Preparation and in vitro evaluation of benzylsulfanyl benzoxazole derivatives as potential antituberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2008.06.027

A set of 2-benzylsulfanyl derivatives of benzoxazole was synthesized and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis, non-tuberculous mycobacteria and multidrug-resistant M. tuberculosis. The activities were

Full text of DOI:10.1016/j.ejmech.2008.06.027

European Journal of Medicinal Chemistry 44 (2009) 2286–2293
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Preparation and in vitro evaluation of benzylsulfanyl benzoxazole
derivatives as potential antituberculosis agents
a
,
a
b
c
ˇ
ˇ
´
ˇ´ a
´
*
Vera Klimesova , Jan Kocı , Karel Waisser , Jarmila Kaustova , Ute Mo¨llmann
a
´
´
´
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, Hradec, Kralove, 500 05, Czech Republic
National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Public Health, Partyzanske nam. 7, Ostrava 702 00, Czech Republic
c Hans-Kno¨ll-Institut fu¨r Naturstofforschung, Beutenbergstrasse 11, D-07745 Jena, Germany
b
´
´
´
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of 2-benzylsulfanyl derivatives of benzoxazole was synthesized and evaluated for their in vitro
antimycobacterial activity against Mycobacterium tuberculosis, non-tuberculous mycobacteria and
multidrug-resistant M. tuberculosis. The activities were expressed as the minimum inhibitory concen-
tration (MIC) in mmol/L. The substances showed similar activity against all tested strains. The lead
compounds in the set, dinitro derivatives exhibited significant activity against both sensitive and
resistant strains of M. tuberculosis and also against non-tuberculous mycobacteria. To facilitate drug
design of benzoxazole as potential antituberculosis agent, we have explored the quantitative structure–
activity relationship (QSAR). We demonstrated that lower lipophilicity has significant contribution to
activity. Dinitrobenzylsulfanyl derivative of benzoxazole represents the promising small-molecule
synthetic antimycobacterials.
Received 7 February 2008
Received in revised form 23 June 2008
Accepted 30 June 2008
Available online 5 July 2008
Keywords:
Benzoxazole
2-Benzylsulfanyl derivatives
Antimycobacterial activity
Mycobacterium tuberculosis
Multidrug-resistant M. tuberculosis
Non-tuberculous mycobacteria
Quantitative structure–activity relationship
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
Previously, we have reported a large number of alkylsulfanyl
derivatives of pyridine [7,8], benzimidazole [9–11], and tetrazole
In 2006 almost 9.2 million new TB cases occurred, 80% of them
in 22 countries and 1.7 million people died from TB. It is estimated
that more than 2 billion people – one third of the world’s
population – are infected with TB bacilli, the microbes that cause
TB. Drugs for treating tuberculosis (TB) have been available for over
half a century, but the total number of deaths and TB cases is still
rising due to population growth. TB annual incidence rates have
peaked globally in 2003–2004, however, they are falling very
slowly in all WHO regions, and stagnating in Eastern Europe. Five
percentage of all TB cases have multidrug-resistant TB (MDR-TB)
and in 2008 WHO ever recorded the highest rates of MDR-TB.
Extensively drug-resistant TB (XDR-TB) cases have been confirmed
in more than 45 countries. Already in 1993, the World Health
Organization recognized this developing situation and declared TB
‘‘a global health emergency’’ [1–3]. In the last decade the number of
TB research publications with a drug discovery focus increased
[4,5], some of new compounds advanced into clinical trials and
development [6].
[12] which showed promising antimycobacterial activity and
detailed QSAR findings supported the hypothesis that an alkylsul-
fanyl group bound to an electron deficient carbon in various
heterocycles was responsible for antimycobacterial activity [13]. In
the course of our ongoing efforts to investigate benzylsulfanyl
derivatives of benzazoles, we decided to replace the atom of
nitrogen of the benzimidazole ring with the corresponding isosteric
atom of oxygen. The activity of some benzylsulfanyl derivatives of
benzoxazole was mentioned in our preliminary communication
[14]. Herein, we report the synthesis of a large set of 2-benzylsul-
fanyl derivatives of benzoxazole, their antimycobacterial activity, as
well as the quantitative structure–activity relationship.
2. Chemistry
The compounds were synthesized following the steps depicted
in Scheme 1. The commercially available benzoxazole-2-thiol (1)
serves as a convenient starting material in the syntheses and it is
alkylated with benzyl halides (2). Thus, thiol 1 was converted to the
corresponding sodium salt by dissolving in an ethanolic solution of
sodium ethanolate, and the resulting salt was subjected to
a nucleophilic substitution upon the addition of a benzyl halide 2.
The reaction was carried out in N,N-dimethylformamide (DMF) at
* Corresponding author. Tel.: þ420 495 067 302; fax: þ420 495 067 166.
ˇ
´
E-mail addresses: vera.klimesova@faf.cuni.cz (V. Klimesova), karel.waisser@
faf.cuni.cz (K. Waisser), jarmila.kaustova@zuova.cz (J. Kaustova´), Ute.Moellmann@
hkijena.de (U. Mo¨llmann).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.06.027

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V. Klimesova et al. / European Journal of Medicinal Chemistry 44 (2009) 2286–2293
2287
Scheme 1. Synthesis of 2-benzylsulfanylbenzoxazoles 3a–z, 3aa and 4a and b. Reagents and conditions: (a) Na, CH3CH2OH, DMF, rt, 2–6 h; (b) H2S (g), Pyridine, TEA, rt, 3–4 h and
45 ꢀC, 1 h.
room temperature, under anhydrous conditions. The reaction
required 2–6 h to complete, depending on the alkylating agent and
furnished products 3 in 53–97% yields. The benzylsulfanyl deriva-
tives bearing the CN group 3z, 3aa were further converted into the
corresponding carbothioamides 4a and 4b by the addition of
hydrogen sulfide in pyridine/triethylamine solution.
3.2. Antiproliferative and cytotoxic effect
The target compounds were assayed against cell lines K-562 and
L-929 for their antiproliferative effects and against HeLa for their
cytotoxic effects. The cells were incubated with 10 concentrations
of the test compounds [15].
The structures of the compounds were fully characterized by 1H
NMR, 13C NMR, and IR spectral data, and their purity by elemental
analysis. The 1H NMR spectra of benzylsulfanyl derivatives have
thetypical singlet of CH2S group lying in the region 4.5–4.9 ppm
and multiplet of aromatic part of molecules occurring in region
between 7.0 and 8.0 ppm. The 13C NMR signal of CH2S group can be
observed at 34.0–36.0 ppm. IR spectra of 2-benzylsulfanyl deriva-
tives were also in agreement with the structures.
Suspension cultures of K-562 in micro plates were analyzed by
an electronic cell analyzer system CASY 1 (SCHARFE, Reutlingen,
¨
Germany) using an aperture of 150
mm. The software for data
¨
evaluation CASYSTAT (SCHARFE) offers fast graphical evaluation of
the measurement parameters, e.g. as diagrams of cell diameter
distributions, overlays of different curves, and cell volume distri-
butions. The 0.2 mL content of each well in the micro plate was
diluted 1:50 with CASYTON (NaCl: 7.93 g/L; Na2EDTA: 0.38 g/L; KCl:
0.4 g/L; NaH2PO4 monohydrate: 0.22 g/L; NaH2PO4 dihydrate:
¨
2.45 g/L; NaF: 0.3 g/L; SCHARFE). Every count/mL was automati-
3. Biology
cally calculated from the arithmetic mean of three successive
counts of 0.4 mL each. From the dose response curves the Gl50
values (concentration which inhibited cell growth by 50%) were
calculated with CASYSTAT. The Gl50 value was defined as being
where the concentration–response curve intersected the 50% line,
determined by means of the cell counts/mL, compared to control.
