On the relationship between the substitution pattern of thiobenzanilides and their antimycobacterial activity

Article abstract of DOI:10.1016/S0014-827X(02)01285-5

The goal of this work was to shed more light on a preliminary finding about the relationship between the substitution in the thioacyl part of thiobenzanilides and their antituberculous effect. Thus, we prepared a set of 14 derivatives, out of which eight

Full text of DOI:10.1016/S0014-827X(02)01285-5

Il Farmaco 57 (2002) 777ꢀ782
/
www.elsevier.com/locate/farmac
Short Communication
On the relationship between the substitution pattern of
thiobenzanilides and their antimycobacterial activity
Jirˇ´ı Kunesˇ a,*, Vojteˇch Balsˇa´nek a, Milan Pour a, Karel Waisser a, Jarmila Kaustova´ b
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, CZ-500 05 Hradec Kra´love´, Czech
Republic
b National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Hygiene, CZ-728 92 Ostrava, Czech Republic
Received 23 January 2002; received in revised form 29 April 2002; accepted 25 May 2002
Abstract
The goal of this work was to shed more light on a preliminary finding about the relationship between the substitution in the
thioacyl part of thiobenzanilides and their antituberculous effect. Thus, we prepared a set of 14 derivatives, out of which eight had
not yet been reported, and the compounds were evaluated for antimycobacterial activity on a panel of four Mycobacteria species,
including Mycobacterium tuberculosis CNCTC My 331/88, Mycobacterium kansasii CNCTC My 235/80, Mycobacterium avium
CNCTC My 330/88 and M. kansasii 6509/96. While the contribution of the substituents with differing electronic and lipophilicity
characteristics in position 3 to the antituberculous activity was negligible, we found that unsubstituted position 4 in the thioacyl part
´
appears to be a prerequisite for a thiobenzanilide derivative to possess appreciable biological activity. # 2002 Editions scientifiques
et me´dicales Elsevier SAS. All rights reserved.
Keywords: Thiobenzanilides; Antimycobacterial activity; Nontuberculous strains of mycobacteria
1. Introduction
The substituents in the thioacyl part of the molecule
were selected in accordance with the approach described
by Topliss [3], which serves as a standard tool for
In the process of optimizing the structure of thioben-
zanilide, Waisser et al. found out that the antimyco-
bacterial activity of its derivatives was favourably
influenced by the introduction of a lipophilic substituent
into position 4? (in the anilide part of the molecule).
Therefore, they prepared a group of compounds with
the isopropyl, butyl [1] and cyclohexyl [2] moieties in this
position and tested their antimycobacterial activity
against Mycobacterium tuberculosis, Mycobacterium
kansasii, Mycobacterium avium and Mycobacterium
fortuitum. In order to quantitatively evaluate the
influence of the alkyl substituents on the antimycobac-
terial activity of the derivatives, the experimental data
determining structureꢀactivity relationships (SARs)
/
from the order of biological activities in a series of
derivatives. Thus, the following functional groups were
introduced into the thioacyl part of the molecule: H, 4-
Cl, 4-CH3, 4-CH3O, 3-Br, 3-NO2. Compared to the
substitution recommended by Topliss (H, 4-Cl, 4-CH3,
4-OCH3 and 3,4-diCl), the 3-bromo compound was
prepared in lieu of the 3,4-dichloro substitution, and the
3-nitro derivative was added to the set. 3-Bromo
derivatives (derived from 3-bromobenzoic acid) are
more easily accessible, and their biological evaluation
can also supply information about the steric hindrance
of position 4 in relationship to the biological activity.
This change falls in line with the original Topliss’s idea
as well, because in comparison with the 4-chloro
derivative, both lipophilicity and electron-accepting
properties are enhanced by introducing the 3-bromo
function, even though not so markedly as in the case of
the 3,4-dichloro compound.
were analyzed according to the FreeꢀWilson approach.
/
The increase of biological activity was most significant
in the case of the cyclohexyl derivative; this effect was
probably linked to lipophilicity.
* Corresponding author
E-mail address: kunes@faf.cuni.cz (J. Kunesˇ).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 2 8 5 - 5

778
J. Kunesˇ et al. / Il Farmaco 57 (2002) 777ꢀ782
/
The order of antimycobacterial activities in the
2. Experimental
subseries of 4?-isopropyl and 4?-butylthiobenzanilides
bearing various functional groups in the thioacyl part
indicated a steric influence of substitution in position 4,
which led to a substantial decrease in the activity.
