Antimicrobial activity of 5-arylidene aromatic derivatives of hydantoin. Part 2

Article abstract of DOI:10.1016/S0014-827X(01)01172-7

Various 5-chloroarylidene-2-amino substituted derivatives of imidazoline-4-one were synthesized and evaluated for their activity in vitro against Mycobacterium tuberculosis and other type strains of bacteria and fungi. 2-Chloro- and 2,4-dichlorobenzylidene substituted hydantoins exhibited antimycobacterial effect. The most potent compounds 3i, 3j, 3o, 3q and 3s were classified for further tests. The antimitotic effect of the investigated hydantoins was also examined.

Full text of DOI:10.1016/S0014-827X(01)01172-7

Il Farmaco 57 (2002) 39–44
www.elsevier.com/locate/farmac
Antimicrobial activity of 5-arylidene aromatic derivatives of
hydantoin. Part 2ꢀ
Ewa Szyman´ska a, Katarzyna Kiec´-Kononowicz a,*, Anna Białecka b,
Andrzej Kasprowicz b
a Department of Chemical Technology of Drugs, Jagiellonian Uni6ersity Medical College, Medyczna 9, Pl 30-688 Krako´w, Poland
b Jan Bober’s Center of Microbiology and Auto6accines, Sławkowska 17, Pl 31-016 Krako´w, Poland
Accepted 13 August 2001
Abstract
Various 5-chloroarylidene-2-amino substituted derivatives of imidazoline-4-one were synthesized and evaluated for their activity
in vitro against Mycobacterium tuberculosis and other type strains of bacteria and fungi. 2-Chloro- and 2,4-dichlorobenzylidene
substituted hydantoins exhibited antimycobacterial effect. The most potent compounds 3i, 3j, 3o, 3q and 3s were classified for
further tests. The antimitotic effect of the investigated hydantoins was also examined. © 2002 Elsevier Science S.A. All rights
reserved.
Keywords: Arylidene hydantoins; Antimycobacterial activity; Antibacterial activity; Antimitotic activity
The antimitotic effect of the obtained hydantoins was
also examined.
1. Introduction
For each compound the octanol–water coefficient
(log P combined) and distribution coefficient (log D)
were calculated with PALLAS program [19].
Antimicrobial activity was stated among hydantoins
possessing aromatic or heterocyclic substituents at imi-
dazolone nitrogen e.g. N-acyl and 5-arylidene deriva-
tives of hydantoin and 2-thiohydantoin [1–3].
Antifungal activity was shown for compounds with
N-aromatic acyl substituents with 5-aromatic, arylidene
or without substituents derivatives [4–8]. Antifungal
and antimicrobial activity was found for the arylidene,
arylhydrazone and aromatic substituted derivatives
(with)out substituents on nitrogen atoms [9–14].
As a result of the analysis of the above presented
literature data and continuing our studies on the bio-
logical activity of hydantoin derivatives [15–18], we
have synthesized and examined the new series of 5-
chloroarylidene-2-amino substituted derivatives of hy-
dantoin. Their antimicrobial effect on the set of type
strains of microorganisms as well as the antimycobacte-
rial activity was investigated.
2. Chemistry
The
starting
5-(2-chlorobenzylidene)-,
5-(2,6-
dichlorobenzylidene)- and 5-(2,4-dichloro-benzylidene)-
2-thiohydantoins were prepared according to the
literature procedure [20]. To prepare target compounds
5-chlorobenzylidene-2-thiohydantoins 1 were treated
with methyl iodide (Scheme 1). Obtained methylthio
derivatives 2 were reacted with 10% excess of amines
possessing (un)substituted aromatic residue to give with
good yields solid products 3a–q (Table 1). Last reac-
tions were carried out in toluene for the benzylamine
derivatives (3f–h, 3n) or in acetic acid for aniline
derivatives (3a–e, 3i–m, 3o–q), with 10% excess of the
appropriate amine. Target compounds were recrystal-
lized from acetic acid, methanol or DMF.
ꢀ Part of this work was presented as a poster communication at the
2nd European Symposium on Antimicrobial Agents, Hradec
Kralove, Czech Republic, 1–4 July 1998.
