Synthesis and tuberculostatic activity evaluation of novel benzazoles with alkyl, cycloalkyl or pyridine moiety

Article abstract of DOI:10.3390/molecules23040985

Compounds possessing benzimidazole system exhibit significant antituberculous activity. In order to examine how structure modifications affect tuberculostatic activity, a series of benzazole derivatives were synthesized and screened for their antitubercul

Full text of DOI:10.3390/molecules23040985

molecules
Article
Synthesis and Tuberculostatic Activity Evaluation of
Novel Benzazoles with Alkyl, Cycloalkyl or
Pyridine Moiety
2
Malwina Krause 1, Henryk Foks 1, Ewa Augustynowicz-Kopec´ , Agnieszka Napiórkowska 2
,
ID
Małgorzata Szczesio 3 and Katarzyna Gobis 1,
*
1
Department of Organic Chemistry, Medical University of Gdan´sk, 107 Gen. Hallera Ave., 80-416 Gdan´sk,
Poland; malwinakrause@gumed.edu.pl (M.K.); hfoks@gumed.edu.pl (H.F.)
2
3
Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, 26 Płocka Str.,
01-138 Warsaw, Poland; e.kopec@igichp.edu.pl (E.A.-K.); araceli@op.pl (A.N.)
˙
Institute of General and Ecological Chemistry, Technical University of Łodz´, Zeromskiego 116 Str.,
90-924 Łódz´, Poland; malgorzata.szczesio@p.lodz.pl
*
Correspondence: kgobis@gumed.edu.pl; Tel.: +48-58-349-16-47; Fax: +48-58-349-12-77
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 March 2018; Accepted: 20 April 2018; Published: 23 April 2018
Abstract: Compounds possessing benzimidazole system exhibit significant antituberculous activity.
In order to examine how structure modifications affect tuberculostatic activity, a series of benzazole
derivatives were synthesized and screened for their antitubercular activity. The compounds 1–20
were obtained by the reaction between o-diamine, o-aminophenol, or o-aminothiophenol with
carboxylic acids or thioamides. The newly synthesized compounds were characterized by IR,
1H-NMR, 13C-NMR spectra, and elemental analysis. Synthesized benzazoles were evaluated
for their tuberculostatic activity toward Mycobacterium tuberculosis strains. Quantum chemical
calculations were performed to study the molecular geometry and the electronic structure of
benzimidazoles GK-151B,
4
,
6, and benzoxazole 11, using the Gaussian 03W software (Gaussian,
Inc., Wallingford, CT, USA). Three-dimensional structure of benzimidazoles
1–3, MC-9, and GK-151B
was determined by ab initio calculation using Gamess-US software. The activity of the received
benzimidazoles was moderate or good. All of the benzoxazoles and benzothiazoles demonstrated
much lower activity. Benzoxazoles were less active by about 50 times, and benzothiazole by
100 times than the benzimidazole analogs. Quantum chemical calculations showed differences in the
distribution of electrostatic potential in the benzazole system of benzimidazoles and benzoxazoles.
Three-dimensional structure calculations revealed how the parity of the alkyl substituent at the C2
position impacts the activity. Benzimidazole system is essential for the antituberculosis activity that
is associated with the presence of the imine nitrogen atom in N-1 position. Its replacement by an
oxygen or sulfur atom results in a decrease of the activity. The parity of the alkyl substituent at the
C-2 position also modifies the activity.
Keywords: benzazole; pyridine; synthesis; tuberculostatic activity; SAR-study; quantum
chemical calculations
1. Introduction
Tuberculosis is a disease caused by the presence of Mycobacterium tuberculosis complex in humans
or animals. This group includes M. tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG,
Mycobacterium africanum, and Mycobacterium microti. Tuberculosis is transmitted via droplets from
one person to another. The disease most commonly affects the lungs, but it can also involve pleura,
lymph nodes, bones and joints, central nervous system, and gastrointestinal tract [1]. Tuberculous
Molecules 2018, 23, 985; doi:10.3390/molecules23040985
www.mdpi.com/journal/molecules

Molecules 2018, 23, 985
2 of 15
meningitis is highly dangerous for children and people with untreated human immunodeficiency
virus infection. It causes death or disability of about half of the patients [ ]. Bacteria M. tuberculosis
initially are present in the lungs, they later penetrate into the brain causing damage [ ]. Infections
with bacteria of the Mycobacterium genus are a concern not only in developing countries, but are a
global problem [ ]. This results in a continuous search for new drugs and diagnostics, as well as the
2,3
4
5
formation of health programs.
Many studies support the effect of a heterocyclic moiety with the nitrogen atom on tuberculostatic
activity. Such systems include chemotherapeutic agents used clinically, such as isoniazid (INH) and
pyrazinamide (PZA) (Figure 1) [
6,7]. This fact led the researchers to synthesize a number of pyrazine
and pyridine derivatives [ ]. Compounds possessing other nitrogen heterocyclic systems, like the
8,9
benzimidazoles, were also obtained [10–13].
Figure 1. Structure and tuberculostatic activity of isoniazid (INH) and pyrazinamide (PZA).
In particular, derivatives that were substituted at the C-2 position with cyclohexylethyl or
cyclohexylpropyl moiety were pretty highly active. The compounds were tested against three strains
of M. tuberculosis: a standard strain called H37Rv and two strains isolated from patients, namely Spec.
210-resistant to p-aminosalicylic acid, isoniazid, rifampicin, and ethambutol and Spec. 192-sensitive to
all medications. Tuberculostatic activity of these compounds was expressed as minimum inhibitory
concentration (MIC) value in the range of 6.2–25 µg/mL [14].
The high activity of compounds with cyclohexylethyl substituent at the benzimidazole structure
encouraged us to synthesize other heterocyclic systems possessing that moiety. Among the compounds that
were obtained were derivatives of 1,3,4-oxadiazole-2-thione, 1,2,4-triazole-5-thione, and benzimidazole-like
systems. However, tuberculostatic activity was shown only by benzimidazole analogs. The most active
were naphtho[d]imidazole, imidazole[d]pyridine, and imidazole[d]phenazine. MIC values for those
compounds ranged from 1.5 to 3.1 µg/mL. All of the obtained compounds exhibited higher activity than
the pyrazinamide, a first line antituberculosis drug [15].
Subsequent studies gave derivatives of 2-(2-cyclohexylalkyl)-1H-benzo[d]imidazole. Among them,
there were the most active compounds that contained two methyl groups in C-5 and C-6 positions of
the benzimidazole system (Figure 2). The MIC values of these compounds were 0.8–6.2
At the same time, the compounds exhibited low cytotoxicity against human epidermal neonatal
fibroblasts (ATCC PCS-201-010) [15 17]. Thus, because of their high tuberculostatic activity and
µg/mL [16,17].
,
low cytotoxicity these compounds were considered as very good candidates for tuberculosis drugs,
although the mechanism of their action remained unknown.
It inspired further studies on the effect of replacing one of the nitrogen atoms of the benzimidazole
by an oxygen or sulfur atom on tuberculostatic activity. In particular, the antituberculosis activity of
some benzoxazoles [18–22] and benzothiazoles [23–26] has been described previously by other authors.
The purpose of this study was to obtain derivatives of 1H-benzo[d]imidazole, benzo[d]oxazole,
and benzo[d]thiazole that were substituted at the C-2 and C-5 positions. The receipt of these compounds
used cyclization reactions of carboxylic acids, 4-substituted pyridine-2-carbothioamide or picolinonitrile
with o-phenylenediamines, o-aminophenols, or o-aminothiophenols. Substituent in the C-2 position
differed in the length of the alkyl chain, in the presence of cyclic ring and in their saturation.

