New 1-(2-chloroquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ols with antituberculosis activity

Article abstract of DOI:10.1007/s11172-019-2646-5

New 4-dimethylamino-2-(naphthalen-1-yl)-1-phenyl-1-(quinolin-3-yl)butan-2-ols with antituberculosis activity were synthesized. (1R*,2S*)-1-(6-Bromo-2-chloroquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ol hydrocitrate exhibiting high

Full text of DOI:10.1007/s11172-019-2646-5

Russian Chemical Bulletin, International Edition, Vol. 68, No. 10, pp. 1908—1918, October, 2019
1908
New 1-(2-chloroquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-
1-phenylbutan-2-ols with antituberculosis activity*
A. V. Omel´kov, V. E. Fedorov, and A. A. Stepanov
Pharm-Sintez,
Build. 134, 29 ul. Vereyskaya, 121357 Moscow, Russian Federation.
E-mail: omelkov@mail.ru, Vfedorov@pharm-sintez.ru
New 4-dimethylamino-2-(naphthalen-1-yl)-1-phenyl-1-(quinolin-3-yl)butan-2-ols with
antituberculosis activity were synthesized. (1R*,2S*)-1-(6-Bromo-2-chloroquinolin-3-yl)-4-
dimethylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ol hydrocitrate exhibiting high antitu-
berculosis activity is in the final stage of clinical trials and is prepared for use in clinical practice.
Key words: lithium diethylamide, diarylquinolines, 1-(2-chloroquinolin-3-yl)-4-dimethyl-
amino-2-(naphthalen-1-yl)-1-phenylbutan-2-ols, 3-benzyl-2-chloroquinolines, 3-dimethyl-
amino-1-(naphthalen-1-yl)propan-1-one, tuberculosis.
A number of 3-benzyl-6-bromo-2-methoxyquinoline
these compounds are of great interest as potential antitu-
berculosis agents.4—6 It is assumed that the tertiary amine
Bedaquiline is an arginine mimetic and blocks the proton
pump for ATP synthase of mycobacteria. Investigations
performed in the Pharm-Sintez resulted in the preparation
of new patentable compounds of this class.2,3
derivatives were found to have high activity against the
mycobacterium M. tuberculosis (Mtb) and its drug-resis-
tant strains. In 2013, the Janssen-Cilag International NV
announced the development of the innovative anti-
tuberculosis drug TMC-207, Bedaquiline® containing
(1R*,2S*)-1-(6-bromo-2-methoxyquinolin-3-yl)-4-di-
methylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ol (1)
as the active substance. This compound, in the form of
hydrofumarate, was approved for medical use in 2014
under the brand name Sirturo®.1 We investigated other
derivatives of this class and synthesized a series of new
compounds, which exhibited different activities against
different M. tuberculosis strains.2,3
X = Br (2), OMe (3), F (4)
Compounds 2 and 3 have passed microbiological test-
ing. Compound 4 is planned to be tested for biological
activity. Compound 2 is currently in clinical trials and is
ready for the pilot production as hydrocitrate.
Due to a fundamentally new mechanism of action of
diarylquinolines on M. tuberculosis based on their interac-
tion with the C subunit of mycobacterial ATP synthase,
* Based on the materials of the IV Interdisciplinary Symposium
and Youth Forum on Medicinal, Organic, and Biological
Chemistry and Pharmaceutics (MOBI-ChemPharma, 2018,
Novy Svet, Crimea).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1908—1918, October, 2019.
1066-5285/19/6810-1908 © 2019 Springer Science+Business Media, Inc.

Diarylqunolines active against Tuberculosis
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
1909
Scheme 1
Reagents and conditions: i. H3BO3, xylene, reflux; ii. Me2NCHO, POCl3, 80 C; iii. MeONa/MeOH, reflux; iv. (CH2O)x, Me2NH•HCl,
CHCl3, reflux; v. NaOH, water, toluene; vi. Pri2NLi, THF, –78 C, then HCl.
Results and Discussion
Unfortunately, this method appeared to be unsuitable
for the synthesis of the target compound 2 because of
instability of the chlorine atom at the quinoline ring and the
formation of compound 9 as a by-product (Scheme 3).
Hence, the synthesis of the target compounds 2—4
using organolithium compounds according to Scheme 1
has no alternative taking into account the limiting condi-
tions on the structure of compounds, the cost of the start-
ing materials, and minimization of the number of synthe-
sis steps (Scheme 4).
The key step is the synthesis of tertiary alcohols 2—4
from 3-dimethylamino-1-(naphthalen-1-yl)propan-1-one
and substituted 3-benzyl-2-chloroquinolines. Organo-
lithium compounds have not only nucleophilic but also
basic properties. The reactions of the resulting organo-
lithium derivative can, in principle, not only give the re-
quired tertiary alcohols, but it also can be accompanied
by proton abstraction from the most acidic carbon atom
in reactant 6 (Scheme 5).
Russia’s demands for hydrocitrate of amine 2 are es-
timated at 1—1.2 tons per year. There is a need to optimize
the synthesis conditions for such compounds in order to
accomplish large-scale production taking into account
high cost of the starting materials.
Two procedures for the synthesis of such compounds
are described in the literature.7,8 One procedure is shown
in Scheme 1. The drawback of this method is the use of
organolithium compounds, which require high-purity
solvents and very low reaction temperatures necessary for
the formation of tertiary alcohols.
Another route to such alcohols (Scheme 2) is covered
by a patent.8 The advantage of this method is that the
reaction can be performed at –35 C (rather than at
–78 C) due to lower acidity of compound 7 compared to
compound 6. Besides, this method affords product 8
in higher yield. The drawback of this approach is an ad-
ditional double bond hydrogenation step.
The latter procedure seemed to us preferable for ap-
plication at the industrial and pilot-plant levels because it
requires less cooling in the step of adding an organolithium
component at the C=O-bond .
