Synthesis of some new S-triazine based chalcones and their derivatives as potent antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2009.10.037

Base catalysed condensation of ketone 5 with different aldehydes give chalcones, 2.4-bis-(phenylamino)-6-[4′-{3″-(4?-substituted phenyl/2?-furanyl/2?-thienyl)-2″-propenon-1″-y l}phenylamino]-s-thriazines 6a-e. These chalcones on cyclization with hydrazine

Full text of DOI:10.1016/j.ejmech.2009.10.037

European Journal of Medicinal Chemistry 45 (2010) 510–518
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some new S-triazine based chalcones and their derivatives
as potent antimicrobial agents
a
a
b
b
c
´
´
´
Anjani Solankee , Kishor Kapadia , Ana Ciric , Marina Sokovic , Irini Doytchinova ,
Athina Geronikaki d
,
*
a Department of Chemistry, B.K.M.Science College, Valsad-396001, India
b Aristotle University, School of Pharmacy, Thessaloniki 54124, Greece
c Mycological Laboratory, Institute for Biological Research, Bulevra despota Stefana 142, Belgrade 11 000, Serbia
d Faculty of Pharmacy, Medical University of Sofia, 2 Dunav st., 1000 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
Base catalysed condensation of ketone 5 with different aldehydes give chalcones, 2.4-bis-(phenylamino)-
6-[40-{300-(4%-substituted phenyl/2%-furanyl/2%-thienyl)-200-propenon-100-yl}phenylamino]-s-thriazines
6a–e. These chalcones on cyclization with hydrazine hydrate in the presence of glacial acetic acid,
guanidine nitrate in the presence of alkali and malononitrile in the presence of ammonium acetate give
the corresponding acetylpyrazolines 7a–e, aminopyrimidines 8a–e and cyanopyridines 9a–e respec-
tively. The products 6a–e, 7a–e, 8a–e and 9a–e were fully characterized by spectroscopic and elemental
analysis and also tested for antibacterial activity.
Received 26 February 2009
Received in revised form
21 September 2009
Accepted 27 October 2009
Available online 30 October 2009
Keywords:
Triazine
Ó 2009 Elsevier Masson SAS. All rights reserved.
Chalcones
Antimicrobial
QSAR
Microdilution
1. Introduction
Pyrimidines play a vital role in many biological processes since
their ring system is present in several vitamins, coenzymes and
The s-triazine based chalcones and their derivatives demon-
strate a range of biological activities and in general have been
studied extensively because of their wide range of biological
activity [1–14]. They are found to be effective as local anaesthetics
[1], antibacterial [2,3], antimalarial [4–6], antiprotozoal [7,8] anti-
tubercular [9], anticancer [10,11] and antifungal agents [12,13].
These diverse properties of chalcones have prompted us to
synthesize them in order to study their biological activities.
The study of pyrazoline derivatives has been a developing field
within the realm of heterocyclic chemistry for the past several decades
because of their ready accessibility through synthesis, wide range of
chemical reactivity and broad spectrum of biological activity [14–21].
Pyrazoline derivatives have been found to be bactericidal [14,15]
fungicidal [10,16,17] and insecticidal agents [18,19]. A survey of more
recent literature reveals that some pyrazoline derivatives possess
cerebroprotective properties [20] and antidepressant activity [21,22].
nucleic acids. Synthetic members of this group are also important as
chemotherapeutic agents [23,24]. Recently much interest has focused
on the synthesis of pyrimidines possessing fungicidal [25], herbicidal
[26], antidepressant [27] and antitumor properties [28,29]. Finally,
within the context of our biological studies, cyanopyridines have
attracted considerable attention as they posses antibacterial [30],
antitubercular [31], antifungal [32] and analgesic [33] activities.
In continuation of work on some novel s-triazine based chal-
cones [34,35] and their derivatives, we herein, report the reaction
of 2,4-bis-(phenylamino)-6-(40-acetylphenylamino)-s-triazine
5
with different aldehydes to form chalcones 6a–e and their subse-
quent conversion to products 7a–e, 8a–e and 9a–e possessing
pyrazoline, pyrimidine and cyanopyridine components.
2. Results and discussion
2.1. Chemistry
* Corresponding author. School of Pharmacy, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece. Tel.: þ30 2310997616; fax: þ30 2310997612.
E-mail addresses: dranjani_solankee@yahoo.com (A. Solankee), mris@ibiss.bg.
ac.rs (M. Sokovic´), doytchinova@gmail.com (I. Doytchinova), geronik@pharm.auth.
gr (A. Geronikaki).
Compound 5 was prepared by the condensation of cyanuric
chloride 1 and aniline 2 at 0–5ꢀ to form 3, which reacts further
with aniline at room temperature to form 4. This, in turn, on
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.037

A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
511
Cl
Cl
H2N
o
N
N
N
N
0-5
H2N
+
Acetone
Room temp.
Acetone
Cl
N
1
Cl
HN
N
3
Cl
2
Cl
NH
N
COCH3
N
N
N
H2N
COCH3
HN
N
NH
HN
N
NH
Reflux
Acetone
4
5
NH
N
CO CH= CH R
R-CHO
N
DMF/KOH
HN
N
NH
6a-e
NH2NH2 H2O
gl. CH3COOH
NH2CNH2 HNO3
NH
CH2(CN)2
R
N
CN
NH2
NH
NH
R
N
N
N
N
N
N
HN
N
NH
COCH3
HN
N
NH
NH2
9a-e
7a-e
N
N
NH
R
N
N
HN
N
NH
8a-e
Scheme 1. Synthetic procedure for preparation of title compounds.
treatment with 4-aminoacetophenone, gives compound
5
group. The aromatic cluster of compounds also support the
synthesis of compounds.
(Scheme 1). Subsequent condensation of ketone 5 with various
aldehydes led to compounds 6a–e. Chalcones 6a–e were cyclised
with hydrazine hydrate in the presence of glacial acetic acid,
guanidine nitrate in the presence of alkali and malononitrile in the
presence of ammonium acetate to form acetylpyrazolines 7a–e,
aminopyrimidines 8a–e and cyanopyridines 9a–e respectively
(Scheme 1).
The structures of the synthesized compounds were assigned on
the basis of elemental analysis (Table 1), IR and 1H NMR (Table 2).
The IR spectrum of compound 6e shows the characteristic band at
1647 cmꢁ1 due to the –C]O group, the IR spectrum of compound
7e shows the characteristic band at 1573 cmꢁ1 due to the –C]N
group and the IR spectrum of compounds 8e and 9e shows the
characteristic bands in the region of 3300–3400 cmꢁ1 which indi-
cate the presence of a primary amine. There are no absorptions in
the region of 1600–1700 cmꢁ1 in IR spectra of compound 8e and 9e,
indicating the absence of a–C]O group in these structures. The 1H
NMR spectrum of compound 6e showed doublet of –CO–CH] at
2.2. Antibacterial activity
All synthesized compounds were screened for their antibacte-
rial activity by using the disc-diffusion method, TLC- bioauto-
graphic and microdilution methods against a panel of selected
Gram-positive and Gram-negative bacteria. DMSO was used as
a solvent while Streptomycin and Ampicillin were used as a control.