* Correspondence and reprints.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
The purity of all obtained compounds was checked
by thin-layer chromatography. The structures were
0014-827X/02/$ - see front matter © 2002 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01172-7

40
E. Szyman´ska et al. / Il Farmaco 57 (2002) 39–44
Scheme 1. Synthesis of 5-arylidene hydantoin derivatives 3a–y.
1
confirmed by elemental and spectral analyzes (IR, H
NMR).
Log D values calculated for 2-chloro compounds
(3o–y) at pH 7.00 varied from 3.23 to 4.97; for 2,4-
dichloro from 4.07 to 5.76. For 2,6-dichloro derivatives
log D varied from 3.95 to 5.68. The comparison of the
lipophilic properties indicates that values of log P for
the benzylamine structures (3f–h, 3n, 3t–y) are lower
than values of log P for the aniline derivatives, what
seems to be correlated with microbiological effects.
However, no exact relation between log P and my-
cobacterial activity of the tested compounds was found.
The present work is the continuation of our studies
on structure—antimicrobial activity relationships
among 5-arylidene hydantoin derivatives. Some of the
compounds with structure 3 (3r–y) mentioned for com-
parison in this work were synthesized and examined
previously [18].
3. Results and discussion
In order to find a potential antimycobacterial activ-
ity, all the compounds were tested according to the
Tuberculosis Antimicrobial Acquisition and Coordinat-
ing Facility (TAACF) screening program using the
BACTEC 460 radiometric system [21] (Table 2).
Log P combined and log D values prediction of the
obtained compounds taken with PALLAS program are
presented in Table 1. The compounds show insignifi-
cant acid–basic properties and pH hardly influences on
log D values, so that log P=log D (at pH 7.00) for all
investigated compounds.
In the tested group, 2-chloro- and 2,4-dichloro ben-
zylidene compounds (3i–y) showed better effect against
M. tuberculosis than 2,6-dichloro group (3a–h). The
occurrence of an additional chlorine atom in the ortho-
position in a benzylidene residue reduced activity, while
the influence of additional para-chloro substituent on
the antitubercular effect is more difficult to specify. On
the basis of the obtained screening results, we can state
that in each series among all aniline derivatives com-
pounds substituted with 4-chloro-aniline residue (3a, 3i,
3o) possessed the highest antimycobacterial activity. On
the whole, with growing distance between the imida-
zolone and aromatic ring the activity seems to decrease.
The most potent compounds 3i, 3j, 3o, 3q and 3s were
classified to further tests.
Table 1
Arylidene hydantoins
Comp.
R1
R2
n
Log P
Log Da
3a
3b
3c
3d
3e
3f
3g
3h
3i
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
4-Cl
2,4-diCl
3-Cl
2-Cl
H
0
0
0
0
0
1
1
1
0
0
0
0
0
1
0
0
0
0
0
1
1
1
1
4.90
5.68
4.96
4.89
4.18
4.72
4.14
3.95
4.98
5.76
5.04
4.97
4.25
4.07
4.19
4.97
4.24
4.18
3.46
3.28
4.00
3.43
3.23
4.90
5.68
4.96
4.89
4.18
4.72
4.14
3.95
4.98
5.76
5.04
4.97
4.25
4.07
4.19
4.97
4.24
4.18
3.46
3.28
4.00
3.43
3.23
4-Cl
4-F
4-OCH3
4-Cl
2,4-diCl
3-Cl
2-Cl
H
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
H
4-Cl
2,4-diCl
3-Cl
2-Cl
H
H
4-Cl
4-F
3u
3w
3y
4-OCH3
a Log D calculated for pH 7.0.

E. Szyman´ska et al. / Il Farmaco 57 (2002) 39–44
41
Table 2
Antibacterial activity against M. tuberculosis H37Rv
Comp.
2,6-dichloro
MIC (mg/mL) Inhibition a
Comp.
2,4-dichloro
MIC (mg/mL) Inhibition a
Comp.
