Heterocyclic benzazole derivatives with antimycobacterial in vitro activity.

Article abstract of DOI:10.1016/S0960-894X(02)00697-2

The series of 2-benzylsulfanyl derivatives of benzoxazole and benzothiazole were synthesized, evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis and non-tuberculous mycobacteria, and the activity expressed as the minimum inhibitory concentration (MIC) in micromol/L. The substances bearing two nitro groups (4e, 4f, 5e, 5f) or a thioamide group (4i, 4j, 5i, 5j) exhibited appreciable activity particularly against non-tuberculous strains. The most active compounds were subjected to the toxicity assay and were evaluated as moderately cytotoxic.

Full text of DOI:10.1016/S0960-894X(02)00697-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3275–3278
Heterocyclic Benzazole Derivatives with Antimycobacterial In
Vitro Activity
Jan Kocı, Vera Klimesova, * Karel Waisser, Jarmila Kaustova
ˇ ˇ ˇ ´ ´
´
,b Hans-Martin Dahsec
a
a,
a
c
¨
and Ute Mollmann
aDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, 50005 Hradec Kr a´ love´,
Czech Republic
National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Hygiene, Partyza´nsk e´ n a´ meˇstı´ 7, 72892 Ostrava 1,
Czech Republic
b
c
Hans-Kno¨ll-Institut fu¨r Naturstoff-Forschung, Beutenbergstr. 11, D-07745 Jena, Germany
Received 7 June 2002; revised 1 August 2002; accepted 20 August 2002
Abstract—The series of 2-benzylsulfanyl derivatives of benzoxazole and benzothiazole were synthesized, evaluated for their in vitro
antimycobacterial activity against Mycobacterium tuberculosis and non-tuberculous mycobacteria, and the activity expressed as the
minimum inhibitory concentration (MIC) in mmol/L. The substances bearing two nitro groups (4e, 4f, 5e, 5f) or a thioamide group
(
4i, 4j, 5i, 5j) exhibited appreciable activity particularly against non-tuberculous strains. The most active compounds were subjected
to the toxicity assay and were evaluated as moderately cytotoxic.
2002 Elsevier Science Ltd. All rights reserved.
#
Worldwide, tuberculosis (TB) still remains a major
public health problem, and it is estimated that new
infections of humans by Mycobacterium tuberculosis are
greater than 8 million annually, and more than 3 million
mycobacterial activity and detailed QSARfindings
which support the hypothesis that an alkylsulfanyl
group bound to an electron deficient carbon atom in
various heterocycles is responsible for antimycobacterial
1
6
people will die from this disease each year. Therefore,
activity. Significant antimycobacterial activity was
observed in a series of 4-benzylsulfanyl derivatives of
the development of specific and potent chemother-
apeutics is an important goal in pharmaceutical
research. An urgent need for novel antituberculous
agents is justified by several important factors: (a) the
incidence and prevalence of opportunistic infections due
to nontuberculous mycobacteria, nowadays especially
the Mycobacterium avium complex, seen particularly in
persons with AIDS; (b) the emergence of multidrug
resistant strains of M. tuberculosis to currently admin-
istered therapeutic agents; (c) immigration from coun-
tries where TB is common (Eastern Europe and Africa),
social problems (war, famine, homelessness); (d) inade-
quate funding for TB control and other public health
7
pyridine-2-carbonitrile/2-carbothioamide. Continuing
our search for new substances containing heterocyclic
moieties other than pyridin, we designed the structure of
benzimidazole. The set of benzimidazole with a different
substituted benzylsulfanyl moiety in position 2 elicited
remarkable activity on both tuberculous and especially
nontuberculous strains of mycobacteria, where it excee-
8
ded the activity of the standard INH. To investigate
the substituent effect at positions 2 and 5, we introduced
a methyl group into position 5 of the benzimidazole
core. A newly prepared set of 2-benzylsulfanyl deriva-
tives of 5-methylbenzimidazole exhibited comparable or
even improved antituberculous potency in comparison
with benzimidazole derivatives. A most significant effect
in both sets was exhibited by mononitro-, dinitro- and
2
efforts.
