Synthesis and antimycobacterial evaluation of new (2-oxo-2H-chromen-3-yl) substituted fluoroquinolones

Article abstract of DOI:10.1016/j.jfluchem.2018.01.007

An efficient method for incorporation of the cyanomethyl fragment into the benzene ring of bi- and tricyclic fluoroquinolones through the nucleophilic substitution of a fluorine atom with carbanions derived from CH-active cyanoacetates, followed by an acidic hydrolysis of the reaction intermediates was developed. The reaction of cyanomethyl derivatives of bi- and tricyclic fluoroquinolones with ortho-hydroxy substituted benzaldehydes proved to give the corresponding condensation products, which can be regarded as the key intermediates in the synthesis of previously unknown (2-oxo-2H-chromen-3-yl) substituted bi- and tricyclic fluoroquinolones. It has been shown that an increase in the reaction temperature and exposition time promotes the competitive process, leading to substitution of one more fluorine atom to give 1,4-dihydro[1]-benzoxepino[2,3-g]quinolones. New fluoroquinolones proved to exhibit anti-mycobacterial activity, thus indicating that a search of biologically active compounds in this family of heterocycles appears to be a reasonable approach.

Full text of DOI:10.1016/j.jfluchem.2018.01.007

Accepted Manuscript
Title: SYNTHESIS AND ANTIMYCOBACTERIAL
EVALUATION OF NEW (2-OXO-2H-CHROMEN-3-YL)
SUBSTITUTED FLUOROQUINOLONES
Authors: Valery N. Charushin, Nataliya N. Mochulskaya,
Fedor V. Antipin, Svetlana K. Kotovskaya, Emiliya V.
Nosova, Marina A. Ezhikova, Mikhail I. Kodess, Marionella
A. Kravchenko
PII:
S0022-1139(17)30402-5
DOI:
Reference:
https://doi.org/10.1016/j.jfluchem.2018.01.007
FLUOR 9106
To appear in:
FLUOR
Received date:
Revised date:
Accepted date:
20-9-2017
13-1-2018
15-1-2018
Please cite this article as: Valery N.Charushin, Nataliya N.Mochulskaya,
Fedor V.Antipin, Svetlana K.Kotovskaya, Emiliya V.Nosova, Marina
A.Ezhikova, Mikhail I.Kodess, Marionella A.Kravchenko, SYNTHESIS
AND ANTIMYCOBACTERIAL EVALUATION OF NEW (2-OXO-2H-
CHROMEN-3-YL) SUBSTITUTED FLUOROQUINOLONES, Journal of Fluorine
Chemistry https://doi.org/10.1016/j.jfluchem.2018.01.007
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1
SYNTHESIS AND ANTIMYCOBACTERIAL EVALUATION
OF NEW (2-OXO-2H-CHROMEN-3-YL) SUBSTITUTED
FLUOROQUINOLONES
Valery N. Charushin,a,b Nataliya N. Mochulskaya,*a,b Fedor V. Antipin,a
Svetlana K. Kotovskaya,a,b Emiliya V. Nosova,a,b Marina A. Ezhikova,b
Mikhail I. Kodess,a,b Marionella A. Kravchenkoc
a Chemical Technology Institute, Ural Federal University, 19 Mira st.,
Ekaterinburg 620002, Russia. E-mail: nataliya.mochulskaya@mail.ru
b Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences
22 S. Kovalevskaya st. /20 Akademicheskaya st., Ekaterinburg 620137, Russia
c Ural Research Institute for Phthisiopulmonology, 50 XXII Parts’ezda St.,
Ekaterinburg 620039, Russia
* corresponding author
Graphical Abstract
Highlights




The cyanomethyl fragment was incorporated into the benzene ring of fluoroquinolones
Cyanomethyl fluoroquinolones was converted to (2-oxo-2H-chromen-3-yl) fluoroquinolones
Substitution of another fluorine atom provides 1,4-dihydro[1]-benzoxepino[2,3-g]quinolones
Some of new fluoroquinolones exhibit antimicobacterial activity
Abstract
An efficient method for incorporation of the cyanomethyl fragment into the benzene ring of bi- and
tricyclic fluoroquinolones through the nucleophilic substitution of a fluorine atom with carbanions derived

2
from CH-active cyanoacetates, followed by an acidic hydrolysis of the reaction intermediates was
developed. The reaction of cyanomethyl derivatives of bi- and tricyclic fluoroquinolones with ortho-
hydroxy substituted benzaldehydes proved to give the corresponding condensation products, which can
be regarded as the key intermediates in the synthesis of previously unknown (2-oxo-2H-chromen-3-yl)
substituted bi- and tricyclic fluoroquinolones. It has been shown that an increase in the reaction
temperature and exposition time promotes the competitive process, leading to substitution of one more
fluorine atom to give 1,4-dihydro[1]-benzoxepino[2,3-g]quinolones. New fluoroquinolones proved to
exhibit anti-mycobacterial activity, thus indicating that a search of biologically active compounds in this
family of heterocycles appears to be a reasonable approach.
Keywords: fluoroquinolones; (2-oxo-2H-chromen-3-yl)-substituted fluoroquinolones; 1,4-dihydro[1]-
benzoxepino[2,3-g]quinolones; tuberculosis; multidrug-resistant tuberculosis; in vitro activity.
1. Introduction
Bacterial infections represent one of the main social concerns, and tuberculosis takes the
second place in the list of mortality from infectious diseases; new multi-drug resistant (MDR) and
extensive-drug resistant (XDR) forms of tuberculosis are spreading rapidly all over the world,
especially in India, China, Russia, and some other African and Asian countries, therefore urgent
measures are needed to be taken by the global health care community. In 2016 the World Health
Organization (WHO) reported that 10.4 million people were diagnosed with tuberculosis (TB)
infection, and a global mortality due to this disease reached up to 1.7 million people during the
same year [1].
Main complications with medical therapy of tuberculosis are associated with a limited
arsenal of anti-TB drugs, as well as with a wide spread of multi-drug resistant strains of
M. tuberculosis (MDR-TB), which are resistant to Isoniazid and Rifampicin, the main two front-
line anti-tuberculosis agents. In 2016, there were 600 000 new cases with resistance to rifampicin,
the most effective first-line drug, of which 490 000 had MDR-TB [1].
Fluoroquinolone drugs are known to show a high bactericidal activity towards tuberculosis
mycobacteria in both in vitro and in vivo experiments. Fluoroquinolones are of considerable
interest as remedies for the combined MDR-TB chemotherapy due to their optimal
pharmacokinetics, high intracellular concentrations, and good adult’s tolerance for a prolonged
use (Figure 1) [2]. Indeed, according to the WHO recommendations, Levofloxacin can be used for
the treatment of MDR-TB, and Moxifloxacin is known to be a suitable drug for application in
cases of the proved extensive drug resistance.
The mechanism of action of fluoroquinolones in mycobacterial cells differs significantly
from that of other known anti-tuberculosis drugs, thus providing a bactericidal effect and activity

3
of fluoroquinolones towards both sensitive and resistant strains [3-5]. Fluoroquinolones proved to
inhibit microbial cells during both dormant and growth periods; the mechanism of their action is
based on inhibition of the DNA gyrase.
Bacterial DNA gyrase represents the proteinaceous tetramer, weighing 400 kDa and
consisting of two subunits A (GyrA) and two subunits B (GyrB). Binding and splitting of DNA is
dependent on GyrA. GyrB consists of the amine-terminus domain, which possesses ATP-ase
activity, and the COOH-terminus domain, which interacts with GyrA and DNA. Most antibiotics,
which are capable of inhibiting bacterial DNA gyrase, proved to be selective towards this enzyme;
at the same time they are inactive to eukaryote topoisomerase type II. Fluoroquinolones are known
to inhibit bacterial DNA gyrase. They bind with GyrA subunit and stabilize the splitting complex,
thus inhibiting the gyrase function in general, that leads to cell death.
Inhibitors which bind GyrB subunit are less known. Several examples include coumarins,
novobiocin and coumermycin A, cyclothialidine, cynodine and clerocidine. In particular, it has
been shown that derivatives of the coumarin family bind GyrB rather strongly [6, 7]. Therefore,
the synthesis of new compounds bearing in one molecule combination of fragments, inhibiting
both DNA gyrase subunits, might be of interest for broadening the range of their action on
pathogens. Notably, that nowadays the creation of 4-quinolone hybrids is considered as one of the
most promising ways for the design of antibacterial agents [8, 9].
The overwhelming majority of publications, concerning structural modification of fluoro-
quinolones, deal with incorporation of nitrogen-containing fragments at C-7 (or C-10) through
nucleophilic substitution reactions, and 3-amino- or 3-aminomethylpyrrolidin-1-yl derivatives
have been used predominantly as N-centered nucleophiles in such cases. On the other hand, not
only 7-amino-6-fluoroquinolones obtained through the amino-defluorination process, but also
carbon-centered nucleophiles and methods to form CC7 (or CC10) bonds are gaining a growing
interest in the chemistry of fluoroquinolones, since a number of C-7 C-substituted quinolones,
such as Rosoxacin and Garenoxacin have been shown to be highly active antibacterial drugs [10,
11].
The aim of this communication is to describe the synthesis of a new series of coumarin-3-
yl (2-oxo-2H-chromen-3-yl) substituted fluoroquinolone derivatives, possessing antimicrobial
activity, including that relative to multi-drug resistant strains of M. tuberculosis, to expand
synthetic opportunities for the development of anti-tuberculosis agents.
2. Results and discussion
The synthesis of bi- and tricyclic fluoroquinolones 4a,b, bearing the cyanomethyl fragment at C-7
(or C-10), was realized through nucleophilic substitution of the fluorine atom at С-7 (or С-10) by action of