2-chloro
MIC (mg/mL) Inhibition a
(%)
(%)
(%)
3a
3b
3c
3d
3e
3f
\12.5
\12.5
\12.5
\6.25
\6.25
\12.5
\12.5
\12.5
17
4
0
0
1
0
5
0
3i
3j
3k
3l
3m
3n
B12.5
B12.5
\6.25
\6.25
\6.25
\12.5
100
96
48
13
47
46
3o
3p
3q
3r
3s
3t
3u
3w
3y
B12.5
\12.5
\12.5
\12.5
\12.5
\12.5
\12.5
\6.25
\6.25
99
37
91
79 b
93
20 b
45 b
10
3g
3h
8
a MIC Rifampin=0.25 mg/mL (98% inhibition).
b MIC Rifampin=0.5 mg/mL (100% inhibition).
Table 3
Minimum inhibitory concentration (MIC) mg/mL
Comp.
M. catarrhalis
H. influenzae
M. luteus
S. aureus
78
B. cereus
S. pneumoniae
3b
3c
3f
3g
78
156
78
20
78
78
39
78
78
3h
156
3i
3o
3p
3q
156
78
39
39
78
156
3s
156
3u
156
0.075
39
Gentamycin a
Nalidixic acid b
1.25
0.62
0.15
0.0045
0.018
625
a MIC (mg/mL) estimated for gentamycin.
b MIC (mg/mL) assigned for nalidixic acid.
No compound has shown antifungal effect against
Candida albicans.
The obtained compounds were also investigated
against type strains of microorganisms listed in Section
4.2.2. For active compounds the minimum inhibitory
concentration (MIC) values were determined using disc
diffusion methods by NCCLS procedures [22,23]. Re-
sults of these tests are collected in Table 3.
Obtained compounds were assayed on cdc2 kinase
and cdc25 phosphatase tests for their antimitotic activ-
ity according to the European Organization for Re-
search and Treatment of Cancer (EORTC) program
[24–26].
All tested compounds were (according to EORTC
program procedure) inactive in these tests (IC50\10
mM).
Obtained compounds were mainly active against
Moraxella catarrhalis and Streptococcus pneumoniae.
Some of them inhibited growth of Haemophilus influen-
zae (3f, 3g), Micrococcus luteus (3i, 3q), Staphylococcus
aureus (3b) and Bacillus cereus (3p). 2,6-Dichloroben-
zylidene derivative 3c showed high activity against M.
catarrhalis with MIC=20 mg/mL lower than MIC of
nalidixic acid (39 mg/mL). The activity against S. pneu-
moniae has been shown only by 2,6-dichloroarylidene
derivatives (3b, 3c, 3f–h). The 2,4-dichloro substituted
compounds (except 3i) were almost totally devoid of
antimicrobial effect (MIC\156 mg/mL). We also ob-
served that the lack of any substituent in amine residue
(3e, 3m, 3n, 3s and 3t) reduced antibacterial activity
against tested strains.
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were determined on
Mel-Temp melting point apparatus (Laboratory
Devices Inc., USA). Thin-layer chromatography was
performed with Merck silica gel GF254 aluminum
sheets, using the following developing system:

42
E. Szyman´ska et al. / Il Farmaco 57 (2002) 39–44
chloroform–acetone 1:1. Spots were detected by their
absorption under UV light. Elemental analyzes agree
with theoretical values within 90.40%, unless other-
wise stated.
H-3%, H-4%, H-5%), 10.05 (br.s, 1H, NHaniline), 10.48 (br.s,
1H, 3-NH). Anal. C16H10ON3Cl3 (C, H, N).
3b: 1H NMR l (ppm)=6.16 (s, 1H, CHꢀ), 7.10 (br.s,
1H, H-6%%), 7.26–7.38 (m, 3H, H-3%, H-5%, H-5%%), 7.47 (s,
1H, H-3%%), 7.50–7.53 (m, 1H, H-4%), 9.84 (br.s, 1H,
NHaniline), 11.26 (br.s, 1H, 3-NH). Anal. C16H9ON3Cl4
(C, H, N).