In our earlier paper, we have reported a large number of
derivatives of pyridine,
3ꢀ5
9
which showed potential anti-
thioamide groups in the benzyl moiety. In our further
efforts to investigate benzylsulfanyl derivatives, we
focused on other heterocyclic compounds. We decided
to simply replace the atom of nitrogen of the benzimi-
dazole ring with the corresponding isosteric atom of
*Corresponding author. Fax:+42-49-551-4330; e-mail: klimeso@faf.
cuni.cz
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00697-2

3276
J. Ko c˘ ı´ et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3275–3278
oxygen and sulfur, respectively. In the present
communication, we wish to report the synthesis and
preliminary antimycobacterial evaluation, including
toxicity of the 2-benzylsulfanyl derivatives of benzox-
azole/benzothiazole. The results mentioned above
demonstrate that the most active derivatives are mod-
ified by nitro and thioamide groups.
comparison, we also included the MIC values of the
starting materials (1, 2) and the standard isoniazide
(INH). A detailed observation of the set of benzoxazole
derivatives (4) showed that MIC values are generally
within a range of 2–500 mmol/L, most often between 8
and 32 mmol/L. The compounds were less active than
INH against M. tuberculosis 331/88 and against M.
kansasii 6509/96. On the other hand, the compounds
possessed a better activity against M. kansasii 235/80
and M. avium 330/88 than INH. The starting benzox-
azole-2-thiol (1) was only slightly active (MIC=250–
1000 mmol/L). The introduction of the benzylsulfanyl
moiety resulted in an increase in the activity. The
unsubstituated benzylsulfanyl derivative (4a) displayed
a range of activities from 32 to 500 mmol/L with a better
activity against non-tuberculous strains. In contrast to
initial expectations, mononitro derivatives (4b, 4c, 4d)
displayed only low potency against all strains tested
(MIC=32–>250 mmol/L). The incorporation of a sec-
ond nitro group, leading to compounds 4e, 4f, generally
produced an enhancement of the activity. Both com-
pounds 4e and 4f exhibited significant activity against
M. kansasii 235/80 (MIC =2–4 mmol/L, MIC =4
The synthetic scheme employed for the preparation of
the desired compounds (4, 5) is depicted in Schemes 1
and 2. The synthesis utilized commercially available
benzoxazole-2-thiol (1) and benzothiazole-2-thiol (2).
Thiols (1, 2) were converted to the corresponding
sodium salts by dissolving in an ethanolic solution of
sodium ethanolate, and the resulting salts were sub-
jected to a nucleophilic substitution upon the addition
of a benzyl halide (3). The reaction was carried out in
N,N-dimethylformamide (DMF) at room temperature
for 2–6 h and furnished products 4a–h and 5a–h
1
0
(
Scheme 1). The benzylsulfanyl derivatives bearing a
CN group (4g, 4h, 5g, 5h) were further converted into
the corresponding thioamide (4i, 4j, 5i, 5j) through the
reaction with hydrogen sulfide in the presence of pyri-
4
e
4f
1
1
dine/triethylamine solution (Scheme 2).
mmol/L) and exceeded the activity of INH
MIC=>250 mmol/L). The activity against M. kansasii
(
All newly synthesized compounds were evaluated for
their antimycobacterial activity against a set of four
mycobacterial strains, M. tuberculosis CNCTC My 331/
88, Mycobacterium kansasii CNCTC My 235/80, M.
kansasii 6509/96, and M. avium CNCTC My 330/88,
6509/96 was 2-fold less than that of INH. The com-
pounds showed only weak activity (MIC=8 mmol/L)
against M. tuberculosis 331/88 and the activity of INH
was not reached (MICINH=0.5–1 mmol/L). MICs
against M. avium 330/88 could not be determined due to
insolubility of compounds in the testing medium. With
regard to cyano compounds (4g, 4h), none of them
showed significant activity. The conversion of the cyano
group into a thioamide group afforded more potent
compounds 4i, 4j against all strains tested than the cor-
responding cyano compounds 4h, 4g but they did not
using the micromethod for the determination of the
1
2
minimum inhibitory concentration (MIC). The MIC
1
3
values are summarized in Table 1. In several cases
denoted>), the minimum inhibitory concentration
(
could not be determined due to limited solubility of the
compounds in the testing medium. For the sake of
Scheme 1. Reagents and conditions: (a) Na, ethanol, DMF.
2
Scheme 2. Reagents and conditions: (a) H S, TEA, pyridine.

