Combination of the topliss approach with the Free-Wilson analysis in the study of antimycobacterial activity of 4-alkylthiobenzanilides

Article abstract of DOI:10.1135/cccc19971503

On the basis of a preliminary study of the antimycobacterial activity of thiobenzanilides, a group of 4′-isopropyl- and 4′-butylthiobenzanilides have been synthesized and tested against Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avi

Full text of DOI:10.1135/cccc19971503

Antimycobacterial Activity of 4-Alkylthiobenzanilides
1503
COMBINATION OF THE TOPLISS APPROACH WITH THE FREE–WILSON
ANALYSIS IN THE STUDY OF ANTIMYCOBACTERIAL ACTIVITY
OF 4-ALKYLTHIOBENZANILIDES*
a
a
a
b
Jiri KUNES , Jiri JACHYM , Petr JIRASKO , Zelmira ODLEROVA
and Karel WAISSERa1,**
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
500 05 Hradec Kralove, Czech Republic; e-mail: 1 waisser@faf.cuni.cz
b Institute of Preventive and Clinical Medicine, 833 01 Bratislava, Slovak Republic
Received January 16, 1997
Accepted April 26, 1997
Dedicated to Professor Dr Vaclav Horak (Georgetown University) on the occasion of his 75th birthday.
On the basis of a preliminary study of the antimycobacterial activity of thiobenzanilides, a group of
4′-isopropyl- and 4′-butylthiobenzanilides have been synthesized and tested against Mycobacterium
tuberculosis, Mycobacterium kansasii, Mycobacterium avium and Mycobacterium fortuitum. The ef-
fect of the substituents on minimum inhibitory concentrations was calculated by the Free–Wilson
method. The separated values were analyzed by the Topliss approach. The substitution of the
thiobenzanilide in position 4′ by the isopropyl group or butyl group increased the antimycobacterial
activity less than the cyclohexyl group. The substitution in position 4 decreased the activity by the
steric effect.
Key words: Thiobenzanilides; Tuberculostatics; Mycobacterium spp.; Structure–activity relation-
ships; Free–Wilson analysis, Topliss approach.
Mycobacterial diseases produced by atypical mycobacterial strains have become a
serious health problem at present. The diseases caused by Mycobacterium avium are
particularly dangerous. Antituberculotic drugs were developed with an assumed effect
against Mycobacterium tuberculosis, and the principal aim of research into antileprotic
agents was development of compounds active against Mycobacterium leprae. Other
mycobacterial strains escaped the attention of research laboratories as they were not
considered to be pathogenic for man. Even nowadays they endanger the human popula-
* Part XII in the series Relationships Between the Chemical Structure of Substances and Their
Antimycobacterial Activity to Atypical Strains; Part XI: see ref.1.
**The author to whom correspondence should be addressed.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

1504
Kunes, Jachym, Jirasko, Odlerova, Waisser:
tion only infrequently but the result of their attack is, as a rule, fatal. The research team
of the present authors is therefore systematically engaged in the study of the relation-
ships between the structure and antimycobacterial activity of drugs with the aim of
revealing general rule which would make possible development of chemotherapeutic
agents against atypical mycobacterial strains.
A wide spectrum of antimycobacterial effect is found in a number of compounds
containing a thioxo group, such as thiobenzamides2, thiooxalamides3, 2-(alkysulfanyl)-
pyridine-4-carbothioamides4, 4-(alkylsulfanyl)pyridine-2-carbothioamides5, and thio-
benzanilides6,7. The latter study has revealed that the antimycobacterial effect of
thiobenzanilides is favorably affected by introducing cyclohexyl into position 4′. The
present paper describes preparation, antimycobacterial evaluation and analysis of the
relationships between the structure and antimycobacterial activity of two additional
groups of compounds containing an alkyl in position 4′, namely 4′-isopropylthiobenz-
anilides and 4′-butylthiobenzanilides.
X
1
R
NH
C
2
R
2
R
H
1, 7, 12, 18
2, 8, 13, 19
3, 9, 14, 20
4-CH
3
4-OCH
3-Br
3
4, 11, 15, 22
5, 10, 16, 21
6, 17
4-Cl
3-NO
2
In formulae 1-11: X = O; 12-22: X = S;
1
1
1-6, 12-17: R = CH(CH ) ; 7-11, 18-22: R = (CH ) CH
3 2
2 3
3
All the anilides under study 1–11 (Table I) were prepared by the reaction of benzoyl
chloride and substituted benzoyl chlorides with corresponding 4-isopropylaniline or
4-butylaniline in pyridine. Thiobenzanilides 12–22 (Table II) were prepared from the
corresponding anilides by the reaction with phosphorus pentasulfide in pyridine. The
selection of substitution modifications on the benzoyl moiety was carried out according
to Topliss8. This approach brings an advantage – in most cases it is possible to deter-
mine the structure–activity relationship from the order of activity of substances. In con-
trast to the original Topliss approach, 3-bromo derivatives 15 and 22 were synthesized
instead of 3,4-dichloro derivatives. This substitution is in accord with the original To-
pliss intention as in comparison with 4-chloroderivative lipophilicity and electron-ac-
ceptor properties are also increased. Nevertheless, 3-bromo derivatives can provide
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