The monolayers of the adherent L-929 and HeLa cells were fixed
by glutaraldehyde and stained with a 0.05% solution of methylene
blue for 15 min. After gently washing, the stain was eluted by
0.2 mL of 0.33 N HCl in the wells. The optical densities were
measured at 630 nm in a DYNATECH MR 7000 micro plate reader.
Comparisons of the different values were performed with Microsoft
Excel.
3.1. Antimycobacterial activity
In vitro antimycobacterial activity of the compounds was eval-
uated against Mycobacterium tuberculosis CNCTC My 331/88,
Mycobacterium kansasii CNCTC My 235/80, M. kansasii 6509/96 and
Mycobacterium avium CNCTC My 330/88 using the micromethod
for the determination of the minimum inhibitory concentration
(MIC). All strains were obtained from the Czech National Collection
of Type Cultures (CNCTC), with the exception of M. kansasii 6509/
96, which was a clinical isolate. The activities of the compounds
ˇ
were determined in the Sula semisynthetic medium (SEVAC,
Prague). The compounds were added to the medium in dime-
thylsulfoxide solutions. The following concentrations were used:
1000, 500, 250, 125, 62, 32, 16, 8, 4 and 2 mmol/L. MICs were
determined after incubation at 37 ꢀC for 14 and 21 days, for M.
kansasii for 7, 14, and 21 days. MIC was the lowest concentration of
a substance, at which the inhibition of the growth of mycobacteria
occurred. INH was used as a standard.
4. Results and discussion
All prepared compounds were tested for antimycobacterial
activities and the values of MICs are summarized in Table 1. In
several cases (denoted >), the minimum inhibitory concentration
(MIC) could not be determined due to the limited solubility of the
compounds in the testing medium.
For the sake of comparison, the values of MICs of 1 and the
standard isoniazide (INH) were also included. All of these compounds
(1, 3, 4) displayed in vitro activity against all mycobacterial strains
The most active compounds 3t and 4a were evaluated against
four multidrug-resistant strains of M. tuberculosis using the
micromethod for the determination of the minimum inhibitory
concentration (MIC) under the same conditions as described above.
The characterization of resistant strains of M. tuberculosis is
following: M. tuberculosis 7357/98 resistant to isoniazid, rifampicin,
ethambutol, streptomycin, ofloxacin, rifabutin, and ciprofloxacin;
M. tuberculosis 4166/04 resistant to isoniazid, and streptomycin;
M. tuberculosis 4977/03 resistant to isoniazid, rifampicin, etham-
butol, streptomycin, ofloxacin, and rifabutin and M. tuberculosis
550/04 resistant to isoniazid, rifampicin, gentamicin, amikacin, and
rifabutin.
tested. The values of MICs are within a range of 2–500
mmol/L, most
often between 32 and 62 mol/L. By comparing their MIC values with
m
those of INH, the compounds under study were less active against M.
tuberculosis 331/88 and M. kansasii 6509/96. On the other hand, they
possessed a more pronounced effect against M. kansasii My 235/80
and M. avium 330/88 than INH. In contrast to INH, the studied

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V. Klimesova et al. / European Journal of Medicinal Chemistry 44 (2009) 2286–2293
2288
Table 1
In vitro antimycobacterial activity of compounds 1, 3a–z, 3aa and 4a and b expressed as MIC (mmol/L)
Compound
Strains
Mycobacterium
tuberculosis My 331/88
Mycobacterium kansasii My 235/80
Mycobacterium kansasii 6 509/96
Mycobacterium avium
My 330/88
14 d
500
250
250
62
>250
250
62
125
>62
125
>500
250
62
125
>62
>62
>250
62
>125
125
8
21 d
500
7 d
500
14 d
21 d
7 d
14 d
21 d
14 d
250
62
62
32
125
62
32
62
32
32
32
62
32
32
>32
>32
>125
62
62
62
>16
>16
62
62
32
>125
62
62
32
62
21 d
500
125
62
32
125
62
32
62
62
32
1
1000
250
62
32
32
62
62
32
62
32
62
125
32
32
1000
250
125
62
250
32
32
32
62
62
32
32
32
32
62
125
32
32
32
32
62
32
62
32
4
500
125
62
32
62
62
32
32
62
32
62
125
32
32
32
32
>62
32
62
62
8
1000
250
125
62
125
125
62
62
125
62
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
500
>500
125
>250
500
125
250
>125
250
>500
500
125
250
>125
125
>250
125
>250
125
8
125
32
32
32
32
32
32
32
32
62
125
32
32
32
32
125
32
32
62
2
62
125
125
62
>62
62
125
500
62
125
250
62
62
125
62
62
62
32
32
>62
>32
>125
62
125
125
4
>32
>32
>62
62
125
125
8
8
>62
62
62
62
>32
62
16
>62
>62
>125
62
>32
125
32
62
125
4
4
>32
62
32
62
62
3s
3t
125
>32
>32
125
62
32
>125
62
3u
3v
3w
3x
3y
3z
3aa
4a
4b
8
8
4
4
4
8
>62
125
62
>62
125
125
125
>62
125
16
>32
32
32
62
32
32
8
>32
125
62
125
>32
125
16
32
16
32
32
16
32
8
>32
32
32
32
32
62
16
62
>62
125
8
32
62
16
32
62
32
62
8
16
8
32
8
32
32
INH
0.5
1
>250
>250
>250
2
2
4
>250
>250
compounds were comparably active against both strains of M.
kansasii.
The starting benzoxazole-2-thiol (1) is characterized by the
activity within a range of 250–1000 mmol/L. Comparisons of the
(MICINH ¼ 0.5–1
mmol/L). MICs against M. avium 330/88 could not
be determined due to insolubility of compounds in the testing
medium. The antimycobacterial activity of compounds 3t and 3u is
probably connected with a nitro group. It is noteworthy that the
many antibacterials that are used for treatment of anaerobic
infections feature a nitro group (chloramphenicole, metronidazole)
in their structure. In currently undergoing clinical trials there are
two antituberculotics marked as PA-824 and OPC-67683 bearing
also a nitro group [4,6,18,19]. The vast array of nitrated anti-
mycobacterials reported that all of them probably being prodrugs
to reduced into the corresponding active species acting on
biochemical targets [20]. The study with PA-824 indicated that the
metabolic activation of PA-824 by M. tuberculosis involves a nitro-
reduction step and the resistant mutants had lost ability to carry
out nitro-reduction [18]. A promising effect was also revealed for
thioamide derivatives (4a, 4b) although they did not reach the
activity of dinitro derivatives (3t, 3u). It is very likely that thioamide
group is responsible for the antimycobacterial activity such as in
the antituberculotics ethionamide. Ethionamide also is prodrug
that is oxidized by EthA (a flavoprotein monooxygenase) to the
sulfinic acid [4].
MIC values for 1 to those of the benzylsulfanyl derivative (3a)
indicate that the antimycobacterial activity is connected with the
presence of the benzyl moiety at 2-position of the benzoxazole ring.