The results of antibacterial activity of titled compounds by disc-
diffusion method are presented in Table 3 as inhibition zones of
tested compounds at 10 mg/disc. The molecular weights of the
newly synthesized triazines and their derivatives varied from
478.55 (6c) to 592.61 (9a) and were significantly higher than that of
Ampicillin (403.45) and different to Streptomycin (581.60). To
eliminate this difference, the
in mol/ml and results are also presented in Table 3a as inhibition
zones gave by 1 mol/disc.
mg/ml concentrations were converted
m
m
d
6.90 ppm and doublet of Ar–CH] at
the presence of chalcone moiety. The 1H NMR spectrum of
compound 7e showed singlet of –COCH3 at 2.42 ppm. Compounds
8e and 9e showed singlet between 5.00–6.00 ppm due to –NH2
d
8.05 ppm, which confirm
It can be seen that the most sensitive bacteria among Gram-
positive in disc-diffusion method is Micrococcus flavus. Compounds
6c, 7a, 7c, 8b, 8c, 8d, 8e, 9a, 9b, 9d and 9e showed lower activity
against this bacteria than Streptomycin but higher than Ampicillin
d
d

512
A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
Table 1
The physicochemical data of compounds 6a–e, 7a–e, 8a–e and 9a–e.
Compd No.
R
M.P. ꢀC
Yield %
Molecular formula
Elemental analysis (Found/calcd) %
C
H
N
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
149–150
181–182
131–133
111–113
173–175
154.5–155
185–186
148–149
148–150
178–180
154–155
153.5–154
133–135
147.5–147
146–148
163–164
114–115
134–136
163.5–164
113–115
78
82
76
70
68
64
67
58
62
64
68
61
59
58
66
64
66
61
63
69
C30H23N7O3
C30H23N6OCl
C28H22N6O2
C28H22N6OS
C31H26N6O2
C32H27N9O3
C32H27N8OCl
C30H26N8O2
(68.04 68.05)
(69.43 69.43)
(70.87 70.89)
(68.55 68.57)
(72.36 72.37)
(65.63 65.64)
(66.84 66.84)
(67.91 67.92)
(65.92 65.93)
(69.46 69.47)
(65.48 65.49)
(66.72 66.73)
(67.82 67.84)
(65.77 65.78)
(69.42 69.44)
(66.88 66.89)
(68.10 68.10)
(69.26 69.27)
(67.25 67.27)
(70.70 70.71)
(4.38 4.35)
(4.47 4.44)
(4.67 4.64)
(4.52 4.49)
(5.09 5.06)
(4.65 4.62)
(4.73 4.70)
(4.94 4.91)
(4.79 4.76)
(5.30 5.27)
(4.25 4.23)
(4.33 4.30)
(4.51 4.48)
(4.38 4.36)
(4.92 4.88)
(4.08 4.05)
(4.16 4.13)
(4.31 4.28)
(4.19 4.17)
(4.71 4.68)
(18.51 18.53)
(16.19 16.20)
(17.71 17.72)
(17.13 17.14)
(16.33 16.34)
(21.53 21.54)
(19.48 19.50)
(21.12 21.13)
(20.50 20.51)
(19.64 19.65)
(24.63 24.65)
(22.59 22.60)
(24.55 24.56)
(23.80 23.82)
(22.77 22.78)
(23.64 23.65)
(21.66 21.67)
(23.45 23.46)
(22.77 22.78)
(21.82 21.84)
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
2-Thienyl
C30H26N8OS
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
C33H30N8O2
C31H24N10O2
C31H24N9Cl
C29H23N9O
C29H23N9S
C32H27N9O
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
2-Thienyl
2-Methoxyphenyl
C33H24N10O2
C33H24N9Cl
C31H23N9O
C31H23N9S
C34H27N9O
except of 8c and 8e which possessed the same inhibition zones.
Compounds 8b and 9c inhibited Staphylococcus. aureus with lower
inhibition zones than Streptomycin and with slightly higher
activity than Ampicillin, while all tested compounds are not active
against Bacillus cereus. In case of Gram-negative bacteria Escherichia
coli is inhibited by 6a, 6c, 8c, 9b and 9e with lower activity than
Streptomycin but better than Ampicillin. Pseudomonas aeruginosa is
not inhibited by standard drugs used, but compounds 6c and 6e
showed inhibition zones. Also, Listeria monocytogenes is inhibited
by 9e while standard drugs were not effective against this bacteria.
All the tested compounds are not active against Salmonella typhi-
murium and Enterobacter cloacae. In general compounds 8b–9e
showed the best activity against Gram-positive bacteria, being
more active than ampicillin while compounds 9b and 9e exhibited
almost the same activity against both Gram-positive and Gram-
negative bacteria. Moreover, compound 9e is active on L. mono-
cytogenes while no other compounds possessed any activity against
this bacteria. It can be noticed that all tested compounds showed
lower activity than Streptomycin but higher than Ampicillin.
The results of TLC-bioautographic method are presented in Table
4. All the compounds tested showed activity against all the bacteria
used. Streptomycin was also active against all the bacteria tested,
while there is no activity observed for Ampicillin in this method. It
can be seen again that the most sensitive bacteria among Gram-
positive in this method, like in previous one, is M. flavus. All
compounds tested exhibited good ability to inhibit this bacteria,
even much better than antibiotic, used as reference drug, except of
6a which showed the same activity with Streptomycin. It was found
that compounds 6c, 6e, 7b, 7c, 8b, 8c, 8d and 8e are active against
S. aureus. It should be noted that compounds 6c, 6e, 7b, 7c and 8c are
more active than Streptomycin, while compound 8b exhibited the
same activity with the reference drug. Compounds 8d and 8e are the
less potent. In the case of another Gram-positive bacteria, B. cereus,
compounds 7a, 8b and 8d are more active, 7b and 7d -equipotent
and 6c and 6e -slightly less active than standard drug. Among Gram-
negative bacteria the most sensitive bacteria are S. typhimurium and
En. cloacae. All the compounds tested are more active against
L. monocytogenes than Streptomycin with exception of 6a and 6b.
The most active compound in this case is 8a, followed by 7a, 7b and
8e. All the compounds, except of 8a, 8b and 8e are also more active
than Streptomycin and against En. cloacae. The best activity was
found for compound 7d, followed by 6a, 7a–7c, as well as 9a. In the
case of S. typhimurium compounds 6b, 7a, 7c and 8d possessed
activity better than Streptomycin with the most potent compound
being 7a. Compounds 7b, 8b and 9c–9e are less active than the
reference drug, while 6a and 9b are equipotent to it. It could be
noticed that E. coli is a less sensitive bacteria compared with ones
previously mentioned. Thus, only five compounds 6c, 8b, 8c and 9b,
and 9d are active against this bacteria. Compounds 6c, 8b, 8c and 9b,
showed the same inhibition zones as Streptomycin while 9d is more
potent against E. coli. The most resistant bacteria is P. aeruginosa
being sensitive only to 9a and 9b which are more potent than
Streptomycin. In general compounds 9a–9e possessed better
activity against Gram-negative bacteria, however only compound
9b is active against all the Gram-negative bacteria tested. From
another side, compounds 8a–8e are the most active against Gram-
positive bacteria. In general, active compounds tested by this
method are more potent against Gram-negative than Gram-positive
bacteria, showing higher inhibition zones than Streptomycin.
