3
N
N
F
N
O
N
OH
N
H
R
2
N
4
a,b
10
17
18 - 20
Ph
R
1
3
H C
H
N
H
F
F
N
N
O
F
N
N
N
F
N
O
O
N
N
N
N
H
3
C
O
N
2
1, 22
23
24, 25
R
R
4
a: R = H; 4b: R = F; 18: R = R = H; 19: R = CH , R = H; 20: R = H, R = CH ; 21: R = H; 22: R = CH ; 24: R = CH ; 25: R = H
1 2 1 3 2 1 2 3 2 3 3
The synthesized compounds were tested for antifungal activity against strains of Trichophyton rubrum,
T. mentagrophytes var. gypseum, T. tonsurans, T. violaceum, T. mentagrophytes var. interdigitale, Epidermophyton floccosum,
Microsporum canis, and Candida albicans. The antifungal drug fluconazole was used as a positive control. The studied
quinoxalines included 18 and 25, which possessed moderate fungicidal activity against two of the six studied strains (Table 2).
516

EXPERIMENTAL
All reagents were commercially available and were used without further purification (Sigma Aldrich, Merck).
1
3
The course of reactions was monitored by TLC on silica gel plates (Merck). PMR and C NMR spectra were recorded in
DMSO-d with TMS internal standard on an Avance-400 instrument. Electron-impact (70 eV) mass spectra were obtained on
6
a MicrOTOF-Q instrument (Bruker Daltonics) at average ionizing potential 70 eV and heating to 250°C. EPR spectra were
recorded on a Bruker Elexsys E 500 EPR spectrometer.
The synthetic methods for 2a,b–6a,b were improved as compared to the previously reported ones [4]. Thus, the
yields of target products 2a,b–6a,b increased by 10–15% if the reaction mixture was purged with air. The reactions of 1a,b
with the used nucleophiles under N gave 12–20% yields of dimers 7 and 8.
2
General Method for the Reactions of Quinoxalin-2-one with Nucleophiles. A mixture of 1c (0.3 mmol) and the
appropriate nucleophile (0.4 mmol) was heated in AcOH (1.5 mL) for 40 h at 110°C and cooled. When the reaction was
finished (TLC), the resulting precipitate was filtered off and rinsed with H O.
2
3
-(3-Methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)quinoxalin-2(1H)-one (9). Yield 54%, mp > 300°C.
1
Í NMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.60 (3H, s, CH ), 7.22 (1Í, t, J = 7.6, CH), 7.32–7.34 (1H, m, CH),
.38–7.45 (4H, m, CH), 7.67 (1H, d, J = 7.6, CH), 7.91 (2H, d, J = 8, CH), 13.08 (1H, s, NH), 15.44 (1H, br.s, NH). C NMR
6
3
1
3
7
spectrum (100 MHz, DMSO-d , δ, ppm): 18.52, 98.29, 115.66, 119.90, 123.24, 124.56, 125.25, 127.67, 128.25, 128.84,
6
+
1
38.26, 147.69, 148.66, 155.80, 159.60. Mass spectrum (EI, 70 eV), m/z (I , %): 318 (M , 100), 226 (45), 157 (29), 77 (28).
rel
1
3
-(2,4-Dihydroxyphenyl)quinoxalin-2(1H)-one (10). Yield 51%, mp > 300°C. Í NMR spectrum (400 MHz,
DMSO-d , δ, ppm, J/Hz): 6.33 (1H, s, CH), 6.38 (1H, d, J = 10.0, CH), 7.27–7.34 (2H, m, CH), 7.50 (1H, t, J = 7.6, CH), 7.74
6
1
3
(
1H, d, J = 7.6, CH), 9.01 (1H, d, J = 10.0, CH), 10.16 (1H, s, OH), 12.65 (1H, br.s, OH), 14.16 (1H, s, NH). C NMR
spectrum (100 MHz, DMSO-d , δ, ppm): 102.98, 106.16, 107.17, 110.53, 115.01, 123.67, 126.25, 129.23, 129.33, 131.08,
6
+
1
32.97, 153.53, 154.83, 161.89, 163.10. Mass spectrum (EI, 70 eV), m/z (I , %): 254 (M , 100), 226 (57), 169 (39).