It was according to our earlier hypothesis that an alkylsulfanyl
group bound to an electron deficient carbon atom is responsible for
antimycobacterial activity [16]. Further modifications of the benzyl
moiety with various electron-accepting or electron-donor substit-
uents improved the activity. It is important to point out that it is not
affected by the electronic properties of the substituents, practically
all compounds substituted on the benzyl moiety exhibited similar
activity within the range 32–125 mmol/L. It is according to our
previously presented group of 2-benzylsulfanylbenzimidazole
derivatives [9,10] and similar 2-substituted 5,7-di-tert-butylben-
zoxazoles [17], but it is on the contrary to what was observed for
the previously synthesized 4-benzylsulfanyl derivatives of pyridine
[13] where the electron-withdrawing substituents cause the
increase of the activity. The incorporation of two nitro groups into
the benzyl moiety led to the most active compounds 3t, 3u with the
The antimycobacterial activities against multidrug-resistant
strains of M. tuberculosis are summed up in Table 2. MIC values for
MICs values ranging from 2 to 32 mmol/L. The same conclusions
were obtained in our previously synthesized benzylsulfanyl deri-
viatives of benzimidazoles [9,10]. Both compounds 3t and 3u
exhibited the same activity against M. tuberculosis and non-tuber-
culous mycobacteria M. kansasii. Their activity against M. kansasii
3t and 4a are within a range of 2–4 mmol/L, and 8–32 mmol/L,
respectively. In comparison to the sensitive strain of M. tuberculosis
compound 3t has been once or twice more active against multi-
drug-resistant strains, while compound 4a exhibits the same or less
efficacies. The level of activity against both sensitive and multidrug-
resistant strains indicates that these compounds may target
biochemical mechanisms different from the one mutated in the
resistant strains.
235/80 (MIC ¼ 2–4
(MICINH ¼ >250 mol/L), the activity against M. kansasii 6509/96
was 2-fold less than that of INH. The activity of INH against
M. tuberculosis 331/88 (MIC ¼ 8 mol/L) was not reached
mmol/L) exceeded the activity of INH
m
m

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V. Klimesova et al. / European Journal of Medicinal Chemistry 44 (2009) 2286–2293
2289
Table 2
In vitro antituberculotic activity against MDR M. tuberculosis (MIC expressed in
m
mol/L)
Compound
Strains
Mycobacterium
tuberculosis 7357/98a
Mycobacterium
tuberculosis 4166/04b
Mycobacterium
tuberculosis 4977/03c
Mycobacterium
tuberculosis 550/04d
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
3t
4a
4
32
4
32
2
8
4
16
2
32
2
32
2
8
4
16
a
Resistant to isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, rifabutin, and ciprofloxacin.
Resistant to isoniazid, and streptomycin.
Resistant to isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and rifabutin.
Resistant to isoniazid, rifampicin, gentamicin, amikacin, and rifabutin.
b
c
d
Compounds 3t, 3u and 4a, 4b were assayed against cell lines K-
molecule resulted in better activity. Extending Eq. (1) with log P
(Eq. (2)) lead to the deterioration of statistic parameters (F ¼ 29.89).
The value of regression coefficient for log P indicates that the
antituberculotic activity increases with diminishing lipophilicity.
The lowest log P exhibited the most active derivatives 4 and 3t. Eqs.
(3) and (4) show a small contribution of electronic parameters to
activity. That is in accordance with our previous finding that it is not
affected by the electronic properties of the substituents. Practically
all compounds substituted with whatever substituent on the
benzyl moiety exhibited similar activity. More significant correla-
tions are obtained by replacing Hammet constant with energies of
HOMO, LUMO orbitals (Eqs. (4) and (5)). HOMO and LUMO orbitals
represent electron properties of the whole molecule and express
the ability of molecule to be subjected to oxidation (energy of
HOMO orbitals) or reduction (energie of LUMO orbitals). As
mentioned above the activity of some compounds requires acti-
vation by reduction/oxidation processes.
562 and L-929 for their antiproliferative effects (GI50: concentration
which inhibited cell growth by 50%), and against HeLa cells for their
cytotoxic effects (CC50: cytotoxic concentration which contains
a specific destructive action by 50%; used particularly in referring to
the lysis of cells). The cells were incubated with 10 concentrations
of the tested compounds. The values expressed as GI50 are ranging
from 5.5 to >50 mg/mL and values of CC50 from 21 to 50 mg/mL. The
lower cytotoxicity demonstrated the nitro derivatives. The most
antimycobacterial compound 3t exhibited the lowest cytotoxic
effect (Table 3).
The formerly prepared compounds 3a, 3b, 3e, 3h, and 3n [21–
24] were evaluated by Cranham for toxicity to red spider mite [21].
Of all tested compounds only 3b was moderately active against the
eggs and larvae of the glasshouse red spider mite.
A set of benzylsulfanyl derivatives of benzoxazole was analyzed
by quantitative structure–activity relationship techniques. Physi-
cochemical and quantum-chemical parameters taken into consid-
logð1=CÞ ¼ 0:08ðꢁ0:01ÞMR ꢂ 1:92ðꢁ0:91Þ r ¼ 0:85; r2
eration were log P for the hydrophobic effects,
constant) and energy of HOMO, LUMO orbitals (3HOMO
s
(Hammet
,
3LUMO) as the
¼ 0:72; s ¼ 0:29; F ¼ 44:94; n ¼ 20
(1)
electronic influences, MR (molar refractivity) for the steric inter-
actions and their values are summarized in Table 4. The biological
activities are expressed as a log 1/MIC (MIC values in mol/L).
Multiple linear regression analysis which involves finding the best
fit of dependent variable (antimycobacterial activity) to a linear
combination of independent variables (descriptors) is used by the
least squares method. Regression analyses were carried out for each
strain separately, however, the statistically significant equations
were obtained for M. tuberculosis.
Mycobacteria are surrounded by a thick and waxy cell wall;
hence, efficient drugs have a reasonable lipophilicity in order to
penetrate this barrier. Newly prepared compounds 3 and 4 are
characterized by a calculated log P within range from 3.5 to 5.6. The
most active compounds 3t, 3u, 4a, and 4b possess values log P 3.8
and 3.5, respectively (see Table 4). An antituberculous drug should
be taken orally, and, according to the ‘‘Lipinski rule of five’’,
a compound with good oral bioavailability generally has log P < 5
[25]. The limit of log P values by benzylsulfanyl derivatives was not
exceeded.