The results of antibacterial activity obtained by microdilution
method are presented in Table 5. The kind of the exerted antibac-
terial activity was investigated by determining the minimal
bactericidal concentrations (MBCs). The experimental data (values
in brackets) presented in Table 4 show that all derivatives 6a–9e
possess bacteriostatic properties, being MBCs twofold or more
higher than the corresponding MICs.
All the compounds tested showed MIC ranging from 4.5 to
78.0
m
mol/ml ꢂ 10ꢁ2 and MBC ranging from 9.0 to 78.0
mmol/
ml ꢂ 10ꢁ2. Only compound 7c, did not show MBC against E.colacae,
S. typhimurium and L. monocytogenes. Standard drugs are also active
against all the bacteria tested and show MIC ranging from 4.3 to
25.8
m
mol/ml ꢂ 10ꢁ2 for Streptomycin and 24.8–74.4
mmol/
mol/
ml ꢂ 10ꢁ2 for Amplicillin. MBC for the antibiotics are 8.6–51.6
m
ml ꢂ 10ꢁ2 and 37.2–124.0
m
mol/ml ꢂ 10ꢁ2, respectively.
The most sensitive bacteria is B. cereus with very low MIC and
MBC, while L. monocytogenes is the most resistant one with high
MIC and MBC. From the group 6a–e, compound 6e exhibited the
best antibacterial activity (MIC of 13.4–26.8
MBC of 26.8–53.6
for compound 6c (MIC of 21.1–42.2
42.2–84.4
m
mol/ml ꢂ 10ꢁ2 and
m
mol/ml ꢂ 10ꢁ2). The worst activity is obtained
m
mol/ml ꢂ 10ꢁ2 and MBC of
m
mol/ml ꢂ 10ꢁ2). The highest antibacterial activity in
group 7a–e, was observed for compound 7a (MIC of 8.5–68.4 mmol/
ml ꢂ 10ꢁ2 and MBC of 17.1–68.4
m
mol/ml ꢂ 10ꢁ2), while 7c showed
the lowest activity with MIC of 37.8–75.6
m
mol/ml ꢂ 10ꢁ2 and MBC

A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
513
Table 2
IR and 1H NMR spectra of synthesized compounds.
Compd No.
IR
1H NMR (CDCl3)
d ppm d C (CDCl3)
6a
1643 (C]O), 1597 (–CH]CH–, str.),
1338 (C–N), 811 (C–N, s-triazine).
6.90 (d, 1H, –CO–CH]), 7.15–7.80 (m, 18 Ar–H and 3NH), 8.05 (d, 1H, Ar–CH]).
119, 120.6, 122.3, 123.9, 126.3, 128.4, 128.9, 129.8, 129.8, 130.4, 139.7, 140.1, 141.4,
145.3, 147.95, 163.9, 164.1, 187.1
6b
6c
1646 (C]O), 1593 (–CH]CH–, str.),
1336 (C–N), 809 (C–N, s-triazine),
786 (C–Cl).
1649 (C]O), 1592 (–CH]CH–, st.),
1342 (C–N), 810 (C–N, s-triazine).
6.92 (d, 1H, –CO–CH]), 7.10–7.75 (m, 18 Ar–H and 3NH), 8.05 (d, 1H, Ar–CH]).
118.9, 120.5,122.3, 122.9, 128.4, 128.9, 129.6, 130.5, 130.7, 133.9, 134.8, 141.5,
145, 163.9, 164, 187.3
6.89 (d, 1H, –CO–CH]), 7.15–7.85 (m, 17 Ar–H and 3NH), 8.05 (d, 1H, Ar–CH]).
112.98, 116.4, 118.9, 119.1, 120.5, 122.9, 126.2, 129.6, 130.8, 136.2, 139.7, 139.9, 144.9,
145.9, 151.3, 163.96, 164.1, 186.8
6d
6e*
7a
7b
7c
1650 (C]O), 1591 (–CH]CH–, str.),
1344 (C–N), 811 (C–N, s-triazine).
6.91 (d, 1H, –CO–CH]), 7.10–7.85 (m, 17 Ar–H and 3NH), 8.05 (d, 1H, Ar–CH]).
119.1, 120.5, 122.3, 128.4, 128.7, 129.4, 130.0, 130.8, 132.4, 135.8, 139.7, 139.9,
144.9, 163.98, 164.1, 186.9
3.95 (s, 3H, –OCH3), 6.90 (d, 1H, –CO–CH]), 7.10–7.80 (m, 18 Ar–H and 3NH),
8.05 (d, 1H, Ar–CH]). 114.2, 120.3, 122.1, 127.3, 128.2, 129.2, 130.4, 130.9, 139.5,
142.7, 160.95, 163.7, 187.
2.42 (s, 3H, –COCH3), 3.15 (dD, 1H, C4-HA), 3.65 (dd, 1H, C4-HB), 5.60 (dd, 1H, –CH–CH2),
6.90–7.80 (m, 18 Ar–H and 3NH). 118.9, 119.7, 120.5, 122.3, 123.9, 125.3, 127,1, 128.4, 128.9,
130.3, 139.7, 144.8, 146.7, 149.9, 154.1.
2.42 (s, 3H, –COCH3), 3.10 (dD, 1H, C4-HA), 3.60 (dd, 1H, C4-HB), 5.62 (dd, 1H, –CH–CH2),
6.90–7.80 (m, 18 Ar–H and 3NH). 118.9, 119.6, 120.4, 122.2, 124.2, 127.0, 127.5, 128.4, 128.6,
131.6, 139.8, 141.5, 142.2, 154.1, 163.9, 164.0, 167.2
1647 (C]O), 1595 (–CH]CH–, str.),
1340 (C–N), 812 (C–N, s-triazine).
1650 (–COCH3), 1575 (C]N),
806 (C–N, s-triazine).
1656 (–COCH3), 1577 (C]N),
806 (C–N, s-triazine),
768 (C–Cl).
1654 (–COCH3), 1576 (C]N),
806 (C–N, s-triazine).
2.42 (s, 3H, –COCH3), 3.14 (dD, 1H, C4-HA), 3.66 (dd, 1H, C4-HB), 5.58 (dd, 1H, –CH–CH2),
6.90–7.80 (m, 17 Ar–H and 3NH). 21.7, 39.9, 40.1, 106.8, 110.4, 119.7, 120.4, 122.2, 124.2, 127.0,
128.4, 139.8, 142.2, 142.3, 152.9, 154.1, 163.9, 164.1, 167.2.
7d
7e*
8a
8b
8c
1651 (–COCH3), 1572 (C]N),
806 (C–N, s-triazine).
2.42 (s, 3H, –COCH3), 3.13 (dD, 1H, C4-HA), 3.62 (dd, 1H, C4-HB), 5.65 (dd, 1H, –CH–CH2),
6.90–7.80 (m, 17 Ar–H and 3NH). 21.6, 40.1, 41.7, 119.9, 120.4, 122,2, 124.2, 124.5, 124.9, 126.7,
127.1, 128.4, 128.7, 131.6, 131.7, 139.8, 142.2, 145,0, 154.3, 163.9, 164.1, 166.9, 167.2.