4
carbon-centered nucleophiles, derived from ethyl cyanoacetate or tert-butyl cyanoacetate, followed by
acidic hydrolysis of the intermediates 3a,b (Scheme 1).
It has been found, that nucleophilic substitution of a fluorine atom with carbon-centered
nucleophiles proceeds in both bicyclic and tricyclic fluoroquinolones 1a,b under the same reaction
conditions (heating at 70-80 С for 4 hours), and the process can be scaled. We have failed to convert
compounds 3a,b into 4a,b according to such methods, as refluxing in toluene in the presence of р-
toluenesulfonic acid [12] or in acetic anhydride [13, 14]. The target fluoroquinolones 4a,b, bearing the
cyanomethyl group at С-7 (or С-10), were synthesized by heating of compounds 3a,b in a mixture of
sulfuric and acetic acids [15]. The reaction is accompanied by protonation of COOR groups, elimination of
ethylene (butylene) and subsequent decarboxylation. It has also been shown that, when refluxing 3a,b in
a mixture of H2SO4/AcOH, the hydrolysis of the ethoxycarbonyl group at С-3 (or С-6) of fluoroquinolones
proved to occur.
Condensation of cyanomethyl derivatives of bi- and tricyclic fluoroquinolones 4a,b with ortho-
hydroxybenzaldehydes was studied. Fluoroquinolone 4a was shown to react smoothly with salicylic
aldehydes 5a-e in DMF solution in the presence of catalytic amounts of piperidine at room temperature.
The reaction was carried out for 1−12 hours according to the known procedure [16]. The synthesis was
accomplished without isolation of the intermediate condensation products 6a-e, which were subjected
to the hydrolysis in 3 % aqueous H2SO4 solution (heating for 5−9 hours at 110 С) to form (2-oxo-2H-
chromen-3-yl) substituted fluoroquinolones 7a-e in 46−77 % yields (Scheme 2).
1
The structure of fluoroquinolones 7a-e was confirmed by Н, 19F and 13C NMR spectroscopy,
1
1
including 2D H–13C HSQC and HMBC experiments, as well as the mass-spectrometry data. In Н NMR
spectra the signals, which are typical for 6-fluoro-7-substituted quinolones, have been observed, namely,
singlets of Н-2 and two doublets of Н-5 and Н-8 with coupling constants 3J(H-5,F-7) = 9.7–9.8 Hz and 4J (H-
8,F-7) = 5.8–5.9 Hz. The 1H NMR spectra of 7a-e reveal a characteristic downfield singlet of H-4 (at 8.39–
8.45 ppm), that indicates at the presence of the coumarin-3-yl fragment. In the low-field area of the 13
C
NMR spectra of compounds 7, along with C-4 and COOH signals from quinolone fragment, coumarinyl
carbonyl signal is appeared at C ~158 ppm. The C-4 carbon is split into doublet (4JC,F = 1.6 Hz) due to the
long-range spin-spin coupling with F-6. The diagnostic cross-peaks for pairs (H-4, C-7) and (H-8, C-3) were
observed in 2D 1H–13C HMBC spectra.
Elucidation of the condensation reaction between cyanomethyl fluoroquinolone 4a and salicylic
aldehydes 5 has shown that both heating of reactants and increase in reaction time promotes the
competitive intramolecular substitution of the fluorine atom at C-6, thus resulting in the formation of
polycyclic structures 8a-c,e,f (Scheme 2). Indeed, heating of 7-cyano-methylfluoroquinolone 4a with
aromatic hydroxyaldehydes 5 at 100 C for 3 hours afforded polycyclic compounds as the only products.

5
The structure of polycyclic derivatives 8 was established on the basis of the data of mass spectrometry,
as well as 1H and 13C NMR spectroscopy. Assignment of 1H and 13C signals for compounds 8 was achieved
by using 2D 1H–1H NOESY and 1H–13C HSQC, HMBC experiments. The key signals of nodal carbons of the
benzoxepinoquinoline core were assigned by using cross-peaks with protons of the neighboring rings in
the HMBC spectra, for example, C-5a with H-13, C-6a with H-10 and H-11, C-12a with H-5 and H-11, C-13a
with H-2 and H-5.
It has been shown that the reaction of cyanomethyl substituted tricyclic fluoroquinolone 4b with
salicylic aldehydes 5a-с proceeds in DMF solution in the presence of catalytic amounts of piperidine at
room temperature, and it takes 12 hours to be completed. Hydrolysis of the condensation products takes
place in aqueous 3−5 % H2SO4 for 7-15 hours at 110 С [14], thus allowing one to isolate (2-oxo-2H-
chromen-3-yl) substituted tricyclic fluoroquinolones 9a-с in 50−67 % yields (Scheme 3).
In compounds 9a-c there are two elements of chirality: the asymmetric C-3 carbon atom and the
chiral axis due to the hindered rotation around the C10–C3' bond. As a sequence, compounds 9a-с can exist
as mixtures of comparable amounts of stable atropisomers, thus giving rise a double set of signals in the
1H, 19F and 13C NMR spectra at room temperature. Heating of compounds 9a,c in a sealed NMR tube up
to 393 K resulted in coalescence of NMR signals of these isomers. The coalescence point for the methyl
group protons of 9a was observed approximately at 80 °C (Figure 2); the same story for F-6 signals, with
the difference it is observed at 100−110 °C. The rotational energy barrier ΔG# at coalescence was
calculated to be 18 kcal/mol.
of the methyl group protons of compound 9a.
(2-Oxo-2H-chromen-3-yl) substituted bi- and tricyclic fluoroquinolones 7, 9 and 1,4-
dihydro[1]benzoxepino[2,3-g]quinolines 8 were tested in vitro against M. tuberculosis H37Rv, avium,
terrae and MDR strains, and the data on the minimum inhibitory concentrations (MICs) of these
compounds are presented in Table 1. All new compounds were compared with the commercially available
drugs Isoniazid and Levofloxacin, tested under the same experimental conditions.
The results obtained show that tricyclic fluoroquinolones 9, bearing the coumarinyl fragment, as
well as 1,4-dihydro[1]benzoxepino[2,3-g]-quinolines 8 have a poor tuberculostatic activity, whereas their
bicyclic analogues, 6-fluoro-7-(2-oxo-2H-chromen-3-yl) substituted fluoroquinolones 7 can be considered
as promising anti-M. tuberculosis agents (Table 1). In particular, the compound 7e exhibits the highest
activity (MIC 0.7-1.5 g/mL) not only against M. tuberculosis H37Rv, but also against M. avium, M. terrae
and MDR strains. It should be noted that, unlike the known anti-tuberculosis drug Isoniazide,

6
fluoroquinolone 7e is active against MDR strains. Acute in vivo toxicity in mice LD50 for the most active
compound 7e was found to be 600 mg/kg.
3. Conclusion
In summary, an efficient synthetic approach to analogues of DNA gyrase inhibitors, Levofloxacin
and Pefloxacin, based on nucleophilic substitution of a fluorine atom in bicyclic and tricyclic
fluoroquinolones with carbon-centered nucleophiles, such as cyanoacetates, and subsequent cyclization
of intermediates with ortho-hydroxy substituted benzaldehydes, has been advanced. As a result, novel (2-
oxo-2H-chromen-3-yl) substituted fluoroquinolones and 1,4-dihydro[1]benzoxepino-[2,3-g]quinolones
have first been obtained.
In the series of novel fluoroquinolones obtained a number of compounds proved to exhibit an
antimycobacterial activity, which can possibly be improved to reach a better potency than the reference
drugs. The results obtained show that novel fluoroquinolones can be regarded as promising anti-M.
tuberculosis agents. Further research studies to get a more detailed information, concerning structure–
activity relationships (QSAR) for this series of compounds are in progress in our laboratory.
4. Experimental
4.1. General
Unless otherwise indicated, all common reagents and solvents were used from commercial
suppliers without further purification.
1H (500.1 MHz), 19F (470.5 MHz) and 13C (125.7 MHz) NMR spectra were recorded on the AVANCE-
1
500 spectrometer in DMSO-d6 solution. H chemical shifts are given in parts per million downfield from
tetramethylsilane, 19F chemical shifts have been measured from internal C6F6 and are reported from
external CFCl3. Carbon chemical shifts were referenced to the carbon resonances of the solvent (DMSO-
d6:  = 39.5 ppm). Peaks are labeled as singlet (s), broad singlet (br), doublet (d), triplet (t), quartet (q),
doublet of doublets (dd), doublet of quartets (dq) and multiplet (m). Full assignment of 1H and 13C signals
of all compounds was achieved by using 2D 1H–1H NOESY and 1H–13C HSQC, HMBC experiments.
Mass spectra were recorded on the SHIMADZU GCMS-QP2010 Ultra instrument with electron
ionization (EI) of the sample. Analytical HPLC of compounds 4a was performed on a Knauer Smartline-
1000 instrument using a Chiralcel OD-H column (250×4.6 mm, 5 µm), detection at 230 nm, 1 mL/min flow

