Synthesis of N,N′-diarylalkanediamides and their antimycobacterial and antialgal activity

Article abstract of DOI:10.3390/50500714

A set of N,N′-diarylalkanediamides was synthesized. The compounds were tested for their antimycobacterial and antialgal activity. The antimycobacterial activity of N,N′-diarylalkanediamides depends on the lipophilicity of the respective acid. Antimycobacteri-ally active substances were found only in the series of N,N′-diarylethanediamides and N,N′-diarylbutanediamides. Other compounds (derivatives of pentane-, hexane-, octane- and nonanediamide) were inactive against various strains of mycobacteria. The compounds inhibited growth and chlorophyll production in Chlorella vulgaris. Their relatively low antial-gal activity is probably connected with their lowered aqueous solubility, and hence by a restricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.

Full text of DOI:10.3390/50500714

Molecules 2000, 5, 714-726
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of N,N’-Diarylalkanediamides and Their
Antimycobacterial and Antialgal Activity
Lenka Kubicova1*, Karel Waisser1, Jiri Kunes2, Katarina Kralova3, Zelmira Odlerova4, Milan
Slosarek5, Jiri Janota5 and Zbynek Svoboda6
1Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of Pharmacy
in Hradec Kralove, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
Tel.: +420 49 5067339, Fax: +420 49 5210002, E-mail: kubicova@faf.cuni.cz
2 Laboratory of Structure and Interactions of Biologically Active Molecules, Charles University in
Prague, Faculty of Pharmacy in Hradec Kralove, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech
Republic
3Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-2, 842
15 Bratislava, Slovak Republic
4Institute of Preventive and Clinic Medicine, Limbova 14, 835 01 Bratislava, Slovak Republic
5National Institute of Public Health, Srobarova 48, 100 42 Praha 10, Czech Republic
6Institute of Experimental Biopharmaceutics, Heyrovskeho 1207, 500 02 Hradec Kralove, Czech
Republic
Received: 2 November 1999 / Accepted: 13 December 1999 / Published: 14 May 2000
Abstract: A set of N,N'-diarylalkanediamides was synthesized. The compounds were tested
for their antimycobacterial and antialgal activity. The antimycobacterial activity of N,N'-
diarylalkanediamides depends on the lipophilicity of the respective acid. Antimycobacteri-
ally active substances were found only in the series of N,N'-diarylethanediamides and N,N'-
diarylbutanediamides. Other compounds (derivatives of pentane-, hexane-, octane- and
nonanediamide) were inactive against various strains of mycobacteria. The compounds in-
hibited growth and chlorophyll production in Chlorella vulgaris. Their relatively low antial-
gal activity is probably connected with their lowered aqueous solubility, and hence by a re-
stricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.
Keywords: N,N'-diarylalkanediamides, antimycobacterial activity, antialgal activity.
© 2000 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.