rel
1
3
-(2,4,6-Trimethoxyphenyl)quinoxalin-2(1H)-one (11). Yield 43%, mp > 300°C. Í NMR spectrum (400 MHz,
DMSO-d , δ, ppm, J/Hz): 3.71 (6Í, s, 2ÑÍ ), 3.86 (3Í, s, ÑÍ ), 6.24 (2Í, s, 2ÑÍ), 7.22 (1Í, t, J = 8.0, ÑÍ), 7.31 (1Í, d,
6
3
3
1
3
J = 8.0, ÑÍ), 7.43 (1Í, t, J = 8.0, ÑÍ), 7.69 (1Í, d, J = 8.0, ÑÍ), 12.17 (1Í, s, NÍ). C NMR spectrum (100 MHz,
DMSO-d , δ, ppm): 55.40, 55.69, 90.86, 107.10, 115.16, 123.01, 128.54, 130.09, 131.89, 132.01, 154.39, 156.11, 158.76,
6
+
1
61.57. Mass spectrum (EI, 70 eV), m/z (I , %): 312 (M , 100), 283 (73), 269 (50).
rel
6
-Fluoro-7-(prop-2-yn-1-yloxy)quinoxaline (12). 6,7-Difluoroquinoxaline (1b, 0.166 g, 1.0 mmol) in propargyl
alcohol (3.0 mL) in the presence of NaOH (0.128 g, 3.2 mmol) was held at 60–70°C for 1 h. When the reaction was finished
TLC), the mixture was cooled and diluted with H O (2.0 mL). The precipitate of 12 was filtered off and rinsed with H O.
(
2
2
1
Yield 0.090 g (58%), mp 128–130°C. Í NMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 3.43 (1H, t, J = 2.4, ÑÍ), 5.06
6
(
2H, d, J = 2.4, ÑÍ ), 7.70 (1H, d, J = 8.8, ÑÍ), 7.76 (1H, d, J = 11.2, ÑÍ), 8.76 (1Í, d, J = 2.0, ÑÍ), 8.81 (1Í, d, J = 2.0, ÑÍ).
2
1
3
C NMR spectrum (100 MHz, DMSO-d , δ, ppm): 56.95, 79.54, 110.88, 113.22, 138.19, 140.52, 144.15, 145.12, 148.00,
6
+
1
52.42, 154.94. Mass spectrum (EI, 70 eV), m/z (I , %): 201 (M , 50), 173 (88), 135 (69), 38 (100).
rel
6
,7-bis(Prop-2-yn-1-yloxy)quinoxaline (13). 6,7-Difluoroquinoxaline (1b, 0.166 g, 1.0 mmol) in propargyl alcohol
(
3.0 mL) in the presence of NaOH (0.128 g, 3.2 mmol) was held at 100–110°C for 1 h. When the reaction was finished (TLC),
the mixture was cooled and diluted with H O (10.0 mL). The precipitate of 13 was filtered off, rinsed with H O, and recrystallized
2
2
1
from aqueous EtOH. Yield 0.070 ã (55%), mp 144–145°C. Í NMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 3.41 (2Í,
6
1
3
t, J = 2.4, ÑÍ), 5.06 (4Í, d, J = 2.4, ÑÍ ), 7.51 (2Í, s, ÑÍ), 8.68 (2Í, s, ÑÍ). C NMR spectrum (100 MHz, DMSO-d , δ,
2
6
+
ppm): 56.37, 79.12, 108.97, 139.25, 143.24, 150.02. Mass spectrum (EI, 70 eV), m/z (I , %): 238 (M , 15), 199 (29), 171 (70),
rel
143 (27), 38 (100).
6
-Ethoxy-7-fluoroquinoxaline (14). 6,7-Difluoroquinoxaline (1b, 0.160 g, 0.96 mmol) in anhydrous EtOH
(
(
1.0 mL) in the presence of NaOH (0.1 g, 2.5 mmol) was held at 45–50°C for 1.0–1.5 h. When the reaction was finished
TLC), the solvent was evaporated. The solid was worked up with H O (8.0 mL). The precipitate of 14 was filtered off, rinsed
2
with H O, and recrystallized from aqueous EtOH. Yield 42%, mp 108–110°C. The spectral characteristics were analogous to
2
those published earlier [17].