7
rate. Melting points were measured on the instrument Boetius. Microanalyses (C, H, N) were performed
using the Perkin–Elmer 2400 elemental analyzer.
4.2. Procedure for the Synthesis of Starting Substrate 4a
Ethyl
7-(1-cyano-2-ethoxy-2-oxoethyl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylate (3a)
Ethyl cyanoacetate (4.80 g, 42.66 mmol) and calcined potassium carbonate (11.79 g) were
added to a solution of ethyl 6,7-difluoro-1-ethyl-4-oxo-1,4-dihydroquinolin-3-carboxylate (1)
(10.00 g, 35.55 mmol) in dimethyl sulfoxide (65 mL). The mixture was refluxed with a calcium
chloride tube at 80 С for 4 h. After cooling the mixture was poured into ice water ( 250 mL) and
then НCl solution (18 %) was added at stirring to рН = 2−3. The product obtained was extracted
with methylene chloride ( 600 mL). Water layer was separated, and organic layer was washed
twice with distilled water (200 mL) and once with saturated sodium chloride solution (2×200 mL),
then dried over anhydrous sodium sulfate. Methylene chloride solution was concentrated at
rotational vacuum evaporator, and diethyl ester (75 mL) was added to a residue. Colorless crystals
of compound 3a were filtered off, dried at air and recrystallized from ethanol. Yield 11.6 g (87
%), mp. 192−194 °С. 1Н NМR: δ = 1.22 (t, J = 7.1 Hz, 3H, H-4'), 1.30 (t, J = 7.1 Hz, 3H, H-3"),
1.41 (t, J = 7.1 Hz, 3H, NCH2CH3), 4.24 (q, J = 7.1 Hz, 2H, H-2"), 4.27 (q, J = 7.1 Hz, 2H, H-3'),
4.41 (dq, J = 14.1, 7.1 Hz, 1H, NCHB), 4.45 (dq, J = 14.1, 7.1 Hz, 1H, NCHA), 6.13 (s, 1H, H-1');
8.00 (d, J = 10.2 Hz, 1H, H-5), 8.09 (d, J = 5.9 Hz, 1H, H-8), 8.77 (s, 1H, H-2). 19F NМR: δ = -
119.93 (dd, J = 10.2, 5.9 Hz, F-6). 13C NMR: δ = 13.8 (C-4'), 14.25 and 14.28 (2∙CH3), 37.9 (C-
1'), 48.5 (NCH2), 59.9 (C-2"), 63.2 (C-3'), 109.9 (C-3), 111.9 (d, 2JCF = 22.3 Hz, C-5), 115.2 (CN),
121.4 (d, 3JCF = 2.7 Hz, C-8), 124.2 (d, 2JCF = 17.7 Hz, C-7), 130.5 (d, 3JCF = 6.6 Hz, C-4a), 135.3
(d, 4JCF = 1.5 Hz, C-8a), 149.5 (C-2), 156.9 (d, 1JCF = 248.5 Hz, C-6), 163.9 (C-2'), 164.4 (C-1"),
171.5 (d, 4JCF = 2.1 Hz, C-4). MS (m/z, Irel %): 375 [M + 1]+ (1), 374 [M]+ (6), 329 (7), 303 (18),
302 (100), 230 (7), 229 (7), 228 (7), 145 (5). Anal. Calc. for С19H19FN2O5, C 60.96, Н 5.12, N
7.48. Found: С 60.92, Н 5.12, N 7.43.
7-(Cyanomethyl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4a)
A mixture sulfuric acid/acetic acid/distilled water (1:20:20) was added to compound 3a (10.00 g,

8
26.71 mmol), and the suspension was heated at 125 °C for 6 h. After cooling the precipitate was filtered
off, washed with distilled water and dried at air. The colorless crystals of the product 4a were recrystallized
from the mixture ethanol/N,N-dimethylformamide. Yield 6.7 g, (91 %), mp. 235−237 °С. 1H NMR: δ = 1.46
(t, J = 7.1 Hz, 3H, CH3), 4.34 (s, 2H, CH2CN), 4.60 (q, J = 7.1 Hz, 2H, NCH2), 8.10 (d, J = 9.5 Hz, 1H, H-5), 8.22
(d, J = 6.0 Hz, 1H, H-8), 9.10 (s, 1H, H-2), 14.89 (s, 1H, COOH). 19F NMR: δ = -118.06 (dd, J = 9.5, 6.0 Hz, F-
2
6). 13C NMR: δ = 14.5 (CH3), 17.8 (CH2CN), 49.4 (NCH2), 107.7 (C-3), 110.9 (d, JCF = 22.8 Hz, C-5), 117.3
(CN), 121.2 (d, 3JCF = 3.6 Hz, C-8), 126.7 (d, 2JCF = 18.8 Hz, C-7), 126.9 (d, 3JCF = 7.7 Hz, C-4a), 135.8 (d, 4JCF =
1.4 Hz, C-8a), 149.4 (C-2), 157.7 (d, 1JCF = 249.9 Hz, C-6), 165.7 (COO), 176.6 (d, 4JCF = 2.6 Hz, C-4). MS (m/z,
Irel %): 275 [M + 1]+ (1), 274 [M]+ (7), 231 (14), 230 (100), 215 (53), 147 (10). Anal. Calc. for С14H11FN2O3, С
61.31, Н 4.04, N 10.22. Found: С 61.24; Н 3.86; N 10.15.
4.3. Procedure for the Synthesis of Starting Substrate 4b
Ethyl
(3S)-10-(2-tert-butoxy-1-cyano-2-oxoethyl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate (3b)
tert-Butylcyanoacetate (0.55 g, 3.88 mmol) and calcined potassium carbonate (1.46 g) were
added to
a
solution of ethyl (3S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-
[1,4]oxazino[2,3,4-ij]-quinoline-6-carboxylate (1b) (1.00 g, 3.23 mmol) in dimethyl sulfoxide (13
mL). The mixture was refluxed with a calcium chloride tube at 70 С for 4 h. After cooling the
mixture was poured into the mixture ethyl acetate/ice (150:100 mL) and then НCl solution (18 %)
was added at stirring to рН= 2. Water layer was separated, and organic layer was washed twice
with distilled water (2 × 100 mL) and once with saturated solution of sodium chloride (100 mL),
than dried over anhydrous sodium sulfate. Ethyl acetate solution was concentrated at rotational
vacuum evaporator, and diethyl ester (30 mL) was added to a residue. Colorless crystals of
compound 3b were filtered off, dried at air (yield 1.3 g, 93 %) and recrystallized from aqueous
ethanol. The sample was isolated as a mixture of two diastereomers (ratio ≈ 1:1), mp. 164−165 °С.
HPLC (Chiralcel OD-H, hexane:iPrOH:MeOH = 2:0.8:0.2, 1 mL/min): 1 = 9.7 min (47.5 %); 2
= 11.7 min (52.2 %). 1H NMR: δ = 1.31 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.41−1.44 (m, 12H, tBu
and CH3-C3), 4.20−4.30 (m, 2H, OCH2CH3), 4.47 and 4.54 (both dd, J = 11.4, 2.3 Hz, 1H, H-2B),
4.65 and 4.68 (both dd, J = 11.4, 1.6 Hz, 1H, H-2A), 4.83 (m, 1H, H-3), 6.03 (s, 1H, H-1'), 7.51
and 7.52 (both d, J = 9.9 Hz, 1H, H-8), 8.71 and 8.72 (both s, 1H, H-5). 19F NMR: δ = -118.34 and
-118.21 (both d, J = 9.9 Hz, F-9). 13C NMR: δ = 14.20 (OCH2CH3), 17.4 and 17.5 (CH3-C3), 27.18
3
and 27.19 ((CH3)3C), 33.03 and 33.08 (both d, JCF = 2.9 Hz, C-1'), 53.6 and 53.8 (C-3), 59.8
(OCH2CH3), 68.9 and 69.1 (C-2), 83.87 and 83.91 ((CH3)3C), 102.78 and 102.85 (both d, 2JCF
=