J. Ko c˘ ı´ et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3275–3278
3277
Table 1. In vitro antimycobacterial activity of compounds 1, 2, 4a–j, 5a–j expressed as MIC (mmol/L)
Compd
Strains
Mycobacterium
tuberculosis
My 331/88
Mycobacterium
kansasii
My 235/80
Mycobacterium
kansasii
6 509/96
Mycobacterium
avium
My 330/88
14 d
21 d
7 d
14 d
21 d
7 d
14 d
21 d
14 d
21 d
1
500
250
>62
>62
>250
8
500
500
>125
125
>250
8
500
125
32
32
125
2
1000
250
32
>32
125
4
4
32
62
16
1000
250
>62
>32
>125
4
4
>32
125
16
250
32
32
32
62
4
500
125
32
32
>62
8
8
32
62
16
1000
250
>32
>32
>62
8
8
>32
62
250
62
>32
>32
>125
>16
>16
62
62
32
62
>62
32
32
62
125
>62
>32
32
500
125
>62
>62
>125
>32
>32
62
62
32
62
>62
62
>62
62
4
4
4
4
4
4
4
4
4
4
2
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
8
8
4
4
>62
125
8
8
62
250
>32
>62
125
2
>62
125
16
16
62
500
>125
>125
500
2
32
32
8
16
32
8
16
32
j
8
32
32
8
32
62
62
32
62
62
16
8
125
125
4
125
500
>62
>125
125
16
>125
>500
>250
>125
500
>62
16
>500
500
8
62
62
32
32
32
>62
125
>32
62
32
32
8
32
62
>62
250
>125
>62
125
>62
>32
32
a
b
c
d
e
f
g
h
i
>250
>62
>32
>32
62
a
NE
8
2
62
62
4
4
62
250
8
8
250
250
8
16
32
4
125
8
16
32
16
32
8
16
32
62
j
8
16
8
16
16
8
INH
0.5
1
>250
>250
>250
2
2
4
>250
>250
aNE, not evaluated; d, days.
reach the activity of dinitro derivatives (4e, 4f). It is
worth noting that the 4-substituted derivative (4i) had
generally greater activity than its 3-substituted analogue
was weak and did not reach the activity of dinitro deri-
vatives of benzoxazole. A similar effect as in the set of
benzoxazole was revealed for cyano and thioamide
derivatives. The thioamide derivatives (5i, 5j) exhibited
promising activity, particularly 5i showed an improve-
ment in the activity against M. avium 330/88
(MIC=16–32 mmol/L) and both strains of M. kansasii
(MIC=4–8 mmol/L).
(4j). The compound 4i showed better activity, particu-
larly against M. avium 330/88, and was the most active
compound against this strain in the set (MIC=32 mmol/
L). In the case of benzothiazole derivatives (5), in vitro
evaluation revealed similar trends as in the benzoxazole
set (4). Thus, the starting benzothiazole-2-thiol (2) and
its 2-benzylsulfanyl derivative (5a) were found to be
only weakly active. Identically, as in the case of the
benzoxazole set, mononitro derivatives (5b, 5c, 5d)
exhibited no pronounced activity against all tested strains.
The derivatives bearing two nitro groups 5e and 5f exhib-
ited the highest activity of both sets against M. tubercu-
losis 331/88 (MIC =2 mmol/L and MIC =2–4 mmol/L)
The most active derivatives of each set were also sub-
jected to cytotoxic effect assay against the HeLa cells.
As reported in Table 2, the values expressed as CC50 are
ranging from 36.7 to >158.0 mmol/L. The values of
CC50 were further compared with the microbial data
and expressed as Safety Indexes (S.I.). The data revealed
1
4
that compounds can considered as moderately toxic.
5
e
5f
and were only 2-fold less active than INH (MIC=0.5–1
mmol/L). The activity against non-tuberculous strains
In conclusion, the results revealed that incorporation of
the benzylsulfanyl moiety into position 2 is important to
the antimycobacterial activity. The substitution of the
phenyl ring enhances the activity (4e, 4f, 4i, 4j, 5e, 5f, 5i,
5j). The further modification of the most active com-
pounds and also mechanism of action are in progress.
Table 2. Cytotoxic (CC50) evaluation of compounds 4e, 4f, 4i, 4j, 5e,
5f, 5i, 5j expressed in mmol/L
Compd
HeLa CC50
S.I.a
4
4
4
4
5
5
5
5
e
f
i
>150.9
136.1
78.6
>18.9
17.0
9.8
Acknowledgements
j
71.9
9.0
e
f
i
>143.9
>143.9
>158.0
36.7
>72.0
>72.0
>39.5
4.6
This work was financially supported by the Grant Agency
of the Czech Republic (grant No. 203/02/0082) and by the
Ministry of Education of the Czech Republic (Project No.