Molecules 2018, 23, 985
3 of 15
Figure 2. Structure and tuberculostatic activity of some benzimidazole derivatives.
C-5 position of the benzazole system occurred in bromine, chlorine or fluorine atom, and in the
methyl group or trifluoromethyl group. Substrates have been selected in such a way that the resulting
products were derivatives or structural analogs of benzimidazoles, for which the highest results of
tuberculostatic activity have been determined [16]. Carboxylic acids with different chain lengths were
used to determine its effect on potency. We wanted to determine, how the exchange of the NH group
in position 1 of the benzimidazole system by an oxygen or sulfur atom would affect tuberculostatic
activity. In addition, we wanted to verify the effect of the presence of various cyclic substituents,
such as cyclohexyl, pyridyl, and phenyl rings on the antitubercular activity, and to identify a cyclic
ring that promotes the occurrence of tuberculostatic activity.
2. Results and Discussion
2.1. Chemistry
Initially, benzimidazoles 1–8 were obtained using the method that was previously developed
by our team [16]. The starting materials were fused at 160–180 ◦C without the presence of a solvent
(Scheme 1). The applied method provided products in good or very good yields of up to 46–80%.
Whereas, benzimidazole
for 12 h.
9
was obtained by heating it in the presence of polyphoshoric acid at 160 ◦C
Subsequently, the synthesis of benzoxazoles 10–11 was performed. The starting materials reacted
in the environment of polyphosphoric acid at a temperature of 160 ◦C. Reactions were performed for
4 h. The yield of the reaction varied widely, ranging from 47% to 77%.
Then, the benzoxazole derivatives 12–13 were obtained. 2-Amino-4-chlorophenol was used
in their synthesis by the method of Kaul, which had previously been used for the synthesis of
benzimidazoles. Contrary to the claims by the authors about the cyclization of reagents to the
benzimidazole or benzoxazole system, only acid amides were obtained [27]. The amides were
then heated with polyphosphoric acid (PPA) analogously for cyclization as compounds 10–11.
The compounds were obtained with a yield of 25–71%.
For the synthesis of compounds 14–16, a method that was developed by researchers at the
University of Shiraz was used [28]. 2-Amino-4-chlorothiophenol or 2-amino-4-trifluoromethylthiophenol
and the appropriate carboxylic acid with methanesulfonic acid (MSA) and the silica gel were heated at
140 ◦C for 72 h. Under these conditions, the yields of intended compounds were 17–65%.

Molecules 2018, 23, 985
4 of 15
Scheme 1. Synthesis of benzimidazoles 1–9, benzoxazoles 10–13, and benzothiazoles 14–16.
Thioamide derivatives were used to obtain benzimidazoles possessing pyridine moiety 17–20
(Scheme 2). When 4,5-dimethylbenzene-1,2-diamine was heated with the appropriate thioamide in the
presence of ethylene glycol for 2 h, pyridine derivatives were obtained with a good yield in the range
70–94%.
Scheme 2. Synthesis of 2-pyridin-2-yl-1H-benzoimidazole derivatives 17–20.
All if the newly synthesized compounds as well as already known [29
elemental analysis, IR, 1H-NMR, and 13C-NMR spectra.
–31] were characterized by
2.2. Tuberculostatic Activity
The obtained benzazole derivatives 1–20 were evaluated for their in vitro tuberculostatic activity
against the M. tuberculosis H37Rv strain and a “wild” strain isolated from tuberculosis patients
(Spec. 210) resistant to p-aminosalicylic acid (PAS), INH, ethambutol (ETB), and rifampicin (RMP).
The MIC values were determined as the minimum concentration inhibiting the growth of tested
tuberculosis strains in relation to the probe with no tested compound. INH, PZA, and RMP were used
as reference drugs. The results were summarized in Table 1.

Molecules 2018, 23, 985
5 of 15
Table 1. In vitro tuberculostatic activity of compounds 1–20.
MIC a [µg/mL (µM)]
M. tuberculosis c
MIC a [µg/mL (µM)]
M. tuberculosis c
Compd.
Compd.
H37Rv
Spec. 210
H37Rv
Spec. 210
1
2
3
4
5
6
7
8
9
3.1 (13)
6.25 (25)
1.5 (5)
3.1 (11)
25 (96)
50 (190)
25 (89)
>100 (>409)
50 (225)
25 (91)
3.1 (13)
6.25 (25)
1.5 (5)
3.1 (11)
12.5 (48)
25 (95)
12.5 (44)
>100 (>409)
50 (225)
25 (91)
50 (194)
50 (190)
13
14
15
16
17
18
19
20
100 (361)
100 (358)
100 (341)
100 (306)
12.5 (56)
12.5 (43)
25 (81)
12.5 (33)
0.125 (0.91)
25 (203)
100 (361)
100 (358)
100 (341)
100 (306)
12.5 (56)
25 (86)
25 (81)
12.5 (33)
12.5 (91)
>400 (3249)
2.5 (2.8)
INH b
PZA
RMP
10
11
12
50 (194)
50 (190)
1.2 (1.4)
a
The values obtained for the most potent compounds are marked in bold. Minimum inhibitory concentrations for
b
mycobacterial strains was determined by two-fold classical test-tube method of successive dilution. INH isoniazid;
PZA pyrazinamide; RMP rifampicin; c M. tuberculosis H37Rv, Spec. 210.
The resulting benzimidazoles
(3.1–100 g/mL equivalent to 10–409
linker (
activity with MIC values that were ranging from 1.5 to 3.1
odd number of carbon atoms ( ) the activity is more than two fold lower (MIC 6.25–25
1
–
9
µ
were characterized by very good to weak tuberculostatic activity
M). The studies revealed that benzimidazole derivatives without
µ
1
) or with even aliphatic chain (
3,
4
) at the C-2 position exhibited the highest antitubercular
g/mL (5–13 M). If the linker contains an
M). Replacement
µ
µ
2
µ
of the cyclohexyl ring to the aliphatic chain does not cause significant changes in the activity.
As we have observed previously, compounds that contained two methyl groups at the C-5 and
C-6 positions demonstrated higher potency. Benzimidazole
showed lower activity (MIC 12.5–25 g/mL equivalent 95–190
the phenyl ring ( ) results in a complete loss of activity. The results of the biological tests that were
obtained for the group of benzimidazole analogs were much lower than the results of corresponding
benzimidazoles [16 17]. Benzoxazoles 10 11 possessing methyl group at C-5 and C-6 position showed
lower activity. These compounds were active at the level corresponding to MIC value of 25–50 g/mL
(91–194 M). Benzoxazole 11, which is an analog of the previously described benzimidazole GK-151B
that exhibits a significant selectivity against M. tuberculosis [16], showed much lower activity. A weaker
activity was also exhibited by benzoxazoles 12 13 substituted by a chlorine atom in the same position
with MICs 50–100 g/mL (190–389 M). Benzothiazoles 14 16 also showed the same level of activity.
Although the antitubercular activity of benzimidazoles 17 20 possessing pyridine ring is moderate
(MIC 12.5–25 g/mL), they exhibit lower MIC value than the derivatives with alkyl chain or cyclohexyl
ring. By comparing the tuberculostatic activity of compounds that differ in the type of the ring at
position C2, a significant increase in the activity was observed for derivative with cyclohexyl ring.
Compounds possessing aryl group ( 17) exhibited weaker activity (MICs 12.5–50 g/mL). However,
6
with high electronegative fluorine atom
µ
µM). The absence of substituents on
8
,
–
µ
µ
–
µ
µ
–
–
µ
1
9
,
µ
derivative 17 with pyridine ring showed four times higher antitubercular activity than the phenyl
derivative (9).
2.3. Quantum Chemical Calculations
Density functional theory calculations for structures of benzimidazole GK-151B, 4, 6,
and benzoxazole 11 were performed (Figure 3). Electrostatic potential surfaces were plotted while
employing 6-31G(d) basis set, using the computer software GaussView and Gaussian 03W. On the
contained maps the red region indicates the region of the greatest electron density, while the structural
fragment with the lowest electron density corresponds to the blue region. All of the tested benzimidazole