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Molecules 2000, 5
Introduction
As the end of the 20th century witnesses a sharp rise in the incidence of mycobacterial infections, the
development of new antimycobacterial drugs is presently of utmost importance and should proceed at
a rapid pace. When exploring a possible link between antituberculous activity and the ability to form
chelates with heavy metals, we prepared a set of N,N'-diarylethane- and -propanediamides, which were
evaluated in vitro against Mycobacterium tuberculosis, and some of them showed significant activity
[1]. Other authors have described various kinds of biological activity of alkanediamide derivatives as
well. For example, some 2-methylcarbonylbutanediamides are active against M. tuberculosis [2], 2,3-
diarylpentanediamides display activity against Gram-positive bacteria [3], and N,N'-substituted 2-
halobutanediamides act as herbicides [4]. Raynes et al. studied the influence of the length of the con-
necting chain on the antimalarial activity of bisquinolines; the derivative of butanediamide was the
most efficient [5]. In our previous study [6] we found that N,N'-bis(3,4-dichlorophenyl)butanediamide
effectively inhibited oxygen evolution rate (OER) in spinach chloroplasts and that this compound in-
teracted with the pigment-protein complexes in photosystem 2. The increase of the length of the con-
necting chain in the series of N,N'-bis(3,4-dichlorophenyl)alkanediamides led to the decrease of OER-
inhibiting activity in spinach chloroplast [7]. The decrease in biological activity with increasing lipo-
philicity of the compounds is probably linked to their lowered aqueous solubility, and hence to a re-
stricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.
This study is focused on the synthesis of a large set of N,N'-diarylalkanediamides and on the study
of antimycobacterial and antialgal activity of these compounds.
Results and Discussion
N,N'-Diarylalkanediamides, with the exception of N,N'-diarylethanediamides, were prepared from
the corresponding anilines by treatment with the appropriate alkanedioyl dichlorides in pyridine at
0°C. The reaction mixtures were allowed to stand at room temperature and after 24 hours they were
poured into water. The products were filtered off, washed with water and crystallized from ethanol.
The alkanedioyl dichlorides were prepared from the corresponding acids by the reaction with phospho-
rus pentachloride (butanedioyl dichloride) or thionyl chloride (all other alkanedioyl dichlorides). N,N'-
Diarylethanediamides were prepared from diethyl oxalate by the reaction with the corresponding ani-
line in the presence of sodium ethanolate. All syntheses are outlined in Scheme 1. The yields, physical
properties and analytical data for compounds 1-46 are given in Table 1 and Table 2.

716
Molecules 2000, 5
CONH
CONH
COOC2H5
+
COOC2H5
Na, C2H5OH
R
R
NH2
R
1 4
-
CONH
COOH
COCl
NH2
PCl5 or SOCl2
(
(
R
)
)
m
H2C
R
R
H2C
m
(
H2C
)
m
COOH
COCl
CONH
m = 2 (5-20), 3 (21-26), 4 (27-35), 6 (36-41), 7 (42-46)
Scheme 1.
Compounds 1-46 were tested for their in vitro antimycobacterial activity against Mycobacterium tu-
berculosis, M. avium, M. fortuitum, and M. kansasii. We found that the antimycobacterial activity of
N,N'-diarylalkanediamides depends on the lipophilicity of the respective acid. Active substances were
found only in the series of N,N'-diarylethanediamides and N,N'-diarylbutanediamides, but their anti-
mycobacterial activity was mostly low. The MIC values of the active compounds are given in Table 3.
All derivatives of pentane-, hexane-, octane- and nonanediamide were inactive against the studied my-
cobacteria strains.
The inhibition of chlorophyll production in statically cultivated algae Chlorella vulgaris by selected
derivatives was investigated at a constant inhibitor concentration of 75 µmol dm-3. The antialgal activ-
ity of the compounds was generally low, and the observed inhibition of algal chlorophyll production
varied in the range of 14.2 (10) to 57.9% (40) (Table 4 and Table 5). The antialgal activity of N,N'-
diarylbutanediamides was relatively low, varying in the range of 14.2 (10) to 43.6% (15) (Table 5).
The most effective inhibitor from the series of N,N'-diarylalkanediamides was N,N'-bis(4-
methoxyphenyl)octanediamide (40), causing 57.9% inhibition of chlorophyll production (Table 4).
Antialgal activity of substituted N,N'-diarylalkanediamides with the same substituent R was propor-
tional to the number of methylene groups in the connecting chain of the molecule (m = 2-4, 6, 7) for
derivatives with R = H and 4-Cl, respectively. For derivatives with R = 4-CH3, 4-OCH3 and 3,4-Cl2, a
quasi-parabolic dependence of the inhibitory activity on m was found, with maximum inhibition for
N,N'-diaryloctanediamides (m = 6; R = 4-CH3 (39) and 4-OCH3 (40)) and N,N'-bis(3,4-
dichlorophenyl)hexanediamide (m = 4; 31). The relatively low biological activity of the compounds is
probably a consequence of their low aqueous solubility, and hence a restricted passage of the inhibitor
through the hydrophilic regions of thylakoid membranes. An efficient inhibition of photosynthetic
electron transport in spinach chloroplasts by N,N'-bis(3,4-dichlorophenyl)butanediamide has been ob-
served previously [6,7].