A general way to increase the yield of compounds 2—4
is to decrease the polarity of the reaction mixture during
the synthesis. This is achieved by the addition of compound
6 to organolithium derivatives 10—12 in a non-polar
solvent (toluene, hexane).

1910
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
Omel´kov et al.
Scheme 2
Reagents and conditions: i. H3BO3, xylene, reflux; ii. Me2NCHO, POCl3, 80 C. iii. MeONa/MeOH, reflux; iv. Me2NCH(OMe)2,
xylene, reflux; v. Pri2NLi, THF, –35 C, then HCl. vi. H2/Pd.
Scheme 3
Reagents and conditions: Et2NLi, THF, –35 C, then HCl.
We succeeded in separating the resulting mixtures of
diastereomers of 2—4 to individual components. Dia-
stereomers of compounds 2 and 4 were separated by their
transformation from hydrochlorides to bases, washing with
boiling acetone, and recrystallization from ethyl acetate.
Compound 3 (in the form of hydrochloride) was washed
with chloroform, and the residue was concentrated and
crystallized from ethanol.
The synthesized compounds were characterized by
NMR spectroscopy, chromatography-mass spectrometry,
and X-ray diffraction (Figs 1—3).
Crystals of compound 4 rapidly decompose in air.
Hence, this compound was studied at 200 K under an
argon atmosphere. The X-ray diffraction study of com-
pound 3 was performed in a similar way. Crystals of
compound 2 were studied at 295 K. The crystals of 2 and
3 are characterized by a similar arrangement of the mol-
ecules. The asymmetric unit of the crystal of 4 contains
an ethanol solvent molecule in a ratio of two molecules 4
per ethanol molecule. The solvent molecules are located
at lattice points (in the vicinity of inversion centers) and
are disordered mainly over two positions. This weak bind-
ing of the solvent molecule to molecules 4 is responsible
for crystal degradation. There is a strong intramolecular
O(1)—H(1)…N(2) hydrogen bond in all molecules 2—4
that has an effect on the molecular conformation.

Diarylqunolines active against Tuberculosis
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
1911
Scheme 4
X = Br (2, 5b), OMe (3, 5c), F (4, 5d)
Reagents and conditions: i. H3BO3, xylene, reflux. ii. Me2NCHO, POCl3, 80 C. iii. (CH2O)x, CHCl3, Me2NH2Cl, reflux. iv. toluene,
water, NaOH. v. Et2NLi, THF, –78 C, then HCl.
Scheme 5
Table 1. Results of assays of diastereomers of 2 hydrocitrate
in the M.tuberculosis H37Rv strain
Compound
MIC/mg mL–1
(1R*,2S*)-2
3.125
6.25
1.56
0.10
0.10
(1R*,2R*)-2
Bedaquiline® hydrocitrate*
Rifampicin
Isoniazid
* (1R*,2S*)-1-(6-Bromo-2-methoxyquinolin-3-yl)-4-
dimethylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ol
hydrocitrate.
10.94 0.09 and 16.00 0.06 days, respectively, and reliably
differed from the control in the absence of the substance
(the delay was ~2.5 and ~7.5 days). Besides, compound 3
taken at a concentration of 2.5 g mL–1 caused a reliable
increase in the duration of the active cell division phase
by ~8 days compared to the control.
To sum up, we synthesized a series of new quino-
line-substituted aminobutanols, separated their dia-
stereomers, and characterized them by mass spectro-
metry, 1H and 13C NMR spectroscopy, and X-ray
diffraction. Hydrocitrates of the derivatives were
subjected to biological assays to evaluate activity
against M. tuberculosis. Hydrocitrate of (1R*,2S*)-2
proved to be twice as active as hydrocitrate of
(1R*,2R*)-2. Compound 3 also exhibited antituberculosis
activity.
X = Br (5b, 10), OMe (5c, 11), F (5d, 12)
The bases of 4-dimethylamino-2-(naphthalen-1-yl)-
1-phenyl-1-(quinolin-3-yl)butan-2-ols 2—4 were trans-
formed to hydrocitrates. Hydrocitrates of amines 2 and 3
were subjected to biological assays. The results of assays
of diastereomers of 2 hydrocitrate in the M.tuberculosis
H37Rv strain are given in Table 1. Hydrocitrate of
(1R*,2S*)-2 proved to be twice as active as hydrocitrate
of (1R*,2R*)-2.
At concentrations of 5 and 10 g mL–1, compound 3
caused the complete suppression of the Mtb growth
throughout the experiment.
In the presence of compound 3 at concentrations of
1.25 and 2.5 g mL–1, the Mtb growth started on the

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Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
Omel´kov et al.
C(31)
C(32)
N(2)
C(30)
Br(1)
C(7)
C(29)
C(8)
O(1)
C(6)
C(9)
C(19)
C(21)
C(26)
N(1)
C(5)
C(18)
C(22)
C(2)
C(10)
C(20)
C(4)
C(3)
C(27)
C(28)
CL(1)
C(23)
C(24)
C(11)
C(17)
C(16)
C(12)
C(25)
C(13)
C(14)
C(15)
Fig. 1. Molecular structure of (1R*,2S*)-1-(6-bromo-2-chloroquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenyl-
butan-2-ol (2).
C(31)
N(2)
C(32)
C(21)
C(30)
C(20)
C(22)
C(29)
C(17)
O(1)
C(28)
C(23)
C(27)
C(19)
C(18)
C(33)
O(2)
C(24)
C(4)
C(5)
C(8)
C(6)
C(7)
C(10)
C(9)
C(3)
C(2)
C(11)
C(26)
C(25)
C(12)
CL(1)
N(1)
C(16)
C(15)
C(13)
C(14)
Fig. 2. Molecular structure of (1R*,2S*)-1-(2-chloro-6-methoxyquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenyl-
butan-2-ol (3).