6
,7-Diethoxyquinoxaline (15). 6,7-Difluoroquinoxaline (1b, 0.083 g, 0.50 mmol) in NaOEt solution (0.05 g,
2
.2 mmol) in anhydrous EtOH (5.0 mL) was refluxed for 2.0 h and cooled. The precipitate of 15 was filtered off and rinsed
with H O. Yield 0.045 g (54%), mp 130–131°C. The spectral characteristics were analogous to those published earlier [14].
2
Reaction of 6,7-Difluoroquinoxalin-2-ones 16a-c with Piperazine and Morpholine (general method). A mixture
of 16a–c (0.2 mmol) and piperazine or morpholine (1.5 mmol) in DMSO (1.0 mL) was heated at 130–135°C for 24 h.
517

When the reaction was finished (TLC), the mixture was cooled and diluted (3×) with H O. The precipitate was filtered off and
2
rinsed with H O.
2
1
6
-Fluoro-3-(1H-indol-3-yl)-7-morpholinoquinoxalin-2(1H)-one (17). Yield 82%, mp 287–288°C. Í NMR spectrum
(
400 MHz, DMSO-d , δ, ppm, J/Hz): 3.11–3.14 (4Í, m, 2CH ), 3.81–3.85 (4Í, m, 2CH ), 6.82 (1Í, d, J = 8.4, CH),
6 2 2
7
1
1
.14–7.20 (2Í, m, ÑÍ), 7.45–7.47 (1Í, m, CH), 7.53 (1Í, d, J = 13.2, CH), 8.78–8.80 (1Í, m, CH), 8.85 (1Í, d, J = 4.0, ÑÍ),
1.57 (1Í, s, NH), 12.22 (1H, s, NH). 13C NMR spectrum (100 MHz, DMSO-d , δ, ppm): 50.33 (2C), 66.01 (2C), 103.22,
6
11.32, 111.74, 113.37, 120.79, 122.41, 122.98, 126.05, 132.43, 136.20, 140.34, 150.26, 154.32. Mass spectrum (EI, 70 eV),
+
m/z (I , %): 364 (M , 100), 306 (37), 278 (40).
rel
1
6
-Fluoro-3-(1H-indol-3-yl)-7-(piperazin-1-yl)quinoxalin-2(1H)-one (18). Yield 68%, mp 258–259°C. Í NMR
spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.95–3.17 (8Í, m, 4CH ), 6.79 (1Í, d, J = 8.4, ÑÍ), 7.14–7.19 (4Í, m, ÑÍ),
6
2
1
3
7
.43–7.50 (2Í, m, ÑÍ), 8.77–8.97 (1Í, m, ÑÍ), 8.85 (1Í, d, J = 2.8, ÑÍ), 11.49 (1H, s, NH). C NMR spectrum (100 MHz,
DMSO-d , δ, ppm): 45.32 (2C), 51.03 (2C), 103.33, 111.34, 111.74, 113.27, 113.40, 120.77, 122.40, 122.98, 126.06, 127.27,
6
+
1
27.57, 132.35, 136.20, 140.58, 150.13, 152.56, 154.34. Mass spectrum (EI, 70 eV), m/z (I , %): 363 (M , 83), 321 (100), 306 (21).
rel
6
-Fluoro-3-(1-methyl-1H-indol-3-yl)-7-(piperazin-1-yl)quinoxalin-2(1H)-one (19). Yield 53%, mp 289–291°C.
1
Í NMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.94–2.98 (4H, m, 2CH ), 3.00–3.10 (4H, m, 2CH ), 3.93 (3H, s,
6
2
2
CH ), 6.80 (1H, d, J = 8.0, CH), 7.21–7.27 (2H, m, CH), 7.47 (1H, m, CH), 7.50 (1H, d, J = 12.0, CH), 8.81 (1H, m, ÑH), 8.83
3
1
3
(
1Í, s, ÑÍ). C NMR spectrum (100 MHz, DMSO-d , δ, ppm): 32.93, 45.48 (2C), 51.32 (2C), 103.24, 110.06, 113.35,
6
1
21.07, 122.50, 123.14, 126.54, 127.17, 127.57, 136.10, 136.77, 141.15, 149.67, 150.19, 152.59, 154.28. Mass spectrum
+
(
EI, 70 eV), m/z (I , %): 377 (M , 97), 335 (100).
rel
6
-Fluoro-3-(2-methyl-1H-indol-3-yl)-7-(piperazin-1-yl)quinoxalin-2(1H)-one (20). Yield 69%, mp 269–270°C.