Table 4
Physicochemical and quantum-chemical parameter values of compounds 3 and 4
Compound
s
log P
MR (Å3)
3HOMO (eV)
3LUMO (eV)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
0
0.23
0.37
3.872
4.390
4.390
4.390
4.012
4.012
4.012
4.664
4.664
4.339
4.339
3.619
3.619
3.826
3.826
3.826
4.530
4.908
4.151
3.779
3.779
3.965
4.755
4.755
5.638
3.908
3.908
3.525
3.525
69.716
74.520
74.520
74.520
69.932
69.932
69.932
77.338
77.338
74.757
74.757
76.179
76.179
77.040
77.040
77.040
74.737
79.325
70.148
84.365
84.365
77.257
75.689
75.689
81.663
75.453
75.453
86.409
86.409
ꢂ8.508
ꢂ8.594
ꢂ8.586
ꢂ8.509
ꢂ8.596
ꢂ8.599
ꢂ8.530
ꢂ8.612
ꢂ8.594
ꢂ8.487
ꢂ8.494
ꢂ8.482
ꢂ8.488
ꢂ8.833
ꢂ8.777
ꢂ8.661
ꢂ8.511
ꢂ8.652
ꢂ8.684
ꢂ9.006
ꢂ8.951
ꢂ8.657
ꢂ8.721
ꢂ8.685
ꢂ8.848
ꢂ8.694
ꢂ8.670
ꢂ8.620
ꢂ8.615
ꢂ0.490
ꢂ0.703
ꢂ0.661
ꢂ0.637
ꢂ0.696
ꢂ0.688
ꢂ0.649
ꢂ0.760
ꢂ0.680
ꢂ0.479
ꢂ0.463
ꢂ0.445
ꢂ0.500
ꢂ1.506
ꢂ1.302
ꢂ1.301
ꢂ0.814
ꢂ0.857
ꢂ0.912
ꢂ2.024
ꢂ2.171
ꢂ1.487
ꢂ1.016
ꢂ0.879
ꢂ1.281
ꢂ1.030
ꢂ0.855
ꢂ1.036
ꢂ0.847
–
–
0.06
0.34
0.23
0.39
ꢂ0.17
ꢂ0.07
ꢂ0.27
0.12
0.78
0.71
–
The QSAR analysis reveals that the parameter molar refractivity
(MR) is the most significant for the potency of studied compounds
(Eq. (1)). Parameter MR has connection with steric interactions of
compound with biomacromolecules. An enlargement of MR of
–
0.60
0.4
1.42
–
3s
3t
3u
3v
3w
3x
3y
3z
3aa
4a
4b
–
Table 3
0.54
0.43
0.86
0.66
0.56
0.4
Antiproliferative and cytotoxic effect expressed in mg/mL
Compound
L-929 GI50
K-562 GI50
HeLa CC50
3t
22.4
>50
23.8
17.3
9.2
10.4
9.4
>50
45.1
23.6
21.6
3u
4a
4b
–
5.5

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V. Klimesova et al. / European Journal of Medicinal Chemistry 44 (2009) 2286–2293
2290
(0.015–0.040 mm, Merck). Silica gel 60 (0.015–0.040 mm, Merck)
was used for column chromatography.
The following compounds had been described in the literature:
3a (R ¼ H) [21–24], 3b (R ¼ 4-Cl) [21,22], 3e (R ¼ 4-F) [21,22], 3h
(R ¼ 4-Br) [21,22], 3n (R ¼ 4-NO2) [21,22], 3t (R ¼ 3,5-(NO2)2) [14],
3z (R ¼ 4-CN) [14], and 4a (R ¼ 4-CSNH2) [14].
logð1=CÞ ¼ 0:08ðꢁ0:01ÞMR ꢂ 0:22ðꢁ0:13Þlog P
ꢂ 1:16ðꢁ1:17Þ
r
¼ 0:85; r2 ¼ 0:72; s ¼ 0:30; F ¼ 22:89; n ¼ 20
(2)
(3)
(4)
(5)
logð1=CÞ ¼ 0:04ðꢁ0:02ÞMR ꢂ 0:32ðꢁ0:12Þlog P
6.1.1. General procedure for the synthesis of compounds 3
Benzoxazole-2-thiol (1) (0.75 g, 5 mmol) in dry DMF (8 mL) was
added to a solution of sodium (0.12 g, 5 mmol) in dryethanol (2.5 mL).
After 10 min of stirring at ambient temperature and under atmo-
sphere of argon, benzyl chloride (2) (5 mmol) was added in 2–3
portions, and the resultant suspension was stirred for 2–6 h. The
reaction mixture was then poured onto crushed ice and left at 4 ꢀC
overnight. The solid was filtered off, washed with cold water
(2 ꢃ 30 mL) and air-dried. The crude products were purified by
preparative TLC or column chromatography using acetone–light
petroleum (1:2, 1:3, 1:4, 1:5, 1:6), followed by crystallization from
aqueous ethanol, toluene or ethylacetate to afford the title compounds
as white or yellowish needles in 53–95% yields. The following
compounds were prepared according this general procedure.
þ 0:62ðꢁ0:20Þ
s
ꢂ 1:64ðꢁ1:36Þ
r
¼ 0:89; r2 ¼ 0:79; s ¼ 0:25; F ¼ 13:55; n ¼ 20
logð1=CÞ ¼ 0:06ðꢁ0:01ÞMR ꢂ 0:32ðꢁ0:11Þlog P
ꢂ 1:51ðꢁ0:45Þ HOMO ꢂ 12:0ðꢁ3:35Þ
r
¼ 0:92; r2 ¼ 0:85; s ¼ 0:24; F ¼ 28:34; n ¼ 20
logð1=CÞ ¼ 0:05ðꢁ0:01ÞMR ꢂ 0:26ðꢁ0:10Þlog P
ꢂ 0:53ðꢁ0:14Þ LUMO þ 0:68ðꢁ1:00Þ
r
6.1.1.1. 2-Benzylsulfanylbenzoxazole (3a). Yield: 61%; mp: 48–49 ꢀC
(51–52.5 ꢀC [21,22], 50 ꢀC [23,24]); IR (KBr) nmax (cmꢂ1) 3432, 3065,
3035 (C–H)Ar, 1602, 1505,1453 (C]Cv)Ar, 1471 (CH2), 1238, 1212,
1129, 1096 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.61 (2H, s), 7.24–
7.37 (5H, m), 7.46–7.52 (2H, m), 7.60–7.68 (2H, m); 13C NMR
(75 MHz, DMSO-d6) 35.4, 110.4, 118.5, 124.6, 124.9, 127.9, 128.8,
129.2, 136.8, 141.4, 151.5, 164.1.
¼ 0:93; r2 ¼ 0:86; s ¼ 0:22; F ¼ 32:42; n ¼ 20
5. Conclusion
We reported a new set of 2-benzylsulfanylbenzoxazoles which
exhibited antimycobacterial activity against M. tuberculosis and
non-tuberculous mycobacteria tested. The activity is connected
with a benzylsulfanyl moiety in position 2 and additional substit-
uents on the phenyl ring increase its level. The introduction of two
nitro groups or thioamide group on benzylsulfanyl moiety,
regardless of position of substitution, increased the anti-
mycobacterial activity. Thus, the substitutions with two nitro
groups are the best substituents identified to date. The most active
compounds 3t exhibited the significant activity against sensitive
strain and multidrug-resistant strains of M. tuberculosis. The QSAR
study indicates that the lower value of lipophilicity and enlarge-
ment of molecule have significant contribution to antituberculous
activity. The ratio between cytotoxicity and minimum inhibitory
concentration for compound 3t is low enough for further study.
6.1.1.2. 2-(4-Chlorobenzylsulfanyl)benzoxazole (3b). Yield: 92%; mp:
58–59 ꢀC (61–62 ꢀC [21,22]); IR (KBr) nmax (cmꢂ1) 3432, 3060, 3058,
3022 (C–H)Ar, 1599, 1500, 1492 (C]Cv)Ar, 1471 (CH2), 1238, 1216,
1133, 1097 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.60 (2H, s), 7.27–
7.34 (2H, m), 7.35–7.42 (2H, m AA0BB0), 7.49–7.56 (2H, m AA0BB0),
7.59–7.68 (2H, m); 13C NMR (75 MHz, DMSO-d6) 34.9, 110.5, 118.5,
124.6, 124.9, 128.8, 131.1, 132.5, 136.2, 141.4, 151.5, 163.9.