IR spectra were recorded with Fourier transform
infrared spectrometer FT/IR-410 (Jasco Co., Japan)
using KBr discs. The following abbreviations were
1
1
used: br (broad), s (sharp). H NMR spectra of com-
3c: H NMR l (ppm)=6.28 (br.s, CHꢀ), 6.39 (br.s,
pounds were determined in DMSO-d6 solution (Varian
Mercury 300 MHz with TMS as an internal standard.
All chemical shifts are quoted in l values.
The predictions of log P combined and log D values
were determined with the PALLAS program [19].
Compounds 1 were prepared according to the litera-
ture procedure [20].
CHꢀ), 7.08 (s, 1H, H-6%%), 7.25–7.55 (m, 5H, H-3%, H-4%,
H-5%, H-4%%, H-5%%), 7.50 (s, 1H, H-2%%), 9.97 (br.s,
NHaniline), 10.30 (br.s, 1-NH), 11.02 (br.s, 3-NH). Anal.
C16H10ON3Cl3 (C, H, N).
1
3d: H NMR l (ppm)=6.14 (s, 1H, CHꢀ), 7.01 (t,
J=8 Hz, 1H, H-4%%), 7.23 (t, J=7.3 Hz, 1H, H-5%%),
7.30–7.48 (m, 5H, H-3%, H-4%, H-5%, H-3%%, H-6%%), 9.82
(br.s, 1H, NHaniline), 10.16 (br.s, 1H, 3-NH). Anal.
C16H10ON3Cl3 (C, H, N).
4.1.1. General procedure for synthesis of compounds
3a–q
1
3e: H NMR l (ppm)=6.31 (s, 1H, CHꢀ), 7.04 (t,
To the stirred solution of sodium (0.04 mol) in 200
mL of ethanol, arylidene-2-tiohydantoin (1) (0.04 mol)
and methyl iodide (0.04 mol) were added. After stirring
at room temperature (r.t.) for 30 min the solid of 2 was
filtered off, washed with water and dried. Methylthio
derivatives 2 were found to be analytically pure Table
4.
J=8 Hz, 1H, H-4%), 7.29–7.38 (m, 4H, H-3%, H-5%,
H-3%%, H-5%%), 7.43–7.53 (m, 3H, H-2%%, H-4%%, H-6%%), 9.94
(br.s, 2H, NHaniline, 3-NH). Anal. C16H11ON3Cl2 (C, H,
N).
3f: 1H NMR l (ppm)=4.39 (s, CH2), 4.54 (d, J=5.4
Hz, 2H, CH2), 6.19 (s, 1H, CHꢀ), 7.31–7.55 (m, 7H,
H
aromat), 8.09 (t, NHꢁCH2), 9.20 (br.s, 1-NH), 9.99
A mixture containing 5 mmol of 2, 5.5 mmol of
amine in 30 mL of toluene (for compounds 3f–h, 3n) or
acetic acid (3a–e, 3i–m, 3o–q) was refluxed for 9 h, and
then allowed to cool. The product was isolated by
suction and recrystallized from acetic acid (3d, 3l, 3o–
q), DMF with addition of H2O (3a–e, 3i–m) or
methanol (3f-h, 3n).
(br.s, 1-NH), 10.13 (br.s, 3-NH), 10.99 (br.s, 3-NH).
Anal. C17H12ON3Cl3 (C, H, N).
1
3g: H NMR l (ppm)=4.39 (s, CH2), 4.53 (d, J=
5.7 Hz, 2H, CH2), 6.19 (s, 1H, CHꢀ), 7.09–7.55 (m, 7H,
H
aromat), 8.08 (t, J=5.5 Hz, NHꢁCH2), 9.16 (br.s,
1-NH), 9.95 (br.s, 1-NH), 10.14 (br.s, 3-NH), 10.89
(br.s, 3-NH). Anal. C17H12ON3Cl2F (C, H, N).