Molecules 2018, 23, 985
6 of 15
derivatives have a similarly distributed positive charge. The positive charge appears to be localized
mostly at the hydrogen atom that was connected with nitrogen atom in the N-1 position. In the case of
benzimidazole 6, strongly electronegative fluorine atom causes the mild effect on the charge distribution
in the ring. Benzoxazole 11 exhibits a significantly different charge distribution when compared to
benzimidazole derivatives. The high positive region does not occur.
Figure 3. Molecular electrostatic potential (MEP) maps on the isodensity surface calculated at the
B3LYP/6-31G(d).
Just the electronegative region that is caused by the presence of oxygen and nitrogen atoms was
observed. Table 2 shows the calculated value of dipole moment (
The lower tuberculostatic activity of benzimidazole as compared to the compound GK-151B may be
related to their higher value of LogP (5.95). The high value of LogP causes stronger interaction between
the cell membrane and impediments in reaching the molecular target [32 33]. The high dipole moment
(4.92 D) of compound could likely be a reason of poor penetration into cells [34]. While comparing the
µ) and the partition coefficient (log P).
4
,
6
last two magnitudes for compounds GK-151B and 11, we can conclude that benzoxazole with a smaller
dipole moment (0.93 D) and with the similar value of log P (5.02), assuming transport by free diffusion,
should penetrate into the cells faster than the benzimidazole. Thus, much lower activity of compound
11 is probably not due to its weak penetration into bacilli, but due to its poor interaction with the
molecular target. Analysis of calculated quantities and the charge distributions allow for implying that
the NH fragment is involved in the target binding and is essential for tuberculostatic activity.
Table 2. Calculated values of absolute energy (E), dipole moment (
benzimidazoles GK-151B, 4, 6, and benzoxazole 11.
µ) and partition coefficient (log P) of
Compd.
MIC (µg/mL)
µ (Debye)
log P
GK-151B
0.75
3.1
25-50
50
3.28
3.57
4.92
0.93
4.95
5.95
5.13
5.02
4
6
11
The three-dimensional structure of benzimidazoles 1–3, GK-151B, and MC-9 was determined
to check how the parity of aliphatic chain at C2 position affect the antitubercular activity. Quantum
chemical calculations were performed using the DFT/M06-2X/6-311G(d,p) method that is available
in the software Gamess-US. Calculations were conducted for five compounds with a length of linker
n = 0–4. A similar arrangement of molecules in the space for compounds possessing even linker has
been observed (1, 3, GK-151B), while compounds having an odd chain are arranged in a different way
(2,
MC-9) (Figure 4). Comparison of the obtained results with antimycobacterial activity confirmed

Molecules 2018, 23, 985
7 of 15
our theory that the lack of a linker or the presence of an even aliphatic chain at the C2 position is
important for increasing the antitubercular activity. Performed studies suggest that the substituent at
the C2 position of the benzimidazole ring participates in the process of adjusting to the cell target.
Figure 4. Spatial arrangement of benzimidazoles with a length of linker n = 0–4.
3. Materials and Methods
3.1. Chemistry
All of the materials and solvents were of analytical reagent grade. Thin-layer chromatography
was performed on Merck (Darmstadt, Germany) silica gel 60F254 plates and visualized with UV.
The stationary phase for column chromatography was silica gel (60 Å, 70–230 mesh). The results of
elemental analyses (%C, H, N) for all of the obtained compounds that were determined on Perkin-Elmer
PE 2400 Series II CHNS analyzer (Perkin-Elmer, Shelton, CT, USA) were in agreement with the
1
calculated values within
±
0.4% range. H- and 13C-NMR spectra in CDCl3 or DMSO-d6 were recorded
on Varian Unity Plus (500 MHz) and Varian Gemini (200 MHz) instruments (Varian, Palo Alto, CA,
USA). 1H-NMR spectra of selected compounds can be found in Supplementary Materials. IR Spectra
(KBr) were determined as KBr pellets of the solids on a Satellite FT-IR spectrophotometer (Mattson
Instruments, Madison, WI, USA). Melting points were determined on a Stuart SMP30 apparatus and
were uncorrected (Bibby Scientific, Staffordshire, UK).
3.1.1. General Procedure for the Synthesis of Benzimidazoles 1–8
Appropriate carboxylic acid (5.5 mmol) and appropriate diamine (5 mmol) were heated on Wood’s
metal bath at 160–180 ◦C for 1 h. While cooling down. A 10% NaOH solution (10 mL) was added and
the reaction mixture was stirred at room temperature for 24 h. Then, the precipitated benzimidazoles
were filtered off, washed with water to the neutral reaction, dried, and purified by recrystallization
from the methanol—water mixture 2:1 with the addition of activated carbon.
2-Cyclohexyl-5,6-dimethyl-1H-benzo[d]imidazole (1). Starting from cyclohexanecarboxylic acid (0.704 g)
and 4,5-dimethylbenzene-1,2-diamine (0.680 g), the title compound
1
was obtained as beige crystals
N–H), 1449 ( C=C), 1307 ( C–C),
1.21–1.78 (m, 8H, 4CH2), 1.95–1.98 (m, 2H, CH2),
◦
(0.812 g, 71%): m.p. 226–228 C; IR (KBr): 2924, 2855 (
ν
C–H), 1539 (
δ
ν
ν
848 (γ
C–H) cm−1; 1H-NMR (500 MHz, DMSO-d6):
δ
2.25 (s, 6H, 2CH3), 2.74–2.78 (m, 1H, CH), 7.19 (s, 2H, ArH), 11.50 (br s, 1H, NH) ppm; Anal. Calcd. for
C15H20N2 (228.16): C, 78.90; H, 8.83; N, 12.27; Found: C, 79.17; H, 8.90; N, 12.34 [29].
2-(Cyclohexylmethyl)-5,6-dimethyl-1H-benzo[d]imidazole (
and 4,5-dimethylbenzene-1,2-diamine (0.680 g), the title compound
(0.784, 65%): m.p. 196–198 ◦C; IR (KBr): 2292, 2849 (
C–H), 1543 (
C–C), 1003 ( C–H), 843 (
C–H) cm−1; 1H-NMR (500 MHz, DMSO-d6):
and CH), 1.57–1.79 (m, 6H, 3CH2), 2.25 (s, 6H, 2CH3), 2.55 (d, 2H, CH2, J = 7 Hz), 7.18 (s, 2H, ArH),
2
). Starting from 2-cyclohexylacetic acid (0.782 g)
was obtained as beige crystals
N–H), 1448, 1410 ( C=C), 1300
0.91–1.21 (m, 5H, 2CH2
2
ν
δ
ν
(ν
δ
γ
δ