6.1.1.3. 2-(3-Chlorobenzylsulfanyl)benzoxazole (3c). Yield: 75%; mp:
32–34 ꢀC; IR (KBr) nmax (cmꢂ1) 3431, 3064 (C–H)Ar, 1637, 1598, 1576,
1500, 1476 (CH2), 1453, 1431 (C]Cv)Ar, 1239, 1214, 1133 (Ar–H); 1H
NMR (300 MHz, DMSO-d6) 4.61 (2H, s), 7.27–7.40 (4H, m), 7.43–7.52
(1H, m), 7.57–7.69 (3H, m); 13C NMR (75 MHz, DMSO-d6) 34.9,
110.5,118.6,124.6,124.9, 127.9,127.9,129.0,130.7,133.2,139.6,141.4,
151.5, 163.8.
6. Experimental protocols
6.1.1.4. 2-(2-Chlorobenzylsulfanyl)benzoxazole (3d). Yield: 77%; mp:
61–62 ꢀC; IR (KBr) nmax (cmꢂ1) 3432, 3054 (C–H)Ar, 1599, 1500, 1453
(C]Cv)Ar, 1471 (CH2), 1237, 1215, 1156, 1132 (Ar–H); 1H NMR
(300 MHz, DMSO-d6) 4.69 (2H, s), 7.26–7.39 (4H, m), 7.45–7.53 (1H,
m), 7.59–7.72 (3H, m); 13C NMR (75 MHz, DMSO-d6) 34.0, 110.5,
118.6, 124.7, 124.9, 127.7, 129.8, 130.1, 131.7, 133.6, 134.0, 141.4, 151.5,
163.6.
6.1. Chemistry
The melting points were determined on a Kofler block and are
uncorrected. Analytical samples were dried over P4O10 at 78 ꢀC or
61 ꢀC and 2.4–2.6 kPa for 8–10 h. Elemental analyses were per-
formed on CHNS–O CE instrument (FISONS EA 1110) and were
within ꢁ0.4% of the theoretical values. IR spectra were obtained on
a Nicolet Impact 400 spectrometer in KBr pellets. NMR spectra were
recorded in DMSO-d6 and acetone-d6 solutions at ambient
temperature on a Varian Mercury-Vx BB 300 spectrometer oper-
ating at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts were
6.1.1.5. 2-(4-Fluorobenzylsulfanyl)benzoxazole (3e). Yield: 95%; mp:
44.5–45.5 ꢀC (44–45 ꢀC [21,22]); IR (KBr) nmax (cmꢂ1) 3432, 3070,
3044 (C–H)Ar,1629,1601,1509,1454,1419 (C]Cv)Ar,1471 (CH2),1238,
1221, 1158, 1133, 1097 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.60
(2H, s), 7.50–7.58 (2H, m), 7.59–7.68 (2H, m), 7.11–7.20 (2H, m), 7.26–
7.37 (2H, m); 13C NMR (75 MHz, DMSO-d6) 34.9 (d, J ¼ 0.8 Hz), 110.4,
115.6 (d, J ¼ 21.4 Hz), 118.5,124.7 (d, J ¼ 22.6 Hz), 133.2 (d, J ¼ 3.2 Hz),
131.3 (d, J ¼ 8.3 Hz), 141.4, 151.5, 161.8 (d, J ¼ 243.9 Hz), 164.0.
recorded as
d values in ppm and were indirectly referred to tetra-
methylsilane (TMS). Coupling constants (J) are given in Hz. The
reactions were monitored and the purity of the products was
checked by TLC (Merck TLC plates silica gel 60 F254, aluminum back)
in acetone–light petroleum. The plates were visualised using UV
light, iodine fumes and/or dipping in a solution of Ce(SO4)2$4H2O,
H3Mo12O40P$xH2O, H2SO4, H2O and subsequent heating. Prepara-
tive thin layer chromatography was carried out on silica gel 60 F254
6.1.1.6. 2-(3-Fluorobenzylsulfanyl)benzoxazole (3f). Yield: 95%; mp:
34.5–36 ꢀC; nmax (cmꢂ1) 3425, 1617, 1590, 1501, 1453 (C]Cv)Ar, 1471
(CH2), 1238, 1132, 1097 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.62

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V. Klimesova et al. / European Journal of Medicinal Chemistry 44 (2009) 2286–2293
2291
(2H, s), 7.05–7.15 (1H, m), 7.26–7.42 (5H, m), 7.59–7.69 (2H, m); 13
C
6.1.1.14. 2-(4-Nitrobenzylsulfanyl)benzoxazole (3n). Yield: 94%; mp:
109–112 ꢀC (114–115 ꢀC [21,22]); nmax (cmꢂ1) 3432, 3080 (C–H)Ar
NMR (75 MHz, DMSO-d6) 35.0 (d, J ¼ 2.0 Hz), 110.4, 114.6, 114.9,
116.0 (d, J ¼ 21.8 Hz), 118.6, 124.7 (d, J ¼ 21.2 Hz), 125.3 (d,
J ¼ 2.9 Hz), 130.7 (d, J ¼ 8.6 Hz), 139.8, 139.9, 141.4, 151.5, 162.2 (d,
J ¼ 243.9 Hz), 163.9.
,
1648, 1608, 1601 (C]Cv)Ar, 1518, 1347 (NO2), 1472 (CH2), 1455, 1432
(C]Cv)Ar, 1238, 1135, 1099 (Ar–H); 1H NMR (300 MHz, DMSO-d6)
4.88 (2H, s), 7.26–7.36 (2H, m AA0BB0) 7.51–7.66 (1H, m overlapped),
7.51–7.60 (2H, m AA0BB0 overlapped), 7.71 (1H, td, J1 ¼1.4 Hz,
J2 ¼ 7.6 Hz), 7.85 (1H, dd, J1 ¼1.1 Hz, J2 ¼ 7.7 Hz), 8.06 (1H, dd,
J1 ¼1.1 Hz, J2 ¼ 8.2 Hz); 13C NMR (75 MHz, DMSO-d6) 34.8, 110.5,
118.6, 123.9, 124.7, 124.9, 130.4, 141.3, 145.3, 147.0, 151.6, 163.6.
6.1.1.7. 2-(2-Fluorobenzylsulfanyl)benzoxazole (3g). Yield: 81%; mp:
36–38 ꢀC; nmax (cmꢂ1) 3432, 1636, 1617, 1604, 1587, 1471 (CH2),
1493, 1453 (C]Cv)Ar, 1237, 1132, 1096 (Ar–H); 1H NMR (300 MHz,
DMSO-d6) 4.64 (2H, s), 7.11–7.40 (2H, m), 7.27–7.39 (3H, m), 7.60–
7.69 (2H, m overlapped), 7.58 (1H, dd, J1 ¼1.7 Hz, J2 ¼ 7.7 Hz); 13C
NMR (75 MHz, DMSO-d6) 29.5 (d, J ¼ 3.2 Hz), 110.5, 115.7 (d,
J ¼ 20.9 Hz), 118.6, 123.6, 123.8, 124.8, 124.8, 124.8 (d, J ¼ 18.3 Hz),
130.4 (d, J ¼ 8.0 Hz), 131.6 (d, J ¼ 3.7 Hz), 141.4, 151.5, 160.6 (d,
J ¼ 246.0 Hz), 163.5.
6.1.1.15. 2-(3-Nitrobenzylsulfanyl)benzoxazole (3o). Yield: 77%; mp:
79–81 ꢀC; nmax (cmꢂ1) 3428, 3098, 3083 (C–H)Ar, 1638, 1617, 1505
(C]Cv)Ar, 1533, 1349 (NO2), 1471 (CH2), 1453, 1408 (C]Cv)Ar, 1239,
1133, 1098 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.74 (2H, s), 7.27–
7.37 (2H, m), 7.59–7.66 (3H, m), 7.98 (1H, bd, J ¼ 7.7 Hz), 8.12 (1H,
dm, J ¼ 8.2 Hz), 8.43 (1H, bt, J ¼ 1.8 Hz); 13C NMR (75 MHz, DMSO-
d6) 34.6, 110.5, 118.6, 122.8, 124.0, 124.7, 124.9, 130.3, 135.9, 139.8,
141.3, 147.9, 151.5, 163.7.