2.42 (s, 3H, –COCH3), 3.15 (dD, 1H, C4-HA), 3.65 (dd, 1H, C4-HB), 3.80 (s, 3H, –OCH3),
5.60 (dd, 1H, –CH–CH2), 6.90–7.80 (m, 18 Ar–H and 3NH). 113.7, 119.5, 120.2,122.0, 124.2,
126.6, 126.8, 128.2, 134.4, 139.5, 141.9, 153.9, 158.2, 163.2, 163.8, 166.9
5.15 (s, 2H, –NH2), 6.95–8.15 (m, 19 Ar–H and 3NH). 102.0, 118.8, 120.5, 122.2, 123.1, 127.4,
128.1, 128.3, 128.5, 128.8, 129.1, 129.8, 130.2, 139.6, 139.7, 139.8, 142.6, 143.6, 144.7, 144.8,
145.8, 152.9, 162.1, 163.9, 164.0, 165.1, 196.3, 196.5
1653 (–COCH3), 1573 (C]N),
806 (C–N, s-triazine).
3398 (–NH2), 1575 (C]N),
806 (C–N, s-triazine).
3398 (–NH2), 1575 (C]N),
806 (C–N, s-triazine),
770 (C–Cl).
3398 (–NH2), 1575 (C]N),
806 (C–N, s-triazine).
5.12 (s, 2H, –NH2), 6.90–8.10 (m, 19 Ar–H and 3NH). 101, 118.8, 119.5, 120.2,120.4, 122.0, 122.2,
127.2, 128.0, 128.2, 128.4, 128.6, 128.7, 128.8, 128.9, 129.4, 130.3, 139.5, 139.7, 163.8, 164.0, 196.8
5.05 (s, 2H, – NH2), 6.90–8.15 (m, 18 Ar–H and 3NH). 110.4, 110.5, 113.0, 116.4, 118.8, 119.0,
120.3, 120.5, 122.2, 128.3, 128.5, 129.0, 129.2, 129.5, 130.7, 139.6, 139.7, 139.8, 144.1, 145.9
144.8, 145.9, 151.2, 163.9, 164.0, 186.8
8d
3398 (–NH2), 1575 (C]N),
806 (C–N, s-triazine).
5.14 (s, 2H, – NH2), 6.85–8.20 (m, 18 Ar–H and 3NH). 118.8, 118.8, 119.0, 119.4, 119.5, 120.4,
122.1, 122.2, 127.1, 128.3, 128.6, 128.8, 128.9, 129.0, 129.3, 130.0, 132.3, 139.6, 139.7, 139.8,
163.9, 164,.0, 186.8
8e*
9a
3398 (–NH2), 1575 (C]N),
806 (C–N, s-triazine).
3396 (–NH2), 2205 (C^N)
803 (C–N, s-triazine).
3.85 (s, 3H, –OCH3), 5.10 (s, 2H, – NH2), 6.90–8.15 (m, 19 Ar–H and 3NH). 114.0, 120.6, 120.7,
122.3, 128.2, 139.9, 140.1, 140.2, 140.3, 164.2
5.20 (s, 2H, – NH2), 6.90–8.25 (m, 19 Ar–H and 3NH).
9b
3386 (–NH2), 2210 (C^N)
805 (C–N, s-triazine), 776 (C–Cl).
5.26 (s, 2H, – NH2), 6.90–8.20 (m, 19 Ar–H and 3NH). 112.9, 113.2, 113.8, 118.8, 118.9, 119.5,
119.7, 119.8, 120.3, 120.4, 120.5, 121.9, 122.0, 122.2, 127.6, 128.2, 128.5, 128.6, 128.8, 130.0,
130.1, 131.1, 132.8, 133.6, 136.8, 139.5, 139.6, 139.7, 163.8, 163.9, 175.2, 194.4
5.22 (s, 2H, – NH2), 6.85–8.15 (m, 18 Ar–H and 3NH). 108.2, 110.6, 112.7, 113.1, 113.8, 114.1,
118.9, 120.5, 122.2, 128.2, 128.5, 128.8, 128.9, 129.0, 139.5, 139.6, 143.1, 144.4, 145.2, 150.9,
163.8, 163.9, 175.2, 194.0
9c
3392 (–NH2), 2209 (C^N)
801 (C–N, s-triazine).
9d
9e
3399 (–NH2), 2204 (C^N)
804 (C–N, s-triazine).
3396 (–NH2), 2200 (C^N)
806 (C–N, s-triazine).
5.25 (s, 2H, – NH2), 6.85–8.18 (m, 18 Ar–H and 3NH).
3.95 (s, 3H, –OCH3), 5.28 (s, 2H, – NH2), 6.90–8.20 (m, 19 Ar–H and 3NH). 108.1, 114.0, 114.3,
117.3, 119.4, 120.2, 122.0, 127.4, 128.2, 129.1, 129.6, 130.6, 139. 6, 154.0, 158.0, 160.2, 160.7,
163.8, 163.9.
of 75.6
m
mol/ml ꢂ 10ꢁ2. In group 8a–e, compound 8b possessed
It is clear that all synthesized compounds are more active against
Gram-positive bacteria than Gram-negative bacteria in this method.
It can be seen that the growth of tested bacteria responded
differently to the compounds tested, which indicates that different
components may have different modes of action or that the
metabolism of some bacteria is able to better overcome the effect of
the agents or adapt to it. Gram-negative bacteria are in general
more resistant than Gram-positive [36].
The inhibition zones obtained from TLC-bioautographic
method are higher than in disc-diffusion method. More
compounds are active in TLC-bioautographic method than in disc-
diffusion and a great number of compounds showed inhibition in
inhibitory activity of 8.95–35.8
17.9–71.6
higher MIC 19.5–78.0
effect at 39.0–78.0
m
mol/ml ꢂ 10ꢁ2 and bactericidal of
m
mol/ml ꢂ 10ꢁ2, while 8c inhibited bacterial growth with
m
mol/ml ꢂ 10ꢁ2 and showed bactericidal
m
mol/ml ꢂ 10ꢁ2. Compound 9d possessed the
best antibacterial activity in group 9a–e with MIC 4.5–18.0 mmol/
ml ꢂ 10ꢁ2 and MBC of 9.0–36.0
m
mol/ml ꢂ 10ꢁ2, while 9b showed
the lowest activity with MIC 17.2–68.8
m
mol/ml ꢂ 10ꢁ2 and MBC of
34.4–68.8
m
mol/ml ꢂ 10ꢁ2
.
Among all the compounds tested, 9d exhibited the best anti-
bacterial activity against B.cereus, with very low MIC and not anti-
bacterial activity, with very low MIC.

514
A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
Table 3
Antibacterial activity of compounds 6a–e, 7a–e, 8a–e and 9a–e (conc. 10
m
g/disc) tested by disc-diffusion method.
Antibacterial activity
Zone of Inhibition in mm
Comp No.
