Antimicrobial activity of some N-alkyl substituted of (E)-4-azachalconium and (E)-3'-hydroxy-4-azachalconium bromides.

Article abstract of DOI:10.1016/S0014-827X(02)01230-2

Twelve new N-substituted (E)-azachalconium bromides were synthesized and tested for antimicrobial and antifungal activities. Compounds 5c, 5d and 5h-5l showed very good antimicrobial activity against Staphylococcus aureus, Enterococcusfaecalis as well as

Full text of DOI:10.1016/S0014-827X(02)01230-2

Il Farmaco 57 (2002) 657–661
www.elsevier.com/locate/farmac
Antimicrobial activity of some N-alkyl substituted of
(E)-4-azachalconium and (E)-3%-hydroxy-4-azachalconium bromides
Zdzisława Nowakowska a, Elzbieta Wyrzykiewicz a,*, Bogdan Ke˛dzia b
;
a Faculty of Chemistry, Adam Mickiewicz Uni6ersity, Grunwaldzka 6, 60-780 Poznan, Poland
b Institute of Medicinal Plants, Libelta 27, 61-707 Poznan, Poland
Received 31 January 2002; accepted 2 March 2002
Abstract
Twelve new N-substituted (E)-azachalconium bromides were synthesized and tested for antimicrobial and antifungal activities.
Compounds 5c, 5d and 5h–5l showed very good antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis as well
as Bacillus subtilis and 5h–5j showed moderate activity against Escherichia coli. In particular, (E)-N-dodecyl-4-azachalconium
bromide (5i) and (E)-N-tetradecyl-4-azachalconium bromide (5j) showed the most intensive activity against all tested microorgan-
´
isms. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Antimicrobial activity; N-Substituted (E)-Azachalconium bromides
1. Introduction
hibitory concentration (MIC) between 1 and 10 mg/ml.
In view of the continuous interest of antimicrobial
agents we deemed it worthwhile to investigate other
N-substituted (E)-azachalconium bromides in this res-
pect, in order to better evaluate the structural require-
ments for activity. We wanted to determine the influ-
ence of the three structural modifications on the
antimicrobial activity of (E)-N-bromoalkyl-4-azachal-
conium bromides. These modifications, i.e. the lack of
the hydrophilic bromine atom at the end of the N-alkyl
substituent, the length of the carbon chain in the N-al-
kyl substituent as well as the introduction of the
strongly hydrophilic hydroxy group on the 3 positions
of the phenyl ring, change both the electronic distribu-
tion and the lipophilic–hydrophilic balance of the
molecules of N-substituted bromides of (E)-chalcones.
It ought to be pointed out that according to Tsuchiya
et al. [17], the presence of hydroxyl groups at C-2, C-4
and C-2% in chalcone is essential to inhibit growth of the
Candida species.
Over the last twenty years, analogs of azachalcones
have been synthetically prepared by several investiga-
tors and tested for antimicrobial [1–10], antituberculo-
static [11,12] and anti-inflammatory activities [13,14].
The 4-azachalcone and their derivatives [15,16] were the
most potent of the chalcone series as inhibitors of
myeloperoxidase (MPO) enzyme release from rat poly-
morphonuclear leukocytes (PMN).
A few years ago we described the antimicrobial acti-
vity of several derivatives of (E)-4-azachalcones with a
structure characterized by the presence of N-bro-
moalkyl, ortho-(meta- and para-)halobenzyl and
alkylthiouracil substituents [2–4]. Among them a few
compounds were of particular interest [as (E)-N-uracil-
(and 6-methyluracil-)thiodecyl substituted 4-azachalco-
nium bromides, (E)-N-halobenzyl-4-azachalconium
bromides and (E)-N-bromodecyl-4-azachalconium bro-
mide], exhibiting a good level of activity against
Staphylococcus aureus, Streptococcus faecalis, Entero-
coccus faecalis and Bacillus subtilis with minimum in-
In this paper, we describe the synthesis and charac-
teristics of 12 new (E)-N-alkyl-4-azachalconium bro-
mides, and (E)-N-alkyl-3%-hydroxy-4-azachalconium
bromides, and report the results of microbiological
screening.
* Corresponding author
E-mail address: wyrzyk@main.amu.edu.pl, zdzisian@main.amu.
edu.pl (E. Wyrzykiewicz).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(02)01230-2