717
Molecules 2000, 5
Table 1. Physical properties and analytical data of compounds 1-46.
Compd.
Formula
(M. w.)
m
R
M.p. (°C)
(Yield, %)
% Calc.
% Found
C
-
H
-
N
-
1a
2a
C16H16N2O2
(268.3)
0
0
0
0
2
2
2
2
2
2
2
2
2
2
2
2
2-CH3
3-CH3
4-NO2
4-Br
211-212
(65)
C16H16N2O2
(268.3)
135-137
(60)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3a
C14H10N4O6
(330.3)
357-358
(61)
4a
C14H10Br2N2O2
(398.1)
329-331
(58)
5a
C16H14Br2N2O2
(426.1)
4-Br
281-282
(92)
6b
C16H12Cl4N2O2
(406.1)
3,4-Cl2
H
258-259
(93)
7a
C16H16N2O2
(268.3)
231-233
(95)
8a
C18H20N2O4
(328.4)
4-OCH3
4-Cl
256-257
(90)
C16H14Cl2N2O2
(337.2)
288-290
(90)
56.99 4.18
57.24 4.15
72.95 6.80
72.87 6.79
8.31
8.38
9.45
9.59
-
9
C18H20N2O2
(296.4)
4-CH3
3-F
273-275
(91)
10
11b
12b
13b
14b
15a
16b
C16H14F2N2O2
(304.3)
206-207
(89)
-
-
-
-
-
-
-
-
-
-
-
-
C16H14F2N2O2
(304.3)
4-F
244-245
(92)
-
-
-
-
-
C16H14N4O6
(358.3)
3-NO2
3,4-(CH3)2
3-Cl
228-230
(88)
C20H24N2O2
(324.4)
231-232
(85)
C16H14Cl2N2O2
(337.2)
233-235
(92)
C24H32N2O2
(380.5)
4-C4H9
232-234
(81)

718
Molecules 2000, 5
Continuation of the Table 1.
Compd.
Formula
(M. w.)
m
R
M.p. (°C)
(Yield, %)
% Calc.
% Found
C
-
H
-
N
-
17b
18b
19a
20
C22H28N2O2
(352.5)
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
4-isoC3H7
234-236
(83)
C24H32N2O2
(380.5)
4-sec-C4H9
178-179
(81)
-
-
-
-
-
-
C20H26N4O2
(354.5)
4-N(CH3)2 282.5-283.5
(88)
c
C18H14N4O2S2
(382.5)
282-285
56.53 3.69 14.65
56.55 3.87 14.88
(92)
C17H16Cl2N2O2
(351.2)
4-Cl
4-OCH3
H
242-243
(70)
58.13 4.59
58.13 4.59
66.65 6.48
66.93 6.45
7.98
7.98
8.18
8.24
-
21
C19H22N2O4
(342.4)
222.5-224
(62)
22
23a
24a
25b
26a
27
C17H18N2O2
(282.3)
224-225
(84)
-
-
-
-
-
-
-
-
C19H22N2O2
(310.4)
4-CH3
3,4-Cl2
4-Br
220-2211
(54)
-
-
-
C17H14Cl4N2O2
(420.1)
266-267
(63)
C17H16Br2N2O2
(440.1)
256-257
(58)
C20H24N2O2
(324.4)
4-CH3
H
258-259
(67)
74.05 7.46
74.21 7.52
8.63
8.66
-
28a
29
C18H20N2O2
(296.4)
237-238
(75)
-
-
C18H18N4O6
(386.4)
3-NO2
3-Cl
238-239
(58)
55.96 4.70 14.50
55.80 4.73 14.67
C18H18Cl2N2O2
(365.3)
197-198
(60)
59.19 4.97
59.14 4.97
7.67
7.90
-
30
31b
32
C18H16Cl4N2O2
(434.2)
3,4-Cl2
2-OCH3
276-277
(59)
-
-
C20H24N2O4
(356.4)
151-152
(40)
67.40 6.79
67.49 6.68
7.86
7.79