Consequently, the most efficient substances against the
tested strains of mycobacteria were those with unsub-
stituted p-position.
Following on from these results, we set out to
synthesise a larger series of 3-substituted 4?-alkylthio-
benzanilides in order to further investigate these find-
ings.
2.1. Chemistry
M.p.s were determined on a Kofler block and are
1
uncorrected. H and 13C NMR spectra were recorded
for CDCl3 solutions at ambient temperature on a Varian
Mercury-Vx BB 300 spectrometer operating at 300 MHz
1
for H. Chemical shifts were recorded as d values in
parts per million (ppm), and were indirectly referenced
to tetramethylsilane (TMS) via the solvent signal (7.26
1
for H, 77.0 for 13C in CDCl3). Infrared spectra were
Table 1
Some characteristic of the benzanilides 1ꢀ14
/
Comp.
Code
Yield (%)
M.p. (8C)
M.p. (8C) [Reference]
IR (CO) (cmꢂ1
)
4?-Isopropylbenzanilide
1
91
75
94
82
81
79
77
81
77
69
73
83
94
95
161ꢀ
141ꢀ
112ꢀ
108ꢀ
146ꢀ
151ꢀ
123ꢀ
125ꢀ
85ꢀ
69ꢀ
/
165
142
114
111
148
152
124
130
159ꢀ
/
162 [1]
1650
1651
1655
1648
1654
1652
1649
1652
1652
1647
1652
1652
1647
1649
3-Chloro-4?-isopropylbenzanilide
3-Methyl-4?-isopropylbenzanilide
3-Methoxy-4?-isopropylbenzanilide
3-Bromo-4?-isopropylbenzanilide
3-Nitro-4?-isopropylbenzanilide
4?-Butylbenzanilide
3-Chloro-4?-butylbenzanilide
3-Methyl-4?-butylbenzanilide
3-Methoxy-4?-butylbenzanilide
3-Bromo-4?-butylbenzanilide
3-Nitro-4?-butylbenzanilide
4?-Cyklohexylbenzanilide
2
/
3
/
4
/
5
/
144ꢀ
143ꢀ
112ꢀ
/
147 [1]
146 [1]
115 [1]
6
/
/
7
/
/
8
/
9
/
87
71
10
11
12
13
14
/
130ꢀ
147ꢀ
204ꢀ
208ꢀ
/
132
149
206
209
128ꢀ
/
131 [1]
210 [2]
/
/
195ꢀ
/
3-Chloro-4?-cyklohexylbenzanilide
/
Table 2
1H NMR spectroscopic data of the benzanilides
Code 1N NMR (300 MHz, CDCl3)
2
d 1.24 (d, 6H, Jꢁ6.87 Hz, CH3), 2.97ꢀ
Jꢁ7.87, 1.76, 1.1 Hz, H4), 7.55ꢀ7.50 (m AA?, BB?, 2H, H2?, H6?), 7.69 (dt, 1H, Jꢁ7.87, 1.76 Hz, H6), 7.81 (t, 1H, Jꢁ1.76 Hz, H2), 8.07 (bs,
1H, NH)
d 1.25 (d, 6H, Jꢁ6.86 Hz, CH3), 2.40 (s, 3H, CH3), 2.99ꢀ
H5), 7.59ꢀ7.53 (m AA?, BB?, 2H, H2?, H6?), 7.65ꢀ7.60 (m, 1H, H6), 7.69ꢀ
d 1.25 (d, 6H, Jꢁ7.14 Hz, CH2), 2.98ꢀ2.82 (m, 1H, CH), 3.83 (s, 3H, OCH3), 7.05 (ddd, 1H, Jꢁ7.42, 2.47, 1.79 Hz, H4), 7.25ꢀ
BB?, 2H, H3?, H5?), 7.44ꢀ7.33 (m, 3H, H2, H5, H6), 7.59ꢀ7.52 (m AA?, BB?, 2H, H2?, H6?), 7.96 (bs, 1H, NH)
d 0.93 (t, 3H, Jꢁ7.55 Hz, CH3), 1.43ꢀ1.28 (m, 2H, CH2), 1.65ꢀ1.52 (m, 2H, CH2), 2.59 (t, 2H, Jꢁ7.55 Hz, CH2), 7.19ꢀ
2H, H3?, H5?), 7.35 (t, 1H, Jꢁ7.83 Hz, H5), 7.46 (dd, 1H, Jꢁ1.97, 0.97 Hz, H4), 7.55ꢀ7.47 (m AA?, BB?, 2H, H2?, H6?), 7.72ꢀ
H6), 7.81 (t, 1H, Jꢁ1.97 Hz, H2), 8.02 (bs, 1H, NH)