6.1.1.8. 2-(4-Bromobenzylsulfanyl)benzoxazole (3h). Yield: 80%;
mp: 69–72 ꢀC (70–72 ꢀC [21,22]); IR (KBr) nmax (cmꢂ1) 3432, 1636,
1454, 1492 (CH2), 1432 (C]Cv)Ar, 1238, 1135 (Ar–H); 1H NMR
(300 MHz, DMSO-d6) 4.58 (2H, s), 7.27–7.37 (2H, m), 7.43–7.49 (2H,
m AA0BB0), 7.50–7.55 (2H, m AA0BB0), 7.60–7.68 (2H, m); 13C NMR
(75 MHz, DMSO-d6) 34.9, 110.4,118.5,121.1,124.6,124.9,131.4,131.7,
136.6, 141.4, 151.5, 163.9.
6.1.1.16. 2-(2-Nitrobenzylsulfanyl)benzoxazole (3p). Yield: 81%; mp:
65–67 ꢀC; nmax (cmꢂ1) 3432, 1636, 1676 (C]Cv)Ar, 1525, 1336 (NO2),
1471 (CH2), 1453, 1414 (C]Cv)Ar, 1238, 1133, 1097 (Ar–H); 1H NMR
(300 MHz, DMSO-d6) 4.88 (2H, s), 7.26–7.36 (2H, m), 7.52–7.66 (3H,
m), 7.71 (1H, td, J1 ¼1.4 Hz, J2 ¼ 7.6 Hz), 7.85 (1H, dd, J1 ¼1.1 Hz,
J2 ¼ 7.7 Hz), 8.06 (1H, dd, J1 ¼1.1 Hz, J2 ¼ 8.0 Hz); 13C NMR (75 MHz,
DMSO-d6) 33.1, 110.5, 118.6, 124.6, 124.9, 125.3, 129.7, 132.4, 132.7,
134.3, 141.2, 148.4, 151.6, 163.8.
6.1.1.9. 2-(3-Bromobenzylsulfanyl)benzoxazole (3i). Yield: 70%; mp:
37–39 ꢀC; nmax (cmꢂ1) 3427, 1636, 1595, 1569, 1473 (CH2), 1453,
1428 (C]Cv)Ar, 1239, 1133, 1097 (Ar–H); 1H NMR (300 MHz, DMSO-
d6) 4.60 (2H, s), 7.26–7.36 (3H, m), 7.42–7.56 (2H, m), 7.58–7.69 (2H,
m), 7.73 (1H, t, J ¼ 1.6 Hz); 13C NMR (75 MHz, DMSO-d6) 34.9, 110.4,
118.5, 121.8, 124.6, 124.9, 128.3, 130.7, 130.9, 131.9, 139.9, 141.4, 151.5,
163.8.
6.1.1.17. 2-(2-Fluoro-6-chlorobenzylsulfanyl)benzoxazole (3q). Yield:
61%; mp: 41–43 ꢀC; nmax (cmꢂ1) 3427, 1636, 1600, 1501, 1453, 1429
(C]Cv)Ar, 1240, 1133, 1097 (Ar–H); 1H NMR (300 MHz, DMSO-d6)
4.74 (2H, d, J ¼ 1.7 Hz), 7.60–7.70 (2H, m), 7.23–7.47 (5H, m); 13C
NMR (75 MHz, DMSO-d6) 27.8, 110.5, 115.0 (d, J ¼ 22.0 Hz), 118.7,
121.9 (d, J ¼ 18.0 Hz), 124.9 (d, J ¼ 8.9 Hz), 126.0 (d, J ¼ 3.4 Hz), 131.2
(d, J ¼ 9.7 Hz), 134.9, 141.4, 151.6, 161.0 (d, J ¼ 250.2 Hz), 162.8.
6.1.1.10. 2-(4-Methylbenzylsulfanyl)benzoxazole (3j). Yield: 95%;
mp: 52–54 ꢀC; nmax (cmꢂ1) 3448, 3095, 3081, 3046, 3023 (C–H)Ar
,
2970, 1395 (CH3), 2919, 1470 (CH2), 1774, 1636, 1617, 1516, 1503,
1453 (C]Cv)Ar, 1236, 1134, 1095 (Ar–H); 1H NMR (300 MHz, DMSO-
d6) 2.24 (3H, s), 4.56 (2H, s), 7.08–7.16 (2H, m AA0BB0 overlapped),
7.26–7.40 (2H, m overlapped), 7.34–7.40 (2H, m AA0BB0 overlapped),
7.58–7.68 (2H, m); 13C NMR (75 MHz, DMSO-d6) 20.9, 35.6, 110.4,
118.5, 124.5, 124.8, 129.1, 129.4, 133.6, 137.2, 141.5, 151.4, 164.1.
6.1.1.18. 2-(3,4-Dichlorobenzylsulfanyl)benzoxazole (3r). Yield: 57%;
mp: 63.5–66 ꢀC; nmax (cmꢂ1) 3432, 3081, 3053, 3047 (C–H)Ar, 1630,
1501, 1471 (CH2), 1455, 1408 (C]Cv)Ar, 1238, 1134, 1099 (Ar–H); 1H
NMR (300 MHz, DMSO-d6) 4.59 (2H, s), 7.26–7.36 (2H, m), 7.50 (1H,
dd, J1 ¼1.9 Hz, J2 ¼ 8.4 Hz), 7.60–7.67 (2H, m), 7.58 (1H, d,
J ¼ 8.2 Hz), 7.78 (1H, d, J ¼ 1.9 Hz); 13C NMR (75 MHz, DMSO-d6)
34.3, 110.4, 118.6, 124.6, 124.9, 129.5, 130.5, 130.9, 131.2, 131.2, 138.5,
141.3, 151.5, 163.7.
6.1.1.11. 2-(3-Methylbenzylsulfanyl)benzoxazole (3k). Yield: 93%; oil,
solidified by 5 ꢀC; nmax (cmꢂ1) 3423, 3051, 3025 (C–H)Ar, 2862, 1378
(CH3), 2919, 1470 (CH2), 1774, 1607, 1591 (C]Cv)Ar, 1236, 1180, 1095
(Ar–H); 1H NMR (300 MHz, DMSO-d6) 2.26 (3H, s), 4.57 (2H, s), 7.08
(1H, bd, J ¼ 7.4 Hz), 7.25–7.40 (4H, m), 7.21 (1H, t, J ¼ 7.4 Hz), 7.58–
7.71 (2H, m); 13C NMR (75 MHz, DMSO-d6) 21.1, 35.7, 110.4, 118.5,
124.5, 124.9, 126.3, 128.6, 128.7, 129.8, 136.5, 138.0, 141.4, 151.4,
164.1.