1
Í NMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.53 (3H, s, CH ), 3.27–3.32 (8H, m, 4CH ), 6.92 (1H, d, J = 8.0,
6
3
2
CH), 7.00 (1H, t, J = 8.0, CH), 7.03 (1H, t, J = 8.0, CH), 7.31 (1H, d, J = 8.0, CH), 7.50 (1H, d, J = 12.0, CH), 7.74 (1H, d,
1
3
J = 8.0, CH), 9.37 (1H, br.s, NH), 11.44 (1H, s, NH), 12.26 (1H, br.s, NH). C NMR spectrum (100 MHz, DMSO-d , δ, ppm):
6
1
1
4.23, 42.69 (2C), 46.86 (2C), 103.86, 109.02, 110.53, 113.72, 113.94, 119.42, 120.77, 127.84, 128.71, 135.09, 139.09,
+
39.80, 149.79, 152.19, 153.16, 154.40. Mass spectrum (EI, 70 eV), m/z (I , %): 377 (M , 87), 335 (100).
rel
Antimicrobial Activity. Antibacterial activity of the compounds was studied using standard bacteria strains from the
American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC, England), and Russian Collection
of Pathogenic Microorganisms (RCPM). Strains N. gonorrhoeae (ATCC 49226/NCTC 12700), E. coli (ATCC 8739),
C. braakii (ATCC 101/57), Shigella flexneri (RCPM 1a8516), Proteus vulgaris [RCPM 160125 (222)], Serratia marcescens
(
ATCC 13880), K. pneumoniae (ATCC 13883), P. aeruginosa (ATCC 9027), S. pyogenes (ATCC 19615), S. aureus
[
ATCC 25923/NCTC 12981 (F-49)], and methicillin-resistant S. aureus (MRSA) (NCTC 12493) were used. One-day microorganism
®
cultures were identified on a VITEK MS analyzer (bioMerieux).
Antibacterial activity of the compounds against obligate pathogen N. gonorrhoeae was determined using double
serial dilutions in agar (gold standard). At least 12 points with dilutions 250–0.06 μg/mL were prepared for each chemical
compound. The solvent was DMSO; diluents, distilled sterile H O (for injection) and agar-based growth medium. The
2
5
inoculation dose (final concentration) of the aliquot of one-day N. gonorrhoeae culture was 10 CFU/mL. Plates were incubated
at 37°C. Results were estimated after 18–24 h [18].
Antibacterial activity of the chemical compounds against the other pathogenic microorganisms was tested using
sequential microdilutions in Mueller—Hinton broth in sterile 96-well plates [19]. The solvent was DMSO; diluents, distilled
sterile H O (for injection) and Mueller–Hinton growth broth. Aliquots of control strains were prepared according to a
2
8
6
0
.5 MacFarland standard (1.5⋅10 CFU/mL) and diluted 100× to a concentration of 10 CFU/mL. Each well of a horizontal
row was charged with an aliquot of the appropriate strain (50.0 μL). Plates were incubated in a thermostat at 37°C for 18–24 h
[
18]. The control drug was spectinomycin (Sigma-Aldrich, USA).
Antifungal Activity. The fungistatic activity was studied using test cultures of dermatophytes from the Russian
Collection of Pathogenic Fungi (RCPF) including Trichophyton rubrum (RCPF 1408), T. mentagrophytes var. gypseum
RCPF 1425), T. tonsurans (RCPF 1458), T. violaceum (RCPF 1393/658), T. mentagrophytes var. interdigitale (RCPF 1229),
T. schoenleinii (RCPF 235/25), Epidermophyton floccosum (RCPF 1174), Microsporum canis (RCPF 1403), and C. albicans
RCPF 401/NCTC-885-653).
Double serial dilutions and Sabouraud broth (Russia) were used [20]. Fungi were cultivated at 27°C. Test compounds
(
(
and the standard were dissolved in DMSO (1:10) and used at various concentrations (from 250 to 1.9 μg/mL). The diluents
were distilled sterile H O (for injection) and growth medium. DMSO was used as a negative control; fluconazole antifungal
2
preparation, as a positive control. Samples were incubated at 27°C for 14 d.