R
B.c
M.f
S.a
E.c
P.a
En.c
S.t
L.m
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6 ꢃ 0.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8 ꢃ 0.6
8 ꢃ 0.3
6 ꢃ 0.6
2-Thienyl
–
–
–
–
–
–
–
–
–
–
–
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
6 ꢃ 0.6
10 ꢃ 0.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10 ꢃ 0.6
–
–
2-Thienyl
–
–
–
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
–
9 ꢃ 0.6
6 ꢃ 0.6
6 ꢃ 0.6
6 ꢃ 0.3
6 ꢃ 0.3
7 ꢃ 1
–
7 ꢃ 0.6
10 ꢃ 1
20 ꢃ 1
8 ꢃ 0.6
7 ꢃ 0.6
–
–
–
–
–
10 ꢃ 1
2-Thienyl
–
–
–
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
9b
9c
9d
9e
–
–
–
–
–
7 ꢃ 1
–
–
–
–
–
–
–
6 ꢃ 0.6
–
2-Thienyl
–
–
–
2-Methoxyphenyl
Streptomycin
Ampicillin
7 ꢃ 0.6
10 ꢃ 0.6
6 ꢃ 0.3
6 ꢃ 0.3
Standard Drugs
18 ꢃ 1
13 ꢃ 1
12 ꢃ 0.6
–
–
10 ꢃ 1
8 ꢃ 0.3
6 ꢃ 0.6
– no inhibition; B.c – Bacillus cereus, M.f – Micrococcus flavus, S.a – Staphylococcus aureus, E.c – Escherichia coli, P.a – Pseudomonas aeruginosa, En.c – Enterobacter cloacae,
S.t – Salmonella typhimurium, L. m – Listeria monocytogenes.
this method. The reason of this could be explained by different
hydrophilic and diffusion capacity of different compounds through
the agar medium used in disc-diffusion method [37–39].
Compounds with higher hydrophilic characteristics possess better
diffusion capacity through the medium and, on the contrary, low
water solubility limits diffusion through the medium. Therefore,
specificity and levels of activity is related to the type of functional
group, but also associated with hydrogen-bonding parameters in
all cases.
compounds had quantitative values for MIC and MBC. As the
range of MIC and MBC values was very narrow (around one log
unit), the QSAR models derived in the present study should be
used with care for prediction and design of new compounds. The
models goodness of fit was assessed by r2, while their predictive
ability was evaluated by leave-one-out cross-validation and
assessed by q2. Only models with r2 > 0.6 and q2 > 0.3 were
considered as significant.
Microdilution method, carried out in microtitre trays, has the
advantage of lower workloads for a larger number of replicates and
the use of small volumes of the test substance and growth medium.
In addition, the results are more informative than in other methods
and could be expressed as quantitative data.
Table 3a
Antimicrobial activity of the studied compounds presented as a inhibition of 1
disc presented as inhibition zones (mm).
mmol/
Compound
Antimicrobial activity presented in mm
As regards to the relationships between the structure and the
detected antibacterial activity, the inhibitory effect appears to be
dependent on the substituent of chalcone. It seems, that for chal-
cones 6a–6e and its pyrazoline (7a–7e), pyrimidine (8a–8e) and
cyanopyridine (9a–9e) derivatives the introduction of different
substituents (R) play different role in antibacterial activity. It is
interesting to point out that for chalcones and its pyrazoline and
pyrimidine derivatives the presence of substituted benzene ring (6,
7, 8a, 8b, 8e) are mostly endowed with higher activity with respect
to five-member heterocyclic rings (6, 7, 8c, 8d). For example, for
6a-6e group the most active is derivative with 2-methoxyphenyl
substituent, for 7a–7e – 4-nitrophenyl- (only in case of Gram-
positive bacteria) while for 8a–8e -the most active is derivative
with 4-chlorophenyl substituent in aromatic ring. In the contrary,
for the antibacterial activity of cyanopyridines important is
substitution with 5-member-ring heterocycles. Thus, the best
activity in this series was observed for 2-thienyl derivative followed
by 2-furanyl one.
B.c
M.f
S.a
E.c
P.a
En.c
S.t
L.m
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.04
–
–
0.04
–
0.05
–
–
–
0.05
0.03
0.04
0.03
0.04
0.04
–
0.04
0.06
0.1
0.03
–
–
–
–
–
–
–
–
–
–
–
0.04
–
–
–
–
–
0.03
–
0.04
–
–
–
–
–
–
–
–
–
0.05
–
–
–
0.04
–
–
–
0.02
–
0.03
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9b
9c
9d
9e
–
–
–
–
0.1
0.04
–
–
–
–
0.07
0.02
–
–
–
0.03
–
0.03
–
–
0.08
0.03
–
–
–
–
–
0.04
0.06
0.02
Streptomycin
Ampicillin
2.3. QSAR study
–
– no inhibition; B.c – Bacillus cereus, M.f – Micrococcus flavus, S.a – Staphylococcus
aureus, E.c – Escherichia coli, P.a – Pseudomonas aeruginosa, En.c – Enterobacter
cloacae, S.t – Salmonella typhimurium, L. m – Listeria monocytogenes.
A
QSAR study was conducted only on the antibacterial
activities obtained by the microdilution method, because all the

A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
515
Table 4
Antibacterial activity of compounds 6a–e, 7a–e, 8a–e and 9a–e tested with TLC method and calculated as inhibition of 1
m
mol/spot presented as inhibition zone (mm).
Comp No.
R
Antibacterial activity
Zone of Inhibition in mm
B.c
M.f
S.a
E.c
P.a
En.c
S.t
L.m
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
–
–
0.05
0.07
0.07
–
–
0.07
–
–
–
–
0.07
0.07
0.07
0.08
0.06
0.07
0.07
–
0.08
0.06
0.05
–
–
–
0.08
–
0.07
–
0.08
0.07
–
–
–
0.04
0.06
0.03
0.03
–
–
–
–
–
–
–
0.08
–
–
–
–
–
–
–
–
0.08
0.08
–
–
–
0.09
–
0.06
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.09
0.08
0.08
0.08
0.08
0.09
0.09
0.09
0.1
0.08
0.09
–
–
–
0.1
0.07
0.09
–
–
–
0.07
0.07
0.08
0.08
0.08
0.09
0.09
0.08
0.08
0.08
0.1
0.08
0.08
0.08
0.09
0.1
0.07
–
0.06
0.1
0.08
–
0.08
–
–
0.09
–
0.09
–
–
–
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
0.08
–
–
0.07
–
0.09
–
0.08
0.08
–
0.09
0.08
0.09
0.08
0.08
0.07
–
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
0.09
0.09
–
–
–
–
9b
9c
9d
9e
0.08
0.07
0.07
0.07
0.08
–
0.08
0.08
0.09
0.09
0.08
–
–
–
–
0.08
–
2-Thienyl
2-Methoxyphenyl
Streptomycin
Ampicillin
Standard drugs
0.04
–
0.06
–
0.08
–
– no inhibition; B.c – Bacillus cereus; M.f – Micrococcus flavus; S.a – Staphylococcus aureus, E.c – Escherichia coli, P.a – Pseudomonas aeruginosa, En.c – Enterobacter cloacae,
S.t – Salmonella typhimurium, L. m – Listeria monocytogenes.
2.3.1. Free-Wilson models
In the present study Free-Wilson models were generated using
nine discontinuous descriptors accounting for the presence or
pMICðS:aureusÞ [ 0:110 ꢄ 2 ꢁ thienyl D0:315 ꢄ 2
ꢁ methoxyphenylL 0:308 ꢄ chalcone L 0:417
ꢄ acetylpyrazoline D 3:717
absence of a substituent or a main structure in a molecule. Genetic
algorithm (GA) was applied preliminary to select the most impor-
tant descriptors, followed by multiple linear regression (MLR) on
n [ 20r2 [ 0:637 SEE [ 0:181 F [ 6:152 p < 0:01 q2
the selected variables. Among the 16 models obtained (8 for MIC
[ 0:303 cvRSS [ 0:884
and 8 for MBC) only one showed a moderate explained and
predictive ability. This was the model, including MIC against
S. aureus and it is given by the following equation:
The chalcone and acetylpyrazoline main structures have nega-
tive contributions in the antimicrobial activity against S. aureus.