6.1.1.19. 2-(3,4-Difluorobenzylsulfanyl)benzoxazole (3s). Yield: 71%;
mp: 52–53 ꢀC; nmax (cmꢂ1) 3432, 3069 (C–H)Ar, 1636, 1610, 1518,
1501, 1454, 1411 (C]Cv)Ar, 1471 (CH2), 1238, 1134, 1097 (Ar–H); 1H
NMR (300 MHz, DMSO-d6) 4.59 (2H, s), 7.27–7.44 (4H, m), 7.55–
7.68 (3H, m); 13C NMR (75 MHz, DMSO-d6) 34.5 (d, J ¼ 1.4 Hz),110.5,
117.8 (d, J ¼ 17.7 Hz), 118.3 (d, J ¼ 17.1 Hz), 118.6, 124.6, 124.9, 126.2
(dd, J1 ¼6.7 Hz, J2 ¼ 3.6 Hz), 134.9 (dd, J1 ¼6.3 Hz, J2 ¼ 3.7 Hz),141.3,
149.1 (dd, J1 ¼245.8 Hz, J2 ¼ 14.8 Hz), 149.3 (dd, J1 ¼245.6 Hz,
J2 ¼ 14.6 Hz), 151.5, 163.8.
6.1.1.12. 2-(4-Methoxybenzylsulfanyl)benzoxazole (3l). Yield: 79%;
mp: 55–58 ꢀC; nmax (cmꢂ1) 2935, 2841, 1470 (CH2), 1610, 1582, 1513,
1497, 1453, 1441 (C]Cv)Ar, 1243, 1182, 1131 (Ar–H), 1214, 1095 (C–O
Ar–O–CH3); 1H NMR (300 MHz, DMSO-d6) 3.71 (3H, s), 4.56 (2H, s),
6.84–6.92 (2H, m AA0BB0), 7.26–7.37 (2H, m), 7.38–7.45 (2H, m
AA0BB0), 7.58–7.69 (2H, m); 13C NMR (75 MHz, DMSO-d6) 35.4, 55.3,
110.4, 114.2, 114.2, 118.5, 124.5, 124.8, 130.5, 141.5, 151.4, 159.0, 164.2.
6.1.1.20. 2-(3,5-Dinitrobenzylsulfanyl)benzoxazole (3t). Yield: 62%;
mp: 96–106 ꢀC (96–106 ꢀC [14]); nmax (cmꢂ1) 3042, 3107, 3086 (C–
H)Ar, 1627, 1596, 1541, 1345 (NO2), 1470 (CH2), 1454 (C]Cv)Ar, 1236,
1134, 1094 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.85 (2H, s), 7.26–
7.37 (2H, m), 7.58–7.66 (2H, m), 8.69 (1H, t, J ¼ 2.2 Hz), 8.87 (2H, d,
J ¼ 2.2 Hz); 13C NMR (75 MHz, DMSO-d6) 34.0, 110.5, 118.0, 118.5,
124.7, 125.0, 129.9, 141.2, 142.2, 148.1, 151.6, 163.5.
6.1.1.13. 2-(3-Methoxybenzylsulfanyl)benzoxazole (3m). Yield: 70%;
mp: 37–41 ꢀC; nmax (cmꢂ1) 2939, 2836, 1470 (CH2), 1636, 1601,1585,
1453, 1438 (C]Cv)Ar, 1239, 1153, 1096 (Ar–H), 1214, 1096 (C–O Ar–
O–CH3); 1H NMR (300 MHz, DMSO-d6) 3.71 (3H, s), 4.58 (2H, s),
6.84 (1H, bd, J1 ¼2.5 Hz J2 ¼ 8.2 Hz), 7.03–7.10 (2H, m), 7.26–7.37
(2H, m), 7.24 (1H, t, J ¼ 7.8 Hz), 7.61–7.69 (2H, m); 13C NMR
(75 MHz, DMSO-d6) 35.7, 55.2, 110.4, 113.4, 114.8, 118.5, 121.3, 124.6,
124.9, 129.9, 138.3, 141.4, 151.5, 159.5, 164.1.
6.1.1.21. 2-(2,4-Dinitrobenzylsulfanyl)benzoxazole (3u). Yield: 58%;
mp: 97.5–102 ꢀC; nmax (cmꢂ1) 3443, 3095, 3080, 3055, 3044 (C–
H)Ar, 1763, 1609 (C]Cv)Ar, 1540, 1346 (NO2), 1471 (CH2), 1453, 1421

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2292
(C]Cv)Ar, 1235, 1135, 1096 (Ar–H); 1H NMR (300 MHz, DMSO-d6)
4.98 (2H, s), 7.23–7.37 (2H, m), 7.55–7.65 (2H, m), 8.15 (1H, d,
J ¼ 8.5 Hz), 8.52 (1H, dd, J1 ¼2.3 Hz, J2 ¼ 8.7 Hz), 8.73 (1H, d,
J ¼ 2.2 Hz); 13C NMR (75 MHz, DMSO-d6) 32.4, 110.6, 118.6, 120.5,
124.8, 125.0, 128.1, 134.3, 139.3, 141.1, 147.1, 148.6, 151.7, 163.3.
TLC chromatography in acetone–light petroleum (1:1 or 1:2) and
crystallization from aqueous ethanol gave the products as yellow
needles in 36–53% yields. The following compounds were prepared
according this general procedure.
6.1.2.1. 4-(2-Benzoxazolylsulfanylmethyl)benzothioamide (4a). Yield:
53%; mp: 155–160 ꢀC (143–160 ꢀC [14]); nmax (cmꢂ1) 3424, 3386,
3289 (NH), 3166 (C–H)Ar, 2925, 2853, 1470 (CH2), 1621, 1607, 1575,
1493, 1452, 1438 (C]Cv)Ar, 1230, 1133, 1098 (Ar–H), 1190 (C]S
–NH–C]S); 1H NMR (300 MHz, acetone-d6) 4.67 (2H, bs), 7.27–7.38
(2H, m), 7.50–7.66 (2H, m overlapped), 7.58–7.66 (2H, m AA0BB0
overlapped), 7.92–7.98 (2H, m AA0BB0), 8.86 (1H, bs), 9.04 (1H, bs);
13C NMR (75 MHz, acetone-d6) 36.1, 110.8, 119.2, 125.0, 125.3, 128.4,
129.5, 140.0, 141.3, 142.6, 152.7, 164.8, 202.1.
6.1.1.22. 2-(2-Fluoro-6-nitrobenzylsulfanyl)benzoxazole (3v). Yield:
53%; mp: 89–93 ꢀC; nmax (cmꢂ1) 3447, 3100, 3052 (C–H)Ar, 1618
(C]Cv)Ar, 1533, 1365 (NO2), 1464 (CH2), 1453, 1411 (C]Cv)Ar, 1239,
1135, 1096 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.89 (2H, d,
J ¼ 1.4 Hz), 7.27–7.37 (2H, m), 7.54–7.74 (4H, m), 7.90 (1H, dt,
J1 ¼1.4 Hz, J2 ¼ 8.0 Hz); 13C NMR (75 MHz, DMSO-d6) 25.9, 110.5,
118.6, 120.3 (d, J ¼ 18.3 Hz), 121.4 (d, J ¼ 20.0 Hz), 124.8, 124.9, 130.8
(d, J ¼ 9.7 Hz), 141.2, 149.8, 151.6, 160.7 (d, J ¼ 250.5 Hz), 163.1.
6.1.1.23. 2-(4-Trifluoromethylbenzylsulfanyl)benzoxazole (3w). Yield:
63%; mp: 48–54 ꢀC; nmax (cmꢂ1) 1617, 1499 (C]Cv)Ar, 1455, 1429,
1414 (C]Cv)Ar, 1332, 1172, 1133 (CF3), 1238, 1071 (Ar–H); 1H NMR
(300 MHz, DMSO-d6) 4.70 (2H, s), 7.30–7.35 (2H, m), 7.62–7.67 (2H,
m), 7.68–7.77 (4H, m); 13C NMR (75 MHz, DMSO-d6) 34.9, 110.5,
118.6, 124.4 (q, J ¼ 272.1 Hz), 125.6, 125.7 (d, J ¼ 3.8 Hz), 128.3 (q,
J ¼ 32.2 Hz), 130.0, 141.4, 142.1, 142.2, 151.5, 163.8.