9
23.5 Hz, C-8), 109.9 (C-6), 110.7 and 110.9 (both d, 2JCF = 17.2 Hz, C-10), 115.0 and 115.1 (CN),
4
3
123.16 and 123.18 (both d, JCF = 1.5 Hz, C-10b), 129.4 (d, JCF = 8.2 Hz, C-7a), 145.04 and
145.07 (both d, 3JCF = 6.3 Hz, C-10a), 146.2 and 146.3 (C-5), 156.5 and 156.6 (both d, 1JCF = 247.0
Hz, C-9), 162.8 and 162.9 (C-2'), 164.18 and 164.19 (COO), 171.11 and 171.13 (C-7). MS (m/z,
Irel %): 431 [M + 1]+ (2), 430 [M]+ (6), 358 (13), 302 (29), 284 (112), 258 (41), 243 (7), 216 (9),
57 (100), 41 (46), 39 (13). Anal. Calc. for С22H23FN2O6, С 61.39, Н 5.39, N 6.51. Found: С 61.27,
Н 5.25, N 6.51.
(3S)-10-(Cyanomethyl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylic acid (4b)
The mixture sulfuric acid/acetic acid/distilled water (1:20:20) was added to compound 3b
(0.31 g, 0.72 mmol), and the mixture was heated at 110 °C for 5 h. After cooling the precipitate
was filtered off, washed with distilled water and dried at air. The colorless crystals of product 4b
1
were recrystallized from aqueous ethanol. Yield 0.12 g (55 %), mp. 237−238 °С. H NMR: δ =
1.47 (d, J = 6.8 Hz, 3H, CH3), 4.14 (AB system, JAB = 17.7 Hz, 2H, H-1'), 4.51 (dd, J = 11.5, 2.4
Hz, 1H, H-2A); 4.71 (dd, J = 11.5, 1.9 Hz, 1H, H-2B), 5.01 (qt, J = 6.8, 2.3 Hz, 1H, H-3), 7.72 (d,
J = 9.6 Hz, 1H, H-8), 9.09 (s, 1H, H-5), 14.89 (s, 1H, COOH). 19F NMR: δ = -115.39 (d, J = 9.6
Hz, F-9). 13C NMR: δ = 11.4 (d, 3JCF = 3.7 Hz, C-1'), 17.7 (CH3), 54.8 (C-3), 68.9 (C-2), 102.3 (d,
2
4
2JCF = 24.0 Hz, C-8), 107.7 (C-6), 111.5 (d, JCF = 21.6 Hz, C-10), 116.8 (CN), 123.9 (d, JCF
=
1.5 Hz, C-10b), 126.2 (d, 3JCF = 9.5 Hz, C-7a), 145.6 (d, 3JCF = 7.5 Hz, C-10a), 146.5 (C-5), 157.7
(d, 1JCF = 248.1 Hz, C-9), 165.6 (COO), 176.5 (d, 4JCF = 3.1 Hz, C-7). MS (m/z, Irel %): 302 [M]+
(15), 259 (18), 258 [M-CO2]+ (100), 243 (19), 216 (63), 53 (15), 41 (17). Anal. Calc. for
С15H11FN2O4, С 59.60, Н 3.67, N 9.27. Found: С 59.48, Н 3.62, N 9.23.
4.4. General synthetic procedure for 7-(2-oxo-2H-chromen-3-yl)-substituted bicyclic
fluoroquinolones (7a-e)
The corresponding salicylaldehyde 5 (1.82 mmol) was added to a suspension of 7-(cyanomethyl)-1-ethyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4a) (0.500 g, 1.82 mmol) in DMF (5 mL), and then
5 drops of piperidine was added. The mixture was heated for 1−2 min, and after that it was stirred at room
temperature for 12 h. The precipitate was filtered off, added to 3 % H2SO4 solution (20 mL), and the mixture
was heated at 110 С for 6 h. After cooling the precipitate was filtered off and washed with distilled water,
then with hexane or heptane. Compounds 7a,b,d were recrystallized from the mixture ethanol/DMF.

10
1-Ethyl-6-fluoro-4-oxo-7-(2-oxo-2H-chromen-3-yl)-1,4-dihydroquinoline-3-carboxylic acid (7a)
Compound was obtained from salicylaldehyde (5a). Yield 0.48 g (69 %), mp. 323−324 C. 1H NMR:
δ = 1.46 (t, J = 7.2 Hz, 3H, CH3), 4.64 (q, J = 7.2 Hz, 2H, NCH2), 7.46 (td, J = 7.5, 1.0 Hz, 1H, H-6'), 7.54 (br.d,
J = 8.3 Hz, 1H, H-8'), 7.74 (ddd, J = 8.3, 7.2, 1.6 Hz, 1H, H-7'), 7.86 (dd, J = 7.7, 1.6, 1H, H-5'), 8.16 (d, J = 9.7
Hz, 1H, H-5), 8.36 (d, J = 5.8, 1H, H-8), 8.45 (s, 1H, H-4'), 9.13 (s, 1H, H-2), 14.97 (s, 1H, COOH). 19F NMR: δ
= -115.06 (dd, J = 9.7, 5.8 Hz, F-6). 13C NMR: δ = 14.6 (CH3), 49.4 (NCH2), 107.7 (C-3), 110.7 (d, 2JCF = 24.0
3
3
Hz, C-5), 116.2 (C-8'), 118.6 (C-4'a), 121.3 (C-3'), 122.2 (d, JCF = 3.3 Hz, C-8), 125.0 (C-6'), 127.1 (d, JCF
=
2
8.1, C-4a), 129.1 (C-5'), 129.8 (d, JCF = 18.2 Hz, C-7), 132.9 (C-7'), 135.6 (C-8a), 144.7 (C-4'), 149.4 (C-2),
153.4 (C-8'a), 157.2 (d, 1JCF = 250.1 Hz, C-6), 158.6 (C-2'), 165.7 (COO), 176.6 (d, 4JCF = 2.3 Hz, C-4). MS (m/z,
Irel %): 379 [M]+ (8), 336 (21), 335 [M-CO2]+ (100), 321 (8), 320 (40), 292 (6), 251 (6), 222 (6), 167 (6). Anal.
Calc. for С21H14FN1O5, С 66.49, Н 3.72, N 3.69. Found: С 66.05; Н 3.71; N 3.65.
7-(6-Bromo-2-oxo-2H-chromen-3-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7b)
Compound was obtained from 5-bromosalicylaldehyde (5b). Yield 0.59 g (77 %), mp. 309−310 C.
1H NMR: δ = 1.46 (t, J = 7.1 Hz, 3H, CH3), 4.63 (q, J = 7.1 Hz, 2H, NCH2), 7.52 (d, J = 8.9 Hz, 1H, H-8'), 7.89
(dd, J = 8.9, 2.4 Hz, 1H, H-7'), 8.11 (d, J = 2.4 Hz, 1H, H-5'), 8.17 (d, J = 9.7 Hz, 1H, H-5), 8.34 (d, J = 5.8 Hz,
1H, H-8), 8.39 (s, 1H, H-4'), 9.13 (s, 1H, H-2), 14.95 (s, 1H, COOH). 19F NMR: δ = -115.21 (dd, J = 9.7, 5.8 Hz,
F-6). 13C NMR: δ = 14.6 (CH3), 49.4 (NCH2), 107.7 (C-3), 110.8 (d, 2JCF = 23.8 Hz, C-5), 116.4 (C-4'a), 118.5
3
2
(C-8'), 120.4 (C-6'), 122.3 (d, JCF = 2.7 Hz, C-8), 122.4 (C-3'), 127.3 (d, 3JCF = 7.8 Hz, C-4a), 129.3 (d, JCF
=
18.1 Hz, C-7), 131.0 (C-5'), 135.2 (C-7'), 135.5 (d, 4JCF = 1.3 Hz, C-8a), 143.4 (d, 4JCF = 1.6 Hz, C-4'), 149.5 (C-
2), 152.5 (C-8'a), 157.1 (d, 1JCF = 250.7 Hz, C-6), 158.1 (C-2'), 165.7 (COO), 176.6 (d, 4JCF = 2.4 Hz, C-4). MS
(m/z, Irel %): 460 [M+2]+ (2),458 [M]+ (2), 416 (22), 415 (100), 414 [M-CO2]+ (24), 413 (100), 400 (33), 398
(32), 369 (14), 222 (11), 207 (12), 206 (10), 194 (11). Anal. Calc. for С21H13BrFNO5, С 55.04, Н 2.86, N 3.06.
Found: С 55.12, Н 2.67, N 3.18.
7-(6,8-Di-tert-butyl-2-oxo-2H-chromen-3-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic
acid (7c)
3,5-di-tert-Butylsalicylaldehyde (5c) (0.436 g, 1.86 mmol) was added to a suspension of 7-
(cyanomethyl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-carboxylic acid (4a) (0.510 g, 1.86 mmol) in