Antimycobacterial Activity of 4-Alkylthiobenzanilides
1505
TABLE I
Characteristic data of 4′-isopropylbenzanilides 1–6 and 4′-butylbenzanilides 7–11
Calculated/Found
M.p., °C
Yield, %
Formula
M.w.
Compound
% C
% H
% N
1
2
152–162a
82
C16H17NO
80.30
80.13
7.16
7.15
5.85
5.79
239.3
155–157
93
C17H19NO
253.3
80.60
80.43
7.56
7.71
5.53
5.71
3
165–167
65
C17H19NO2
269.3
75.81
75.63
7.11
7.38
5.20
5.22
4
144–147
83
C16H16BrNO
318.2
60.39
60.07
5.07
4.60
4.40
4.47
5
239–241
98
C16H16ClNO
273.8
70.20
70.12
5.89
5.88
5.12
5.15
6
143–146
85
C16H16N2O3
284.3
67.59
67.66
5.67
5.32
9.85
9.85
7
112–115b
86
C17H19NO
253.3
80.60
80.27
7.56
7.35
5.53
5.80
8
118–121
86
C18H21NO
267.37
80.86
80.93
7.92
7.77
5.24
5.43
9
144–147c
82
C18H21NO2
283.8
76.30
76.40
7.47
7.47
4.94
4.67
10
11
193–196
90
C17H18ClNO
287.8
70.95
71.26
6.30
6.43
4.87
4.58
128–131
87
C17H18BrNO
332.2
61.46
61.62
5.46
4.99
4.22
4.72
a Ref.10 gives m.p. 161.4–162 °C. b Ref.11 gives m.p. 126 °C. c Ref.12 gives m.p. 145.5–146 °C.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Kunes, Jachym, Jirasko, Odlerova, Waisser:
TABLE II
Characteristic data of 4′-isopropylthiobenzanilides 12–17 and 4′-butylthiobenzanilides 18–22
Calculated/Found
M.p., °C
Yield, %
Formula
M.w.
Compound
% C
% H
% N
% S
12
13
66–69
58
C16H17NS
255.4
75.25
74.84
6.71
6.49
5.48
5.57
12.55
12.26
105–108 C17H19NS
75.79
75.70
7.11
7.22
5.20
4.81
11.90
11.95
43
269.4
14
15
99–101 C17H19NOS
71.54
71.79
6.71
6.58
4.91
5.11
11.23
5.21
41
285.4
109–111 C16H16BrNS
54 334.3
57.49
57.48
4.82
4.68
4.19
4.23
11.06
10.85
16
17
18
19
20
21
22
195–197 C16H16ClNS
66.31
66.30
5.56
5.50
4.83
4.82
9.59
9.38
65
289.8
96–99
39
C16H16N2O2S
300.4
63.98
63.84
5.37
5.36
9.33
9.14
10.67
9.24
69–72
57
C17H19NS
269.4
75.79
75.48
7.11
7.09
5.20
5.14
11.90
11.82
71–74
63
C18H21NS
283.4
76.28
76.29
7.47
7.58
4.94
4.80
11.31
11.09
105–108 C18H21NOS
48 299.4
72.20
71.92
7.07
7.18
4.68
4.59
10.71
10.75
121–124 C17H18ClNS
67.20
67.61
5.97
5.94
4.61
4.77
10.55
10.66
61
303.9
75–78
44
C17H18BrNS
348.3
58.62
58.51
5.21
5.18
4.02
4.21
9.20
8.84
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