d 0.93 (t, 3H, Jꢁ7.51 Hz, CH3), 1.44ꢀ1.29 (m, 2H, CH2), 1.66ꢀ
7.12 (m, AA?, BB?, 2H, H3?, H5?), 7.36ꢀ7.30 (m, 2H, H4, H5), 7.59ꢀ
1H, NH)
d 0.93 (t, 3H, Jꢁ7.51 Hz, CH3), 1.43ꢀ
(ddd, 1H, Jꢁ7.97, 2.47, 1.37 Hz, H4), 7.19ꢀ
H2?, H6), 8.04 (bs, 1H, NH)
d 0.93 (t, 3H, Jꢁ7.41 Hz, CH3), 1.42ꢀ
2H, H3?, H5?), 7.58ꢀ7.48 (m AA?, BB?, 2H, H2?, H6?), 7.65 (t, 1H, Jꢁ7.97 Hz, H5), 8.28ꢀ
8.69ꢀ8.66 (m, 1H, H2)
d 1.48ꢀ1.21 (m, 5H, CH2), 1.97ꢀ
H5), 7.56ꢀ7.48 (m AA?, BB?, 2H, H2?, H6?), 7.75ꢀ
/
2.81 (m, 1H, CH), 7.23ꢀ
/
7.17 (m AA?, BB?, 2H, H3?, H5?), 7.35 (t, 1H, Jꢁ7.87 Hz, H5), 7.47 (ddd, 1H,
/
3
4
8
/2.82 (m, 1H, CH), 7.24ꢀ
/
7.18 (m AA?, BB?, 2H, H3?, H5?), 7.35ꢀ
/
7.31 (m, 2H, H4,
7.18 (m AA?,
/
/
/
7.65 (m, 1H, H2), 7.96 (bs, 1H, NH)
/
/
/
/
/
/
/
7.11 (m AA?, BB?,
7.67 (m, 1H,
/
/
9
/
/
1.53 (m, 2H, CH2), 2.39 (s, 3H, CH3), 2.59 (t, 2H, Jꢁ7.51 Hz, CH2), 7.21ꢀ
/
/
/
7.50 (m AA?, BB?, 2H, H2?, H6?), 7.70ꢀ7.59 (m, 2H, H2, H6), 7.98 (bs,
/
10
12
14
/
1.28 (m, 2H, CH2), 1.66ꢀ
/1.52 (m, 2H, CH2), 2.59 (t, 2H, Jꢁ7.51 Hz, CH2), 3.81 (s, 3H, OCH3), 7.04
/7.12 (m AA?, BB?, 2H, H3?, H5?), 7.43ꢀ
/
7.27 (m, 3H, H2, H5, H6), 7.58ꢀ7.50 (m AA?, BB?, 2H,
/
/
1.27 (m, 2H, CH2), 1.65ꢀ
/
1.51 (m, 2H, CH2), 2.59 (t, 2H, Jꢁ7.41 Hz, CH2), 7.21ꢀ/7.11 (m, AA?, BB?,
/
/
8.18 (m, 2H, H6, NH), 8.40ꢀ/8.32 (m, 1H, H4),
/
/
/
1.69 (m, 5H, CH2), 2.59ꢀ
/
2.43 (m, 1H, CH), 7.25ꢀ7.19 (m AA?, BB?, 2H, H3?, H5?), 7.42 (t, 1H, Jꢁ7.83 Hz,
/
/
/7.70 (m, 2H, H4, H6), 7.84 (t, 1H, Jꢁ1.79 Hz, H2)

J. Kunesˇ et al. / Il Farmaco 57 (2002) 777ꢀ
/
782
779
Table 3
13C NMR spectroscopic data of the benzanilides
Code 13C NMR (75 MHz, CDCl3)
2
d 24.0, 33.6, 120.6, 125.1, 126.9, 127.4, 129.9, 131.6, 134.8,
135.2, 136.8, 145.6, 164.5
3
d 21.3, 24.0, 33.6, 120.4, 123.9, 126.9, 127.8, 128.1, 132.4, 135.0,
135.6, 138.5, 145.1, 165.9
4
d 24.0, 33.6, 55.4, 112.4, 117.9, 118.7, 120.3, 126.9, 129.6, 135.5,
136.5, 145.3, 159.9, 165.5
8
d 13.9, 22.2, 33.6, 35.0, 120.5, 125.1, 127.4, 128.9, 129.9, 131.6,
134.8, 135.1, 136.8, 139.6, 164.5
9
d 13.9, 21.3, 22.2, 33.6, 35.0, 120.3, 123.9, 127.8, 128.5, 128.8,
132.4, 135.0, 135.5, 138.5, 139.1, 165.9
10
12
14
d 13.9, 22.2, 33.6, 35.0, 55.3, 112.3, 117.9, 118.7, 120.3, 128.8,
129.6, 135.5, 136.5, 139.2, 159.8, 165.6
d 13.9, 22.3, 33.6, 35.1, 120.7, 121.8, 126.2, 129.0, 130.0, 133.4,
134.8, 136.6, 140.1, 148.1, 163.4
d 26.1, 26.8, 34.5, 44.0, 120.4, 125.0, 127.3, 127.4, 130.1, 131.8,
135.0, 135.2, 136.9, 145.0, 164.2
recorded in CDCl3 on a Nicolet Impact 400 spectro-
photometer. Low resolution mass spectra were mea-
sured on
a Magnum Finnigan Mat apparatus.