11
DMF (5 mL), then 5 drops of piperidine were added. The mixture was stirred at room temperature for 12
h. The precipitate was filtered off and washed with small amount of ethanol, then with hexane. To solid
obtained 3 % H2SO4 solution (20 mL) was added, and the mixture was heated at 110 С for 6 h. After
cooling the precipitate was filtered off and washed with distilled water, then with hexane or heptane and
recrystallized from acetonitrile. Yield 0.41 g (46 %), mp. 304−305 °С. 1H NMR: δ = 1.36 (s, 9H, tBu-C6'), 1.47
(t, J = 7.1 Hz, 3H, CH3), 1.51 (s, 9H, tBu-C8'), 4.65 (q, J = 7.1 Hz, 2H, NCH2), 7.65 (d, J = 2.3 Hz, 1H, H-7'), 7.72
(d, J = 2.3 Hz, 1H, H-5'), 8.15 (d, J = 9.7 Hz, 1H, H-5), 8.36 (d, J = 5.9 Hz, 1H, H-8), 8.43 (s, 1H, H-4'), 9.12 (s,
1H, H-2), 14.97 (s, 1H, COOH). 19F NMR: δ = -115.13 (dd, J = 9.7, 5.9, F-6). 13C NMR: δ = 14.6 (CH3), 29.5
((CH3)3C-C6'), 31.0 ((CH3)3C-C8'), 34.4 ((CH3)3C-C6'), 34.6 ((CH3)3C-C8'), 49.4 (NCH2), 107.6 (C-3), 110.7 (d, 2JCF
= 24.0 Hz, C-5), 118.6 (C-4’a), 120.1 (d, 3JCF = 0.8 Hz, C-3'), 122.2 (d, 3JCF = 3.5, C-8), 123.9 (C-5'), 127.1 (d,
3JCF = 7.8, C-4a), 127.3 (C-7'), 129.8 (d, 2JCF = 18.1, C-7), 135.6 (d, 4JCF = 1.5, C-8a), 136.1 (C-6'), 146.0 (d, 4JCF
= 1.5, C-4'), 146.6 (C-8'), 149.4 (C-2), 150.3 (C-8'a), 157.2 (d, 1JCF = 250.4, C-6), 158.3 (C-2'), 165.7 (COO),
176.6 (d, 4JCF = 2.4, C-4). MS (m/z, Irel %): 491 [M]+ (5), 448 (31), 447 [M-CO2]+ (100), 432 (8), 216 (13), 202
(9), 57 (25), 41 (12). Anal. Calc. for С29H30FNO5, С 70.86, Н 6.15, N 2.85. Found: С 70.52, Н 6.25, N 3.02.
7-(6-Chloro-2-oxo-2H-chromen-3-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7d)
Compound was obtained from 5-chlorosalicylaldehyde (5d). Yield 0.47 g (62 %), mp. 325−326 C.
1H NMR: δ = 1.46 (t, 3H, J = 7.1 Hz, CH3), 4.63 (q, J = 7.1 Hz, 2H, NCH2), 7.59 (d, J = 8.9 Hz, 1H, H-8'), 7.77
(dd, J = 8.9, 2.5 Hz, 1H, H-7'), 7.98 (d, J = 2.5 Hz, 1H, H-5'); 8.17 (d, J = 9.8 Hz, 1H, H-5), 8.34 (d, J = 5.8 Hz,
1H, H-8), 8.39 (s, 1H, H-4'), 9.13 (s, 1H, H-2), 14.95 (s, 1H, COOH). 19F NMR: δ = -115.24 (dd, J = 9.7, 5.8 Hz,
F-6). 13C NMR: δ = 14.6 (CH3), 49.4 (NCH2), 107.7 (C-3), 110.8 (d, 2JCF = 24.0 Hz, C-5), 118.3 (C-8'), 120.0 (C-
4'a), 122.3 (d, 3JCF = 3.1 Hz, C-8), 122.4 (C-3'), 127.3 (d, 3JCF = 7.8 Hz, C-4a), 128.0 (C-5'), 128.6 (C-6'), 129.3
(d, 2JCF = 18.1 Hz, C-7), 132.4 (C-7'), 135.5 (d, 4JCF = 1.5 Hz, C-8a), 143.4 (d, 4JCF = 1.5 Hz, C-4'), 149.5 (C-2),
152.1 (C-8'a), 157.1 (d, 1JCF = 250.5 Hz, C-6), 158.2 (C-2'), 165.7 (COO), 176.6 (d, 4JCF = 2.3 Hz, C-4). MS (m/z,
Irel %): 415 [M+2]+ (2), 413 [M]+ (6), 372 (7), 371 (38), 370 (24), 369 [M-CO2]+ (100), 354 (41), 348 (8), 285
(7), 194 (7). Anal. Calc. for С21H13ClFNO5, С 60.96, Н 3.17, N 3.39. Found: С 60.68, Н 3.02, N 3.59.
7-(8-Ethoxy-2-oxo-2H-chromen-3-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7e)
3-Ethoxysalicylaldehyde (5e) (0.303 g, 1.82 mmol) was added to a suspension of 7-(cyanomethyl)-
1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4a) (0.500 g, 1.82 mmol) in DMF (5 mL),

12
then 5 drops of piperidine were added. The mixture was stirred at room temperature for 1 h. Then ethanol
(10 mL) was added to a mixture, the precipitate was filtered off and washed with small amount of ethanol,
then with heptane. To solid obtained 5 % H2SO4 solution (20 mL) was added, and the mixture was heated
at 110 С for 6 h. After cooling the precipitate was filtered off and washed with distilled water, then with
hexane or heptane, and recrystallized from the mixture DMF/water. Yield 0.375 g (49 %), mp. 280−281 °С.
1H NMR: δ = 1.43 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.47 (t, J = 7.1 Hz, 3H, NCH2CH3), 4.23 (q, J = 7.0 Hz, 2H,
OCH2), 4.64 (q, J = 7.1 Hz, 2H, NCH2), 7.34−7.41 (m, 3H, H-5', 6', 7'), 8.14 (d, J = 9.7 Hz, 1H, H-5), 8.35 (d, J
= 5.9 Hz, 1H, H-8), 8.42 (s, 1H, H-4'), 9.12 (с, 1H, H-2), 14.97 (с, 1H, COOH). 19F NMR: δ = -114.99 (dd, J =
9.7, 5.9, F-6). 13C NMR: δ = 14.59 and 14.65 (2 CH3), 49.5 (NCH2), 64.5 (OCH2), 107.7 (C-3), 110.8 (d, 2JCF =
24.0 Гц, C-5), 116.0 (C-7'), 119.3 (C-4'a), 121.5 (C-3'), 122.2 (d, 3JCF = 3.1, C-8), 125.0 (C-6'), 127.2 (d, 3JCF =
7.8, C-4a), 129.8 (d, 2JCF = 18.1, C-7), 135.6 (d, 4JCF = 1.2, C-8a), 142.9 (C-8'a), 145.0 (d, 4JCF = 1.5, C-4'), 145.7
(C-8'), 149.5 (C-2), 157.2 (d, 1JCF = 250.5, C-6), 158.4 (C-2'), 165.8 (COO), 176.7 (d, 4JCF = 2.7, C-4). MS (m/z,
Irel %): 424 [M+1]+ (2), 423 [M]+ (7), 380 (26), 379 (100), 336 (18), 176 (11). Anal. Calc. for С23H18FNO6, С
65.25, Н 4.29, N 3.31. Found: С 64.89, Н 4.28, N 3.36.
4.5. General synthetic procedure for 12-cyano-8-R-1-ethyl-4-oxo-1,4-dihydro[1]benzoxepino-
[2,3-g]quinoline-3-carboxylic acid (8a-c,e,f)
The corresponding salicylaldehyde 5 (2.19 mmol) was added to a suspension of 7-(cyanomethyl)-1-ethyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4a) (0.400 g, 1.46 mmol) in DMF (4 mL), then
piperidine (0.152 mL, 1.46 mmol) was added, and the mixture was heated at 100 С for 3 h. After cooling
ethanol (10 mL) was added to a reaction mass, the precipitate was filtered off and washed with small
amount of ethanol, then with heptane. Compounds 8a,b,e,f were recrystallized from the mixture
ethanol/DMF, whereas compound 8c was recrystallized from ethanol.
12-Cyano-1-ethyl-4-oxo-1,4-dihydro[1]benzoxepino[2,3-g]quinoline-3-carboxylic acid (8a)
Compound was obtained from salicylaldehyde (5a). Yield 0.24 g (45 %), mp. 318−319 С. 1H NMR:
δ = 1.46 (t, J = 7.2 Hz, 3H, CH3), 4.64 (q, J = 7.2 Hz, 2H, NCH2), 7.37 (td, J = 7.5, 1.4 Hz, 1H, H-9), 7.58−7.60
(m, 2H, H-7, H-10); 7.64 (ddd, J = 8.2, 7.2, 1.7 Hz, 1H, H-8), 7.96 (s, 1H, H-13), 8.21 (s, 1H, H-11), 8.28 (s,
1H, H-5), 9.12 (s, 1H, H-2), 14.89 (s, 1H, COOH). 13C NMR: δ = 14.2 (CH3), 49.3 (NCH2), 107.8 (C-3), 111.3
(C-12), 117.8 (C-13), 117.9 (CN), 118.0 (C-5), 121.8 (C-7), 126.2 (C-9), 127.2 (C-10a), 127.9 (C-4a), 131.5 (C-
10), 131.9 (C-12a), 134.2 (C-8), 136.4 (C-13a), 146.4 (C-11), 149.9 (C-2), 154.5 (C-5a), 157.4 (C-6a), 165.6