Diarylqunolines active against Tuberculosis
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
1913
C(31)
C(21)
C(22)
C(20)
N(2)
C(28)
C(19)
C(32)
C(23)
C(30)
O(1)
C(27)
C(29)
C(26)
C(24)
C(18)
C(25)
C(13)
C(14)
C(11)
C(15)
C(4)
C(12)
C(17)
C(3)
C(10)
C(5)
C(16)
C(5)
F(1)
C(2)
CL(1)
C(9)
N(1)
C(7)
C(8)
Fig. 3. Molecular structure of (1R*,2S*)-1-(2-chloro-6-fluoroquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenyl-
butan-2-ol (4).
grade, Khimmed); toluene (special purity grade, EKOS) was
distilled over sodium metal. The water content in the solvents
was determined by coulometric titration using hydronal (Coulo-
mat AG, Fluka). The following reagents and solvents were
utilized: THF (stabilized ABCR GmBh, Reatorg), 2.5 M BunLi
(98%, Acros Organics), 2.5 M BunLi (98%, ABCR), 1.6 M BunLi
(98%, Acros Organics), diethylamine* (99%, Aldrich),
N,N,N´,N´-tetramethylethylenediamine (99%, Aldrich), and
hexane (reagent grade, Khimmed). Diethylamine, N,N,N´,N´-
tetramethylethylenediamine, hexane, and toluene were dried and
distilled over sodium metal; THF was dried over sodium metal
followed by distillation or drying over activated alumina using
a Pure Solve 400 solvent purification system.
Synthesis of 3-dimethylamino-1-(naphthalen-1-yl)propan-1-
one (6). 3-Dimethylamino-1-(naphthalen-1-yl)propan-1-one
hydrochloride (11.2 g, 0.042 mol) was dissolved in distilled water
(10 mL) in a 250 mL one-neck round-bottom flask equipped
with a magnetic stirrer. Toluene (100 mL) and a solution of
sodium hydroxide (1.8 g, 0.045 mol) in distilled water (10 mL)
were added to the resulting solution. The emulsion thus formed
was vigorously stirred for 10 min at room temperature. The aque-
Experimental
The reaction mixtures and target compounds were analyzed
by HPLC on an Agilent 1200 Series system equipped with
a ReproSil-Pur Basic chromatographic column (modified silica
gel C18 with a granule diameter 5 m, geometric sizes of the
column were 4.6×250 mm). The conditions of HPLC: the elution
gradient (95% phase A + 5% phase B)100% phase B during
20 min. The phase A consisted of H2O and 0.01% trifluoroacetic
acid (TFA); the phase B, of acetonitrile and 0.01% TFA. The
rate of elution was 1 mL min–1. The analyzed compounds were
detected using a spectrophotometric detector at  = 220 nm.
Electrospray ionization mass spectra (ESI-MS) were re-
corded on an Agilent MSD IonTrap 6310 mass spectrometer in
positive ion mode. The pressure of the nebulizer gas was 275 MPa,
the drying gas flow rate was 7 L min–1, the drying gas temperature
was 350 C.
1
The H NMR spectra were measured on a Bruker AM-360
spectrometer (working frequency 360.13 MHz) in CDCl3 using
the signal of the nondeuteratred solvent as the internal standard.
The melting points of the products were determined on a PTP
(M) capillary-type analyzer (KlinLabPribor) and on a Mettler
Toledo FP62 melting point apparatus.
* We used diethylamine (pKb  35) instead of diisopropylamine
(pKb  34) because of its higher basic properties and since it is
more readily available on the industrial scale, being a by-product
of the synthesis of triethylamine.
3-Dimethylamino-1-(naphthalen-1-yl)propan-1-one hydro-
chloride was transformed into the base employing sodium hydr-
oxide (reagent grade, Khimmed) and sodium bicarbonate (reagent

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Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
Omel´kov et al.
ous layer was separated, and the toluene layer was dried over
magnesium sulfate, filtered, and concentrated. A mobile oily
yellowish liquid was obtained in a yield of 9.6 g. The chromato-
graphic retention time tchr = 10.34 min. The yield of 6 was 99%
according to HPLC data and 1H NMR spectra. 1H NMR
(CDCl3), δ: 2.95 (s, 6 H, 2-CH3); 3.47 (t, 2 H, CH2—CH2,
J = 7.0 Hz); 3.75 (t, 2 H, CH2—CH2, J = 7.0 Hz); 7.60—8.50
(m, 7 H, CHarom). MS, found: m/z 228.1 [M + H]+; C15H17NO;
calculated M + H = 228.1.
CHarom); 7.80—7.85 (m, 1 H, CHarom); 7.95—8.15 (m, 3 H,
CHarom); 8.65—8.71 (m, 1 H, CHarom); 9.35 (s, 1 H, —OH).
13C NMR of the base (1R*,2S*)-2 (CDCl3), δ: 33.0 (1 C), 44.5
(2 C), 53.5 (1 C), 56.0 (1 C), 82.0 (1 C), 124.5 (1 C), 124.8 (2 C),
125.6 (1 C), 126.0 (1 C), 126.8 (1 C), 127.2 (1 C), 127.2 (1 C),
127.3 (1 C), 127.5 (1 C), 127.8 (1 C), 128.0 (1 C), 128.2 (2 C),
129.6 (1 C), 129.8 (2 C), 134.0 (1 C), 134.5 (2 C), 140.0 (2 C),
141.0 (1 C), 146.0 (1 C), 152.0 (1 C).
MS, found: m/z 559.92 [M + H]+; C31H28BrClN2O; calcu-
lated M + H = 560.00.