Elemental analysis was carried out on a CHNS-OCE
FISONS EA 1110 instrument. Analytical thin-layer
chromatography (TLC) was conducted on E. Merck
TLC plates (silica gel 60 F254, aluminum back), and the
plates were visualised under UV light and in iodine
Scheme 1.
recrystallised from EtOH or purified by chromatogra-
phy (Tables 1ꢀ3).
/
vapours. Silica gel 60 (230ꢀ400 mesh) for column
/
chromatography was purchased from E. Merck.
2.3. Preparation of thiobenzanilides
A benzanilide (0.5ꢀ1 g) was mixed with a solution of
an equivalent amount of P2S5 in Py, and the reaction
2.2. Preparation of benzanilides
/
A p-substituted aniline (1ꢀ
/
3 g) was dissolved in Py
(5 ml), and an equivalent amount of an m-substituted
benzoyl chloride was added to the solution upon cool-
ing. The reaction mixture was allowed to stand for 12 h,
and a saturated aq. solution of Na2CO3 was then added.
The crude compound was filtered off and either
mixture was heated at reflux for 10 h. The solution was
poured into water (25ꢀ30 ml), and the resultant mixture
/
was allowed to stand until the initially formed oily
material turned into a solid. The crude product was
purified by chromatography (Tables 4ꢀ6).
/
Table 4
Some characteristic of the thiobenzanilides 1ꢀ14
/
Comp.
Code
Yield (%)
M.p. (8C)
M.p. (8C) [Reference]
66ꢀ69 [1]
LRMS
+ ꢃ
4?-Isopropylthiobenzanilide
15
16
17
18
19
20
21
22
23
24
25
26
27
28
10
26
5
67ꢀ
103ꢀ105
79ꢀ
81ꢀ
/
70
/
255 [M
289 [MꢃꢂH]
269 [M
285 [M
334 [M
299 [M
268 [M
]
3-Chloro-4?-isopropylthiobenzanilide
3-Methyl-4?-isopropylthiobenzanilide
3-Methoxy-4?-isopropylthiobenzanilide
3-Bromo-4?-isopropylthiobenzanilide
3-Nitro-4?-isopropylthiobenzanilide
4?-Butylthiobenzanilide
3-Chloro-4?-butylthiobenzanilide
3-Methyl-4?-butylthiobenzanilide
3-Methoxy-4?-butylthiobenzanilide
3-Bromo-4?-butylthiobenzanilide
3-Nitro-4?-butylthiobenzanilide
4?-Cyklohexylthiobenzanilide
/
+ ꢃ
/
85
84
]
]
]
]
]
+ ꢃ
+ ꢃ
+ ꢃ
+ ꢃ
+ ꢃ
42
6
/
109ꢀ/112
109ꢀ111 [1]
/
45
6
100ꢀ/102
96ꢀ/99 [1]
77ꢀ
93ꢀ
29ꢀ
59ꢀ
79ꢀ
74ꢀ
/
80
96
32
61
82
78
69ꢀ/72 [1]
7
/
302 [M ꢂH]
+ ꢃ
30
41
11
15
9
/
283 [M
299 [M
348 [M
]
]
]
+ ꢃ
+ ꢃ
+ ꢃ
/
/
75ꢀ/78 [1]
/
313 [M ꢂH]
294 [M ꢂH]
329 [M ꢂH]
+ ꢃ
115ꢀ/119
122 [2]
+ ꢃ
3-Chloro-4?-cyklohexylthiobenzanilide
35
175ꢀ/178

780