13
(COO), 176.7 (C-4). MS (m/z, Irel %): 358 [M]+ (16), 315 (22), 314 [M-CO2]+ (100), 299 (25), 286 (8), 271 (8),
258 (8), 229 (8), 201 (8). Anal. Calc. for С21H14N2O4, С 70.39, Н 3.94, N 7.82. Found: С 70.39, Н 3.91, N 7.87.
9-Bromo-12-cyano-1-ethyl-4-oxo-1,4-dihydro[1]-benzoxepino[2,3-g]quinoline-3-carboxylic acid (8b)
Compound was obtained from 5-bromosalicylaldehyde (5b). Yield 0.53 g (83 %), mp. 342−343 С.
1H NMR: δ = 1.46 (t, J = 7.2 Hz, 3H, CH3), 4.65 (q, J = 7.2 Hz, 2H, NCH2), 7.85 (d, J = 8.5 Hz, 1H, H-7), 7.81
(dd, J = 8.5, 2.5 Hz, 1H, H-8), 7.83 (d, J = 2.5 Hz, 1H, H-10), 7.97 (s, 1H, H-13), 8.14 (s, 1H, H-11), 8.31 (s, 1H,
H-5), 9.12 (s, 1H, H-2), 14.85 (s, 1H, COOH). 13C NMR: δ = 14.2 (CH3), 49.3 (NCH2), 107.9 (C-3), 112.6 (C-
12), 117.6 (CN), 118.06, 118.11 and 118.20 (C-5, C-10a and C-13), 124.0 (C-7), 128.1 (C-4a), 129.2 (C-9),
131.4 (C-12a), 133.5 (C-10), 136.4 (C-8), 136.5 (C-13a), 144.7 (C-11), 150.0 (C-2), 154.2 (C-5a), 156.5 (C-
6a), 165.5 (COO), 176.7 (C-4). MS (m/z, Irel %): 439 [M+2]+ (3), 438 [M+1]+ (14), 437 [M]+ (4), 436 [M-1]+
(15), 395 (23), 394 (45), 393 (25), 392 (100), 379 (26), 377 (22), 229 (13), 201 (21). Anal. Calc. for
С21H13BrN2O4, С 57.69, Н 3.00, N 6.41. Found: С 57.74, Н 3.03, N 6.39.
7,9-Di-tert-butyl-12-cyano-1-ethyl-4-oxo-1,4-dihydro[1]-benzoxepino[2,3-g]quinoline-3-carboxylic acid
(8c)
Compound was obtained from 3,5-di-tert-buthylsalicylaldehyde (5c). Yield 0.56 g (81 %); mp.
311−312 С. 1H NMR: δ = 1.27 (s, 9H, (tBu-C9), 1.46 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.54 (s, 9H, (tBu-C7), 4.63
(q, J = 7.2 Hz, 2H, NCH2), 7.46 (d, J = 2.5 Hz, 1H, H-10), 7.56 (d, J = 2.5 Hz, 1H, H-8), 7.89 (s, 1H, H-13), 8.04
(s, 1H, H-11), 8.23 (s, 1H, H-5), 9.10 (s, 1H, H-2), 14.77 (s, 1H, COOH). 13C NMR: δ = 14.2 (NCH2CH3), 30.8
((CH3)3C-C9), 31.1 ((CH3)3C-C7), 34.3 ((CH3)3C-C9), 35.1 ((CH3)3C-C7), 49.2 (NCH2), 107.9 (C-3), 110.8 (C-12),
118.0 (C-5, C-13 and CN), 127.1 (C-4a), 127.8 (C-10a), 128.0 (C-10), 128.9 (C-8), 132.6 (C-12a), 136.2 (C-
13a), 141.5 (C-7), 147.6 (C-9), 148.6 (C-11), 149.8 (C-2), 154.7 (C-6a), 155.1 (C-5a), 165.5 (COO), 176.5 (C-
4). MS (m/z, Irel %): 470 [M]+ (25), 427 (31), 426 (100), 411 (26), 206 (20), 57 (21), 41 (16). Anal. Calc. for
С29H30N2O4, С 74.02, Н 6.43, N 5.95. Found: С 74.04, Н 6.56, N 5.91.
12-Cyano-8-ethoxy-1-ethyl-4-oxo-1,4-dihydro[1]-benzoxepino[2,3-g]quinoline-3-carboxylic acid (8e)
Compound was obtained from 3-ethoxysalicylaldehide (5e). Yield 0.30 g (51 %), mp. 311−312 С.
1H NMR: δ = 1.46 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.48 (t, J = 6.9 Hz, 3H, OCH2CH3), 4.18 (q, J = 6.9 Hz, 2H,

14
OCH2), 4.65 (q, J = 7.2 Hz, 2H, NCH2), 7.12 (dd, J = 7.9, 1.2 Hz, 1H, H-10), 7.27 (t, J = 7.9 Hz, 1H, H-9), 7.36
(dd, J = 8.3, 1.2 Hz, 1H, H-8), 7.98 (s, 1H, H-13), 8.18 (s, 1H, H-5), 8.22 (s, 1H, H-11), 9.12 (s, 1H, H-2), 14.83
(s, 1H, COOH). 13C NMR: δ = 14.2 (NCH2CH3), 14.7 (OCH2CH3), 49.2 (NCH2), 64.5 (OCH2), 107.8 (C-3), 111.4
(C-12), 117.7 (C-5, C-8, C-13), 117.8 (CN), 122.0 (C-10), 126.4 (C-9), 127.8 (C-4a), 128.4 (C-10a), 132.1 (C-
12a), 136.3 (C-13a), 145.1 (C-6a), 146.2 (C-11), 149.9 (C-2), 150.7 (C-7), 154.4 (C-5a), 165.6 (COO), 176.7
(C-4). MS (m/z, Irel %): 402 [M]+ (15), 359 (26), 358 [M-CO2]+ (100), 301 (9), 273 (19). Anal. Calc. for
С23H18N2O5, С 68.65, Н 4.51, N 6.96. Found: С 68.52, Н 4.54, N 6.86.
12-Cyano-8-diethylamino-1-ethyl-4-oxo-1,4-dihydro[1]-benzoxepino[2,3-g]quinoline-3-carboxylic acid (8f)
Compound was obtained from 4-diethylaminosalicylaldehyde (5f). Yield: 0.50 g (81 %), mp.
250−251 С. 1H NMR: δ = 1.12 (t, J = 7.0 Hz, 6H, N(CH2CH3)2), 1.46 (t, J = 7.2 Hz, 3H, N1CH2CH3), 3.44 (q, 4H,
J = 7.0 Hz, N(CH2CH3)2), 4.61 (q, J = 7.2 Hz, 2H, N1CH2), 6.57 (dd, J = 9.0, 2.5 Hz, 1H, H-9), 6.83 (d, J = 2.5 Hz,
1H, H-7), 7.28 (d, J = 9.0 Hz, 1H, H-10), 7.77 (s, 1H, H-13), 7.82 (s, 1H, H-11), 8.24 (s, 1H, H-5), 9.08 (s, 1H,
H-2), 15.02 (s, 1H, COOH). 13C NMR: δ = 12.4 (N(CH2CH3)2), 14.1 (N1CH2CH3), 44.1 (N(CH2CH3)2), 49.2
(N1CH2), 102.8 (C-12), 103.6 (C-7), 107.5 (C-3), 108.4 (C-9), 114.1 (C-10a), 116.1 (C-13), 118.4 (C-5), 119.3
(CN), 126.9 (C-4a), 133.6 (C-10), 133.8 (C-12a), 136.4 (C-13a), 146.4 (C-11), 149.6 (C-2), 152.9 (C-8), 153.2
(C-5a), 159.4 (C-6a), 165.8 (COO), 176.8 (C-4). MS (m/z, Irel %): 430 [M+1]+ (19), 429 [M]+ (63), 414 (10),
385 (22), 371 (27), 370 (100), 342 (20), 314 (13). Anal. Calc. for С25H23N3O4, С 69.92, Н 5.40, N 9.78. Found:
С 69.57, Н 5.45, N 9.69.
4.6. General synthetic procedure for 7-(2-oxo-2H-chromen-3-yl)-substituted tricyclic
fluoroquinolones (9a-c)
The corresponding salicylaldehyde 5 (1.65 mmol) was added to a suspension of (-)-(S)-10-
cyanomethyl-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-d,e]1,4-benzoxazin-6-
carboxylic acid (4b) (0.500 g, 1.65 mmol) in DMF (5 mL), then 5 drops of piperidine were added.
The mixture was stirred at room temperature for 12 h. The precipitate was filtered off, added to
3 % H2SO4 solution (15 mL), and the mixture was heated at 110 С for 7−15 h. After cooling the
precipitate was filtered off and washed with distilled water, then with hexane or heptane.
Compounds 9a-c were recrystallized from the mixture ethanol/DMF.
(3S)-9-Fluoro-3-methyl-7-oxo-10-(2-oxo-2H-chromen-3-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-
6-carboxy-lic acid (9a)