Synthesis of a diastereomeric mixture of 1-(2-chloroquinolin-
3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ol
hydrochlorides (2—4). Two four-neck flasks were evacuated at
120 C for 1 h. The flasks were filled with argon and cooled to
25 C. Diethylamine (1.46 g, 0.02 mol), THF (50 mL), and
N,N,N´,N´-tetramethylethylenediamine (2.32 g, 0.02 mol) were
placed in the first flask equipped with a thermometer and cooled
to –20 C. Then 2.5 M n-butyllithium (8 mL, 0.02 mol) was
added to the resulting solution, and the mixture was stirred for
30 min and cooled to –78 C. The corresponding 3-benzyl-2-
chloroquinoline 5 (0.015 mol) was placed in the second flask and
dissolved in THF (50 mL). Then the mixture was cooled to
–50 C. The first flask was connected to the second flask by
a rubber tube, and the content of the first flask was poured into
the second one. After the completion of the ixing of the reagents,
the reaction mixture was allowed to stand for 1 h and cooled to
–78 C. Compound 6 was placed in the third flask and then it
was diluted with toluene to 50 mL and cooled to –78 C. The
resulting cold solution of compound 6 in toluene was added to
the reaction mixture. The mixture was kept for 3 h at –78 C,
treated with concentrated hydrochloric acid (10 mL, 0.108 mol),
and heated to 25 C. The reaction mixture was concentrated to
dryness, dissolved in chloroform (100 mL), and washed with water
(3×30 mL). The aqueous layer was separated, and the chloroform
layer was dried over magnesium sulfate and concentrated. The
dry residue was twice suspended in toluene (50 mL) and then
filtered off. The resulting product was dried to constant weight.
1-(6-Bromo-2-chloroquinolin-3-yl)-4-dimethylamino-2-(naphth-
alen-1-yl)-1-phenylbutan-2-ol (2). The yield of a mixture of two
diastereomers of 3•HCl was 3.62 g (40.5%), the (1R*,2R*)/
(1R*,2S*) diastereomeric ratio was 60 : 40.
1-(2-Chloro-6-methoxyquinolin-3-yl)-4-dimethylamino-2-
(naphthalen-1-yl)-1-phenylbutan-2-ol (3). The yield of a mixture
of two diastereomers of 2•HCl was 4.62 g (56.2%), the (1R*,2R*)/
(1R*,2S*) diastereomeric ratio was 40 : 60. A mixture of two
diastereomer hydrochlorides was obtained as white or beige
crystals with m.p. 239.1—242.9 C (with decomp.). The chrom-
atographic retention time tchr((1R*, 2R*)-3) = 13.6 min,
tchr((1R*, 2S*)-3) = 14.6 min.
1H NMR of the base (1R*,2R*)-3 (CDCl3), δ: 1.95—2.00
(s, 6 H, 2-CH3); 2.05—2.15 (m, 2 H, CH2—CH2); 2.25—2.35
(m, 1 H, CH2—CH2); 2.50—2.55 (m, 1 H, CH2—CH2);
3.80—3.90 (s, 3 H, OMe); 5.80—5.85 (s, 1 H, CH); 7.00—7.05
(m, 1 H, CHarom); 7.11—7.20 (m, 1 H, CHarom); 7.25—7.32
(m, 2 H, CHarom); 7.36—7.43 (m, 3 H, CHarom); 7.55—7.62
(m, 3 H, CHarom); 7.73—7.78 (m, 1 H, CHarom); 7.81—7.88
(m, 2 H, CHarom); 8.26—8.34 (d, 1 H, —CHarom, J = 7.2 Hz);
8.52—8.59 (m, 2 H, CHarom); 9.10—9.15 (s, 1 H, OH). 13C NMR
of the base (1R*,2R*)-3 (CDCl3), δ: 34.0 (1 C), 44.5 (2 C), 55.0
(1 C), 55.5 (1 C), 56.2 (1 C), 82.0 (1 C), 105.4 (1 C), 121.9 (1 C),
124.6 (1 C), 124.7 (1 C), 125.2 (1 C), 125.5 (1 C), 126.7 (1 C),
127.4 (1 C), 127.7 (2 C), 128.1 (1 C), 128.4 (1 C), 129.0 (1 C),
129.6 (1 C), 129.7 (1 C), 131.7 (2 C), 134.1 (1 C), 134.9 (1 C),
138.6 (1 C), 139.7 (1 C), 141.5 (1 C), 141.7 (1 C), 149.2 (1 C),
157.6 (1 C).
1H NMR of the base (1R*,2S*)-3 (CDCl3), δ: 1.95—2.00
(s, 6 H, 2-CH3); 2.00—2.15 (m, 2 H, CH2—CH2), 2.40—2.45
(m, 2 H, CH2—CH2); 3.95—4.00 (m, 3 H, OMe); 6.00—6.05
(s, 1 H, CH); 6.85—6.95 (m, 3 H, CHarom); 7.15—7.40 (m, 8 H,
CHarom); 7.45—7.55 (m, 1 H, CHarom); 7.60—7.70 (m, 2 H,
CHarom); 7.85—7.90 (m, 1 H, CHarom); 7.90—7.95 (m, 1 H,
CHarom); 8.65—8.70 (s, 1 H, OH). 13C NMR of the base
(1R*,2S*)-3 (CDCl3), δ: 33.0 (2 C), 44.0 (2 C), 53.0 (1 C), 54.0
(1 C), 56.0 (1 C), 82.6 (1 C), 105.3 (1 C), 122.6 (1 C), 124.5
(1 C), 124.8 (1 C), 125.2 (1 C), 125.9 (1 C), 127.2 (1 C), 128.0
(1 C), 128.4 (1 C), 128.6 (1 C), 129.3 (1 C), 129.4 (1 C), 129.5
(1 C), 129.6 (1 C), 129.8 (2 C), 134.2 (1 C), 134.5 (1 C), 139.9
(1 C), 140 (1 C), 140.5 (1 C), 142.5 (1 C), 150 (1 C), 158 (1 C).
MS, found: m/z 511.0 [M + H]+; C32H31ClN2O2; calcu-
lated M + H = 511.1.