Table 5
Minimal inhibitory (MIC
m
mol/ml ꢂ 10ꢁ2) and bactericidal concentration (MBC
m
mol/ml ꢂ 10ꢁ2) of compounds 6a–e, 7a–e, 8a–e and 9a–e tested in microdilution method.
Comp No.
R
Antibacterial activity
Zone of Inhibition in mm
B.c MIC MBC
M.f MIC MBC
S.a MIC MBC
E.c MIC MBC
P.a MIC MBC
En.c MIC MBC
S.t MIC MBC
L.m MIC MBC
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
18.9 37.8
19.3 38.6
21.1 42.2
20.4 40.8
13.4 26.8
8.5 17.1
69.6 69.6
37.8 75.6
18.8 37.6
17.5 35.0
17.6 35.2
8.95 17.9
19.5 39.0
18.9 37.8
9.05 18.1
16.9 33.8
17.2 34.4
9.3 18.6
18.9 37.8
19.3 38.6
42.2 84.4
20.4 40.8
26.8 53.6
17.1 34.2
69.6 69.6
37.8 75.6
18.8 37.6
17.5 35.0
35.2 70.4
17.9 35.8
19.5 39.0
18.9 37.8
9.05 18.1
33.8 67.6
17.2 34.4
9.3 18.6
37.8 75.6
38.6 77.2
42.2 84.4
40.8 81.6
13.4 26.8
17.1 34.2
69.6 69.6
75.6 75.6
37.6 73.2
35.0 70.0
17.6 35.2
17.9 35.8
19.5 39.0
18.9 37.8
9.05 18.1
33.8 67.6
17.2 34.4
18.6 37.2
9.0 18.0
18.9 37.8
77.2 77.2
21.1 42.2
40.8 81.6
13.4 26.8
17.1 34.2
34.8 69.6
37.8 75.6
18.8 37.6
35.0 70.0
17.6 35.2
8.95 17.9
19.5 39.0
18.9 37.8
18.1 36.2
33.8 67.6
17.2 34.4
9.3 18.6
37.8 75.6
38.6 77.2
21.1 42.2
40.8 81.6
26.8 53.6
68.4 68.4
69.6 69.6
75.6 75.6
37.6 73.2
35.0 70.0
35.2 70.4
17.9 35.8
78.0 78.0
75.6 75.6
18.1 36.2
67.6 67.6
68.8 68.8
37.2 74.4
9.0 18.0
18.9 37.8
19.3 38.6
21.1 42.2
20.4 40.8
26.8 53.6
68.4 68.4
69.6 69.6
75.6 –
73.2 73.2
17.5 35.0
17.6 35.2
8.95 17.9
19.5 39.0
18.9 37.8
18.1 36.2
16.9 33.8
17.2 34.4
18.6 37.2
9.0 18.0
37.8 75.6
38.6 77.2
21.1 42.2
40.8 81.6
26.8 53.6
68.4 68.4
34.8 69.6
75.6 –
37.6 73.2
35.0 70.0
35.2 70.4
17.9 35.8
19.5 39.0
18.9 37.8
18.1 36.2
67.6 67.6
17.2 34.4
18.6 37.2
4.5 9.0
37.8 75.6
38.6 77.2
42.2 84.4
40.8 81.6
26.8 53.6
68.4 68.4
69.6 69.6
75.6 –
73.2 73.2
70.0 70.0
70.4 70.4
35.8 71.6
78.0 78.0
75.6 75.6
72.4 72.4
67.6 67.6
68.8 68.8
37.2 74.4
18.0 36.0
34.6 69.2
25.8 51.6
37.2 74.4
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
2-Thienyl
2-Methoxyphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Furanyl
9b
9c
9d
9e
2-Thienyl
4.5 9.0
8.65 17.3
4.3 8.6
4.5 9.0
8.65 17.3
8.6 17.2
4.5 9.0
2-Methoxyphenyl
Streptomycin
Ampicillin
17.3 34.6
17.2 34.4
24.8 37.2
17.3 34.6
17.2 34.4
37.2 49.2
34.6 69.2
17.2 34.4
74.4 124.0
17.3 34.6
17.2 34.4
37.2 49.2
17.3 34.6
17.2 34.4
24.8 49.2
Standard drugs
24.8 37.2
24.8 37.2
– no inhibition; B.c – Bacillus cereus, M.f – Micrococcus flavus, S.a – Staphylococcus aureus, E.c – Escherichia coli, P.a – Pseudomonas aeruginosa, En.c – Enterobacter cloacae,
S.t – Salmonella typhimurium, L.m – Listeria monocytogenes.

516
A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
Among the substitutents, 2-methoxyphenyl and 2-thienyl works
positively against S. aureus.
more active against Gram-positive bacteria than Gram-negative
bacteria in microdilution method. Compound 9d exhibited the best
antibacterial activity against all the bacteria tested with very low
MIC and MBC, much lower than Ampicillin and almost 1.5 times
lower in most cases than Streptomycin.
2.3.2. LFER models
Continuous descriptors accounting for the molecular shape,
electronic properties, polarity, lipophilicity and other molecular
properties were selected by GA and included in the QSAR models
for antimicrobial activity of the investigated compounds based on
the linear free energy relationship (LFER). Only models with
r2 > 0.6 and q2 > 0.3 were considered as significant. These were
models describing the antimicrobial activities against B. cereus, M.
flavus, S. aureus, En. cloacae and L. monocytogenes (Tables 2 and 3).
Molecular shape descriptors, like ovality of molecule, surface,
volume, refractivity or parahor, take part in these models. Elec-
tronic properties, like the polarity indices Qv and Qs, dipole
moments, polarizabilities and specific polarizabilities are impor-
tant against B. cereus, M. flavus, S. aureus and En. cloacae. Lipophilic
compounds are more active against B. cereus, S. aureus and En.
cloacae. Compounds with more rings in their structure are more
active against S. aureus and less active against En. cloacae.
Compounds with less hydrogen-bond acceptors are more active
against M. flavus and S. aureus, while more hydrogen-bond accep-
tors work well against B. cereus. The nitro group in 4-nitrophenyl
brings two more hydrogen-bond acceptors. Hydrogen-bond donors
are important for the antimicrobial activity against En. cloacae.
Compounds 6a–e and 7a–e can form three hydrogen bonds, in
which they act as donors, while compounds 8a–e and 9a–e have an
amino group and can form two additional hydrogen bonds. Flexible
compounds with many free rotatable bonds have high activity
against S. aureus.
4. Experimental
4.1. Chemistry
All melting points were determined in an open capillary and are
uncorrected. Elemental analyses were performed on a Carlo Erba
1108 machine Quoted values are ꢃ0.4% of the theoretical ones.