J. Kunesˇ et al. / Il Farmaco 57 (2002) 777ꢀ782
/
Table 5
1H NMR spectroscopic data of the thiobenzanilides 15ꢀ
28
/
Code 1H NMR (300 MHz, CDCl3)
15
16
17
18
19
20
d 1.27 (d, 6H, Jꢁ6.87 Hz, CH3), 3.03ꢀ
H2?, H6?), 7.88ꢀ7.79 (m, 2H, H2, H6), 9.01 (bs, 1H, NH)
d 1.27 (d, 6H, Jꢁ6.86 Hz, CH3), 3.03ꢀ2.85 (m, 1H, CH), 7.33ꢀ
(m, 1H, H5), 7.70ꢀ7.62 (m, AA?, BB?overlaped, 3H, H6, H2?, H6?), 7.80 (t, 1H, Jꢁ1.65 Hz, H2), 9.01 (bs, 1H, NH)
d 1.27 (d, 6H, Jꢁ7.14 Hz, CH3), 2.42 (s, 3H, CH3), 3.04ꢀ2.86 (m, 1H, CH), 7.36 (m, 4H, H4, H5, H3?, H5?), 7.73ꢀ7.56 (m, 4H, H2, H6, H2?,
H6?), 8.97 (bs, 1H, NH)
d 1.27 (d, 6H, Jꢁ6.87 Hz, CH3), 3.03ꢀ
7.45ꢀ7.41 (m, 1H, H2), 7.73ꢀ7.63 (m, AA?, BB?, 2H, H2?, H6?), 9.02 (bs, 1H, NH)
d 1.27 (d, 6H, Jꢁ6.86 Hz, CH3), 3.03ꢀ2.82 (m, 1H, CH), 7.35ꢀ7.26 (m, 3H, H4, H3?, H5?), 7.70ꢀ
H6?), 7.74 (d, 1H, Jꢁ7.70 Hz, H6), 7.96 (t, 1H, Jꢁ1.65 Hz, H2), 8.97 (bs, 1H, NH)
d 1.27 (d, 6H, Jꢁ6.86 Hz, CH3), 3.05ꢀ2.83 (m, 1H, CH), 7.35ꢀ7.27 (m AA?, BB?2H, H3?, H5?), 7.61 (t, 1H, Jꢁ7.97 Hz, H5), 7.72ꢀ7.64 (m,
/
2.87 (m, 1H, CH), 7.34ꢀ
/
7.26 (m, 2H, H3?, H5?), 7.55ꢀ
/
7.38 (m, 3H, H3, H4, H5), 7.72ꢀ
/
7.64 (m, 2H,
/
/
/
7.26 (m, AA?, BB?, 2H, H3?, H5?), 7.26 (d, 1H, Jꢁ7.96 Hz, H4), 7.48ꢀ
/
7.42
/
/
/
/
2.85 (m, 1H, CH), 3.86 (s, 3H, OCH3), 7.07ꢀ
/6.98 (m, 1H, H4), 7.38ꢀ7.26 (m, 4H, H5, H6, H3?, H5?),
/
/
/
/
/
/
7.57 (m, AA?, BB? overlaped, 3H, H5, H2?,
/
/
/
AA?, BB?, 2H, H2?, H6?), 8.20 (d, 1H, Jꢁ7.97 Hz, H6), 8.31 (dd, Jꢁ7.97 Hz, Jꢁ1.38 Hz, H4), 8.59 (t, 1H, Jꢁ1.38 Hz, H2), 9.18 (bs, 1H,
NH)
21
22
23
24
0.94 (t, 3H, Jꢁ7.41 Hz, CH3), 1.45ꢀ
H3?, H5?), 7.55ꢀ7.38 (M 3H, H3, H4, H5), 7.71ꢀ
d 0.94 (t, 3H, Jꢁ7.41 Hz, CH3), 1.46ꢀ1.24 (m, 2H, CH2), 1.69ꢀ
H5?), 7.41ꢀ7.31 (m, 1H, H5), 7.53ꢀ7.42 (m, 1H, H4), 7.72ꢀ7.59 (m, 3H, H6, H2?, H6?), 7.84ꢀ
d 0.94 (t, 3H, Jꢁ7.41 Hz, CH3), 1.46ꢀ1.28 (m, 2H, CH2), 1.68ꢀ1.55 (m, 2H, CH2), 2.64 (t, 2H, Jꢁ7.41 Hz, CH2), 7.28ꢀ
H5?), 7.34ꢀ7.28 (m, 2H, H4, H5), 7.71ꢀ7.56 (m, 4H, H2, H6, H2?, H6?), 8.99 (bs, 1H, NH)
d 0.94 (t, 3H, Jꢁ7.41 Hz, CH3), 1.45ꢀ1.29 (m, 2H, CH2), 1.69ꢀ1.55 (m, 2H, CH2), 2.63 (t, 2H, Jꢁ7.41 Hz, CH2), 3.86 (s, 3H, OCH3), 7.07ꢀ
6.99 (m, 1H, H4), 7.28ꢀ7.21 (m, 2H, H3?, H5?), 7.37ꢀ7.30 (m, 2H, H5, H6), 7.45ꢀ7.40 (m, 1H, H2), 7.70ꢀ7.62 (m, 2H, H2?, H6?), 9.01 (bs, 1H,
NH)