Molecules 2018, 23, 985
8 of 15
11.50 (br s, 1H, NH) ppm; Anal. Calcd. for C16H22N2 (242.18): C, 79.29; H, 9.15; N, 11.56; Found: C,
79.03; H, 8.99; and, N, 11.41.
2-(4-Cyclohexylbutyl)-5,6-dimethyl-1H-benzo[d]imidazole (3). Starting from 5-cyclohexylpentanoic acid
(1.056 mL) and 4,5-dimethylbenzene-1,2-diamine (0.680 g), the title compound
3
was obtained as beige
N–H), 1463, 1447 ( C=C),
C–H) cm−1; 1H-NMR (500 MHz, DMSO-d6):
0.76–0.83 (m, 2H,
◦
crystals (1.104 g, 78%): m.p. 112–113 C; IR (KBr): 2922, 2850 (
ν
C–H), 1539 (
δ
ν
1308 (
ν
C–C), 1020, 999 (
δ
C–H), 852 (
γ
δ
CH2), 1.05–1.29 (m, 8H, 4CH2), 1.56–1.71 (m, 7H, 3CH2 and CH), 2.25 (s, 6H, CH3), 2.71 (t, 2H, CH2,
J = 7 Hz), 7.18 (s, 2H, ArH), 11.90 (br s, 1H, NH) ppm; Anal. Calcd. for C19H28N2 (284.23): C, 80.23; H,
9.92; N, 9.85; Found: C, 79.97; H, 10.02; and, N, 9.71.
5,6-Dimethyl-2-nonyl-1H-benzo[d]imidazole (
benzene-1,2-diamine (0.680 g), the title compound
71–73 ◦C; IR (KBr): 2927, 2851 (
C–H), 1542 ( N–H), 1466, 1411 (
854, 724 ( 0.80–0.86 (m, 3H, CH3), 1.22 (s, 12H, 6CH2),
C–H) cm−1; 1H-NMR (200 MHz, DMSO-d6):
4
). Starting from decanoic acid (0.946 g) and 4,5-dimethyl-
was obtained as beige crystals (1.091 g, 80%): m.p.
C=C), 1307 ( C–C), 998 ( C–H),
4
ν
δ
ν
ν
δ
γ
δ
1.68–1.74 (m, 2H, CH2), 2.26 (s, 6H, 2CH3), 2.73 (t, 2H, CH2, J = 7 Hz), 7.15 (s, 1H, ArH), 7.25 (s, 1H,
ArH), 11.89 (s, 1H, NH) ppm; Anal. Calcd. for C18H28N2 (272.23): C, 79.36; H, 10.36; N, 10.28; Found:
C, 79.47; H, 10.45; and, N, 10.16.
5-Methyl-2-nonyl-1H-benzo[d]imidazole (
1,2-diamine (0.610 g), the title compound
74–75 ◦C; IR (KBr): 2921, 2852 (
C–H), 1547 (
cm−1; 1H-NMR (200 MHz, DMSO-d6):
0.81–0.86 (m, 3H, CH3), 1.22 (s, 12H, 6CH2), 1.73–1.76 (m, 2H,
5
). Starting from decanoic acid (0.946 g) and 4-methylbenzene-
was obtained as brown crystals (0.801 g, 62%): m.p.
N–H), 1448, 1421 ( C=C), 1281 ( C–C), 803 ( C–H)
5
ν
δ
ν
ν
γ
δ
CH2), 2.37 (s, 3H, CH3), 2.75 (t, 2H, CH2, J = 7 Hz), 6.88 (d, 1H, ArH, J = 8 Hz), 7.22–7.33 (m, 2H, ArH),
12.00 (s, 1H, NH) ppm; Anal. Calcd. for C17H26N2 (258.21): C, 79.02; H, 10.14; N, 10.84; Found: C,
79.15; H, 10.40; and, N, 11.00 [30].
5-Fluoro-2-nonyl-1H-benzo[d]imidazole (
1,2-diamine (0.630 g), the title compound
59–60 ◦C; IR (KBr): 2923, 2852 (
C–H), 1539 (
843, 801 (
C–H) cm−1; 1H-NMR (200 MHz, DMSO-d6):
6
). Starting from decanoic acid (0.946 g) and 4-fluorobenzene-
was obtained as light beige crystals (0.732 g, 56%): m.p.
N–H), 1487, 1456, 1421 ( C=C), 1139, 958 ( C–H),
0.80–0.83 (m, 3H, CH3), 1.22–1.26 (m, 12H,
6
ν
δ
ν
δ
γ
δ
6CH2), 1.70–1.73 (m, 2H, CH2), 2.77 (t, 2H, CH2, J = 7 Hz), 6.89–6.98 (m, 1H, ArH), 7.24–7.41 (m, 2H,
ArH), 12.27 (s, 1H, NH) ppm; Anal. Calcd. for C16H23N2F (262.18): C, 73.25; H, 8.84; N, 10.68; Found:
C, 73.28; H, 9.06; and, N, 10.59.
5-Chloro-2-nonyl-1H-benzo[d]imidazole (
1,2-diamine (0.713 g), the title compound
66–68 ◦C; IR (KBr): 2953, 2923 (
C–H), 1540 (
C–H), 854, 801 ( δ
C–H) cm−1; 1H-NMR (200 MHz, DMSO-d6):
7
). Starting from decanoic acid (0.946 g) and 4-chlorobenzene-
was obtained as light brown crystals (0.896 g, 64%): m.p.
N–H), 1468, 1447, 1413 ( C=C), 1292 ( C–C), 924
0.80–0.83 (m, 3H, CH3), 1.22 (s, 12H,
7
ν
δ
ν
ν
(δ
γ
6CH2), 1.70–1.73 (m, 2H, CH2), 2.78 (t, 2H, CH2, J = 7 Hz), 7.10 (d, 1H, ArH, J = 8 Hz), 7.39–7.51 (m, 2H,
ArH), 12.35 (s, 1H, NH) ppm; Anal. Calcd. for C16H23N2Cl (278.15): C, 68.92; H, 8.31; N, 10.05; Found:
C, 69.04; H, 8.36; and, N, 9.85 [30].
2-Nonyl-1H-benzo[d]imidazole (
(0.540 g), the title compound
IR (KBr): 2924, 2852 ( C–H), 1541 (
1H-NMR (200 MHz, DMSO-d6):
0.81–0.84 (m, 3H, CH3), 1.23–1.27 (m, 12H, 6CH2), 1.71–1.78 (m, 2H,
8
). Starting from decanoic acid (0.946 g) and benzene-1,2-diamine
was obtained as light brown crystals (0.564 g, 46%): m.p. 124–125 ◦C;
N–H), 1454, 1421 ( C=C), 1271 ( C–C), 752, 739 (
C–H) cm−1
8
ν
δ
ν
ν
γ
;
δ
CH2), 2.78 (t, 2H, CH2, J = 7 Hz), 7.03–7.14 (m, 2H, ArH), 7.43–7.45 (m, 2H, ArH), 12.16 (s, 1H, NH)
ppm; Anal. Calcd. For C16H24N2 (244.19): C, 78.64; H, 9.90; N, 11.46; Found: C, 78.69; H, 10.14; and, N,
11.48 [30].