719
Molecules 2000, 5
Continuation of the Table 1.
Compd.
Formula
(M. w.)
m
R
M.p. (°C)
(Yield, %)
% Calc.
% Found
H
C
N
C20H24N2O4
(356.4)
4
4
4
6
6
6
6
6
6
7
7
7
7
7
4-OCH3
4-Cl
233-234
(65)
67.40 6.79
67.62 6.78
59.19 4.97
58.91 4.82
65.44 6.41
65.70 6.33
7.86
7.88
7.67
7.79
6.36
6.17
-
33
34
C18H18Cl2N2O2
(356.3)
238-239
(67)
C24H28N2O6
(440.5)
4-
216-217
(54)
35
COOC2H5
3,4-Cl2
36b
37
C20H20Cl4N2O2
(462.2)
167-168
(35)
-
-
C20H22Cl2N2O2
(393.3)
4-Cl
H
197-198
(81)
61.11 5.60
61.08 5.64
7.12
7.12
-
38a
39a
40
C20H24N2O2
(324.4)
186-188
(57)
-
-
C22H28N2O2
(352.5)
4-CH3
4-OCH3
4-Br
224-226
(55)
-
-
-
C22H28N2O4
(384.5)
220-221
(63)
68.78 7.24
68.60 7.15
7.29
7.19
-
41a
42b
43
C20H22Br2N2O2
(482.2)
251-253
(54)
-
-
C21H22Cl4N2O2
(476.2)
3,4-Cl2
4-Cl
170-171
(52)
-
-
-
C21H24Cl2N2O2
(407.3)
196-197
(61)
61.92 5.94
62.06 5.85
6.88
6.99
-
44a
45a
46
C21H26N2O2
(338.5)
H
180-181
(63)
-
-
C23H30N2O2
(366.5)
4-CH3
4-OCH3
197-198
(61)
-
-
-
C23H30N2O4
(398.5)
194-196
(44)
69.32 7.59
69.61 7.73
7.03
7.07
aM. p. values from literature: Compound, value (°C) [ref.]: 1, 209 [8]; 2, 133 [8]; 3, 359 [9]; 4, 321-322
[10]; 5, 284 [11]; 7, 230,5 [12]; 8, 256 [13]; 15, 232 [14]; 19, 277-280 [15]; 23, 223 [11]; 24, 218 [11];
26, 256 [11]; 28, 235 [11]; 38, 186-7 [16]; 39, 219 [11]; 41, 248 [11]; 44, 186-7 [16]; 45, 198 [11].
bThe data of the compound were taken from [7].
cThe compound is N,N'-bis(2-benzothiazolyl)butanediamide.

720
Molecules 2000, 5
Table 2. 1H NMR and IR spectroscopic data.
Compd.
1H NMR
IR
n(C=O) (cm-1)
1668
d (ppm)
-
-
-
-
-
-
-
-
1
2
3
4
5
6
7
8
1666
1706
1666
1652
1659
1663
1648
2.63 (s, 4H), 7.35-7.28 (m, 4H) , 7.63-7.56 (m, 4H), 10.14 (s,
1652
9
2H)
2.21 (s, 6H), 2.60 (s, 4H), 7.11-7.03 (m, 4H) , 7.48-7.40 (m,
1655
10
4H), 9.89 (s, 2H)
-
1663
1651
1675
1651
1668
1659
1656
1655
1645
1694
11
12
13
14
15
16
17
18
19
20
-
-
-
-
-
-
-
-
2.88 (s, 4H), 7.32-7.23 (m, 2H) , 7.46-7.37 (m, 2H), 7.76-7.69
(m, 2H), 7.97-7.90 (m, 2H)
1.95-1.81 (m, 2H), 2.36 (t, J=7.4 Hz, 4H), 7.35-7.29 (m, 4H) ,
1664
1659
21
22
7.64-7.57 (m, 4H), 10.05 (s, 2H)
1.93-1.81 (m, 2H), 2.31 (t, J=7.4 Hz, 4H), 3.69 (s, 6H), 6.89-
6.79 (m, 4H) , 7.53-7.43 (m, 4H), 9.76 (s, 2H)
-
1673
1664
1679
1664
1659
23
24
25
26
27
-
-
-
1.63-1.55 (m, 4H),2.21 (s, 6H), 2.33-2.24 (m, 4H), 7.10-7.03
(m, 4H) , 7.48-7.41 (m, 4H), 9.79 (s, 2H)
-
1660
28