15
Compound was obtained from salicylaldehyde (5a), reaction mixture was heated at 110 С in 3 %
H2SO4 solution for 7 h. Yield: 0.38 g (56 %) (mixture of atropoisomers, ratio ≈ 1:1); mp. 322-323 C with
decomposition. 1H NMR (tamb): δ = 1.46 and 1.52 (both d, J = 6.7 Hz, 3H, CH3), 4.42 and 4.50 (both dd, J =
11.6, 2.3 Hz, 1H, H-2B), 4.59−4.62 (m, 1H, H-2A), 5.01 (m, 1H, H-3), 7.45 (br.t, J = 7.5 Hz, 1H, H-6'), 7.53 (br
d, J = 8.4 Hz, 1H, H-8'), 7.72-7.76 (m, 2H, H-7', H-8), 7.84 (dd, J = 7.7, 1.9 Hz, 1H, H-5'), 8.40 and 8.42 (both
s, 1H, H-4'), 9.11 and 9.12 (both s, 1H, H-5), 14.95 and 14.96 (both s, 1H, COOH). 19F NMR (tamb): δ = -
113.44 and -112.94 (both d, J = 9.4 Hz, F-9). 13C NMR (tamb): δ = 17.7 and 18.0 (CH3), 54.9 and 55.0 (C-3),
68.6 and 68.8 (C-2), 102.1 and 102.3 (both d, 2JCF = 24.6 Hz, C-8), 107.6 and 107.7 (C-6), 115.5 and 115.8
2
(both d, JCF = 22.1 Hz, C-10), 116.4 (C-8'), 117.1 and 117.1 (C-3'), 118.37 and 118.40 (C-4'a), 123.9 and
4
3
124.1 (both d, JCF = 1.2 Hz, C-10b), 125.04 and 125.05 (C-6'), 126.6 (d, JCF = 9.7 Hz, C-7a), 128.97 and
128.99 (C-5'), 133.0 (C-7'), 145.7 and 146.0 (C-4'), 145.9 and 146.0 (both d, 3JCF = 7.1 Hz, C-10a), 146.5 and
146.7 (C-5), 153.40 and 153.41 (C-8'a), 157.4 (d, 1JCF = 247.0 Hz, C-9), 158.09 and 158.12 (C-2'), 165.73 and
165.75 (COO), 176.6 (C-7).
1H NMR (t = 120 ºC): δ = 1.52 (d, J = 6.8 Hz, 3H, CH3-C3), 4.45 (br d, J = 11.6 Hz, 1H, H-2B), 4.51 (dd, J = 11.6,
2.5 Hz, 1H, H-2A), 4.93 (qt, J = 6.8, 2.6 Hz, 1H, H-3), 7.40 (td, J = 7.5, 1.0 Hz, 1H, H-6'), 7.45 (br d, J = 8.6 Hz,
1H, H-8'), 7.68 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H, H-7'), 7.71 (d, J = 9.6 Hz, 1H, H-8), 7.77 (dd, J = 7.8, 1.6 Hz, 1H,
H-5'), 8.23 (s, 1H, H-4'), 8.93 (s, 1H, H-5), 14.52 (br. s, 1H, COOH). 19F NMR (t = 120 ºC): δ = -113.18 (br. s,
F-9). 13C NMR (t = 120 ºC): δ = 16.8 (CH3), 54.4 (C-3), 68.4 (C-2), 101.6 (d, 2JCF = 24.7 Hz, C-8), 107.6 (C-6),
115.3 (d, 2JCF = 22.1 Hz, C-10), 115.6 (C-8'), 116.9 (C-3'), 117.9 (C-4'a), 123.5 (d, 4JCF = 1.9 Hz, C-10b), 124.2
3
3
(C-6'), 126.3 (d, JCF = 9.6 Hz, C-7a), 128.2 (C-5'), 132.0 (C-7'), 144.7 (C-4'), 145.4 (d, JCF = 7.1 Hz, C-10a),
145.4 (C-5), 153.0 (C-8'a), 157.0 (d, 1JCF = 247.7 Hz, C-9), 157.3 (C-2'), 164.7 (COO), 176.0 (C-7). MS (m/z,
Irel %): 408 [M+1]+ (5), 407 [M]+ (19), 364 (22), 363 [M-CO2]+ (100), 349 (13), 182 (12). Anal. Calc. for
С22H14FN1O6, С 64.87, Н 3.46, N 3.44. Found: С 64.87, Н 3.43, N 3.58.
(3S)-10-(6-Bromo-2-oxo-2H-chromen-3-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-
ij]quinoline-6-carboxylic acid (9b)
Compound was obtained from 5-bromosalicylaldehyde (5b), reaction mixture was heated at 110
С in 3 % H2SO4 solution for 15 h. Yield 0.40 g (50 %) (mixture of atropisomers, ratio ≈ 1:1), mp. 326-327
C. 1H NMR (tamb): δ = 1.46 and 1.51 (both d, J = 6.7 Hz, 3H, CH3), 4.41 and 4.50 (both dd, J = 11.7, 2.2 Hz,
1H, H-2B), 4.59−4.62 (m, 1H, H-2A), 5.01 (m, 1H, H-3), 7.49 and 7.50 (both d, J = 8.8 Hz, 1H, H-8'), 7.74 (d,
J = 9.4 Hz, 1H, H-8), 7.87 and 7.88 (both dd, J = 8.8, 2.3 Hz, 1H, H-7'), 8.09 (d, J = 2.3 Hz, 1H, H-5'), 8.34 and
8.36 (both s, 1H, H-4'), 9.11 and 9.12 (both s, 1H, H-5), 14.93 (br. s, 1H, COOH). 19F NMR (tamb): δ = -113.44

16
and -112.94 (both d, J = 9.4 Hz, F-9). 13C NMR (tamb): δ = 17.6 and 18.0 (CH3), 54.8 and 55.0 (C-3), 68.7 and
68.8 (C-2), 102.2 and 102.3 (both d, 2JCF = 24.5 Hz, C-8), 107.67 and 107.73 (C-6), 115.0 and 115.3 (both d,
2JCF = 22.0 Hz, C-10), 116.49 and 116.51 (C-4'a), 118.23 and 118.27 (C-3'), 118.6 (C-8'), 120.1 and 120.2 (C-
6')), 123.8 and 124.1 (both d, 4JCF = 1.2 Hz, C-10b), 126.7 (d, 3JCF = 9.6 Hz, C-7a), 130.90 and 130.92 (C-5'),
135.2 (C-7'), 144.4 and 144.7 (C-4'), 145.8 and 145.9 (both d, 3JCF = 7.0 Hz, C-10a), 146.5 and 146.7 (C-5),
152.4 (C-8'a), 157.54 and 157.57 (C-2'), 157.3 (d, 1JCF = 248.8 Hz, C-9), 165.64 (COO), 176.50 (C-7). MS (m/z,
Irel %): 488 [M+2]+ (4), 487 [M+1]+ (18), 486 [M]+ (4), 485 [M-1]+ (17), 444 (22), 443 (95), 442 (26), 441
(100), 428 (48), 426 (51), 221 (16), 53 (22), 41 (23). Anal. Calc. for С22H13BrFN1O6, С 54.34, Н 2.69, N 2.88.
Found: С 54.25, Н 2.79, N 2.98.
(3S)-10-(6,8-Di-tert-butyl-2-oxo-2H-chromen-3-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (9c)
Compound was obtained from 3,5-di-tert-butylsalicylaldehyde (5c), reaction mixture was heated
at 110 С in 3 % H2SO4 solution for 10 h. Yield: 0.58 g (67 %) (mixture of atropisomers, ratio ≈ 1:1), mp.
262−263 C. 1H NMR (tamb): δ = 1.35 (s, 9H, tBu), 1.46−1.52 (m, 12H, tBu, CH3-C3), 4.42 and 4.49 (both dd, J
= 11.6, 2.3 Hz, 1H, H-2B), 4.60 and 4.61 (both dd, J = 11.6, 1.8 Hz, 1H, H-2A), 5.01 (m, 1H, H-3), 7.65 (d, J =
2.4 Hz, 1H, H-7'), 7.69 and 7.70 (both d, J = 2.4 Hz, 1H, H-5'), 7.75 and 7.76 (d, J = 9.4 Hz, 1H, H-8), 8.36
and 8.38 (both s, 1H, H-4'), 9.11 and 9.12 (both s, 1H, H-5), 14.96 (br. s, 1H, COOH). 19F NMR (tamb): δ = -
113.39 and -112.82 (both d, J = 9.4 Hz, F-9). 13C NMR (tamb): δ = 17.7 and 17.9 (CH3-C3), 29.5 ((CH3)3C-C6'),
31.0 ((CH3)3C-C8'), 34.4 ((CH3)3C-C6'), 34.6 ((CH3)3C-C8'), 54.9 and 55.0 (C-3), 68.6 and 68.8 (C-2), 102.1 and
2
2
102.2 (both d, JCF = 24.6 Hz, C-8), 107.6 and 107.7 (C-6), 115.6 and 115.8 (both d, JCF = 22.3 Hz, C-10),
116.00 and 116.02, 118.37 and 118.40 (C-4'a, C-3'), 123.71 and 123.73 (C-5'), 123.9 and 124.1 (both d, 4JCF
= 1.2 Hz, C-10b), 126.6 (d, 3JCF = 9.6 Hz, C-7a), 127.4 (C-7'), 136.15 and 136.16 (C-6'), 145.8 and 145.9 (both
d, 3JCF = 7.0 Hz, C-10a), 146.5 and 146.6 (C-5), 146.70 and 146.71 (C-8'), 146.9 and 147.2 (C-4'), 150.1 (C-
8'a), 157.48 (d, 1JCF = 247.4 Hz, C-9), 157.79 and 157.84 (C-2'), 165.7 (COO), 176.54 (C-7).
1H NMR (t = 110 ºC): δ = 1.36 (s, 9H, tBu), 1.52 (d, J = 6.7 Hz, 3H, CH3-C3), 1.52 (s, 9H, tBu), 4.45 (br. d, J =
11.6 Hz, 1H, H-2B), 4.53 (dd, J = 11.6, 2.4 Hz, 1H, H-2A), 4.94 (qt, J = 6.7, 2.5 Hz, 1H, H-3), 7.62 (d, J = 2.4 Hz,
1H, H-7'), 7.66 (d, J = 2.4 Hz, 1H, H-5'), 7.71 (d, J = 9.5 Hz, 1H, H-8), 8.22 (s, 1H, H-4'), 8.97 (s, 1H, H-5),
14.56 (br. s, 1H, COOH). 19F NMR (t = 110 ºC): δ = -113.06 (br. s, F-9). 13C NMR (t = 110 ºC): δ = 16.9 (CH3-
C3), 29.2 ((CH3)3C-C6'), 30.5 ((CH3)3C-C8'), 33.8 ((CH3)3C-C6'), 34.1 ((CH3)3C-C8'), 54.4 (C-3), 68.4 (C-2), 101.7
2
(d, 2JCF = 24.7 Hz, C-8), 107.6 (C-6), 115.4 (d, JCF = 22.3 Hz, C-10), 115.8, 118.0 (C-4'a, C-3'), 123.0 (C-5'),
3
3
123.6 (unres. d, C-10b), 126.3 (d, JCF = 9.6 Hz, C-7a), 126.7 (C-7'), 136.0 (C-6'), 145.5 (d, JCF = 6.9 Hz, C-
10a), 145.6 (C-5), 146.1 (C-8'), 146.4 (C-4'), 149.7 (C-8'a), 157.2 (C-2'), 157.2 (d, 1JCF = 247.4 Hz, C-9), 164.8