Molecules 2018, 23, 985
9 of 15
3.1.2. General Procedure for Synthesis of Benzimidazole 9
Benzoic acid (1.1 mmol, 0.134 g) and 1,2-dia◦mine-4,5-dimethylbenzen (1 mmol, 0.136 g) and
PPA (2 g) were heated in the oil bath at the 160 C for 12 h. While cooling down, a 10% NaOH
solution (5 mL) was added. The precipitated product was filtered off, washed with water to the neutral
reaction, dried, and purified by recrystallization from the dioxin—water mixture 1:1 with the addition
of activated carbon.
5,6-dimethyl-2-phenyl-1H-benzo[d]imidazole (
(0.162 g, 73%) m.p. 250–252 C; IR (KBr): 3057 (
(ν C=N), 1493 (ν C=C), 730 (γ δ
C–H) cm−1; 1H-NMR (500 MHz, DMSO-d6 + D2O):
9
). The title compound was obtained as beige crystals
N–H), 2973, 2925 ( C–H), 1599 ( N–H), 1560
2.31 (s, 6H, CH3),
7.37 (s, 2H, ArH), 7.46–7.54 (m, 3H, ArH), 8.13 (d, 2H, ArH, J = 7 Hz) ppm; 13C-NMR (125 MHz,
DMSO-d6): 20.49 (2C), 126.76 (2C), 129.03 (2C), 129.38 (2C), 129.72 (2C), 130.17, 131.35, 133.33, 150.56,
◦
ν
ν
δ
δ
167.79 ppm; Anal. Calcd. for C15H14N2 (222.12): C, 81.05; H, 6.35; N, 12.60; Found: C, 81.36; H, 6.09;
and, N, 12.24 [29].
3.1.3. General Procedure for Synthesis of 4,5-Dimethylbenzoxazoles 10, 11
Appropriate carboxylic acid (5.5 mmo◦l), 2-amino-4,5-dimethylphenol (5 mmol, 0.690 g) and PPA
(10 g) were heated in the oil bath at 160 C for 4 h. While cooling down, a 10% NaOH solution
(10 mL) was added. The mixture was extracted with benzene (3
evaporated. Crude oily products were purified by column chromatography with methylene chloride
×
30 mL), dried with MgSO4, and
(9) or chloroform (10) as the eluent.
5,6-Dimethyl-2-nonylbenzo[d]oxazole (10). Starting from decanoic acid (0.946 g), the title compound 10
was obtained as light beige crystals (1.044 g, 76%): m.p. 25–26 ◦C; IR (KBr): 2925, 2854 (
ν
C–H), 1574
C–H) cm−1; 1H-NMR (500 MHz,
0.82 (t, 3H, CH3, J = 7 Hz), 1.20–1.24 (m, 12H, 7CH2), 1.71–1.74 (m, 2H, CH2), 2.26 (s, 3H,
CH3), 2.28 (s, 3H, CH3), 2.84 (t, 2H, CH2, J = 7 Hz), 7.39 (s, 2H, ArH) ppm; 13C-NMR (125 MHz,
DMSO-d6): 14.61, 20.31, 20.57, 22.78, 26.85, 28.37, 29.12, 29.31, 29.33, 29.53, 31.94, 111.23, 119.85, 133.02,
(δ
N–H), 1466 (ν C=C), 1282 (ν C–O–Ar), 1148 (δ C–H), 868, 849 (γ
DMSO-d6):
δ
δ
133.83, 139.76, 149.50, 166.69 ppm; molecular weight for C18H27NO (273.21); and, MS (+): m/z = 274
(100%, [M + H]+), m/z = 275 (20%, [M + 2H]+);
2-(2-Cyclohexylethyl)-5,6-dimethylbenzo[d]oxazole (11). Starting from 3-cyclohexylpropionic acid (0.942 mL),
the title compound 11 was obtained as light beige solid (0.869 g, 68%): m.p. 49–51 ◦C; IR (KBr): 2923,
2850 (
1H-NMR (500 MHz, DMSO-d6):
3CH2 and CH), 2.27 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.86 (t, 2H, CH2, J = 7.8 Hz), 7.40 (s, 2H, ArH) ppm;
13C-NMR (125 MHz, DMSO-d6):
20.3, 20.6, 26.0, 26.4 (2C), 26.7, 33.1 (2C), 34.3, 37.2, 111.3, 119.9, 133.1,
ν
C–H), 1572 (
ν
C=N), 1464, 1451 (
ν C=C), 1276 (ν C–O–Ar), 1146 (δ C–H), 871 (γ ;
C–H) cm−1
δ
0.87–0.89 (m, 2H, CH2), 1.12–1.16 (m, 4H, 2CH2), 1.62–1.72 (m, 7H,
δ
133.9, 139.8, 149.5, 167.0 ppm; Anal. Calcd. for C17H23NO (257.18): C, 79.33; H, 9.01; N, 5.44; Found: C,
79.34; H, 9.29; and, N, 5.12 [31].
3.1.4. General Procedure for the Synthesis of 5-Chlorobenzoxazoles 12, 13
Into the pressure equalizing dropping funnel 5 mL of anhydrous toluene, 1 mL of anhydrous
DMF (13 mmol) and 0.8 mL of thionyl chloride (11 mmol) were poured. After 5 min, the separated
lower layer (N,N-dimethylchlorosulfitemethaniminium chloride) was added drop wise to a solution
of the carboxylic acid (5 mmol) in 10 mL of anhydrous methylene chloride. After a further 15 min,
2-amino-4-chlorophenol (5 mmol, 0.718 g) was scattered to a solution followed by drop wise adding
of 6 mL of methylene chloride and 2.5 mL of anhydrous pyridine (15 mmol) mixture. The solution
was stirred at room temperature for 18 h. The solution was washed with distilled water (3
and dried over anhydrous MgSO4. The drying agent was filtered off and the solvent evaporated.
The resulting amide was cyclized without further purification by adding PPA (10 g) and heating on
×
15 mL)

Molecules 2018, 23, 985
10 of 15
an oil bath at 160 ◦C for 4 h. Then, cooled and basified with 10% NaOH. The alkalized solution was
extracted with benzene (3 30 mL). The combined organic layers were dried over anhydrous MgSO4.
×
The drying agent was filtered off and the solvent evaporated. The resulting product was purified by
column chromatography eluting with chloroform (12) or methylene chloride (13).
5-Chloro-2-(2-cyclohexylethyl)benzo[d]oxazole (12). Starting from 3-cyclohexylpropionic acid (0.857 mL),
◦
the title compound 12 was obtained as colourless crystals (0.401 g, 30%): m.p. 43–44 C; IR (KBr):
3096–3047 (
ν
C–H), 2925, 2852 (
γ
ν
C–H), 1567 (
ν
C=N), 1453, 1426 (
ν C=C), 1257 (ν C–O–Ar), 1051
1
(ν
C–Cl), 810 (
C–H) cm−1; H-NMR (200 MHz, DMSO-d6):
δ
0.87–1.29 (m, 6H, 3CH2), 1.63–1.74
(m, 7H, 3CH2 and 1H CH), 2.94 (t, 2H, CH2, J = 7 Hz), 7.36–7.42 (m, 1H, ArH), 7.68–7.79 (m, 2H, ArH)
ppm; Anal. Calcd. for C15H18ClNO (263.11): C, 68.30; H, 6.88; N, 5.31; Found: C, 68.30; and, H, 6.73; N,
5.26 [3].
5-Chloro-2-(3-cyclohexylpropyl)benzo[d]oxazole (13). Starting from 4-cyclohexylbutyric acid (0.850 g), the
title compound 13 was obtained as rufous crystals (0.943 g, 68%): m.p. 33–35 ◦C; IR (KBr): 2923, 28−510
;
(
ν
C–H), 1568 (
ν
C=N), 1451 (
ν
C=C), 1256 (
ν
C–O–Ar), 1168 (
δ
C–H), 1054 (
ν
C–Cl), 801 (
γ
C–H) cm
1H-NMR (200 MHz, CDCl3):
δ 0.90–1.45 (m, 8H, 4CH2), 1.67–1.90 (m, 7H, 3CH2 and 1H CH), 2.89
(t, 2H, CH2, J = 8 Hz), 7.23–7.64 (m, 3H, ArH) ppm; Anal. Calcd. for C16H20ClNO (277.12): C, 69.18; H,
7.26; N, 5.04; Found: C, 68.98; H, 7.63; and, N, 5,30.
3.1.5. General Procedure for the Synthesis of Benzothiazoles 14, 15
2-Amino-4-chlorothiophenol (5 mmol, 0.798 g) and the appropriate carboxylic acid (5 mmol) were
heated with silica gel (1.5 g) and methanesulfonic acid (5 mL) at 140 ◦C for 72 h. After this time, the
cooled reaction mixture was diluted with 100 mL of ethyl acetate and silica gel was filtered off by
gravity. The solution was washed with 5% NaHCO3 (3
×
30 mL) until alkaline. The organic layer
was dried over anhydrous MgSO4. The drying agent was filtered off and the solvent evaporated.
The resulting product was purified by column chromatography eluting with chloroform (14) or
methylene chloride (15).
5-Chloro-2-(2-cyclohexylethyl)benzo[d]thiazole (14). Starting from 3-cyclohexylpropionic acid (0.857 mL),
the title compound 14 was obtained as green crystals (0.240 g, 17%): m.p. 43–45 ◦C; IR (KBr): 2923,
2851 (
DMSO-d6):
ArH), 7.99–8.09 (m, 2H, ArH) ppm; 13C-NMR (50 MHz, DMSO-d6):
ν
C–H), 1587 (
1.31–1.76 (m, 13H, 12H 6CH2 and 1H CH), 3.11 (t, 2H, CH2, J = 8 Hz), 7.41–7.47 (m, 1H,
25.7 (2C), 26.0, 31.0, 32.4 (2C),
ν C=N), 1516, 1440 (ν C=C), 1074 (ν C–Cl), 799 (γ
C–H) cm−1; 1H-NMR (200 MHz,
δ
δ
36.4, 36.5, 121.5, 123.5, 124.8, 130.7, 133.3, 153.7, 174.7 ppm; Anal. Calcd. for C15H18ClNS (279.08):
C, 64.38; H, 6.48; N, 5.01; Found: C, 64.50; H, 6.49; N, 4.87.
5-Chloro-2-(3-cyclohexylpropyl)benzo[d]thiazole (15). Starting from 4-cyclohexylbutyric acid (0.850 g),
◦
the title compound 15 was obtained as colourless crystals (0.937 g, 64%): m.p. 42–46 C; IR (KBr): 2922,
2849 (
0.81–1.28 (m, 9H, 8H 4CH2 and 1H CH), 1.63–1.87 (m, 8H, 4CH2), 7.42–7.48 (m, 1H, ArH), 8.00–8.11
(m, 2H, ArH) ppm; 13C-NMR (50 MHz, DMSO-d6):
26.2 (2C), 26.5, 26.7, 33.1 (2C), 34.1, 36.6, 37.1,
ν C–H), 1517, 1436 (ν C=C), 1070 (ν C–Cl), 797 (γ
C–H) cm−1; 1H-NMR (200 MHz, DMSO-d6):
δ
δ
122.0, 123.9, 125.2, 130.4, 133.4, 154.1, 174.9 ppm; Anal. Calcd. for C16H20ClNS (293.10): C, 65.40; H,
6.86; N, 4.77; Found: C, 65.44; H, 6.86; and, N, 4.76.
3.1.6. General Method for the Synthesis of 5-Trifluoromethylbenzothiazoles 16
2-Amino-4-trifluoromethylthiophenol hydrochloride (5 mmol, 1.148 g) was dissolved in 30 mL of
distilled water and an equimolar amount of DBU (5 mmol, 0.745 mL) was added. The mixture was
stirred with magnetic stirrer for 1 h and was then extracted with chloroform (3
organic layers were dried over anhydrous MgSO4. The drying agent was filtered off and the solvent
×
10 mL). The combined