658
Z. Nowakowska et al. / Il Farmaco 57 (2002) 657–661
2. Chemistry
13C NMR spectra, as well as by elemental analyses
(Tables 1 and 2).
Scheme 1 illustrates the synthetic approach chosen
for the preparation of (E)-N-alkyl-3%-hydroxy-4-
azachalconium bromides (5a–5f) and (E)-N-alkyl-4-
azachalconium bromides (5g–5l). (E)-3%-hydroxy-4-
azachalcone (3a) and (E)-4-azachalcone (3b) were se-
lected as the starting materials together with 1-bromo-
hexane (4a), 1-bromodecane (4b), 1-bromododecane
(4c), 1-bromotetradecane (4d), 1-bromohexadecane (4e)
and 1-bromooctadecane (4f). 3a and 3b were synthe-
sized by condensation of corresponding acetophenone
(1a–1b) with 4-pyridinecarboxyaldehyde in pyridine.
The reactions of 3a–3b with 4a–4f were carried out in
boiling acetonitrile. Twelve new (E)-N-alkyl substituted
3%-hydroxy-4-azachalconium bromides (5a–5f) and (E)-
N-alkyl-4-azachalconium bromides (5g–5l) were ob-
tained in these reactions of nucleophylic substitution.
The structures of all obtained compounds were deter-
It should be mentioned that analysis of IR spectra
revealed (E)-configuration for all obtained compounds
due to the presence of strong bonds of out-of-plane
trans olefinic CꢀH bending vibrations between 960 and
980 cm−1. The geometry at the ethylene bridge of
(E)-N-alkyl-4-azachalconium bromides (5a–5l) was
1
also assigned as E based on the olefin H NMR cou-
pling constants (J=15.6 Hz) [18].
3. Results and discussion
The new obtained compounds 5a–5l were assayed
against the following nine strains of microorganisms:
Gram-positive cocci: S. aureus, E. faecalis; aerobic
bacilli: B. subtilis; Gram-negative rods: Escherichia coli,
Klebsiella pneumoniae, Pseudomonas aeruginosa; yeasts:
Candida albicans; moulds: Aspergillus fumigatus; and
1
mined by examining their UV–Vis, IR, H NMR and
Scheme 1.