1-(2-Chloro-6-fluoroquinolin-3-yl)-4-dimethylamino-2-(naphth-
alen-1-yl)-1-phenylbutan-2-ol (4). The yield of a mixture of two
diastereomer hydrochlorides of 4•HCl was 2.62 g (32.6%), the
(1R*,2R*)/(1R*,2S*) diastereomeric ratio was 70 : 30. A dia-
stereomeric mixture of the hydrochloride was obtained as white
or beige crystals with m.p. 220—229.1 C (with decomp.). The
chromatographic retention time tchr((1R*, 2R*)-2) = 13.72 min,
tchr((1R*, 2S*)-2) = 14.44 min. 1H NMR of the base (1R*,2R*)-4
(CDCl3), δ: 1.95—2.05 (m, 8 H, CH2—CH2, Me); 2.11—2.16
(m, 1 H, CH2—CH2); 2.24—2.31 (m, 1 H, CH2—CH2);
5.90—5.95 (s, 1 H, CH); 7.15—7.32 (m, 5 H, CHarom); 7.35—7.47
(m, 3 H, CHarom); 7.50—7.57 (m, 2 H, CHarom); 7.60—7.68
(m, 1 H, CHarom); 7.70—7.95 (m, 3 H, CHarom); 8.07—8.15 (d, 1 H
A mixture of diastereomer hydrochlorides was obtained as
white or beige crystals with m.p. 219.4—228.1 C (with decomp.).
The chromatographic retention time tchr((1R*,2R*)-2) = 16.03 min,
tchr((1R*,2S*)-2 = 17.35 min.
1H NMR of the base (1R*,2R*)-2 (CDCl3), δ: 1.95—2.05
(s, 7 H, CH2—CH2, Me); 2.07—2.16 (m, 1 H, CH2—CH2);
2.23—2.32 (m, 1 H, CH2—CH2); 2.48—2.57 (m, 1 H, CH2—
CH2); 5.8—5.82 (s, 1 H, CH); 7.36—7.46 (m, 5 H, CHarom);
7.48—7.53 (m, 2 H, CHarom); 7.56—7.62 (m, 2 H, CHarom);
7.74—7.84 (m, 3 H, CHarom); 7.77—7.91 (m, 1 H, CHarom);
8.24—8.30 (d, 1 H, CHarom, J = 7.2 Hz); 8.50—8.58 (m, 2 H,
CHarom); 9.17 (s, 1 H, OH). 13C NMR of the base (1R*,2R*)-2
(CDCl3), δ: 34.0 (1 C), 44.5 (2 C), 55.5 (1 C), 56.0 (1 C), 82.0
(1 C), 119.3 (1 C), 124.6 (2 C), 125.0 (1 C), 125.4 (1 C), 126.7
(1 C), 127.3 (1 C), 127.7 (2 C), 128.2 (1 C), 128.4 (1 C), 129.3
(1 C), 129.6 (2 C), 131.6 (2 C), 132.6 (1 C), 134.8 (1 C), 135.0
(2 C), 138.5 (1 C), 139.2 (1 C), 141.1 (1 C), 144.0 (1 C), 152.1 (1 C).
1H NMR of the base (1R*,2S*)-2 (CDCl3), δ: 1.95—2.00
(s, 5 H, CH2—CH2, Me); 2.00—2.10 (m, 5 H, CH2—CH2);
6.00—6.05 (s, 1 H, CH); 6.85—6.95 (m, 3 H, CHarom); 7.15—7.20
(m, 2 H, CHarom); 7.25 (s, 1 H, CHarom); 7.45—7.50 (m, 1 H,
CHarom); 7.55—7.6 (m, 1 H, CHarom); 7.65—7.75 (m, 3 H,

Diarylqunolines active against Tuberculosis
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
1915
CHarom, J = 7.2 Hz); 8.45—8.65 (d, 1 H, CHarom, J = 7.2 Hz);
9.05—9.15 (s, 1 H, —OH). 13C NMR of the base (1R*,2R*)-4
(CDCl3) δ: 33.9 (1 C), 44.5 (2 C), 54.9 (1 C), 56.0 (1 C), 81.9
under vigorous stirring. The resulting suspension was kept at
5—10 C overnight, filtered, and washed with ethanol (50 mL).
The product was dried to constant weight at 40—45 C overnight.
Hydrocitrate 2•C6H8O7 was obtained as a white crystalline
powder in a yield of 35.7 g. The filtrate, which was obtained
after the isolation of the product, was concentrated to 2/3 of the
initial volume and kept at 5—10 C overnight. The precipitate
that formed was filtered off. The yield of the second crop was 1.8 g.
The total yield was 35.6 g (93%), the chromatographic purity
98%.
(1 C), 110.6 (d, 1 C, CHarom,
2JC,F = 21.7 Hz); 119.2 (d, 1 C,
CHarom, 2JC,F =21.7 Hz); 124.5 (1 C), 124.7 (1 C), 125.0 (1 C),
125.5 (1 C), 126.7 (1 C), 127.3 (2 C), 127.7 (2 C), 128.3 (1 C),
129.6 (2 C), 130.0 (2 C), 131.7 (1 C), 134.5 (1 C) 135.0 (1 C),
139.2 (1 C), 139.2 (1 C), 141.0 (1 C), 143.5 (1 C), 151.0 (1 C),
158.8 (d, 1 C, CFarom, 1JC,F = 248 Hz).