Molecules 2018, 23, 985
11 of 15
evaporated. To the free thiophenol, the appropriate carboxylic acid (5 mmol) was added and the
reagents were heated with silica gel (1.5 g) and methanesulfonic acid (5 mL) at 140 ◦C for 72 h, then
treated as in procedure described for benzothiazoles 14, 15, and purified by column chromatography
with chloroform as the eluent.
2-(3-Cyclohexylpropyl)-5-(trifluoromethyl) benzo[d]thiazole (16). Starting from 4-cyclohexylbutyric acid
(0.850 g), the title compound 16 was obtained as pink crystals (0.551 g, 34%): m.p. 33–35 ◦C; IR (KBr):
3056 (
813 (
3CH2 and 1H CH), 3.11 (t, 2H, CH2, J = 7 Hz), 7.71 (d, 1H, ArH, J = 9 Hz), 8.27–8.32 (m, 2H, ArH) ppm;
13C-NMR (50 MHz, DMSO-d6):
26.1 (2C), 26.5, 26.6, 27.6, 33.1 (2C), 34.1, 36.5, 37.1, 119.2, 121.1, 123.9,
ν
C–H), 2926, 2846 (
ν
C–H), 1509, 1422 (
ν
C=C), 1329 (
ν F3C–Ar), 1173, 1149, 1123 (ν C–F), 918,
γ
C–H) cm−1; 1H-NMR (200 MHz, DMSO-d6):
δ
0.79–1.27 (m, 8H, 4CH2), 1.42–1.88 (m, 7H, 6H
δ
127.0, 139.2, 152.7, 175.3 ppm; Anal. Calcd. for C17H20F3NS (327.13): C, 62.36; H, 6.16; N, 4.28; Found:
C, 62.48; H, 6.13; and, N, 4.16.
3.1.7. General Method for the Synthesis of 2-Pyridinebenzimidazoles 17–20
4-Substituted picolinothioamide (1 mmol) and the 4,5-dimethyldiamine (1.3 mmol) were refluxed
in ethylene glycol (3 mL) until hydrogen sulfide evolution ceased (3–5 h). Then, ice (20 g) was added
and the precipitated product was collected by filtration and purified by recrystallization with the
addition of activated carbon.
5,6-Dimethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole (17) Starting from pyridine-2-carbothioamide
(0.138 g), the title compound 17 was obtained as light brown crystals (0.156 g, 70%): m.p. 198–200 ◦C;
1
IR (KBr): 3373 (
(200 MHz, DMSO-d6):
J = 7 Hz), 8.29 (d, 1H, pyridine, J = 8 Hz), 8.70 (d, 1H, pyridine, J = 9 Hz), 12.87 (s, 1H, NH) ppm;
13C-NMR (50 MHz, DMSO-d6):
20.33 (2C), 112.00, 118,5, 121.40 (2C), 124.57 (2C), 137.65 (2C), 149.08,
ν
N–H), 2921 (
ν
C–H), 1593 (
δ
N–H), 1451 (
ν
C=C), 995 (
γ
C–H) cm−1; H-NMR
δ
2.37 (s, 6H, CH3), 7.34–7.50 (m, 3H, 2ArH and 1pyridine), 7.97 (d, 1H, pyridine,
δ
149.48 (2C), 150.15 ppm; Anal. Calcd. for C14H13N3 (223.11): C, 75.31; H, 5.87; N, 18.82; Found: C, 75.28;
H, 5.73; and, N, 18.43.
5,6-Dimethyl-2-(4-(pyrrolidin-1-yl)pyridin-2-yl)-1H-benzo[d]imidazole (18) Starting from 4-(pyrrolidin-
1-yl)pyridine-2-carbothioamide (0.207 g), the title compound 18 was obtained as white crystals (0.250 g,
◦
86%): m.p. 293–294 C; IR (KBr): 3050 (
ν
CAr–H), 2970, 2849 (
ν
C–H), 1606 (
δ N–H), 1513, 1458 (ν C=C),
1012, 985, 853 (
γ
C–H) cm−1; 1H-NMR (500 MHz, DMSO-d6):
δ
1.98 (t, 4H, CH2, J = 6 Hz), 2.29 (s, 6H,
CH3), 3.33–3.36 (m, 4H, CH2), 6.53 (dd, 1H, pyridine, J1 = 2 Hz, J2 = 3 Hz), 7.25 (s, 1H, ArH), 7.35
(d, 1H, pyridine, J = 2 Hz), 7.41 (s, 1H, ArH), 8.19 (d, 1H, pyridine, J = 6 Hz), 12.62 (s, 1H, NH) ppm;
Anal. Calcd. for C18H20N4 (292.19): C, 73.94; H, 6.89; N, 19.16; Found: C, 73.76; H, 6.87; and, N, 18.99.
4-(2-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)pyridin-4-yl)morpholine (19) Starting from 4-morpholinopyridine-
◦
2-carbothioamide (0.223 g), the title compound 19 was obtained as white solid (0.290 g, 94%): m.p. >300 C;
IR (KBr): 2963, 2854 (
ν
C–H), 1598 (
2.30 (s, 6H, CH3), 3.36–3.38 (m, 4H, CH2), 3.74 (t, 4H, CH2, J = 4 Hz), 6.92
δ N–H), 1489, 1445 (ν C=C), 1118 (δ C–H), 989, 946 (γ ;
C–H) cm−1
1H-NMR (500 MHz, DMSO-d6):
δ
(dd, 1H, pyridine, J1 = 2 Hz, J2 = 3 Hz), 7.26 (s, 1H, ArH), 7.41 (s, 1H, ArH), 7.70 (d, 1H, pyridine, J = 2 Hz),
8.30 (d, 1H, pyridine, J = 6 Hz), 12.69 (s, 1H, NH) ppm; Anal. Calcd. for C18H20N4O (308.16): C, 70.11; H,
6.54; 18.17; Found: C, 70.47; H, 6.32; and, N, 18.15.
5,6-Dimethyl-2-(4-(4-phenylpiperazin-1-yl)pyridin-2-yl)-1H-benzo[d]imidazole (20) Starting from 4-(4-
phenylpiperazin-1-yl)pyridine-2-carbothioamide (0.298 g), the title compound 20 was obtained as
light brown solid (0.350 g, 91%): m.p. 267–268 ◦C; IR (KBr): 3413 (
ν
N–H), 1598 (
2.31–2.34 (m, 6H, CH3), 3.39 (t, 8H, CH2,
J = 21 Hz), 6.81–7.46 (m, 8H, 6ArH and 2pyridine), 7.77 (s, 1H, ArH), 8.30 (d, 1H, pyridine, J = 5 Hz);
δ N–H), 1494, 1447
(ν C=C), 1228 (δ δ
C–H) cm−1; 1H-NMR (200 MHz, DMSO-d6):