IR spectra were recorded on Perkin–Elmer 237 spectropho-
tometer and the reported wave numbers are given in cmꢁ1. 1H NMR
spectra, were recorded in CDCl3 solution on a Bruker Avance DPX
200 MHz spectrometer Chemical shifts are reported as
relative to TMS as internal standard.
The progress of the reactions as well as purity of compounds
was monitored by thin layer chromatography with F254 silica-gel
precoated sheets (Merck, Darmstadt, Germany) using benzene/
ethanol 8/2 as eluent; UV light was used for detection.
d (ppm)
4.1.1. 2-Phenylamino-4,6-dichloro-s-triazine (3)
Aniline 2 (0.93 g,0.01 mole) was added slowly to cyanuric
chloride 1 (1.845 g,0.01 mole) in acetone (30 ml) with constant
stirring for 4 h at 0–5 ꢀC. Sodium carbonate solution (10%) was
added to neutralize HCl evolved during the reaction. Finally, the
contents were poured into crushed ice. The solid separated, was
filtered, washed with water, dried and recrystallized from ethanol
to give compound 3. M.p 196 ꢀC, yield 86%. IR: 690 (C–Cl), 774
2.3.2.1. Models for MIC. QSAR models with good explained and
predictive ability regarding MIC were obtained for B. cereus,
S. aureus, En. cloacae and L. monocytogenes (Table 2). Oval molecules
with small surface, high polarity and many hydrogen-bond accep-
tors are good inhibitors of B. cereus growth. Compounds with low
polarity, high lipophilicity, many rings and few hydrogen-bond
acceptors work well against S. aureus. Lipophilic molecules with big
surface, low polarity, few rings and many hydrogen-bond donors
are good inhibitors of En. cloacae. Bulky lipophilic molecules have
high inhibitory activity against L. monocytogenes.
(C–Cl), 1357 (C–N), 805 (C–N, s-triazine). 1H NMR (CDCl3) d ppm:
7.20–7.80 (m, 5 Ar–H and 1 NH).
4.1.2. 2,4-bis-(phenylamino)-6-chloro-s-triazine (4)
Aniline 2 (0.93 g, 0.01 mole) was added slowly to compound 3
(2.41 g, 0.01 mole) in acetone (35 ml) with constant stirring for 4 h
at room temperature. Sodium carbonate solution (10%) was added
to neutralize HCl evolved during the reaction. Finally, the contents
were poured into crushed ice. The solid separated, was filtered,
washed with water, dried and recrystallized from ethanol to give
compound 4. M.p 179 ꢀC, yield 80%. IR (KBr) cmꢁ1: 772 (C–Cl), 1359
2.3.2.2. Models for MBC. QSAR models with q2 > 0.3 regarding MBC
were obtained only for B. cereus, M. flavus and S. aureus. Oval,
lipophilic molecules with small surface and low polarity have
bactericidal activity against B. cereus. Similarly, oval molecules with
small surface, low polarity and few hydrogen-bond acceptors are
bactericidal against M. flavus. The bactericidal activity against
S. aureus has the same structural requirements as its inhibitory
activity. Compounds with wide surface, low polarity, many rings
and few hydrogen-bond acceptors have high MBC values.
Generally, the QSAR study on the compounds investigated here
shows that lipophilic molecules with small surface and low polarity
have good antimicrobial activities. However, the great variety of
descriptors involved in the models are indicative for the absence of
a uniform mechanism of antimicrobial action against the different
bacteria.
(C–N), 805 (C–N, s-triazine). 1H NMR (CDCl3)
(m, 10 Ar–H and 2 NH).
d ppm: 7.20–7.80
4.1.3. 2,4-bis-(phenylamino)-6-(40-acetylphenylamino)
s-triazine (5)
4-Aminoacetophenone (1.35 g, 0.01 mole) and compound 4
(2.975 g, 0.01 mole) were dissolved in acetone (40 ml). The reaction
mixture was refluxed for 6 h. Periodically sodium carbonate was
added to neutralize HCl evolved during the reaction. Finally, the
reaction mixture was cooled and poured into crushed ice. The solid
separated, was filtered, washed with water, dried and recrystallized
from alcohol to give compound 5. M.p 218 ꢀC, yield 75%. IR (KBr)
cmꢁ1: 1662 (C]O), 1355 (C–N), 805 (C–N, s-triazine). 1H NMR
(CDCl3) d ppm: 2.6 (s, 3H, –COCH3), 7.0–7.95 (m, 14 Ar–H and 3 NH).
4.1.4. 2,4-bis-(phenylamino)-6-[40-{300-(4000-methoxyphenyl)-200-
propenon-100-yl}-phenylamino]-s-triazine (6e)
3. Conclusion
Compound 5 (3.96 g, 0.01 mole) was dissolved in DMF (30 ml)
and 4-methoxybenzaldehyde (1.36 g, 0.01 mole) was added with
constant stirring at room temperature. Then KOH solution (40 wt%)
was added to reaction mixture which was stirred for 24 h at room
temperature. Finally the reaction mixture was poured into crushed
ice and neutralized with HCl. The product separated out, was
According to obtained results from disc-diffusion and TLC-bio-
autographic methods it can be concluded that compounds 8a–8e
showed better antibacterial activity against Gram-positive bacteria.
Compounds 9a–9e exhibited higher activity against Gram-negative
bacteria in both methods. All the compounds tested, 6a–9e are

A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
517
filtered, washed with water, dried and recrystallized from ethanol
to give compound 6e. Mp. 174 ꢀC, yield 68%. IR (KBr) cmꢁ1: 1647
(C]O), 1595 (–CH]CH–, str.), 1340 (C–N), 812 (C–N, s-triazine). 1H
on solid medium to verify the absence of contamination and to
check the validity of the inoculum.
NMR (CDCl3)
d
ppm: 3.95 (s, 3H, –OCH3), 6.90 (d, 1H, –CO–CH],
4.2.1. Disc-diffusion test
J ¼ 15 Hz), 7.15–7.80 (m, 18 Ar–H and 3NH), 8.05 (d, 1H, Ar–CH],
Compounds were investigated by the disc-diffusion using 4 mm
filter discs. Bacteria were cultured overnight at 28 ꢀC in LB medium
and then adjusted with sterile saline to a concentration of
1.0 ꢂ 105 cfu/ml. The suspension was added to the top of agar (6 ml)
and dissolved in Petri dishes (2 ml/agar plate) with solid peptone
agar (Institute of Immunology and Virology, Torlak, Belgrade,
J ¼ 15 Hz).
Similarly the remaining compounds 6a–d were prepared.