d 0.94 (t, 3H, Jꢁ7.41 Hz, CH3), 1.45ꢀ
H3?, H5?), 7.67ꢀ7.58 (m, 3H, H5, H2?, H6?), 7.74 (d, 1H, Jꢁ7.97 Hz, H6), 7.96 (t, 1H, Jꢁ1.81 Hz, H2), 8.97 (bs, 1H, NH)
d 0.94 (t, 3H, Jꢁ7.42 Hz, CH3), 1.46ꢀ1.29 (m, 2H, CH2), 1.69ꢀ1.55 (m, 2H, CH2), 2.64 (t, 2H, Jꢁ7.42 Hz, CH2), 7.29ꢀ7.22 (m, AA?, BB?,
2H, H3?, H5?), 7.69ꢀ7.57 (m, 3H, H5, H2?, H6?), 8.20 (d, 1H, Jꢁ7.96 Hz, H6), 8.31 (dd, 1H, Jꢁ7.96, 1.37 Hz, H4), 8.59 (t, 1H, Jꢁ1.37 Hz,
H2), 9.17 (bs, 1H, NH)
d 1.51ꢀ1.16 (m, 5H, CH2), 1.97ꢀ
H4, H5), 7.73ꢀ7.63 (m, AA?, BB?, 2H, H2?, H6?), 7.88ꢀ
d 1.53ꢀ1.16 (m, 5H, CH2), 1.98ꢀ1.68 (m, 5H, CH2), 2.62ꢀ
(m, 1H, H4), 7.72ꢀ7.62 (m, 3H, H6, H2?, H6?), 7.84ꢀ7.79 (m, 1H, H2), 8.96 (bs, 1H, NH)
/
1.29 (m, 2H, CH2), 1.69ꢀ
7.62 (m, 2H, H2?, H6?), 7.89ꢀ
1.51 (m, 2H, CH2), 2.63 (t, 2H, Jꢁ7.41 Hz, CH2), 7.29ꢀ
7.78 (m, 1H, H2), 9.02 (bs, 1H, NH)
7.21 (m, 2H, H3?,
/
1.53 (m, 2H, CH2), 2.64 (t, 2H, Jꢁ7.41 Hz, CH2), 7.30ꢀ
/
7.21 (m, AA?, BB?, 2H,
/
/
/7.79 (m, 2H, H2, H6), 9.01 (bs, 1H, NH)
/
/
/7.21 (m, 2H, H3?,
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
25
26
/
1.30 (m, 2H, CH2), 1.69ꢀ
/
1.54 (m, 2H, CH2), 2.63 (t, 2H, Jꢁ7.41 Hz, CH2), 7.33ꢀ7.22 (m, 3H, H4,
/
/
/
/
/
/
27
28
/
/
1.68 (m, 5H, CH2), 2.61ꢀ
7.79 (m, 2H, H2, H6), 9.00 (bs, 1H, NH)
2.44 (m, 1H, CH), 7.31ꢀ7.24 (m, 2H, H3?, H5?), 7.40ꢀ
/2.46 (m, 1H, CH), 7.32ꢀ/7.24 (m, AA?, BB?, 2H, H3?, H5?), 7.54ꢀ/7.38 (m, 3H, H3,
/
/
/
/
/
/
/
7.32 (m, 1H, H5), 7.50ꢀ7.42
/
/
/
Table 6
13C NMR spectroscopic data of the thiobenzanilides 15ꢀ
/28
Code
13C NMR (75 MHz, CDCl3)
15
16
17
18
19
20
21
22
23
24
25
26
27
28
d 23.9, 33.8, 123.6, 126.6, 127.0, 128.6, 131.2, 136.6, 143.1, 147.8, 198.0
d 23.9, 33.8, 123.5, 124.7, 127.0, 129.8, 131.0, 134.6, 136.4, 144.6, 148.0, 196.1
d 21.4, 23.9, 33.8, 123.4, 123.6, 127.0, 127.7, 128.5, 132.0, 136.7, 138.6, 143.3, 147.8, 198.3
d 23.9, 33.8, 55.5, 112.6, 117.3, 118.1, 123.6, 127.0, 129.6, 136.6, 144.6, 147.8, 159.6, 197.7
d 23.9, 33.8, 122.7, 123.5, 125.2, 127.0, 129.7, 130.1, 133.9, 136.4, 144.8, 148.1, 196.0
d 23.8, 33.8, 121.1, 123.5, 125.4, 127.1, 129.7, 133.1, 136.2, 144.2, 147.9, 148.3, 194.6
d 13.9, 22.3, 33.4, 35.2, 123.6, 126.6, 126.9, 128.6, 128.7, 128.9, 131.2, 136.6, 142.0, 143.2, 198.1
d 13.9, 22.3, 33.4, 35.3, 123.5, 124.7, 127.0, 129.0, 129.8, 131.0, 134.6, 136.3, 142.2, 144.6, 196.1