721
Molecules 2000, 5
Continuation of the Table 2.
Compd.
1H NMR
IR
n(C=O) (cm-1)
1667
d (ppm)
1.70-1.60 (m, 4H), 2.43-2.33 (m, 4H), 7.57 (t, J=8.1, 2H),
7.91-7.83 (m, 4H), 8.63 (t, J=2.1 Hz, 2H), 10.41 (s, 2H)
29
1.65-1.55 (m, 4H), 2.38-2.27 (m, 4H), 7.10-7.03 (m, 2H) , 7.30
1663
30
(t, J=8.1, 2H), 7.45-7.38 (m, 2H), 7.80 (t, J=1.9, 2H), 10.09 (s,
2H)
-
1670
1659
31
32
1.65-1.54 (m, 4H), 2.44-2.33 (m, 4H), 3.79 (s, 6H), 6.93-6.82
(m, 2H), 7.10-6.96 (m, 4H), 7.97-7.84 (m, 2H), 9.03 (s, 2H)
1.64-1.52 (m, 4H), 2.33-2.20 (m, 4H), 3.69 (s, 6H), 6.89-6.79
1648
1656
33
34
35
(m, 4H), 7.52-7.42 (m, 4H), 9.74 (s, 2H)
1.65-1.55 (m, 4H), 2.37-2.26 (m, 4H), 7.35-7.29 (m, 4H), 7.63-
7.56 (m, 4H), 10.03 (s, 2H)
1.28 (t, J=7.1 Hz, 6H), 1.68-1.57 (m, 4H), 2.42-2.31 (m, 4H),
1708, 1693
4.25 (q, J=14.2, J=7.1 Hz, 4H), 7.74-7.67 (m, 4H), 7.91-7.84
(m, 4H), 10.25 (s, 2H)
-
1671
1659
36
37
1.36-1.25 (m, 4H), 1.64-1.49 (m, 4H), 2.27 (t, J=7.4, 4H),
7.35-7.27 (m, 4H), 7.63-7.55 (m, 4H), 10.00 (s, 2H)
-
1659
1656
1652
38
39
40
-
1.34-1.24 (m, 4H), 1.64-1.48 (m, 4H), 2.24 (t, J=7.4, 4H), 3.68
(s, 6H), 6.87-6.79 (m, 4H), 7.50-7.42 (m, 4H), 9.72 (s, 2H)
-
1664
1680
1660
41
42
43
-
1.32-1.22 (m, 6H), 1.63-1.48 (m, 4H), 2.27 (t, J=7.3 Hz, 4H),
7.34-7.28 (m, 4H), 7.62-7.55 (m, 4H), 10.00 (s, 2H)
-
1671
1662
1655
44
45
46
-
1.35-1.20 (m, 6H), 1.63-1.47 (m, 4H), 2.23 (t, J=7.3 Hz, 4H),
3.68 (s, 6H), 6.88-6.80 (m, 4H), 7.50-7.42 (m, 4H), 9.71 (s,
2H)