3a: 1H NMR l (ppm)=6.33 (s, 1H, CHꢀ), 7.32–7.43
(m, 4H, H-2%%, H-3%%, H-5%%, H-6%%), 7.49–7.54 (m, 3H,
3h: H NMR l (ppm)=4.31 (s, CH2), 4.43 (d, J=
1
12.6 Hz, 2H, CH2), 6.15 (s, 1H, CHꢀ), 6.88 (d, J=8.2
Table 4
Physical and IR data of compounds 3a–q
Comp. Yield (%)
m.p. °C (solvent)
TLC Molecular formula IR (cm−1
)
3a
3b
3c
3d
84
43
72
51
301–303 (DMF+water)
289–291 (DMF+water)
305–306 (DMF+water)
267–269 (DMF+water,
CH3COOH)
0.65
0.86
0.70
0.85
C16H10ON3Cl3
C16H9ON3Cl4
C16H10ON3Cl3
1640 (ArCHꢀ), 1688, 1694 (CꢀO), 3192 (NꢁH)
1640 (ArCHꢀ), 1688, 1704 (CꢀO), 3196, 3336 (NꢁH)
1646 (ArCHꢀ), 1693 (CꢀO), 3196 (NꢁH)
C
16H10ON3Cl3
1610 (ArCHꢀ), 1703 (CꢀO), 3231, 3428 (NꢁH)
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
74
88
90
70
63
55
67
86
55
83
89
74
85
298–300 (DMF+water)
237–239 (methanol)
239–240 (methanol)
0.47
0.16
0.23
0.30
0.16
0.67
0.22
0.77
0.42
0.24
0.23
0.67
0.18
C16H11ON3Cl2
C17H12ON3Cl3
C17H12ON3Cl2F
1639 (ArCHꢀ), 1704 (CꢀO), 3365 (NꢁH)
1696, 1706 (CꢀO), 3308 (NꢁH)
1648 (ArCHꢀ), 1694, 1704 (CꢀO), 3320 (NꢁH)
1710 (CꢀO), 3309 (NꢁH)
1656 (ArCHꢀ), 1714 (CꢀO), 3149, 3353 (NꢁH)
1650 (ArCHꢀ), 1683, 1729 (CꢀO), 3176 (NꢁH)
1654 (ArCHꢀ), 1716 (CꢀO), 3276, 3446, 3542 (NꢁH)
1651 (ArCHꢀ), 1724 (CꢀO), 3338, 3426 (NꢁH)
1641 (ArCHꢀ), 1699 (CꢀO), 3347 (NꢁH)
1662 (ArCHꢀ), 1718 (CꢀO), 3083 (NꢁH)
1656 (ArCHꢀ), 1686, 1696 (CꢀO), 3164, 3348 (NꢁH)
1644 (ArCHꢀ), 1680 (CꢀO), 3324 (NꢁH)
1640 (ArCHꢀ), 1678, 1688 (CꢀO), 3172 (NꢁH)
221–223 (methanol)
C18H15O2N3Cl2
291–293 (DMF+water)
304–309 (DMF+water)
296–297 (DMF+water)
276–277 (DMF, CH3COOH)
304–306 (DMF+water)
219–221 (methanol)
297–299 (acetic acid)
279–281 (acetic acid)
292–295 (acetic acid)
C16H10ON3Cl3
C16H9ON3Cl4
C16H10ON3Cl3
C16H10ON3Cl3
C16H11ON3Cl2
C17H13ON3Cl2
C16H11ON3Cl2
C16H10ON3Cl3
C
16H11ON3Cl2

E. Szyman´ska et al. / Il Farmaco 57 (2002) 39–44
43
Hz, 2H, H-3%%, H-5%%), 7.22–7.53 (m, 5H, H-3%, H-4%,
4.2. Biological test procedures
H-5%, H-2%%, H-6%%), 7.98 (br.s, NHꢁCH2), 9.16 (br.s,
1-NH), 9.84 (br.s, 3-NH), 10.10 (br.s, 3-NH). Anal.
C17H13ON3Cl2 (C, H, N).