6.1.2.2. 3-(2-Benzoxazolylsulfanylmethyl)benzothioamide (4b). Yield:
36%; mp: 163–167 ꢀC; nmax (cmꢂ1) 3425, 3242 (NH), 3081 (C–H)Ar,
2925, 1471 (CH2), 1664, 1601, 1489, 1454, 1438, 1414 (C]Cv)Ar, 1231,
1140, 1099 (Ar–H); 1H NMR (300 MHz, acetone-d6) 4.68 (2H, bs),
7.26–7.47 (2H, m overlapped), 7.39 (1H, t overlapped, J ¼ 7.7 Hz),
7.50–7.67 (2H, m), 7.71 (1H, bd, J ¼ 7.7 Hz), 8.19 (1H, bt, J ¼ 1.7 Hz),
8.92 (1H, bs), 9.07 (1H, bs); 13C NMR (75 MHz, acetone-d6) 36.3,
110.8, 119.2, 125.0, 125.3, 127.1, 129.1, 129.2, 132.6, 137.8, 141.1, 142.7,
152.7, 161.8, 202.3.
6.1.1.24. 2-(3-Trifluoromethylbenzylsulfanyl)benzoxazole (3x). Yield:
55%; mp: 32–36 ꢀC; nmax (cmꢂ1) 3070 (C–H)Ar, 1773, 1664, 1505,
1471 (CH2), 1454 (C]Cv)Ar, 1332, 1167, 1132 (CF3), 1239, 1096
(Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.69 (2H, s), 7.01–7.14 (1H,
m), 7.23–7.37 (2H, m), 7.50–7.73 (3H, m), 7.83 (1H, bd, J ¼ 7.4 Hz),
7.90 (1H, bs); 13C NMR (75 MHz, DMSO-d6) 34.9, 109.9, 110.4, 118.5,
122.0, 123.9, 124.3 (q, J ¼ 272.4 Hz), 124.5 (d, J ¼ 3.8 Hz), 124.9,
128.7, 129.4 (q, J ¼ 31.5 Hz), 133.4, 138.8, 139.1, 141.3, 143.5, 151.5,
163.8.
6.2. QSAR study
Quantum-chemical calculations were run on a PC computer
using software HyperChem Suite for Windows (release 5.1) The
semi-empirical AM1 (Austin Model 1) method was used for all
molecular modelling calculations. The most stable conformations
of the molecules were found by the molecular dynamics, by
simulated heating to 7000 K followed by the Monte Carlo method
(293 K, 500 steps). Calculations of logP values were carried out on
the software HyperChem Suite for Windows (release 5.1), the
module ChemPlus 1.6 using atomic parameters derived by Ghose
et al. [26] and Viswanadhan et al. [27]. The MR was estimated by the
same method as log P. Atomic contributions for these calculations
were published by Ghose and Crippen [28] and Viswanadhan et al.
6.1.1.25. 2-[3,5-Bis(trifluoromethyl)benzylsulfanyl]benzoxazole
(3y). Yield: 54%; mp: 47–48.5 ꢀC; nmax (cmꢂ1) 1623, 1507, 1472
(CH2), 1467, 1454 (C]Cv)Ar, 1280, 1173, 1129 (CF3), 1239, 1097
(Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.76 (2H, s), 7.26–7.36 (2H,
m), 7.58–7.63 (2H, m), 7.99 (1H, bs), 8.27 (2H, bs); 13C NMR
(75 MHz, DMSO-d6) 34.3, 121.5, 121.6, 123.4 (q, J ¼ 270.0 Hz), 130.4,
130.4 (q, J ¼ 32.8 Hz), 141.1, 141.1, 141.2, 141.2, 151.5, 163.5.
ˇ
s were taken from the table given by Kuchar and
[27] Parameters
Rejholec [29].
6.1.1.26. 2-(4-Cyanobenzylsulfanyl)benzoxazole (3z). Yield: 80%;
mp: 102–106 ꢀC (102–106 ꢀC [14]); nmax (cmꢂ1) 3048 (C–H)Ar, 2227
(C^N), 1636, 1604, 1509, 1470 (CH2), 1453, 1419 (C]Cv)Ar, 1242,
1139, 1098 (Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.67 (2H, s), 7.26–
7.36 (2H, m), 7.59–7.67 (2H, m), 7.68–7.73 (2H, m AA0BB0), 7.77–7.82
(2H, m AA0BB0); 13C NMR (75 MHz, DMSO-d6) 35.1, 110.5, 110.6,
118.6, 118.9, 124.7, 124.9, 130.2, 132.7, 141.3, 151.6, 163.7.
Correlation and regression analyses of the QSAR study were run
on a PC computer using the Microsoft Excel program. In the
equations, the figures in the parentheses are the standard errors of
the regression coefficients, n is the number of compounds, r is the
multiple correlation coefficient, r2 is the determination coefficient,
F is the significance test (F-test) and s is the standard error of
estimate. F-test values are for all equations statistically significant
at the 1% level of probability.
6.1.1.27. 2-(3-Cyanobenzylsulfanyl)benzoxazole (3aa). Yield: 72%;
mp: 66–79 ꢀC; nmax (cmꢂ1) 3078, 3057 (C–H)Ar, 2228 (C^N), 1608,
1600, 1508, 1471 (CH2), 1453, 1431 (C]Cv)Ar, 1242, 1139, 1098
(Ar–H); 1H NMR (300 MHz, DMSO-d6) 4.65 (2H, s), 7.25–7.38 (2H,
m), 7.54 (1H, t, J ¼ 7.8 Hz), 7.59–7.68 (2H, m), 7.74 (1H, dt, J1 ¼1.4 Hz,
J2 ¼ 8.0 Hz), 7.86 (1H, dt, J1 ¼1.5 Hz, J2 ¼ 8.0 Hz), 7.97 (1H, bt,
J ¼ 1.5 Hz); 13C NMR (75 MHz, DMSO-d6) 34.7, 110.4, 111.6, 118.5,
118.7, 124.6, 124.9, 130.0, 131.6, 132.8, 134.1, 139.0, 141.3, 151.5, 163.7.
Acknowledgements
This work was financially supported by project No. MSM
0021620822 of the Ministry of Education of the Czech Republic. We
are indebted to Pharm Dr. Karel Palat, PhD (Department of Inorganic
and Organic Chemistry, Faculty of Pharmacy, Charles University) for
his help by finding of physicochemical and quantum-chemical
´
´
ˇ ´
parameters and to Ms I. Vencovska and P. Jancarek (Department of
Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles
University) for recording IR spectra and NMR spectra, respectively.
6.1.2. General procedure for the synthesis of compounds 4
Dry hydrogen sulfide was passed through the solution of a cyano
compound (3z, 3aa) (0.5 g, 2 mmol) dissolved in a mixture of dry
pyridine (7 mL) and triethylamine (0.7 mL). The reaction mixture
was maintained at room temperature for 3–4 h and then heated to
45 ꢀC for an additional hour. After cooling, the mixture was poured
onto crushed ice with intensive stirring, the precipitated product
was filtered off, washed with cold water and air-dried. Preparative
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