Molecules 2018, 23, 985
12 of 15
12.75 (s, 1H, NH) ppm; Anal. Calcd. for C24H25N5 (383.21): C, 75.17; H, 6.57; N, 18.26; Found: C, 75.19;
H, 6.82; and, N, 17.87.
3.2. Antimycobacterial Activity Assay
The synthesized compounds were examined in vitro for their tuberculostatic activity against
the Mycobacterium tuberculosis H37Rv and “wild” strain that was isolated from tuberculosis patients
(Spec. 210) resistant to PAS, INH, ETB and RMP. M. tuberculosis strain H37RV ATCC 25618 was
purchased from LGC Standards (Middlesex, UK), which is an intermediary for the sale of reference
strains from the American Type Culture Colection (ATCC). Tuberculosis bacteria cultures and
sputum samples from tuberculosis positive patients, were cultured, stored, prepared, and used for
experiments in microbiological laboratory of National Institute of Tuberculosis and Lung Diseases
in Warsaw, Poland. Investigations were performed by a classical test-tube method of successive
dilution in Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine
serum [35,36]. Bacterial suspensions were prepared from 14 days old cultures of slowly growing strains
and from 48 h old cultures of saprophytic strains [37,38]. Solutions of compounds in ethylene glycol
were tested. Stock solutions contained 10 mg of compounds in 1 milliliter. Dilutions (in geometric
progression) were prepared in Youmans’ medium. The medium containing no investigated substances
and containing INH, PZA or RMP as reference drugs were used for comparison. Incubation was
performed at a temperature of 37 ◦C. The MIC values were determined as minimum concentration
inhibiting the growth of tested tuberculosis strains in relation to the probe with no tested compound.
The influence of the compound on the growth of bacteria at a certain concentration 3.1, 6.2, 12.5, 25, 50,
and 100 µg/mL were evaluated.
3.3. Quantum Chemical Calculations
Quantum chemical calculations were carried out to study the molecular geometry and electronic
structure of benzimidazoles GK-151B, 4, 6, and benzoxazole 11 using the Gaussian 03W software
(Gaussian 03, Revision A.1., Gaussian, Inc., Wallingford, CT USA) The full optimized geometries of
both compounds in vacuum were calculated by density functional theory-B3LYP method using diffuse
functions 6-31G(d) basis set. Three-dimensional structure of benzimidazoles 1–3, MC-9, and GK-151B
was determined by ab initio calculation using Gamess-US [39] software (Iowa State University, Ames,
IA, USA). DFT/M06-2X functional with 6-311G(d,p) basis set was used.
4. Conclusions
In conclusion, a series of 20 derivatives of benz[d]imidazole, benzo[d]oxazole, and benzo[d]thiazole
were successfully synthesized. Their tuberculostatic activity in vitro was evaluated against M. tuberculosis
H37Rv and Spec. 210 strains. The activity of the obtained benzimidazoles toward M. tuberculosis was
good to weak (3.1–100
activity than that was observed previously for their benzimidazole analogs. The determined MIC values
for tested compounds were in the range of 25–100 g/mL, while for the most active benzimidazole
analogs, it was at the level of 0.75 g/mL [16]. The quantum chemical calculations performed for
µg/mL). Whereas, the benzoxazoles and benzothiazoles exhibited much lower
µ
µ
selected benzazoles demonstrated significant differences in the distribution of electrostatic potential.
These results prove that benzimidazole system is essential for the antituberculosis activity that was
associated with the presence of the imine nitrogen atom in N-1 position. Its replacement by an oxygen
or sulfur atom results in a dramatic decrease of activity, which probably is a consequence of the weaker
interaction with the cell target. In addition, the parity of the alkyl substituent at the C-2 position
influences the spatial arrangement of the molecule. Compounds without linker or with even alkyl chain
exhibited higher antitubercular activity, which may be associated with better alignment to the cell target.
The cyclohexyl or pyridine ring promotes the occurrence of tuberculostatic activity, while no activity
was observed in the presence of phenyl ring.

Molecules 2018, 23, 985
13 of 15
Supplementary Materials: Supplementary Materials are available online.
Acknowledgments: This project was funded by the National Science Centre (Cracow, Poland) on the basis of
decision number DEC-2017/25/B/NZ7/00124.
Author Contributions: Malwina Krause performed the synthetic experiments and quantum chemical calculations,
analyzed the chemical and biological data. Katarzyna Gobis and Henryk Foks created the concept and designed
the study. Katarzyna Gobis coordinated the work and wrote the manuscript. Ewa Augustynowicz-Kopec´ and
Agnieszka Napiórkowska conducted the tuberculostatic assay. Małgorzata Szczesio carried out quantum chemical
calculations. All the authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Talbot, E.A.; Raffa, B.J. Mycobacterium tuberculosis. In Molecular Medical Microbiology, 2nd ed.; Academic
Press: Boston, MA, USA, 2015; Volume 3, pp. 1637–1653.
2.
Krauss, M.R.; Harris, D.R.; Abreu, T.; Ferreira, F.G.; Ruz, N.P.; Worrell, C.; Hazra, R. Tuberculosis in
HIV-infected infants, children and adolescents in Latin America. Braz. J. Infect. Dis. 2015, 19, 23–29.
[CrossRef] [PubMed]
3.
Post, F.A.; Grint, D.; Werlinrud, A.M.; Panteleev, A.; Riekstina, V.; Malashenkov, E.A.; Skrahina, A.;
Duiculescu, D.; Podlekareva, D.; Karpov, I.; et al. Multi-drug-resistant tuberculosis in HIV positive patients
in Eastern Europe. J. Infect. 2014, 68, 259–263. [CrossRef] [PubMed]
4.
5.
Thwaites, G.E.; van Toorn, R.; Schoeman, J. Tuberculous meningitis: more questions, still too few answer.
Lancet Neurol. 2013, 12, 999–1010. [CrossRef]
Van der Werf, T.S.; van der Graaf, W.T.A.; Tappero, J.W.; Asiedu, K. Mycobacterium ulcerans infection. Lancet
1999, 354, 1013–1018. [CrossRef]
6.
7.
8.
Isoniazid. Tuberculosis 2008, 88, 112–116. [CrossRef]
Pyrazinamide. Tuberculosis 2008, 88, 141–144. [CrossRef]
Olczak, A.; Główka, M.L.; Gołka, J.; Szczesio, M.; Bojarska, J.; Kozłowska, K.; Foks, H.; Orlewska, C.
Is planarity of pyridin-2-yl- and pyrazin-2-yl-formamide thiosemicarbazones related to their tuberculostatic
activity? X-ray structures of two pyrazine-2-carboxamide-N’-carbonothioyl-hydrazones. J. Mol. Struct. 2007
830, 171–175. [CrossRef]
,
9.
Olczak, A.; Szczesio, M.; Gołka, J.; Orlewska, C.; Gobis, K.; Foks, H.; Główka, M.L. Planarity of
heteroaryldithiocarbazic acid derivatives showing tuberculostatic activity. II. Crystal structures of
3-[amino(pyrazin-2-yl)methylidene]-2-methylcarbazic acid esters. Acta Cryst. C 2011, C67, o37–o42. [CrossRef]
[PubMed]
10. Kazimierczuk, Z.; Andrzejewska, M.; Kaustova, J.; Klimešova, V. Synthesis and antimycobacterial activity of
2-substitute halogenobenzimidazoles. Eur. J. Med. Chem. 2005, 40, 203–208. [CrossRef] [PubMed]
11. Shingalapur, R.V.; Hosamani, K.M.; Keri, R.S. Derivatives of benzimidazole pharmacophore: Synthesis,
anticonvulsant, antidiabetic and DNA cleavage studies. Eur. J. Med. Chem. 2010, 45, 1753–1759. [CrossRef]
[PubMed]
12. Jadhav, G.R.; Shaikh, M.U.; Kale, R.P.; Shiradkar, M.R.; Gill, C.H. SAR study of clubbed [1,2,4]-triazolyl with
fluorobenzimidazoles as antimicrobial and antituberculosis agents. Eur. J. Med. Chem. 2009, 44, 2930–2935.
[CrossRef] [PubMed]
13. Klimešova, V.; Kocˇi, J.; Waisser, K.; Kaustova, J. New benzimidazole derivatives as antimycobacterial agent.
Farmaco 2002, 57, 259–265. [CrossRef]
14. Foks, H.; Pancechowska-Ksepko, D.; Kuz´mierkiewicz, W.; Zwolska, Z.; Augustynowicz-Kopec´, E.;
Janowiec, M. Synthesis and tuberculostatic activity of new benzimidazole derivatives. Chem. Heterocycl. Compd.
2006, 42, 611–614. [CrossRef]
15. Gobis, K.; Foks, H.; Bojanowski, K.; Augustynowicz-Kopec´, E.; Napiórkowska, A. Synthesis of novel
3-cyclohexylpropanoic acid-derived nitrogen heterocyclic compounds and their evaluation for tuberculostatic
activity. Bioorg. Med. Chem. 2012, 20, 137–144. [CrossRef] [PubMed]
16. Gobis, K.; Foks, H.; Serocki, M.; Augustynowicz-Kopec´, E.; Napiórkowska, A. Synthesis and evaluation
of in vitro antimycobacterial activity of novel 1H-benzo[d]imidazole derivatives and analogues. Eur. J.
Med. Chem. 2015, 89, 13–20. [CrossRef] [PubMed]