722
Molecules 2000, 5
Table 3. Antimycobacterial activity of the active compounds expressed as MIC (mmol dm-3).
(m = number of methylene groups in the connecting chain of the compounds).
Compound
m
R
MIC (mmol dm-3)
M. tuberculosis
M. avium
M. kansasii M. fortuitum
a)
a)
a)
0
0
2
2
2
2
2
2
2
2
4-NO2
4-Br
37
4.1
-
-
-
-
-
3
a)
a)
a)
-
4
3,4-Cl2
H
250
500
250
500
500
500
>1000
500
250
500
500
500
6
500
500
7
4-F
1000
>1000
>1000
63
1000
1000
12
14
15
16
17
18
3,4-(CH3)2
3-Cl
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
4-C4H9
4-isoC3H7
4-sec-C4H9
63
63
a)not tested
Table 4. Inhibition of chlorophyll production in Chlorella vulgaris by N,N'-diarylalkanediamides
(m = number of methylene groups in the connecting chain of the compounds; concentrations of com-
pounds were constant, 75 µmol dm-3).
Compound
m
2
3
4
6
7
% of inhibition
R= H
7
4-CH3
10
4-OCH3
8
4-Cl
9
3,4-Cl2
6
22.8
23
14.2
24
30.8
22
33.2
21
19.8
25
41.8
28
26.2
27
41.6
33
34.3
34
28.7
31
41.0
38
37.7
39
46.0
40
41.4
37
33.5
36
44.2
44
41.4
45
57.9
46
45.4
43
22.6
42
48.5
37.9
53.9
45.5
15.1

723
Molecules 2000, 5
Table 5. Inhibition of chlorophyll production in Chlorella vulgaris by N,N'-diarylbutanediamides
(m = 2; concentrations of compounds were constant, 75 µmol dm-3).
Compound
R
H
% of inhibition Compound
R
% of inhibition
27.1
22.8
25.2
43.6
25.4
35.4
33.2
14.2
30.8
4-Br
7
5
3-F
4-isoC3H7
4-C4H9
36.5
11
15
13
12
9
17
16
18
19
6
3-Cl
3-NO2
4-F
32.6
4-sec-C4H9
4-N(CH3)2
3,4-Cl2
36.5
28.8
4-Cl
4-CH3
4-OCH3
19.8
3,4-(CH3)2
26.7
10
8
14
Experimental
General
The melting points were determined on a Kofler block and are uncorrected. The samples for ele-
mental analyses and biological tests were dried over P2O5 at 61°C and 66 Pa for 24 h. Elemental analy-
ses were performed on a C,H,N,S analyzer (FISONS AE 1110, Milano). The IR spectra were measured
in KBr on a Nicolet Impact 400 apparatus. The purity of the compounds was checked by TLC, using
petroleum ether:ethyl acetate (1:1) and chloroform:acetone (9:1) as the mobile phases. 1H NMR spectra
of new compounds were recorded for DMSO-d6 solutions at ambient temperature on a Varian Mer-
cury-Vx BB 300 spectrometer (operating at 300 MHz). Chemical shifts were recorded as d values in
ppm, and were indirectly referenced to tetramethylsilane via the solvent signal (2.49 for 1H).
Synthesis of alkanedioyl dichlorides
Butanedioyl dichloride
Phosphorus pentachloride (0.328 mol) was added to a finely powdered succinic acid (0.400 mol),
and the mixture was heated at 120°C for 5 hours. Phosphorus oxychloride was then distilled off, and
the crude product was purified by vacuum distillation (yield 75%, b.p. 104-105°C / 3.33 kPa, lit. [17]
b.p. 103-104°C / 3.33 kPa).
Octanedioyl dichloride
Suberic acid (0.287 mol) was heated with thionyl chloride (1.260 mol) at 120°C for 4 hours. After

