Accepted Manuscript Spectroscopic and biological studies of new mononuclear metal complexes of a bidentate NN and NO hydrazone-oxime ligand derived from egonol Ilknur Babahan, Safiye Emirdağ-Öztürk, Esin Poyrazoğlu-Çoban PII: DOI: Reference:
S1386-1425(14)01854-X http://dx.doi.org/10.1016/j.saa.2014.12.074 SAA 13124
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
19 August 2014 13 December 2014 17 December 2014
Please cite this article as: I. Babahan, S. Emirdağ-Öztürk, E. Poyrazoğlu-Çoban, Spectroscopic and biological studies of new mononuclear metal complexes of a bidentate NN and NO hydrazone-oxime ligand derived from egonol, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa. 2014.12.074
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1
4
Spectroscopic and biological studies of new mononuclear metal complexes of a bidentate NN and NO hydrazone-oxime ligand derived from egonol
5
Ilknur Babahan1,*, Safiye Emirdağ-Öztürk2, Esin Poyrazoğlu-Çoban3
1 2 3
6 7 8 9 10
1
11
Abstract
12
A novel ligand vicinal dioxime ligand (egonol hydrazone glyoxime (LH2) was synthesized
13
and characterized using 1H-NMR, 13C-NMR, MS, AAS, infrared spectroscopy, and magnetic
14
susceptibility measurements. Mononuclear nickel (II), copper (II) and cobalt (II) complexes
15
with a metal:ligand ratio of 1:2 for LH2 were also synthesized. Zn(II) forms complex
16
[Zn(LH)Cl2] with a metal to ligand ratio of 1:1. IR spectrum shows that the ligand act in a
17
bidentate manner and coordinates N4 donor groups of the ligands to NiII, CuII, CoII and ZnII
18
ions. The detection of H-bonding (O–H⋅⋅⋅O) in the [M(LH)2] metal complexes by IR spectra
19
supported the square-planar MN4 coordination of Ni(II), Cu(II) and Co(II) complexes.
20
The antimicrobial activities of compounds LH2 and their Ni(II), Cu(II), Co(II) and Zn(II)
21
complexes were evaluated using the disc diffusion method against 16 bacteria and 5 yeasts.
22
The minimal inhibitory concentrations (MICs) against all the bacteria and yeasts were also
23
determined. Among the test compounds attempted, it is showed that all the compounds (L,
24
LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2], [Zn(LH)Cl2]) were effect against used test
25
micrroorganisms.
Adnan Menderes University, Faculty of Science and Art, Department of Chemistry, 09010, Aydin, Turkey Ege University, Faculty of Sciences, Department of Chemistry, 35100, Bornova, İzmir, Turkey 3 Adnan Menderes University, Faculty of Science and Art, Department of Biology, 09010, Aydin, Turkey 2
__________________________________________________________________________________________
26 27
Key words: Styrax officinalis; vic-Dioxime; Hydrazone; Egonol; Transition metal complex;
28
Spectroscopy; Antimicrobial activity
29 30 31 32 33
___________________________________________________________________________ ___________ * Corresponding author: Tel.: 90-256-212-8498; fax: 90-256-213-5379 E-mail address:[email protected] (I. Babahan)
2 34
1. Introduction
35
Egonol, a natural 2-aryl benzofuran, is known to be an effective pyrethrum synergist
36
[1]. Egonol and its derivatives attracted the attention of synthetic chemists due to their
37
antibacterial and antifungal [2], anti-complement [3] activities besides their considerable
38
cytotoxic activities against human leukaemic HL-60 cells [4]. It was also reported that
39
significant activities were observed for egonol against C6 (rat glioma) and Hep-2 (larynx
40
epidermoid carcinoma) cell lines [5-6].
41
Hydrazones possessing an azomethine -NHN=CH- proton constitute an important class
42
of compounds for new drug development. Therefore, many researchers have synthesized these
43
compounds as well as their metal complexes as target structures and evaluated their biological
44
activities. These observations have been guiding for the development of new hydrazones that
45
possess varied biological activities [7]. Hydrazones have been demonstrated to possess,
46
among other, antimicrobial, anticonvulsant, analgesic, anti inflammatory, antiplatelet,
47
antitubercular and antitumoral activities [7].