d 13.9, 21.4, 22.3, 33.4, 35.2, 123.4, 123.6, 127.6, 128.5, 128.9, 132.0, 136.6, 138.5, 141.9, 143.2, 198.3
d 13.9, 22.3, 33.4, 35.2, 55.5, 112.6, 117.3, 118.1, 123.5, 128.9, 129.6, 136.5, 141.9, 144.6, 159.6, 197.7
13.9, 22.3, 33.4, 35.3, 122.7, 123.5, 125.3, 129.0, 129.7, 130.1, 133.9, 136.3, 142.2, 144.8, 196.0
d 13.9, 22.3, 33.4, 35.3, 121.1, 123.5, 125.4, 129.0, 129.7, 133.2, 136.1, 142.5, 144.2, 147.9, 194.6
d 26.0, 26.8, 34.3, 44.2, 123.5, 126.6, 127.3, 128.6, 131.2, 136.6, 143.2, 147.0, 197.9
d 26.0, 26.8, 34.3, 44.2, 123.4, 124.7, 127.0, 127.4, 129.9, 131.0, 134.6, 136.4, 144.7, 147.3, 196.0
2.4. Biology
My 330/88), and a clinical isolate (M. kansasii 6509/96)
were used for the evaluation of the biological activity of
the target compounds. The tests were carried out in the
Three CNCTC (Czech National Collection of type
cultures) strains (M. tuberculosis CNCTC My 331/88,
M. kansasii CNCTC My 235/80 and M. avium CNCTC
ˇ
semisynthetic Sula medium (SEVAC, Prague). Twofold
serial dilutions of the compounds in Me2SO (250ꢀ8
/

J. Kunesˇ et al. / Il Farmaco 57 (2002) 777ꢀ
/
782
781
Table 7
Antimycobacterial activity of thiobenzanilides (MIC, mmol lꢂ1
)
Comp.
M. tuberculosis My 331/88
M. avium My 330/88
M. kansasii My 235/80
M. kansasii 6509/96
15
32
62.5
62.5
62.5
32
32
62.5
62.5
32
62.5
62.5
32
16
17
18
19
20
21
22
23
24
25
26
27
28
32
32
32
62.5
62.5
62.5
32
62.5
62.5
32
32
62.5
62.5
62.5
62.5
62.5
32
62.5
62.5
32
32
62.5
32
62.5
62.5
62.5
32
32
32
62.5
32
32
32
62.5
62.5
32
62.5
16
16
32
62.5
62.5
62.5
62.5
32
32
32
32
62.5
62.5
62.5
32
62.5
62.5
32
62.5
62.5
32
32
62.5
62.5
62.5
62.5
62.5
16
62.5
62.5
16
32
32
32
32
32
32
32
32
32
32
32
32
16
32
16
32
32
16
32
16
32
32
32
32
32
32
62.5
62.5
16
32
32
16
32
8
16
16
16
32
16
32
32
16
32
32
32
16
32
16
32
32
32
16
16
32
62.5
62.5
62.5
Isoniazide
0.5
1.0
ꢀ250
250
ꢀ250
ꢀ250
ꢀ250
0.125
25
2.0
4.0
4.4
Rifampicine
Clofazimine
0.25
16
0.125
0.125
0.25
0.06
0.06
0.06
25
0.06
0.125
0.125
0.25
0.25
0.25
0.06
0.06
mmol lꢂ1) were used. Minimum inhibitory concentra-
tions (MICs) were read after 14 and 21 days of
incubation at 37 8C for M. tuberculosis and M. avium,
, and after 7, 14 and 21 days for M. kansasii. The results
are shown in Table 7.