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Molecules 2000, 5
the removal of the excess reagent by distillation, the crude product was purified by distillation at re-
duced pressure (yield 69.20%, b. p. 145°C / 1.33 kPa, lit. [18] b.p. 149-150°C / 1.60 kPa). The same
protocol was used for the preparation of the other alkanedioyl dichlorides (yield; b.p.; b.p. [ref]): pen-
tanedioyl dichloride (90.7%; 104°C / 2.53 kPa; 100°C / 2.00 kPa [19]); hexanedioyl dichloride (81.2%;
130-132°C / 2.40 kPa; 126°C / 1.60 kPa [19]); nonanedioyl dichloride (71.4%; 162°C / 1.20 kPa; 158-
159°C / 1.60 kPa [20]).
Synthesis of N,N’-diarylalkanediamides
N,N’-Diarylethanediamides (1-4)
Sodium (1 g) was dissolved in absolute ethanol (100 ml), a substituted aniline (0.300 mol) followed
by diethyl oxalate (0.150 mol) were added to the solution, and the reaction mixture was heated at re-
flux for 1 hour. After cooling, the crude product was filtered off, washed with water, and crystallized
from ethanol. The experimental data of compounds 1-4 are given in Table 1.
Other N,N’-diarylalkanediamides (5-46)
Butanedioyl dichloride (0.016 mol) was added dropwise to a stirred solution of an appropriate ani-
line (0.032 mol) in pyridine (20 ml) at 0°C. The reaction mixture was allowed to stand at ambient tem-
perature for 24 hours, and then poured into water (100 ml). The product was filtered off and crystal-
lized from ethanol. The yields, melting points, IR and NMR spectral data as well as elemental analyses
are summarized in Table 1 and Table 2.
Biological assays
Antimycobacterial activity
Antimycobacterial evaluation of N,N'-diarylalkanediamides (m = 0, 3, 4, 6, 7) was carried out in a
semisynthetic liquid protein-containing Sula medium (IMUNA, Sarisske Michalany), buffered to
pH=7.2. The following mycobacterial strains were used: Mycobacterium tuberculosis H37Rv, M. kansa-
sii PKG8, M. avium No. 80/72 and M. fortuitum 1021. The MICs were determined after 14 days of in-
cubation at 37°C. The compounds were added to the medium in dimethyl sulfoxide (DMSO) solutions.
The final concentrations were 1000; 333; 111; 37; 12.3; and 4.1 µmol dm-3.
Antimycobacterial activity of N,N'-diarylbutanediamides (m = 2) was determined in Sula semisyn-
thetic medium (SEVAC, Prague). For evaluation of their in vitro antimycobacterial activity, the fol-
lowing strains were used: M. tuberculosis CNCTC My 1/47, M. kansasii CNCTC My 235/80, M.
avium CNCTC My 80/72 and M. fortuitum CNCTC My 187/73 from the National Institute of Public
Health, Prague. The compounds were added to the medium in DMSO solutions. The final concentra-

725
Molecules 2000, 5
tions were 1000; 500; 250; 125; 62; 31; 16; 8; 4 µmol dm-3. The minimum inhibitory concentrations
were determined after incubation at 37°C for 21 days.
MIC was the lowest concentration of a substance (on the above-stated concentration scale), at which
inhibition of the growth of mycobacteria occurred. The compound is considered as active, when its
MIC is lower than 1000 µmol dm-3. DMSO concentration in the medium was maximum for concentra-
tion 1000 µmol dm-3 of the tested compound and the control samples contained the same DMSO
amount (10 v/v %). The applied DMSO content did not affect the antimycobacterial activity.
Antialgal activity
The inhibitory effect of selected N,N'-diarylalkanediamides on algal chlorophyll (Chl) production
has been investigated in statically cultivated Chlorella vulgaris (96 hours; photoperiod 16 h light / 8 h
dark; illumination: 5 000 lx; pH = 7.2; Chl content at the beginning of cultivation: 0.5 mg dm-3) at
room temperature and a constant inhibitor concentration 75 µmol dm-3 according to Kralova et al. [21].
Chl content of algal suspensions was determined spectrophotometrically following its extraction into
N,N-dimethylformamide according to Inskeep and Bloom [22]. The compounds were dissolved in
DMSO as their solubility in water was insufficient. DMSO concentration in the algal suspensions did
not exceed 0.75 v/v % and the control samples contained the same DMSO amount as the suspensions
treated with the tested compounds. The antialgal activity was expressed as the percentage of inhibition
of the untreated control.
Acknowledgements: The authors wish to thank Mrs. J. Zizkova from the Department of Inorganic and
Organic Chemistry for measurements of the IR spectra and Mrs. D. Karlickova from the Department of
Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University, for elemental
analyses. This study was supported by the Ministry of Education of the Czech Republic (CEZ J 13/98:
11600001), and by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic
(grant No. 1/4013/97).
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Samples Availability: available from the authors.
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