Z. Nowakowska et al. / Il Farmaco 57 (2002) 657–661
659
Table 1
Physico-chemical data of compounds 5a–5l
a
Compound
Formula
Yield (%)
m.p. (°C)
13C NMR (DMSO-d6) l ppm
TLC (Rf)
CꢁO
NꢀCH2
Ca
Cb
5a
5b
5c
5d
5e
5f
5g
5h
5I
5j
C20H24NO2Br (390.32)
65
41
59
55
62
65
39
45
49
47
46
49
184–186
123–125
139–141
155–158
153–156
149–152
141–143
159–161
147–149
152–154
146–149
130–131
188.62
188.61
188.59
188.60
188.58
188.58
188.73
188.75
188.77
188.77
188.75
188.73
60.37
60.37
60.35
60.35
60.37
60.35
60.34
60.34
60.33
60.34
60.33
60.34
136.98
136.97
136.95
136.97
136.95
136.95
136.53
136.54
136.54
136.53
136.52
136.54
132.25
132.25
132.23
132.23
132.23
132.24
134.13
134.08
134.04
134.06
134.04
134.06
0.31
0.31
0.32
0.31
0.32
0.31
0.35
0.34
0.35
0.34
0.33
0.33
C
24H32NO2Br (446.43)
C26H36NO2Br (474.48)
28H40NO2Br (502.54)
C
C30H44NO2Br (530.59)
C32H48NO2Br (558.64)
C20H24NOBr (374.32)
C24H32NOBr (430.43)
C26H36NOBr (458.48)
C28H40NOBr (486.54)
5k
5l
C30H44NOBr (514.59)
C32H48NOBr (542.64)
a Analyses for C, H and N are within 90.4% of the theoretical values.
Table 2
Physico-chemical data of compounds 5a–5l
Compound IR (KBr) (cm−1, wCꢁO)
1H NMR (DMSO-d6) l ppm, J Hz a
UV–Vis unm (log m)
CH3
CH2ꢀN OH
CaꢀH
CbꢀH
5a
5b
5c
5d
5e
5f
5g
5h
5I
5j
1667.9
1667.0
1666.8
1666.8
1666.3
1660.9
1664.8
1661.9
1664.7
1662.5
1661.0
1663.2
0.86 t
0.85 t
0.85 t
0.85 t
0.85 t
0.85 t
0.87 t
0.87 t
0.85 t
0.85 t
0.85 t
0.85 t
4.60 t
4.59 t
4.60 t
4.60 t
4.59 t
4.60 t
4.62 t
4.61 t
4.61 t
4.58 t
4.59 t
4.59 t
9.96 s
9.94 s
9.97 s
9.95 s
9.93 s
9.94 s
–
–
–
–
–
–
7.83 d 8.43 d 291.0 (4.46)
7.82 d 8.43 d 291.0 (4.46)
7.82 d 8.43 d 291.0 (4.44)
7.83 d 8.42 d 290.5 (4.46)
7.82 d 8.42 d 291.0 (4.45)
7.83 d 8.42 d 291.0 (4.45)
7.86 d 8.52 d 289.5 (4.37)
7.85 d 8.52 d 289.0 (4.40)
7.86 d 8.53 d 290.0 (4.48)
7.86 d 8.51 d 289.0 (4.47)
7.86 d 8.51 d 290.5 (4.49)
7.87 d 8.52 d 290.5 (4.48)
210.0 (4.37)
206.5 (4.38)
208.5 (4.37)
209.5 (4.37)
209.0 (4.34)
206.5 (4.37)
203.0 (4.36)
203.0 (4.37)
203.0 (4.40)
203.0 (4.36)
202.5 (4.39)
203.0 (4.37)
222.0 (4.01)
222.0 (4.03)
220.5 (4.05)
220.5 (4.01)
220.0 (4.05)
222.0 (4.04)
5k
5l
a The 3J (HH) of 15.6 Hz between H-a and H-b.
dermatophytes: Microsporum gypseum K1. (Table 3).
Many of the obtained compounds showed very good
antibacterial and antifungal activity. The effect on
Gram-positive cocci was stronger than on Gram-nega-
tive rods. The strongest effect on Gram-positive bacte-
ria were observed for (E)-N-dodecyl-3%-hydroxy-
4-azachalconium bromide (5c) (MIC 5.0 mg/ml) and
(E)-N-tetradecyl-3%-hydroxy-4-azachalconium bromide
(5d) (MIC 5.0–7.5 mg/ml) as well as for (E)-N-dodecyl-
4-azachalconium bromide (5i), (E)-N-tetradecyl-4-
azachalconium bromide (5j) and (E)-N-hexadecyl-4-
azachalconium bromide (5k) (MIC 5.0 mg/ml). It
should be pointed out that all of these compounds are
also very effective against B. subtilis (MIC 0.25–7.5
mg/ml). Moderate effects on E. coli were observed for
compounds 5h–5j (MIC 10–25 mg/ml). It is interesting
that the all N-substituted (E)-4-azachalconium bro-
mides (5g–5l) discussed herein are effective against M.
gypseum (MIC 10–100 mg/ml). In the series of the
derivatives of 3%-hydroxy-4-azachalcone (5a–5f), only
(E)-N-dodecyl-3%-hydroxy-4-azachalconium bromide
(5c) showed moderate effects on M. gypseum (MIC 100
mg/ml). Among the (E)-azachalcone derivatives (5g–5l)
examined, (E)-N-dodecyl-4-azachalconium bromide (5i)
and (E)-N-tetradecyl-4-azachalconium bromide (5j)
showed the strongest activity against all tested microor-
ganisms. It should be noted that 5c and 5i–5k break
the evolution of S. aureus and E. faecalis in the same
range of concentrations as Chloramphenicol. Similarly
5c, 5j and 5k affect B. subtilis, but it ought to be
pointed out that 5i is stronger and it is active in lower
concentration (MIC 0.25 mg/ml). Only (E)-N-octadecyl-
3%-hydroxy-4-azachalconium bromide (5f) showed no
antibacterial or antifungal activity. The comparison of
the antibacterial and antifungal activity of (E)-N-pen-
tyl-4-azachalconium bromide (5g) and (E)-N-bro-
mopentyl-4-azachalconium bromide (3c lit.4) showed
that the lack of the bromine atom at the end of