4.2.1. In 6itro e6aluation of antimycobacterial acti6ity
against M. tuberculosis H37R6
1
Primary screening was conducted at 12.5 or 6.25
mg/mL against M. tuberculosis H37Rv (ATCC 27294;
American Type Culture Collection, Rockville, MD) in
BACTEC 12B medium using the BACTEC 460-radio-
metric system [21]. Compounds demonstrating at least
90% inhibition were retested against M. tuberculosis
H37Rv at lower concentration to determine the actual
minimum inhibitory concentration (MIC) in the Mi-
croplate Alamar Blue Assay (MABA). The MIC was
defined as the lowest concentration inhibiting 99% of
the inoculum. Rifampin (Sigma Chemical Compound,
St. Louis, MO) or isoniazid were included as a positive
drug control.
3i: H NMR l (ppm)=6.70 (s, 1H, CHꢀ), 7.42–7.66
(m, 4H, H-3%, H-5%, H-2%%, H-6%%), 7.77 (d, J=8.7 Hz,
2H, H-3%%, H-5%%), 8.80 (d, J=9.2 Hz, 1H, H-6%), 10.10–
11.20 (br.s, 2H, NHaniline, 3-NH). Anal. C16H10ON3Cl3
(C, H, N).
1
3j: H NMR l (ppm)=6.59 (s, 1H, CHꢀ), 7.38–7.47
(m, 4H, H-3%, H-5%, H-5%%, H-6%%), 7.76 (br.s, 1H, H-3%%),
8.25 (br.s, 1H, H-6%), 10.24 (br.s, 2H, NHaniline, 3-NH).
Anal. C16H9ON3Cl4 (C, H, N).
1
3k: H NMR l (ppm)=6.72 (s, 1H, CHꢀ), 7.14 (d,
J=8 Hz, 1H, H-6%%), 7.36–7.46 (m, 2H, H-4%%, H-5%%),
7.59 (d, J=7.5 Hz, 1H, H-5%), 7.66 (s, 1H, H-3%), 8.06
(s, 1H, H-2%%), 8.81 (d, J=8 Hz, 1H, H-6%), 10.38 (br.s,
1H, NHaniline), 11.00 (br.s, 1H, 3-NH). Anal.
C16H10ON3Cl3 (C, H, N).
4.2.2. Antimicrobial acti6ity
1
The activity of the obtained compounds was investi-
gated, using disc diffusion method [22,23], against type
strains of microorganisms as follows: S. aureus (ATCC
25923), S. pneumoniae (ATCC 49619), Enterococcus
faecalis (ATCC 29212), Pseudomonas aeruginosa
(ATCC 27853), Escherichia coli (ATCC 25922), B.
cereus (ATCC 11778), M. catarrhalis (CBM 5), M.
luteus (CBM 4), Streptococcus pyogenes (CBM 7), H.
influenzae (CBM 15) and yeast fungi: C. albicans (CBM
26). As test medium for aerobic bacteria Mueller–Hin-
ton agar (Difco Laboratories USA) was used (for H.
influenzae Mueller–Hinton agar supplemented with he-
matin and yeast extract). Tests for fungi were per-
formed on Yeast Nitrogen Base medium (Difco).
Gentamycin and nalidixic acid were used as positive
drug controls.
The compounds studied were solubilized using
DMSO. The basic concentration was 10 000 mg/mL.
From such a solution a series of dilutions with concen-
trations ranging from 5 to 10 000 mg/mL were prepared
(for reference substances the concentrations ranged
from 0.0045 to 10 000 mg/mL). Minimum inhibitory
concentration (MIC) values of the compounds were
determined with reference to standard microorganism.
The corresponding solution (20 ml) with a different
concentration was put on the sterile paper disc (9 mm
of diameter). Discs were placed on the solid medium
with suspension of a tested microorganism at 0.9%
NaCl. The MIC breakpoints were determined: after 24
h at 37 °C for bacteria and after 48 h at 28 °C for
fungi.
3l: H NMR l (ppm)=6.61 (s, 1H, CHꢀ), 7.15 (t,
J=7.4 Hz, 1H, H-4%%), 7.38 (t, J=7.4 Hz, 1H, H-5%%),
7.46–7.52 (m, 2H, H-5%, H-6%%), 7.65 (s, 1H, H-3%), 7.94
(br.s, 1H, H-3%%), 8.58 (br.s, 1H, H-6%), 10.97 (br.s, 2H,
NHaniline, 3-NH). Anal. C16H10ON3Cl3 (C, H, N).