Molecules 2018, 23, 985
14 of 15
17. Gobis, K.; Foks, H.; Suchan, K.; Augustynowicz-Kopec´, E.; Napiórkowska, A.; Bojanowski, K.
Novel 2-(2-phenalkyl)-1H-benzo[d]imidazoles as antitubercular agents. Synthesis, biological evaluation and
structure–activity relationship. Bioorg. Med. Chem. 2015, 23, 2112–2120. [CrossRef] [PubMed]
18. Koci, J.; Klimešova, V.; Waisser, K.; Kaustova, J.; Dahsec, H.M.; Mollmann, U. Heterocyclic benzazole
derivatives with antimycobacterial in vitro activity. Bioorg. Med. Chem. Lett. 2002, 12, 3275–3278. [CrossRef]
19. Klimešova, V.; Koci, J.; Waisser, K.; Kaustova, J.; Mollmann, U. Preparation and in vitro evaluation of
benzylsulfanyl benzoxazole derivatives as potential antituberculosis agents. Eur. J. Med. Chem. 2009, 44,
2286–2293. [CrossRef]
20. Zahajska, L.; Klimešova, V.; Koci, J.; Waisser, K.; Kaustova, J. Synthesis and antimycobacterial activity
of pyridylmethylsulfanyl and naphthylmethylsulfanyl derivatives of benzazoles, 1, 2, 4-triazole, and
pyridine-2-carbothioamide/-2-carbonitrile. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 549–555. [CrossRef]
[PubMed]
21. Arisoy, M.; Temiz-Arpaci, O.; Kaynak-Onurdag, F.; Ozgen, S. Antimycobacterial properties of some
2,5-disubstituted-benzoxazole derivatives. J. Fac. Pharm. Ankara 2010, 39, 155–162.
22. Pichota, A.; Duraiswamy, J.; Yin, Z.; Keller, T.H.; Alam, J.; Liung, S.; Lee, G.; Ding, M.; Wang, G.; Chan, W.L.;
et al. Peptide deformylase inhibitors of Mycobacterium tuberculosis: Synthesis, structural investigations, and
biological results. Bioorg. Med. Chem. Lett. 2008, 18, 6568–6572. [CrossRef] [PubMed]
0
23. Telvekar, V.N.; Bairwa, V.K.; Satardekar, K.; Bellubi, A. Novel N -benzylidene benzofuran-3-carbohydrazide
derivatives as antitubercular and antifungal agents. Bioorg. Med. Chem. Lett. 2012, 22, 2343–2346. [CrossRef]
[PubMed]
24. Sidoova, E.; Odlerova, Z. 2-Alkylthio-6-(3-nitrobenzoylamino)benzothiazoles and their antimycobacterial
activity. Chem. Pap. 1986, 40, 801–806.
25. Cho, Y.; Ioerger, T.R.; Sacchettini, J.C. Discovery of novel nitrobenzothiazole inhibitors for Mycobacterium
tuberculosis ATP phosphoribosyl transferase (HisG) through virtual screening. J. Med. Chem. 2008, 51,
5984–5992. [CrossRef] [PubMed]
26. Patel, R.V.; Patel, P.K.; Kumari, P.; Rajani, D.P.; Chikhalia, K.H. Synthesis of benzimidazolyl-1,3,4-
oxadiazol-2ylthio-N-phenyl (benzothiazolyl) acetamides as antibacterial, antifungal and antituberculosis
agents. Eur. J. Med. Chem. 2012, 53, 41–51. [CrossRef] [PubMed]
27. Kaul, S.; Kumar, A.; Sain, B.; Bhatnagar, A.K. Simple an convenient one-pot synthesis of benzimidazoles and
benzoxazoles using N,N-dimethylchlorosulfitemethaniminium chloride as condensing agent. Synth. Commun.
2007, 37, 2457–2460. [CrossRef]
28. Sharghi, H.; Asemani, O. Methanesulfonic acid/SiO2 as an efficient combination for the synthesis of
2-substituted aromatic and aliphatic benzothiazoles from carboxylic acids. Synth. Commun. 2009, 39,
860–867. [CrossRef]
29. Jerchel, D.; Fischer, H.; Kracht, M. Zur darstellung der benzimidazole. Justus Liebigs Annalen der Chemie 1952
575, 162–173. [CrossRef]
,
30. Martinez-Palou, R.; Zepeda, L.G.; Hoepfl, H.; Montoya, A.; Guzman-Lucero, D.J.; Guzman, J. Parallel and
automated library synthesis of 2-long alkyl chain benzoazoles and azole[4,5-b]pyridines under microwave
irradiation. Mol. Divers 2005, 9, 361–369. [CrossRef] [PubMed]
31. Yalcin, I.; Oren, I.; Sener, E.; Akin, A.; Ucarturk, N. The synthesis and the structure-activity relationships of
some substituted benzoxazoles, oxazolo(4,5-b)pyridines, benzothiazoles and benzimidazoles as antimicrobial
agents. Eur. J. Med. Chem. 1992, 27, 401–406. [CrossRef]
32. Di, A.L.; Kerns, E.H. Profiling drug-like properties in discovery research. Curr. Opin. Chem. Biol. 2003, 7,
402–408. [CrossRef]
33. Di, L.; Kerns, E.H. Lipophilicity. In Drug-Like Properties: Concepts, Structure Design and Methods from ADME to
Toxicity Optimization, 2nd ed.; Academic Press: Boston, MA, USA, 2016; Volume 5, pp. 43–47.
34. Oyaga, S.C.; Valdes, J.C.; Paez, S.B.; Marquez, K.H. DFT description of intermolecular forces between
9-aminoacridines and DNA base pairs. J. Theor. Comput. Chem. 2013, 2013. [CrossRef]
35. Youmans, G.P. Test tube evaluation of tuberculostatic agents. Am. Rev. Tuberc. 1947, 56, 376. [PubMed]
36. Youmans, G.P.; Youmans, A.S. A method for the determination of the rate of growth of Tubercle bacilli by
the use of small inocula. J. Bactriol. 1949, 58, 247–255.
37. Atlas, R.M.; Snyder, J.W. Handbook of Media for Clinical Microbiology, 2nd ed.; CRC Press: Boca Raton, FL,
USA, 1995.

Molecules 2018, 23, 985
15 of 15
38. Foks, H.; Buraczewska, M.; Manowska, W.; Sawlewicz, J. Investigation on pyrazine derivatives.
Part 1. Synthesis and tuberculostatic action of pyrazinamidrazone 6-chloropyrazinamidrazone and their
condensation products with aldehydes and ketones. Diss. Pharm. Pharmacol. 1971, 23, 49–58.
39. Dykstra, C.; Frenking, G.; Kim, K.; Scuseria, G. Advances in electronic structure theory: GAMESS a decade
later. In Theory and Applications of Computational Chemistry: The First Forty Years, 1st ed.; Elsevier: Amsterdam,
The Netherlands, 2005; pp. 1167–1189.
Sample Availability: Samples of the compounds are not available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).