Antimycobacterial Activity of 4-Alkylthiobenzanilides
1507
information about the steric hindrance of position 4 if it occurs (in connection with
biological activity). The structures of the new products prepared in the present study
were supported by their infrared spectra.
In the spectra of anilides 1–11 (Table III), absorptions of the amide carbonyl were
clearly observed in the region between 1 645 and 1 655 cm–1 but they disappeared after
replacement of the oxygen atom by the sulfur atom. The anilides absorb in the region
of 3 280 and 3 300 cm–1, whereas thiobenzanilides 12–22 in the region of 3 158–3 282 cm–1.
In all spectra it was possible to observe aromatic C–H vibration between 3 024 and
3 064 cm–1 and several absorption bands of alkyls, e.g. in the regions of 2 952–2 967 cm–1,
TABLE III
Infrared spectra of compounds 1–22
Compound
ν(N–H)
ν(C–H)arom
ν(C–H)alif
ν(C=O)
δ(C–H)
1
2
3 300
3 338
3 325
3 340
3 351
3 315
3 325
3 336
3 331
3 336
3 324
3 166
3 186
3 158
3 187
3 189
3 282
3 201
3 244
3 158
3 187
3 187
3 050
3 048
3 050
3 050
3 036
3 050
3 050
3 044
3 050
3 044
3 044
3 044
3 064
3 034
3 051
3 050
3 050
3 061
3 050
3 024
3 055
3 056
2 978, 2 958, 2 927, 2 894
2 961, 2 922, 2 887, 2 870
2 967, 2 929, 2 907, 2 869
2 964, 2 927, 2 894, 2 872
2 958, 2 922, 2 891, 2 866
2 963, 2 928, 2 900, 2 871
2 961, 2 926, 2 874, 2 853
2 959, 2 925, 2 873, 2 858
2 959, 2 929, 2 872, 2 858
2 952, 2 929, 2 874, 2 858
2 961, 2 925, 2 874, 2 853
2 963, 2 927, 2 889, 2 871
2 987, 2 959, 2 932, 2 868
2 962, 2 934, 2 871, 2 839
2 963, 2 927, 2 893, 2 872
2 960, 2 931, 2 895, 2 869
2 963, 2 930, 2 896, 2 871
2 961, 2 927, 2 871, 2 856
2 958, 2 926, 2 867, 2 856
2 960, 2 931, 2 872, 2 858
2 960, 2 927, 2 871, 2 859
2 958, 2 930, 2 870, 2 857
1 649
1 655
1 652
1 651
1 652
1 652
1 651
1 654
1 652
1 651
1 654
–
831
827
827
828
827
827
825
822
823
824
828
831
825
837
826
840
832
826
831
833
829
826
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
–
–
–
–
–
–
–
–
–
–
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Kunes, Jachym, Jirasko, Odlerova, Waisser:
2 922–2 929 cm–1, and 2 853–2 894 cm–1. The absorption maximum in the region of
822–840 cm–1, which occurs in the spectra of all compounds, was assigned to the out-
of-plane vibration of aromatic hydrogen atoms in the anilide moiety. The structure of
the newly prepared compounds was then further confirmed by elemental analysis.
Antimycobacterial activity of 4′-isopropylthiobenzanilides 12–17 and 4′-butyl-
thiobenzanilides 18–22 against Mycobacterium tuberculosis, Mycobacterium kansasii,
Mycobacterium avium, and Mycobacterium fortuitum was evaluated. The antimycobac-
terial activity against M. fortuitum was not significant and it was not therefore further
analyzed. The survey of antimicrobial activities of the compounds under study is given
in Table IV. The experimentally found data were then further separated by the Free–
Wilson method9 to determine the effects of modifying substituents. The calculation also
included the data of the activities of 4′-cyclohexylthiobenzanilides against M. tuberculosis
and M. kansasii which were taken from the previous study7. The results are sum-
marized in Table V. In the evaluation of the relationships between the structure and
activity against M. avium, only the data from the present study could be used as this
activity was not assayed in recent years.
TABLE IV
Minimum inhibitory concentration (MIC, µmol/l) of 4′-isopropylthiobenzamides 12–17 and 4′-bu-
tylthiobenzanilides 18–22
MIC, µmol/l
Compound
M. tuberculosis
M. kansasii
M. avium
M. fortuitum
12
13
14
15
16
17
18
19
20
21
22
60
125
60
60
250
60
125
1 000
250
500 (250)a
>1 000
>1 000
>1 000
>1 000
>1 000
250
30
60
125
500
30
>1 000
250
60
>1 000
1 000 (500)a
250
30
250
60
500
60
500
500
1 000
>1 000
250
1 000
125
125
250
125
>1 000
500
a The values in brackets represent concentrations of partial inhibition.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