1H NMR of the base (1R*,2S*)-4 (CDCl3), δ: 1.95—2.05
(s, 5 H, CH2—CH2, Me); 2.05—2.20 (m, 5 H, CH2—CH2, Me);
6.00—6.05 (s, 1 H, CH); 6.85—6.95 (s, 3 H, CHarom); 7.15—7.25
(m, 2 H, CHarom); 7.30—7.47 (m, 1 H, CHarom); 7.45—7.55
(m, 3 H, CHarom); 7.60—7.75 (m, 2 H, CHarom); 7.85—7.92 (d, 1 H,
CHarom, J = 7.2 Hz); 8.00—8.10 (m, 2 H, CHarom); 8.64—8.70
(d, 2 H, CHarom, J = 7.2 Hz); 9.30—9.35 (s, 1 H, OH). 13C NMR
of the base (1R*,2S*)-4 (CDCl3), δ: 33.4 (1 C), 44.5 (2 C), 53.9
(1 C), 56.2 (1 C), 82.5 (1 C), 111.0 (d, 1 C, CHarom, 2JC,F = 21.7 Hz);
Isolation and separation of diastereomers of 1-(2-chloro-6-
methoxyquinolin-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-
phenylbutan-2-ol (3). Step 1. Preparation of (1R*,2R*)-1-(2-
chloro-6-methoxyquinolin-3-yl)-4-dimethylamino-2-(naphthalen-
1-yl)-1-phenylbutan-2-ol hydrochloride, (1R*,2R*)-3•HCl.
A diastereomeric mixture of 3 hydrochloride (1.8 g, 0.0033 mol)
was suspended in chloroform (30 mL) for 1 h. The suspension
was filtered off. The yield of diastereomer (1R*,2R)-3•HCl was
0.7 g; the chromatographic purity 88%. The precipitate that
formed was repeatedly separated. The yield of (1R*,2R*)-3•HCl
with a chromatographic purity 98% was 0.5 g. The residues were
concentrated to constant weight and were used for the prepara-
tion of the diastereomer (1R*,2S*)-3.
Step 2. Preparation of hydrochloride of (1R*,2S*)-3. A dia-
stereomeric mixture of 3 hydrochloride (1.2 g, 0.0022) was dis-
solved in ethanol (40 mL) under reflux. The solution was cooled
to 2 C and allowed to stand overnight. The precipitate that
formed was filtered off, washed with ethanol (40 mL) cooled to
0 C, and dried to constant weight at room temperature. The
yield of hydrochloride of (1R*,2S*)-3 with a chromatographic
purity 95% was 0.6 g. The precipitate that formed was repeat-
edly crystallized. The yield of hydrochloride of (1R*,2S*)-3 with
a chromatographic purity >98% was 0.3 g. The residues were
concentrated to constant weight and were used for the prepara-
tion of the diastereomer (1R*,2R*)-3.
Preparation of the base of diastereomer (1R*,2S*)-3. Hydro-
chloride of (1R*,2S*)-3 (0.5 g, 0.001 mol) was suspended in
chloroform (20 mL). Then a 25% aqueous ammonia solution
(3 mL) was added to the suspension at 25 C, and the mixture
was stirred for 15—20 min. The resulting suspension was sepa-
rated using a separatory funnel. The lower (chloroform) layer
was washed with distilled water (50 mL). The aqueous extracts
were combined and washed with a fresh portion of chloroform
(50 mL). The organic layers were combined and concentrated
using a rotary evaporator at 25 C. The resulting beige powder
was dried in vacuo at room temperature.
X-ray diffraction study of compounds 2—4. Single crystals of
all compounds were grown from chloroform. The X-ray diffrac-
tion data sets for compounds 2—4 were collected on a STOE
diffractometer equipped with a Pilatus100K semiconductor
detector, a microfocus X-ray source (Cu-Kα,  = 1.54086 Å),
and a focusing multilayered thin-film monochromator. The X-ray
diffraction data were processed using the STOE X-AREA 1.67
software (STOE & Cie GmbH, Darmstadt, Germany, 2013).
The integrated intensities were processed with the LANA program
(implemented in the X-Area software) to minimize the differ-
ences of intensities of symmetry-equivalent reflections (multi-
scan method).
120.0 (d, 1 C, CHarom,
2JC,F = 21.7 Hz); 124.4 (1 C), 124.7
(1 C), 125.4 (1 C), 125.8 (1 C), 127.0 (1 C), 127.6 (2 C), 127.7
(3 C), 127.8 (1 C), 128.0 (1 C), 130.0 (1 C), 130.5 (2 C), 135.0
(2 C), 140.0 (1 C), 140.5 (1 C), 141.0 (1 C), 143.5 (1 C), 151.8
(1 C), 159.0 (d, 1 C, CFarom, 1JC,F = 248 Hz).
MS, found: m/z 499.5 [M + H]+; C31H28FClN2O; calcu-
lated M + H = 499.5.
Isolation and separation of diastereomers of 1-(2-chloroquinol-
in-3-yl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-
ols. Step 1. Preparation of diastereomers of bases 2 or 4. A 25%
aqueous ammonia solution (25 mL) was added to a suspension
of a mixture of diastereomers of the appropriate hydrochloride
(20 g, 0.034 mol) in chloroform (200 mL), and the mixture was
stirred for 15—20 min. The resulting emulsion was separated
using a separatory funnel. The lower (chloroform) layer was
washed with distilled water (50 mL). The aqueous extracts were
combined and washed with chloroform (50 mL). The organic
layers were combined and concentrated using a rotary evapora-
tor at room temperature. The resulting beige powder was thor-
oughly ground and dried in vacuo at 25 C.
Step 2. Separation of diastereomers of bases 2 or 4. The dried
base (100 g) of a diastereomeric mixture of 2 or 4 was suspended
in acetone (1 L). The suspension was refluxed for 15—20 min
with moderate stirring, cooled to 5 C, and kept in a refrigerator
at 5—10 C overnight. The white precipitate of the (1R*,2R*)-
diastereomer was suction-filtered, washed with ice-cold acetone
(70 mL), and dried to constant weight at 25 C. The yield of
(1R*,2R*)-2 with a chromatographic purity >98% was 45.4 g.
The yield of (1R*,2R*)-4 with a chromatographic purity 98%
was 50.4 g.