3. Results and discussion
The compounds were prepared via a three-step
process (Scheme 1). Commercially available substituted
benzoic acids were treated with thionyl chloride to

782
J. Kunesˇ et al. / Il Farmaco 57 (2002) 777ꢀ782
/
afford the corresponding acyl chlorides, which were
further converted into benzanilides by the reaction with
various substituted anilines in pyridine. Finally, the oxo
group was replaced with the thioxo moiety by heating
the benzanilides with phosphorus pentasulphide in
pyridine.
do not significantly influence the activity. As regards
substitution in the anilide moiety, derivatives with the
isopropyl group in position 4? are more active than those
bearing the butyl chain. Further increase in the lipophi-
licity of the substituents in the anilide part does not lead
to derivatives possessing higher activity (4?-cyclohexyl
derivatives).
In summary, it appears that the presence of unsub-
stituted position 4 in the thioacyl part of the thiobenza-
nilide molecule seems to have a positive influence on the
antimycobacterial activity, with the compounds posses-
sing higher activity than that of the standard INH
against some of the nontuberculous strains of myco-
bacteria. The compounds, which do not fit this require-
ment, usually display substantially lower activities [1].
The structures of the compounds were confirmed by
1H and 13C NMR as well as by IR spectroscopy. The
signal of the carbonyl group at 164ꢀ166 ppm was
/
apparent in the 13C NMR spectra of benzanilide
derivatives, while the carbons of the thiocarbonyl
function in the corresponding thiobenzanilides reso-
nated at substantially lower field, in the range 194ꢀ
199 ppm. In the IR spectra of benzanilides, the typical
vibration bands of the CÄO group were observed
between 1647 and 1655 cmꢂ1. On the other hand, the
vibrations of the CÄS group in the thiobenzanilides
appeared at lower frequencies, in the range 1198ꢀ1221
/
/
/
/
cmꢂ1. The structures of the target compounds were also
corroborated by low resolution mass spectra; the
spectrum of each compound contained a peak at m/z
corresponding to the molecular ion. Finally, the purity
of the compounds was confirmed by elemental analysis.
To test the assumption about the steric properties of
the substituents in position 4 having been the cause of
reduced activity of the 4-substituted thiobenzanilides [1],
the target compounds were evaluated for antimycobac-
terial activity. The results, displayed in Table 7, clearly
show that the activities of the compounds against
nontuberculous strains of mycobacteria (M. avium My
330/88 and M. kansasii My 235/80) are superior to that
of the standard antituberculotic isoniazid (INH), but
lower than those of rifampicine and clofazimine. Hence,
it is important for the antimycobacterial effect of
thiobenzanilides that position 4 in the thioacyl part be
unsubstituted. Interestingly, substituents with various
electronic parameters and lipophilicity attached to C(3)
Acknowledgements
This work was supported by the Ministry of Educa-
tion of the Czech Republic (Project No. MSM
11160001).
References
ˇ
[1] J. Kunesˇ, J. Ja´chym, P. Jira´sko, Z. Odlerova´, K. Waisser,
Combination of the Topliss approach with the FreeꢀWilson
analysis in the study of antimycobacterial activity of 4-alkylthio-
benzanilides, Collect. Czech. Chem. Commun. 62 (1997) 1503ꢀ
1510.
[2] K. Waisser, L. Kubicova´, Z. Odlerova´, Antituberculotic 4?-
cyclohexylthiobenzanilides-combination of FreeꢀWilson method
in QSAR with Topliss approach, Collect. Czech. Chem. Commun.
58 (1993) 205ꢀ212.
[3] J.G. Topliss, A manual method for applying the Hansch approach
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ˇ
/
/
/