MSM 111600001). We are indebted to D. Karlı
(Department of Pharmaceutical Chemistry and Drug
j
´
ekova
` ´
aS.I.=CC50/MICM. tuberculosis My 331/88, 14d
.

3278
J. Ko c˘ ı´ et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3275–3278
Control, Faculty of Pharmacy, Charles University) for
ˇ
performing elemental analyses, and J. Ziz
and crystallization from ethanol (95%) yielded products 4i, 4j,
5i, 5j as yellow crystals in 35–66% yield.
ˇ kova and P.
´
1
2. All strains were obtained from the Czech National Col-
Janc rek (Department of Inorganic and Organic Chem-
a
ˇ ´
lection of Type Cultures (CNCTC), with the exception of M.
kansasii 6509/96, which was a clinical isolate. The anti-
mycobacterial activities of the compounds were determined in
istry, Faculty of Pharmacy, Charles University) for
recording the IRspectra and NMRspectra, respectively.
ˇ
the Sula semisynthetic medium (SEVAC, Prague). The com-
pounds were added to the medium in dimethylsulfoxide solu-
tions. The following concentrations were used: 500, 250, 125,
References and Notes
6
2, 32, 16, 8, 4, and 2 (mmol/L). MICs were determined after
ꢂ
incubation at 37 C for 7, 14, and 21 days. MIC was the lowest
concentration of the substance, at which the inhibition of the
growth of mycobacteria occurred.
1
2
. Rouhi, A. M. Chem. Eng. News 1999, 77, 52.
. Bottari, B.; Maccari, R.; Monforte, F.; Ottana, R .; R otondo,
´
E.; Vigorita, M. G. Bioorg. Med. Chem. Lett. 2000, 10, 657.
. Klimesova, V.; Otcena ek, O.; Waisser, K. Eur. J. Med.
Chem. 1996, 31, 389.
. Klimesova, V.; Svoboda, M.; Waisser, K.; Macha
Buchta, V.; Odlerova
13. All prepared compounds were fully characterized by
spectral methods, and their purity checked by elemental ana-
lyses. Representative data for the most potent derivatives:
3
ˇ
´
ˇ
s
´ ˇ
4
ˇ
´
´
c
ˇ
ek, M.;
compound 4e: yellow crystals, yield 62.31%, M =331.30,
r
ˇ
ꢂ
´
, Z. Arch. Pharm. Pharm. Med. Chem.
mp=96–106 C, IR(KBr)
nmax 3107, 3086 (C–H)Ar, 1627,
1
5
996, 329, 438.
. Klimesova, V.; Svoboda, M.; Waisser, K.; Kaustova
lova, K. Eur. J. Med. Chem. 1999, 34, 433.
. Waisser, K.; Klimes
Univ. Carol. 1995, 18, 31.
. Klimesova, V.; Svoboda, M.; Waisser, K.; Pour, M.;
Kaustova, J. Collect. Czech. Chem. Commun. 1999, 64, 417.
. Klimesova, V.; Koc , J.; Pour, M.; Stachel, J.; Waisser, K.;
Kaustova, J. Eur. J. Med. Chem. 2002, 37, 409.
. Klimesova, V.; Koe , J.; Waisser, K.; Kaustova
002, 57, 259.
0. General procedure for the preparation of compounds 4a–
1596, 1454 (C¼C)Ar, 1541, 1345 (NO
2
), 1505 (C¼N), 1470
ꢀ
1
1
ˇ
´
´
, J.;
(CH ), 1236 (Ar–H), 863, 731 (Ar–H 1H) cm
,
H NMR
) d 4.85 (2H, s), 7.26–7.37 (2H, m), 7.58–
7.66 (2H, m), 8.69 (1H, t, J=2.2 Hz), 8.87 (2H, d, J=2.2 Hz),
2
Buchta, V.; Kra
6
´
´
(300 MHz, DMSO-d
6
ˇ
ova, V.; Odlerova, Z. Folia Pharm.