518

ACKNOWLEDGMENT
We thank the RFBR (Grants 18-33-00727 mol_a and 18-03-00715 A) for sponsoring the research.
REFERENCES
1.
A. Dell, D. H. William, H. R. Morris, G. A. Smith, J. Feeney, and G. C. K. Roberts, J. Am. Chem. Soc.,
7 (9), 2497 (1975).
9
2
3.
.
Y. B. Kim, Y. H. Kim, J. Y. Park, and S. K. Kim, Bioorg. Med. Chem. Lett., 14, 541 (2004).
M. D. Mashkovskii, Drugs [in Russian], Meditsina, Moscow, 1993, 564 pp.
4.
O. N. Chupakhin, V. N. Charushin, and H. C. van der Plas, Nucleophilic Aromatic Substitution of Hydrogen,
Academic Press, San Diego, 1994, 367 pp.
5
6.
.
O. N. Chupakhin, E. O. Sidorov, S. M. Shein, and I. I. Bil′kis, Zh. Obshch. Khim., 12, 2464 (1976).
J. Prinson and J. Armand, Collect. Czech. Chem. Commun., 36, 585 (1971).
7.
Yu. A. Azev, O. S. Ermakova, V. A. Bakulev, I. S. Kovalev, A. N. Tsmokalyuk, A. N. Kozitsina, M. G. Pervova,
and V. I. Filyakova, Zh. Obshch. Khim., No. 7, 1111 (2015).
8
9.
.
Yu. A. Azev, B. V. Golomolzin, O. S. Ermakova, and V. A. Bakulev, Pharm. Chem. J., 47 (3), 172 (2013).
Yu. A. Azev, M. I. Kodess, M. A. Ezhikova, A. M. Gibor, V. I. Baranov, O. S. Ermakova, and V. A. Bakulev, Pharm.
Chem. J., 47 (9), 498 (2013).
1
0.
O. N. Chupakhin, E. O. Sidorov, and I. Ya. Postovskii, Khim. Geterotsikl. Soedin., 10, 867 (1976).
O. N. Chupakhin, E. O. Sidorov, A. L. Kozerchuk, and Yu. I. Beilis, Khim. Geterotsikl. Soedin., 13, 557 (1976).
A. G. Makarov, N. Yu. Selikhova, A. Yu. Makarov, V. S. Malkov, I. Yu. Bagryanskaya, Y. V. Gatilov, A. S. Knyazev,
Yu. G. Slizhov, and A. V. Zibarev, J. Fluorine Chem., 165, 123 (2014).
1
1.
1
2.
3.
1
T. F. Mikhailovskaya, A. G. Makarova, N. Yu. Selikhova, A. Yu. Makarova, E. A. Pritchina, I. Yu. Bagryanskaya,
E. V. Vorontsova, I. D. Ivanov, V. D. Tikhova, N. P. Gritsan, Yu. G. Slizhov, and A. V. Zibarev, J. Fluorine Chem.,
1
83, 44 (2016).
V. N. Charushin, E. V. Nosova, G. N. Lipunova, and O. N. Chupakhin, Fluoroquinolones. Synthesis and Application
in Russian], Fizmatlit, Moscow, 2013, 320 pp.
Yu. A. Azev, O. S. Ermakova, V. S. Berseneva, V. A. Bakulev, M. A. Ezhikova, and M. I. Kodess, Zh. Org. Khim.,
3, 90 (2017).
T. Wi and E. Alirol, Antibiotic-resistant Gonorrhea on the Rise, New Drugs Needed;
http://www.who.int/mediacentre/news/releases/2017/Antibiotic-resistant-gonorrhoea, 2017.
V. N. Charushin, G. A. Mokrushina, and A. V. Tkachev, J. Fluorine Chem., 107, 71 (2001).
1
1
1
4.
5.
6.
[
5
17.
8.
1
CLSI Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard,
th
9
Ed., Clinical and Laboratory Standards Institute, Wayne, PA, 2012, 86 pp.
1
2
9.
0.
ISO 20776-1:2006, Clinical Laboratory Testing and In vitro Diagnostic Test Systems, 2006, p. 17.
R. U. Khabriev, Handbook for Experimental (Preclinical) New Drug Trials [in Russian], Meditsina, Moscow,
2005, 832 pp.
519