Antimycobacterial Activity of 4-Alkylthiobenzanilides
1509
It follows from the found data that the effect of the butyl and isopropyl groups in
position 4′ of thiobenzanilides on the increase of the activity of compounds under study
is smaller than the effect of the cyclohexyl group. The effect could be ascribed to a
decrease in the lipophilicity of the substituent. The order of activity of thioanilides
substituted in the thiobenzoyl moiety indicates steric effect of substituents in position 4,
which is manifested by a decrease in the activity. In order to demonstrate this effect,
3-nitro-4′-isopropylthiobenzanilide 17 was synthesized. Its activity against M. tuberculosis
was one of the highest in the group of isopropylthiobenzanilides. However, its activity
against the other mycobacterial strains under study was not significant. The obtained
data contribute to further development of antimycobacterial thiobenzanilides.
EXPERIMENTAL AND CALCULATIONS
The melting points were determined with a Kofler apparatus and are uncorrect. The samples for ana-
lyses and antimycobacterial tests were dried over phosphorus pentoxide at 61 °C and 66 Pa for 24 h.
Elemental analyses were performed on a C,H,N analyzer (Laboratorni pristroje, Prague). The IR
spectra were measured in KBr on a Nicolet Impact 400 apparatus, wavelengths are given in cm–1
(Table III).
4′-Isopropylbenzanilides 1–6 and 4′-Butylbenzanilides 7–11
A benzoyl chloride (23 mmol) was added dropwise into a stirred mixture of substituted aniline (22.8 mmol)
and pyridine (20 ml) under cooling in an ice bath. The reaction mixture was allowed to stand at room
TABLE V
Results of the Free–Wilson structure–antimycobacterial activity analysis, contributions of fragments
to log MIC values (∆ log MIC)
∆ log MIC
Fragment
M. tuberculosisa
M. kansasiib
M. aviumc
H
–0.22
0.18
–0.30
0.30
–0.53
0.22
–0.23
0.52
0.02
0.04
–0.04
–
4-CH3
4-OCH3
4-Cl
–0.12
0.38
–0.30
0.50
3-Br
–0.22
0.16
–0.20
0.06
4′-i-C3H7
4′-C4H9
4′-C6H11
Common skeleton
0.16
0.12
–0.32
1.82
–0.18
2.00
2.78
Statistical evidence: a r = 0.853, s = 0.28, F = 4.80, t = 2.31, n = 16; b r = 0.846, s = 0.31, F = 4.53,
t = 2.31, n = 16; c r = 0.966, s = 0.15, F = 11.25, n = 10.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Kunes, Jachym, Jirasko, Odlerova, Waisser:
temperature for 1 day, whereafter it was poured into a saturated sodium carbonate solution (100 ml).
The separated product was filtered off, washed consecutively with a saturated sodium carbonate solution
and water, and recrystallized from ethanol. The results are given in Table I.
4′-Isopropylthiobenzanilides 12–17 and 4′-Butylthiobenzanilides 18–22
To a refluxing mixture of a 4′-alkylbenzanilide (14 mmol) and pyridine (10 ml), phosphorus penta-
sulfide (7.1 mmol) was added portionwise and the reaction mixture was refluxed for 3–8 h. After
cooling, it was poured into water (100 ml). On the following day, the raw product was filtered off
and recrystallized from ethanol. The results are given in Table II.
Microbiological Evaluation
The microbiological tests were carried out on a semisynthetic liquid medium with proteins by Sula
(SEVAC, Prague). The following mycobacterial strains were used: Mycobacterium tuberculosis
H37Rv, Mycobacterium kansasii PKG 8, Mycobacterium avium No. 80/72 and Mycobacterium fortuitum
No. 1 023. The substances were added after dissolution in dimethyl sulfoxide. The resulting concen-
trations of the compounds in the substrate were: 7, 15, 30, 60, 125, 250, 500 and 1 000 µmol/l. The
minimum inhibition concentrations (MIC) were read after 15 days of incubation at 37 °C. The results
are summarized in Table IV.
Calculations
All calculations were carried out using the Multireg program for Microsoft Excel. Table V presents
a survey of the calculated contributions of the varied structural fragments to the activity.
The authors wish to thank Mrs D. Karlickova from the Department of Pharmaceutical Chemistry and
Drug Control, Faculty of Pharmacy, Charles University, for CHN elemental analysis and Mrs J. Zizkova
from the Department of Inorganic and Organic Chemistry for elemental analysis of sulfur and IR
spectra measurements. The work was supported by grant No. 35/95 of the Grant Agency of the Charles
University and by grant No. 203/96/0262 of the Grant Agency of the Czech Republic.
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