1
3m: H NMR l (ppm)=6.68 (s, 1H, CHꢀ), 7.10 (t,
J=7.3 Hz, 1H, H-4%%), 7.38 (t, J=7.8 Hz, 2H, H-3%%,
H-5%%), 7.52 (d, J=8.6 Hz, 1H, H-5%), 7.64 (s, 1H, H-3%),
7.74 (d, J=8 Hz, 2H, H-2%%, H-6%%), 8.85 (br.s, 1H,
H-6%), 10.17 (br.s, 1H, NHaniline), 10.80 (br.s, 1H, 3-
NH). Anal. C16H11ON3Cl2 (C, H, N).
1
3n: H NMR l (ppm)=4.57 (s, 2H, CH2), 6.51 (s,
1H, CHꢀ), 7.22–7.47 (m, 6H, H-5%, H-2%%, H-3%%, H-4%%,
H-5%%, H-6%%), 7.59 (s, 1H, H-3%), 8.32 (br.s, 1H,
NHꢁCH2), 8.90 (d, J=8.1 Hz, 1H, H-6%), 11.03 (br.s,
1H, 3-NH). Anal. C17H13ON3Cl2 (C, H, N).
3o: 1H NMR l (ppm)=6.81 (s, 1H, CHꢀ), 7.27–7.52
(m, 5H, H-3%, H-4%, H-5%, H-3%%, H-5%%), 7.80 (d, J=8.7
Hz, 2H, H-2%%, H-6%%), 8.80 (d, J=7.6 Hz, 1H, H-6%),
10.25 (br.s, 1H, 1-NH), 10.98 (br.s, 1H, 3-NH). Anal.
C16H11ON3Cl2 (C, H, N).
3p: 1H NMR l (ppm)=6.63 (s, 1H, CHꢀ), 7.26–7.52
(m, 6H, H-3%, H-4%, H-5%, H-3%%, H-5%%, H-6%%), 8.30 (br.s,
1H, H-6%), 10.20 (br.s, 1H, NHaniline), 10.74 (br.s, 1H,
3-NH). Anal. C16H11ON3Cl2 (C, H, N).
1
3q: H NMR l (ppm)=6.83 (s, 1H, CHꢀ), 7.14 (d,
J=6.8 Hz, 1H, H-6%%), 7.27–7.43 (m, 5H, H-3%, H-4%,
H-5%, H-4%%, H-5%%), 8.19 (s, 1H, H-2%%), 8.83 (d, J=6.9
Hz, 1H, H-6%), 10.34 (br.s, 1H, NHaniline), 11.05 (br.s,
1H, 3-NH). Anal. C16H11ON3Cl2 (C, H, N).
In the 1H NMR spectra of the compounds 3f–h
double signals assigned to 1-NH and 3-NH protons are
observed. The splits are probably connected with the
phenomenon of coexistence of two (or more) tau-
tomeric forms. The discussion of dynamic nature of the
described compounds is going to be presented in the
separate article [27].
All antimicrobial potency tests, the medium and mi-
croorganisms suspensions were performed according to
NCCLS procedures. In each assay the control of both
microorganism culture sterility and standard microor-
ganism growth was performed. It was found that
DMSO showed neither antibacterial nor fungicidal
activity.

44
E. Szyman´ska et al. / Il Farmaco 57 (2002) 39–44
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protein kinase, affinity-purified on p9CKShs-sepharose
beads. The enzyme activity is assayed, in the presence
of potential inhibitors, using histone H1 and 32P-la-
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The second screening test uses a highly purified hu-
man recombinant glutathione-S-transferaze/cdc25 fu-
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Facility (TAACF) through a research and development
contract with US National Institute of Allergy and
Infectious Diseases. The authors are grateful to Dr
Laurent Meijer and Dr Sophie Leclerc for the evalua-
tion of the antimitotic activity of the synthesized com-
pounds according to the European Organization for
Research and Treatment of Cancer (EORTC) program.
This work was partly supported by the Polish State
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