After the filtration, the mother liquor was concentrated at
30 C. The beige-brown flocculent precipitate (50 g) was dissolved
in boiling ethyl acetate (330 mL). The resulting dark-red solution
was slowly cooled to room temperature and kept at 0—5 C
overnight. The white coarsely-crystalline precipitate that formed
was suction-filtered, washed with ice-cold acetone (50 mL), and
dried to constant weigh. The yield of (1R*,2S*)-2 with a chro-
matographic purity >98% was 33 g. The yield of (1R*,2S*)-4
with a chromatographic purity >98% was 25.3 g.
Synthesis of (1R*,2S*)-diastereomers of 1-(6-bromo-2-
chloro-3-quinolyl)-4-dimethylamino-2-(naphthalen-1-yl)-1-phen-
ylbutan-2-ol hydrocitrate (2•C6H8O7). Citric acid monohydrate
(12.4 g, 0.059 mol) was dissolved in ethanol (150 mL). The solu-
tion was mixed with the base of (1R*,2S*)-2 (30 g, 0.054 mol)
Crystallographic data and the X-ray data collection statistics
for compounds 2—4 are summarized in Table 2.
The structures were solved and refined with the SHELX(1)
program package. The positional parameters of nonhydrogen

1916
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
Omel´kov et al.
Table 2. Crystallographic data and the X-ray data collection statistics for compounds 2—4
Parameter
2
3
4
Molecular formula
C
31H28BrClN2O
559.91
C32H31ClN2O2
511.04
C31H28ClFN2O
498.00
М
T/K
293(2)
293(2)
293–(2)
Space group
P21/c
P21/c
P1
Crystal system
Monoclinic
15.6337(5)
9.5020(2)
19.0902(7)
108.347(2)
4
Monoclinic
15.8639(3)
9.3739(2)
18.8059(6)
108.347(2)
4
Triclinic
9.7451(3)
12.1285(5)
13.1246(5)
68.184(3)
2
a/Å
b/Å
c/Å
β/deg
Z
V/Å3
2691.72(15)
1.382
3.194
1.54086
2654.41(12)
1.279
3.194
1.54086
1387.75(10)
1.194
1.469
1.54086
dx/mg m–3
μ/mm–1
λ/Å
Number of reflections
measured
unique
18802
4646
0.052
18733
4954
0.1186
5330
2388
0.0488
Rint
atoms were refined by the full-matrix method with anisotropic
displacement parameters. The crystal structure of 4 was refined
using the SQUEEZE procedure. The hydrogen atoms (except
for the proton H(1) at the oxygen atom (O(1)) were positioned
geometrically and refined using a riding model. In all structures,
the hydrogen atom H(1) was located in difference Fourier maps
and its parameters were freely refined. The molecular geometry
was calculated using the SHELX program package. The graphi-
cal representations and figures were made using the DIAMOND(2)
software. The crystallographic data were deposited with the
Cambridge Crystallographic Data Centre and are available, free
of charge, at www.ccdc.cam.ac.uk/data_request/cif (CADS
1920402 (2), 1920404 (3), 1920399 (4)).
Synthesis of diastereomeric 1-(2-chloroquinolin-3-yl)-4-di-
methylamino-2-(naphthalen-1-yl)-1-phenylbutan-2-ol hydro-
citrates (2—4) (general procedure). Citric acid monohydrate
(12.4 g, 0.059 mol) was dissolved in rectified ethanol (150 mL).
The solution was mixed with the base of the (1R*,2S*)- or
(1R*,2R*)-diastereomer of compounds 2—4 (0.054 mol) under
vigorous stirring. The resulting suspension was kept at 5—10 C
overnight, filtered, and washed with ethanol (50 mL). The reac-
tion product was dried to constant weight at 40—45 C overnight.
The yields of hydrocitrates of diastereomers (1R*,2R*)-2 and
(1R*,2S*)-2 were 34.5 g (83%) and 30.3 g (77%), respectively; the
yield of hydrocitrate of diastereomer (1R,2S)*-3 was 33.2 g (86%);
the yields of hydrocitrates of diastereomers (1R*,2R*)-4 and
(1R*,2S*)-4 were 34 g (89%) and 35.3 g (93.9%), respectively.
In vitro evaluation of inhibitory concentrations of compound
2 for mycobacteria in liquid Dubos medium from changes in the
optical density at 600 nm. To evaluate the dynamic effect of
hydrocitrates of diastereomers of 2 on the growth of Mycobacterium
tuberculosis (Mtb), the periodic culturing was performed in 200 L
of the liquid medium (Dubos broth). The Dubos broth was pre-
pared according to the manufacturer's instructions from Difco
(USA). After the autoclaving of the salt base diluted in distilled
water, the flasks containing the medium were cooled to 52—54 C,
and then 5% sterile bovine serum albumin (BSA) and sodium
oleate were added. The solution was distributed in wells of
a 96-well flat-bottom plate. The evaluation was performed using
the laboratory M. tuberculosis H37Rv strain sensitive to all anti-
tuberculosis agents. A suspension of single mycobacterial cells
in the same growth phase was seeded in wells containing Dubos
broth supplemented with the substances under study at different
Table 3. Growth of the control cultures M.tuberculosis
H37Rv in the Bactec MGIT 960 system (without the
addition of the substances and with exposure to ri-
fampicin)
/day Without the substance
Rifampicin
(1 g mL–1
)
Average
SD*
Average SD*
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
—
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
0
0
4
0
0
5
0
0
6
0
0
7
0
0
8
0
0
9
78
6
10
11
12
13
14
15
16
17
18—42
585
2680
6541
11273
15419
17931
18859
19103
19131
42
626
1901
3132
3772
3837
3557
3358
3318
* SD is a standard deviation.