660
Z. Nowakowska et al. / Il Farmaco 57 (2002) 657–661
the alkyl chain increases the activity against S. aureus.
The comparison of antibacterial and antifungal activity
of (E)-N-alkyl-3%-hydroxy-4-azachalconium bromides
(5a–5f) and (E)-N-alkyl-4-azachalconium bromides
(5g–5l) showed that the presence of hydroxy subs-
tituent in the 3% position of the phenyl ring of the
skeleton of (E)-4-azachalcone clearly influences the di-
minishing of the activity against M. gypseum in the
series 3%-hydroxy substituted derivatives of chalcone
(5a–5f). The same effect is seen in the cases of E. coli
(5a–5e), K. pneumoniae (5d), P. aeruginosa (5c and 5d),
C. albicans (5c), S. aureus (5a, 5b, 5e and 5f), E. faecalis
(5b and 5d–5f) and B. subtilis (5b–5f).
It is also clear that the length of the alkyl chain
influences the broadening of the spectrum of antimicro-
bial activity and the value of MIC of investigated
compounds 5a–5l. The optimum length of the alkyl
chain for better and broader activity is situated in the
range of 12–16 carbon atoms in the series of 5g–5l,
and 12–14 carbon atoms in the series of 5a–5f.
on a Varian Gemini VT 300 spectrometer at 300.075
MHz, using Me4Si as an internal standard and DMSO-
d6 as solvent. The standard resolution was 0.2 Hz per
1
point for H spectra. All chemical shifts are quoted in l
(ppm) values. UV–Vis spectra were recorded on a
Specord UV–Vis spectrophotometer in methanol solu-
tion. (E)-4-azachalcone and (E)-3%-hydroxy-4-azachal-
cone were prepared according to the literature [19].
1-bromohexane (4a), 1-bromodecane (4b), 1-bromo-
dodecane (4c), 1-bromotetradecane (4d), 1-bromohexa-
decane (4e) and 1-bromooctadecane (4f) were obtained
from Aldrich.
4.1. General procedure for synthesis of compounds
5a–5l
A mixture of (E)-3%-hydroxy-4-azachalcone or (E)-4-
azachalcone (0.001 mol) and bromoalkanes (1-bromo-
hexane, 1-bromodecane, 1-bromododecane, 1-bromo-
tetradecane, 1-bromohexadecane; 0.003 mol) in acetoni-
trile (50 ml) was refluxed for 30 h. Then half a volume
of acetonitrile was removed using a rotatory evapora-
tor, and the precipitated solid was purified by column
chromatography (column, length 30 cm, diameter 2 cm)
on silica gel (25 g, Merck, 0.063–0.1 mm). The column
was eluted successively with the following solvent mix-
ture: chloroform–methanol, A [50:1, 100 ml] and B
[20:1, 150 ml]. Fractions of 20 ml were collected and
monitored by analytical TLC. The desired products
5a–5l were obtained from fractions 5–11. The isolated
crude product was recrystallized from chloroform–
methanol 1:1.
4. Experimental
The melting points were determined on a Melt-Temp
II melting point apparatus and are uncorrected. Rf
values refer to TLC plates with silica gel F254 (Merck)
developed with chloroform–methanol (5:1) and ob-
served under UV light (u=254 nm). Infrared spectra
were recorded as KBr pellets on a Bruker IFS 113
1
FT-IR spectrometer. H NMR spectra were recorded
Table 3
Antimicrobial activity of 5a–5f and 5g–5l
Compound
Minimum inhibitory concentration a (mg/ml)
1
2
3
4
5
6
7
8
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
5a
5b
5c
5d
5e
5f
5g
5h
5I
5j
5k
5l
100
100
5
100
100
5
7.5
100
–
100
10
5
5
5
100
100
5
7.5
100
–
100
10
0.25
5
5
5
5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
100
–
–
–
100
100
100
–
100
100
–
–
–
b
b
b
b
b
b
b
5
b
b
100
–
b
b
b
–
10
10
5
5
5
100
10
10
25
100
–
–
100
100
50
10
50
10
100
100
50
–
100
75
–
100
–
–
b
b
b
b
b
b
b
b
100
5
5
5
–
–
–
Chloramphenicol (Polfa-Ło´dz´)
5
50
Amfotericin
0.5
10
1
A, Chloramphenicol (Polfa–Ło´dz´); B, Amfotericin. 1, S. aureus FDA 209 P; 2, E. faecalis ATCC 8040/1; 3, B. subtilis ATCC1633; 4, E. coli PZH
026B6; 5, K. pneumoniae 231; 6, P. aeruginosa SR1; 7, C. albicans PCM 1409 PZH; 8, M. gypseum K1.
a MIC, the minimum inhibitory concentration is the lowest value of concentration of the investigated compound which brakes the evolution of
the microorganism.
b The values of MIC are situated in the range 250–1000 mg/ml.

Z. Nowakowska et al. / Il Farmaco 57 (2002) 657–661
661
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4.2. Biological test procedures
The activity of the compounds was investigated
against the following strains: Gram-positive cocci (S.
aureus FDA209P, E. faecalis ATCC 8040), aerobic
bacilli (B. subtilis ATCC1633), Gram-negative rods (E.
coli PZH 026B6, K. pneumoniae 231, P. aeruginosa
S85/2), yeasts (C. albicans PCM 1409 PZH), moulds (A.
fumigatus C1) and dermatophytes (M. gypseum K1).
4.3. Determination of MIC
Compounds were dissolved using DMSO (Serva);
concentration was 1000 mg/ml. The MIC values of the
compounds were determined, with reference to stan-
dard microorganisms, by introducing 1 ml of the corres-
ponding solutions at various concentrations into a se-
ries of tubes (each 12×100 mm), then 0.1 ml of a
standardized 1:1000 diluted suspension of a microor-
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after 18 h of incubation at 37 °C. As a test medium for
bacteria the fluid medium Penassay Broth (Difco) was
used. In each assay the control of both the bacterial
culture sterility and standard bacteria growth was per-
formed. Sabouraud dextrise broth (Difco) was used as a
test medium for fungi; MIC values were determined
after 3–7 days of incubation at 25 °C. In all assays
both fungi culture sterility and standard fungi growth
were checked.
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