4.1.5. 2, 4-bis-(phenylamino)-6-[40-{500-(4%-methoxyphenyl)-100-acety-
lpyrazolin-3 00-yl}-phenylamino]-s-triazine (compound 7e)
A mixture of compound 6e (5.14 g, 0.01 mole) and hydrazine
hydrate (0.5 g, 0.01 mole) in dioxane (25 ml) in presence of glacial
acetic acid (15 ml) was refluxed for 6 h. The reaction mixture after
cooling was poured into crushed ice and the product separated out
was filtered, washed with water, dried and recrystallized from
Serbia). Filter discs with components (10.0 mg/disc) were placed on
agar plates. After 24 h of incubation at 28 ꢀC the diameter of the
growth inhibition zones was measured. Streptomycin and Ampli-
cillin were used as a positive control, and 10 ml was applied to the
discs from stock solution (1 mg/ml). All tests were done in dupli-
cate; replicates were done for each compound.
ethanol to give compound 7e. Mp. 180 ꢀC, yield 64%. IR (KBr) cmꢁ1
:
1650 (–COCH3), 1573 (C]N), 806 (C–N, s-triazine). 1H NMR (CDCl3)
d
ppm: 2.42 (s, 3H, –COCH3), 3.15 (dd, 1H, C4–HA, JAB ¼ 13.68 Hz,
4.2.2. Bioautographic assay test
JAx ¼ 4.60 Hz), 3.65 (dd, 1H, C4–HB, JAB ¼ 13.68 Hz, JBx ¼ 6.68 Hz),
3.80 (s, 3H, –OCH3), 5.60 (dd, 1H, –CH–CH2, JAx ¼ 4.60 Hz,
JBx ¼ 6.68 Hz), 6.90–7.80 (m, 18 Ar–H and 3NH).
Ten microliters (10 mg/spot) of each sample were applied on TLC
plates (Kieselgel 60 F254, Merck, Art. 5721) and sprayed with
freshly prepared bacterial (1.0 ꢂ 105 cfu/ml) in nutrient broth
(Tryptic Soy Broth; Biolife Italiana S.r.l., Milano-Italia). The plates
were incubated for 18 h at 37 ꢀC and then sprayed with aqueous sol.
3% of p-iodonitrotetrazolium violet [2-(4-iodophenyl)-3-(4-nitr-
phenyl)-5-phenyltetrazolium chloride; Sigma], stored for another
3 h and sprayed with 70% EtOH to stop bacterial and fungal growth.
Streptomycin and Amplicillin were used as a positive control, and
Similarly the remaining compounds 7a–d were prepared.
4.1.6. 2, 4-bis-(phenylamino)-6-[40-{200-amino-600-(4%-
methoxyphenyl)-pyrimidine-400-yl}-phenylamino]-s-triazine (8e)
A mixture of compound 6e (5.14 g, 0.01 mole) in alcohol (25 ml),
guanidine nitrate (1.22 g, 0.01 mole) and 1–2 ml of 40% KOH solu-
tion was refluxed for 10 h. The reaction mixture was then cooled,
was poured into crushed ice and the product separated out was
filtered, washed with water, dried and recrystallized from ethanol
to give compound 8e. Mp. 147 ꢀC, yield 66%. IR (KBr) cmꢁ1: 3398
10 ml was applied to TLC plates from stock solution (1 mg/ml).
White inhibition zones on a pinkish background were indicative of
antimicrobial activity of tested extracts. The widths of these zones
(mm) are the measure of efficiency [44] .The activity was evaluated
as the diameters of the inhibition zones with standard errors.
(–NH2), 1575 (C]N), 806 (C–N, s-triazine). 1H NMR (CDCl3)
d ppm:
3.85 (s, 3H, –OCH3), 5.10 (s, 2H, –NH2), 6.90–8.15 (m, 19 Ar–H and
3NH).
4.2.3. Microdilution test
Similarly the remaining compounds 8a–d were prepared.
The minimum inhibitory and bactericidal concentrations (MICs
and MBCs) were determined using 96-well microtitre plates. The
bacterial suspension was adjusted with sterile saline to a concen-
tration of 1.0 ꢂ 105 cfu/ml. Compounds to be investigated were
4.1.7. 2, 4-bis-(phenylamino)-6-[40-{200-amino-300-cyano-400-
(4%-methoxyphenyl)-pyridine-500-yl}-phenylamino]s-triazine (9e)
A mixture of 6e (5.14 g, 0.01 mole) in alcohol (40 ml), malono-
nitrile (0.66 g, 0.01 mole) and ammonium acetate (1.54 g,
0.02 mole) was refluxed for 8 h. After cooling the reaction mixture
was poured into crushed ice. The product separated out was
filtered, washed with water, dried and recrystallized from ethanol
to give compound 9e. Mp. 115 ꢀC, yield 69%. IR (KBr) cmꢁ1: 3396
dissolved in broth LB medium (100 ml) with bacterial inoculum
(1.0 ꢂ 104 cfu per well) to achieve the desired concentrations
(5.0–30.0 m
g/ml). The microplates were incubated for 24 h at 28 ꢀC.
The lowest concentrations without visible growth (at the binocular
microscope) were defined as concentrations that completely
inhibited bacterial growth (MICs). The MBCs were determined by
(–NH2), 2200 (C^N), 806 (C–N, s-triazine). 1H NMR (CDCl3)
d
ppm:
serial sub-cultivation of 2 ml into microtitre plates containing 100 ml
3.95 (s, 3H, –OCH3), 5.28 (s, 2H, –NH2), 6.90–8.20 (m, 18 Ar–H and
3NH).
of broth per well and further incubation for 72 h. The lowest
concentration with no visible growth was defined as the MBC,
indicating 99.5% killing of the original inoculum. The optical density
of each well was measured at a wavelength of 655 nm by Micro-
plate manager 4.0 (Bio-Rad Laboratories) and compared with
a blank and the positive control. Streptomycin and Amplicillin were
used as a positive control (1 mg/ml DMSO). Two replicates were
done for each compound.
Similarly the remaining compounds 9a–d were prepared.
4.2. Microbiology
The following Gram-negative bacteria were used: E. coli (ATCC
35210), P. aeruginosa (ATCC 27853), S. typhimurium (ATCC 13311),
En. cloacae (human isolate) and the following Gram-positive
bacteria: B. cereus (clinical isolate), M. flavus (ATCC 10240), and S.
aureus (ATCC 6538). The organisms were obtained from the
Mycological Laboratory, Department of Plant Physiology, Institute
4.3. QSAR study
4.3.1. Antimicrobial activity presentation
ˇ
´
for Biological Research ‘‘Sinisa Stankovic’’, Belgrade, Serbia.
The antibacterial assays were carried out by the disc-diffusion
[40] and microdilution method [41–43] in order to determine the
antibacterial activity of components against the human pathogenic
bacteria.
For the purpose of the QSAR study the antimicrobial activities
obtained by microdilution method (MIC and MBC) were presented
in p-values (negative log values). The range of both MIC and MBC
was very narrow (less than one log unit).
The bacterial suspensions were adjusted with sterile saline to
a concentration of 1.0 ꢂ 105 cfu/ml. The inocula were prepared daily
and stored at þ4 ꢀC until use. Dilutions of the inocula were cultured
4.3.2. Molecular descriptors
Two types of descriptors were used in the present study:
continuous and discontinuous. The continuous descriptors were

518
A. Solankee et al. / European Journal of Medicinal Chemistry 45 (2010) 510–518
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molecule nrings, number of graph circuits ncirc, Kappa flexibility
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donors numHBd, free rotatable bonds FRB, Lepinski’s rule of 5) were
calculated and used in LFER models to relate the molecular struc-
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ence of a certain substituent or main structure in the molecule.
These descriptors take 1 when the substituent is present and
0 when it is absent. The set of studied compounds consists of four
main structures (chalcone, acetylpyrazoline, aminopyrimidine and
cyanopyridine) and five substitutents (4-nitrophenyl, 4-chlor-
ophenyl, 2-furanyl, 2-thienyl and 2-methoxyphenyl). Thus, nine
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