Diarylqunolines active against Tuberculosis
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
1917
concentrations (50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.2,
and 0.1 g mL–1) and the control agents rifampicin (R) and
isoniazid (H) at the same concentrations. Each concentration of
the substances was tested in triplicate; of the control samples, in
duplicate. The Mtb culture grown in a medium without the ad-
dition of substances, served as the positive control. The incuba-
tion was performed at 37 C for 18 days (the results were
recorded until the positive control of the stationary growth phase
or the maximum bacterial concentration was reached). The
growth of the periodic culture of Mtb was monitored by changes
in optical density (D) in the suspension, which was measured
every 1—2 days with a Sigma plate reader at 600 nm, after the
Table 4. Growth of the control cultures M.tuberculosis H37Rv in the Bactec MGIT 960 system in the presence of compound 3 at
concentrations of 0.15—10 g mL–1
/day
0.15
0.312
0.625
1.25
2.5
5
10
Average SD* СAverage SD* Average SD* Average SD* Average SD* Average SD* Average SD*
1
2
3
4
5
6
7
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
51
0
0
0
0
0
0
0
0
0
0
0
69
305
1124
2028
2572
2575
2168
1681
1367
1223
1149
1128
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
1119
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
48
427
4
49
87
48
469
23
102
607
0
88
577
0
61
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
1545
3779
6686
9693
11840
12986
13520
13687
13718
13723
13728
13729
13731
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
13732
2355
6269
10989
14994
17366
18177
18327
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
18334
351
1459
2984
4278
4910
4850
4446
4129
3961
3876
3836
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
3834
189
279
368
350
332
326
280
282
283
287
288
291
292
292
292
292
292
292
292
292
292
292
292
292
292
292
292
292
292
1576
2257
2382
2179
1908
1780
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
1771
2059
4665
7699
10287
11938
12727
13026
13144
13205
13233
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
13234
412
1555
3452
5779
7953
9500
10375
10806
10992
11066
11085
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
11091
0
0
0
1
0
0
0
1
63
88
236
696
1614
2960
4374
5657
6624
7345
7824
8139
8309
8409
8481
8541
8589
8624
8644
8648
333
891
1906
3184
4098
4420
4221
3761
3352
3120
2951
2823
2724
2642
2583
2543
2517
2512
40—42 13732
* SD is a standard deviation.

1918
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 10, October, 2019
Omel´kov et al.
preliminary stirring of the well content. The statistically signifi-
cant differences between the average optical densities in the wells
containing the test substances and the optical densities in the
control wells and the percentage of inhibition of the culture
growth by the substances were determined in the maximum
growth phase using the optical densities measured on the last day
of observations (18th day).
The statistically significant differences between the average
values were estimated using the Student t-criterion. The results
were processed with the BIOSTAT 3.03 program (Praktika,
Moscow, 1998). The reliable differences were with p  0.01.
The percentage of inhibition was determined by the formula:
The bacteriostatic activity was evaluated based on specific
growth inhibition in the medium containing a particular con-
centration of the substance during 42 days. The minimum con-
centration of compound 3, at which the culture growth was not
detected, was taken as the minimum inhibitory concentration
(MIC). At concentrations lower than MIC, the negative effect
on the growth of M.tuberculosis H37Rv was evaluated. This effect
may be manifested as follows:
1) retardation of the onset of the culture growth compared
to the concentrations in the absence of the test substances;
2) an increase in the duration of the phase of active division
compared to the control, which is indicative of a decrease in the
mycobacterial cell division rate in the culture.
It should be noted that the RFU value recorded with the
Epicenter program once the mycobacterial growth curve reach-
es a plateau has no value in evaluating antituberculosis activity
of the substance because it depends on the instrumental param-
eters, which are not related to the dynamics of Mtb growth and
does not reflect the dependence on the increase in cfu of Mtb.
The results of detection of mycobacterial growth in the con-
trol and test samples are given in Tables 3 and 4.
,
where I (%) is the inhibition
density in the wells containing the test substancees on the last
day of observations (18th day), and are
the average optical densities in the control wells at the beginning
of observations (first day) and on the last day of observations
(18th day), respectively.
is the average optical
The optical densities were recalculated to find the minimum
inhibitory concentrations (MIC) (see Table 1).
References
Evaluation of bacteriostatic activity (growth inhibitory con-
centrations) of hydrocitrate of compound 3 by monitoring the growth
of the M. tuberculosis H37Rv strain in the liquid Middlebrook 7H9
broth base. The antimycobacterial activity of hydrocitrate of 3
was studied by monitoring the growth dynamics of the H37Rv
strain in the enriched Middlebrook 7H9 broth medium in the
presence of different concentrations of hydrocitrate of compound
3 (0.15, 0.312, 0.625, 1.25, 2.5, 5, and 10 g mL–1) compared
to the growth of this strain in the medium containing the first-line
drug (rifampicin) at the critical concentration. Suspensions of
mycobacteria were seeded at a concentration of 105 cfu mL–1
(cfu is a colony-forming unit that is used to estimate the number
of viable mycobacterial cells; one colony corresponds to one
cell). Each concentration, including the control test tubes without
substances and the test tubes containing rifampicin, was analyzed
in triplicate.
The growth of mycobacteria in culture was detected using
a Bactec MGIT 960 automated growth detection system (BD,
USA) in special MGIT tubes containing bound fluorophore
under a semipermeable membrane on the bottom of the tube.
The fluorophore release and light emission at a particular wave-
length are directly proportional to the oxygen uptake of myco-
bacterial cells.
The growth of mycobacteria in culture media was detected
at one-hour intervals using the Epicenter software (BD, USA).
The dynamics of cell division of mycobacteria was expressed in
relative fluorescence units (RFU). The duration of the experiment
was 42 days according to the Becton Dickinson protocol. Then
all grown cultures were tested for species specificity (for belong-
ing to the Mycobacterium tuberculosis). The specificity of growth
of mycobacteria was controlled by the visual inspection of posi-
tive test tubes (medium transparence, the presence of stippling
and culture cloud on the bottom of test tube), Ziehl—Neelsen
microscopy, seeding on blood agar (no growth on blood agar),
and DNA identification (positive results of IS6110 PCR. assay).
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Received November 30, 2018;
in revised form July 2, 2019;
accepted July 10, 2019