17
(COO), 176.1 (C-7). MS (m/z, Irel %): 520 [M+1]+ (5), 519 [M]+ (16), 476 (33), 475 [M-CO2]+ (100), 460 (19),
376 (14), 230 (20), 216 (10), 57 (30), 41 (17). Anal. Calc. for С30H27FNO6, С 69.35, Н 5.82, N 2.70. Found: С
69.37, Н 5.79, N 2.58.
4.7. Antimycobacterial assay
To evaluate the inhibitory efficiency of molecules on Mycobacterium tuberculosis (MTB), M.
tuberculosis H37Rv, which is susceptible to all classical anti-tuberculosis drugs, was used. The
minimal inhibitory concentration (MIC) for M. tuberculosis H37Rv for each compound was
determined by a micro broth dilution method. All molecules tested were dissolved in dimethyl
sulfoxide and their 1/2 dilutions were prepared in 5 mL tubes using Löwenstein-Jensen medium.
A few colonies from freshly grown M. tuberculosis H37Rv were suspended in Löwenstein-Jensen
medium to obtain 1.0 McFarland turbidity and diluted ten times using the same medium and the
tubes were incubated at 37 °C medium with a different concentration of the tested molecule and
to a positive control tube containing only clear growth medium. After 24 hours the tubes were
placed in a vertical position and the free edge of the buried 0.3 mL of the substance in the test
compounds concentrations: 12.5, 6.25, 3.1, 1.5, 0.7, 0.37, 0.15 μg/mL. The tubes were then placed
in thermostat at a temperature of 37 °C and incubated for 10 days. Growth estimate for the MTB
were determined by standard methods, where the appearance of zones of growth retardation MTB
(over 10 mm) indicated the presence of tuberculostatic properties in concentration of the
compounds under study. Penetration size stunting MTB (in mm) is proportional to the degree of
tuberculostatic activity. Growth delay of 100 mm or more is considered as a complete growth
inhibition MTB. The multi-drug-resistant (MDR) tuberculosis strain was isolated from
tuberculosis patients in Ural Research Institute for Phthisiopulmonology (Russia). The minimal
inhibitory concentrations against Mycobacterium tuberculosis avium, Mycobacterium tuberculosis
terrae, and MDR tuberculosis strains were evaluated similarly.
4.8. Evaluation of acute toxicity in vivo
All experimental protocols were carried out in accordance with the standard protocol approved by the
Committee for the Ethical Use of Animals of the Ural Research Institute for Phthisiopulmonology
(CEUA/URIP). The synthesized compounds were evaluated for their approximate LD50 in white healthy
mice (17−20 g body weight) divided into 3 groups of 5 animals each for testing of one compound [18, 19].
Toxicity tests were carried out via a single per oral introduction of compound in a 1 % starch aqueous

18
solution. The volume introduced did not exceed 0.5 mL for mice. The observation period was 14 days. The
median lethal doses LD50 were used as the criteria of toxicity.
Acknowledgment
The work was carried out with financial support from the Russian Scientific Foundation
(grant 15-13-00077) and from the Ministry of Education of Russian Federation (State Contract
4.6351.2017/8.9, research of 1,4-dihydro[1]benzoxepino-[2,3-g]quinolones 8).
The authors are thankful to PhD Andrey A. Tumashov (Postovsky Institute of Organic Synthesis,
Ekaterinburg) for assistance with HPLC analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j. jfluchem.
References
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Global
tuberculosis
report
2017;
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[8] G.-F. Zhang, S. Zhang, B. Pan, X. Liu, L.-S. Feng. 4-Quinolone derivatives and their activities against
Gram positive pathogens. Eur. J. Med. Chem. 143 (2018) 710-723.

19
[9] Y.-Q. Hu, S. Zhang, Z. Xu, Z.-S. Lv, M.-L. Liu, L.-S. Feng. 4-Quinolone hybrids and their antibacterial
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application. Moscow, Fizmatlit, 2013, 320 pp.
[11] V.N. Charushin, E.V. Nosova, G.N. Lipunova, O.N. Chupakhin. Fluoroquinolones: synthesis and
application. In Fluorine in Heterocyclic Chemistry (Ed. V. Nenaydenko). Springer, 2014, v. 2, chapter
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[12] Y. Todo, H. Takagi, F. Iino, K. Hayashi, M. Takata, H. Kuroda, K. Momonoi, H. Narita.
Practical synthesis of T-3761, (S)-10-(1-aminocyclopropyl)-9-fluoro-2,3-dihydro-3-methyl-
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(1994) 2629-2632.
[13] Y. Todo, J. Nitta, M. Miyajima, Y. Fukuoka, Y. Yammashiro, N. Nishida, I. Saikawa, H. Narita.
Pyridonecarboxylic Acids as Antibacterial Agents. VIII. Synthesis and Structure-Activity Relationship
of
7-(1-Aminocyclopropyl)-4-oxo-1,8-naphthyridine-3-carboxylic
Acids
and
7-(1-
Aminocyclopropyl)-4-oxoquinoline-3-carboxylic Acids. Chem. Pharm. Bull. 42(10) (1994) 2063-
2070.
[14] J.C. Barrow, P.G. Nantermet, H.G. Selnick, K.L. Glass, , K.E. Rittle, K.F. Gilbert, T.G. Steele, C.F.
Homnick, R.M. Freidinger, R.W. Ransom, P. Kling, D. Reiss, T.P. Broten, T.W. Schorn, R.S. Chang,
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[18] J.T. Litchfield, F. Wilcoxon. A simplified method of evaluating dose-effect experiments. J.
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Pharmacological Screening Tests, Butterworths, 1961, 1-33.

20
Figure 1. Fluoroquinolones recommended for the treatment of tuberculosis.
T (°C)
120
100
80
60
50
40
30
1.5
ppm
Figure 2. Variable-temperature 1H NMR spectra (500 MHz, DMSO-d6)

21
Scheme 1. Synthesis of cyanomethyl fluoroquinolones 4.

22
Scheme 2. Synthesis of coumarinyl substituted bicyclic fluoroquinolones 7
and polycyclic quinolones 8.
Scheme 3. Synthesis of coumarinyl substituted tricyclic fluoroquinolones 9.

23
Table 1
In vitro antimycobacterial activity of (2-oxo-2H-chromen-3-yl)-substituted
bi- and tricyclic fluoroquinolones 7a-c,e and 9a-c.
Com-
pound
Antimycobacterial activity against different strains of M.
tuberculosis (MIC in g/mL)
LD50 in mice
(mg/kg)
H37RV
M. avium
M. terrae
MDR-TB a
12.5
6.2
n.d.
6.2
n.d.
6.2
n.d.
6.2
3.1
1.5
n.d.
n.d.
n.d.
n.d.
–
n.d.
7a
n.d.
7b
3.1
3.1
1.5
n.d.
7c
1.5
1.5
0.7
600
7e
12.5
12.5
12.5
0.5 b
0.1
n.d.
n.d.
n.d.
n.d.
0.1
n.d.
n.d.
n.d.
n.d.
0.1
n.d.
9a
n.d.
9b
n.d.
9c
1500-2000 b
149c 151d
LEV
INH
n.d. – not determined; INH – Izoniazid; LEV – Levofloxacin;
a MDR-TB – Rifampin and Izoniazid resistant Mycobacterium tuberculosis strain having Beijing genotype
with a combination of mutations Ser 531-Leu 315 and Ser-Thr in rpoB and katC genes, respectively.
b LD50 oral administration in mice and rats [17].
c LD50 i.v. administration in mice [17].
d LD50 i.p. administration in mice [17].