ˇ ´ ´
1
3
C NMR(75 MHz, DMSO- d
124.7, 125.0, 129.9, 141.2, 142.2, 148.1, 151.6, 163.5. Anal.
calculated for C14 S: C 50.76, H 2.74, N 12.68, O
6
) 34.0, 110.5, 118.0, 118.5,
7
ˇ
´
´
9 3 5
H N O
8
ˇ
´
ˇ
ı
´
24.15, S 9.68. Found: C 50.65, H 2.77, N 12.53, S 9.70. 4g:
ꢂ
´
white crystals, yield 79.67%, M
=266.32, mp=102–106 C,
r
9
2
1
ˇ
´
`
ı
´
´
, J. Farmaco
IR(KBr) nmax 3048 (C–H)Ar, 2227 (CꢃN), 1604, 1509, 1453
ꢀ1 1
2
(C¼C)Ar, 1470 (CH
NMR(300 MHz, DMSO- d
7.59–7.67 (2H, m), 7.68–7.73 (2H, m AA BB ), 7.77–7.82 (2H,
), 1242 (Ar–H), 831 (Ar–H 2H) cm , H
6
) d 4.67 (2H, s), 7.26–7.36 (2H, m),
0
0
h, 5a–h. Benzazole-2-thiol (1, 2) (5 mmol) in dry DMF (8 mL)
was added to a solution of sodium (5 mmol) in dry ethanol
0
0
13
m AA BB ), C NMR(75 MHz, DMSO- d ) 35.1, 110.5, 110.6,
6
(
2.5 mL). After 10 min of stirring at ambient temperature,
118.6, 118.9, 124.7, 124.9, 130.2, 132.7, 141.3, 151.6, 163.7.
benzyl halide (3) (5mmol) was added in 2–3 portions, and the
resultant suspension was stirred for 2–6 h. The reaction mix-
ture was then poured into an ice bath and left at it overnight.
The solid was filtered off, washed with cold water (2ꢁ30 mL)
and air-dried. The crude product was purified by preparative
TLC using acetone–light petroleum (1:2, 1:3, 1:4, 1:5, 1:6),
followed by crystallization from ethanol (95%) to afford the
crystals of the pure product 4a–h, 5a–h in 35–97% yield.
10 2
Anal. calculated for C15H N OS: C 67.65, H 3.78, N 10.52, O
6.01, S 12.04. Found: C 67.80, H 3.85, N 10.92, S 11.72. 5i:
ꢂ
yellow crystals, yield 66.37%, M
r
=316.45, mp=143–160 C,
2
IR(KBr) nmax 3425 (NH), 3061 (C–H)Ar, 2924, 1426 (CH ),
1629, 1455 (C¼C)Ar, 1273 (Ar–H), 1126 (C¼S –NH—C¼S),
ꢀ
1 1
850 (Ar–H 2H) cm , H NMR(300 MHz, DMSO- d ) d 4.67
6
(2H, s), 7.32–7.40 (1H, m), 7.42–7.58 (1H, m overlapped),
0
0
7.50–7.58 (2H, m AA BB overlapped), 7.77–7.92 (1H, m
0 0
1
4
1. General procedure for the preparation of compounds 4i,
j, 5i, 5j. Dry hydrogen sulfide was passed through a solution
overlapped), 7.77–7.85 (2H, m AA BB overlapped), 7.96–8.06
1
3
(1H, m), 9.47 (1H, s), 9.86 (1H, s), C NMR(75 MHz,
DMSO–d ) 36.3, 121.4, 122.1, 124.8, 126.6, 127.7, 128.8,
of cyano compounds 4g, 4h, 5g, 5h (2 mmol) dissolved in a
mixture of dry pyridine (7 mL) and triethylamine (0.7 mL).
The reaction mixture was maintained at ambient temperature
6
134.9, 138.9, 140.2, 152.8, 166.0, 199.8. Anal. calculated for
C H N S : C 56.93, H 3.82, N 8.85, S 30.39. Found: C
1
5
12
2 3
ꢂ
for 3 h and then heated at 45 C for additional h. After cool-
56.76, H 3.95, N 8.65, S 30.27.
¨
14. Dahse, H.-M.; Schlegel, B.; Grafe, U. Differentiation
between inducers of apoptosis and nonspecific cytotoxic drugs
by means of cell analyzer and immunoassay. Pharmazie 2001,
56, 489.
ing, the mixture was poured onto crushed ice with intensive
stirring, the precipitated product was filtered off, washed with
cold water (2ꢁ35 mL) and air-dried. Performed preparative
TLC chromatography using acetone–light petroleum (1:1, 1:2)