48
Nowadays, vic-dioximes are appreciated as coordination compounds in lots of usage areas
49
such as analytical, biologically, pigment and medicinal chemistry. Many researchers have
50
studied vic-dioximes which have important role of the complexes especially 1,2-dioximes in
51
coordination chemistry [8-10]. Their complexes have been the source, through the decades, of
52
a never-ending series of interesting reports. Owing to their importance as stable MN4 core-
53
containing coordination compounds, vic-dioxime complexes have been much investigated
54
[11]. The exceptional stability and unique electronic properties of these complexes can be
55
attributed to their planar structure, stabilized by hydrogen bonding [12–15].
56 57 58
3 59
2. Experimental
60
2.1. Materials and measurements
61
All reagents used were purchased from Merck. 1H N.M.R-13C N.M.R spectra (Bruker
62
400 MHz), I.R spectra (Varian 900), melting points (Buchi SPM-20) and pH measurements
63
(Orion Expandable Ion Analyzer EA 940) were used to elucidate the structures of the
64
products. The magnetic moments of the complexes were measured by the Gouy method with
65
a Newport type D-104 instrument magnet power supply. Mass spectrometry (MS) spectra
66
were recorded on a Bruker LC/MS/MS-8030 Triple Quadrupole Mass Spectrometer.
67
The
starting
materials,
Egonol
(5-[300-(hydroxy)propyl]-7-methoxy-2-(30,40-
68
methylene dioxyphenyl)benzofuran; Scheme 1, 1a) was isolated from the seeds of S.
69
Officinalis and its isolation procedure and egonol aldehyde (3-[2-(1,3-Benzodioxol-5-yl)-7-
70
methoxy-1-benzofuran-5-yl]propanal; Scheme 1, 1b) were explained in our previous work
71
[6a-b].
72 73
2.2 Synthesis
74
2.2.1 Synthesis of (L)
75
A solution of egonol aldehyde [6a-b] (Scheme 1, 1b) (1 mmol) in absolute ethanol 30 mL was
76
added dropwise to a solution of hydrazine hydrate (1 mmol) with 1 mL AcOH in absolute
77
ethanol 10 mL for a 30 min period. The reaction mixture was stirred overnight at room
78
temparature. After the end of the period, yellow precipitated solid was filtered, washed
79
thoroughly with distilled water and dried. The chemical reaction and molecular structure are
80
shown in Scheme 1(1c).
81
2.2.1 Synthesis of (LH2)
82
A solution of egonol hydrazone (1c) (1 mmol) in absolute ethanol 30 mL was added
83
dropwise to a solution of anti-chloroglyoxime (1 mmol) in absolute ethanol 10 mL for a 30
84
min period. The reaction mixture was stirred overnight at room temparature. After cooling to
4 85
0 0C the pH of the mixture was raised to 5.0-5.5 with treatment with NaHCO3 dissolved in 5
86
mL distilled water, and stirring was continued for one hour. The solution was poured into 100
87
mL cold water with stirrring. After the end of the period, yellow precipitated solid was
88
filtered, washed thoroughly with distilled water and dried. The chemical reaction and
89
molecular structure are shown in Scheme 1(1d).
90
2.2.2 Synthesis of the Ni(II), Cu(II) and Co(II) Complexes of LH2
91
A solution of a metal salt (1 mmol of NiCl2.6H2O, CoCl2.6H2O and CuCl2.2H2O) in 20
92
mL of water were added to 2 mmol of the ligand solution (0.848 g LH2 in 30 mL of ethanol)
93
with stirring. An initial sharp decrease in the pH of the solution from 5.5 to about 3-3.5 is
94
observed. After raising the pH to 5.0-5.5 using 1% aqueous NaOH solution, the reaction
95
mixture was kept in a hot water bath (60 ◦C) for 2 h to complete the precipitation. Then the
96
precipitated complex compounds were filtered, washed with water and ethanol, and dried at
97
room temperature in a vacuum oven. The structure of the prepared complexes are shown in
98
Scheme 1.
99
2.2.3 Synthesis of the Zn(II) Complex of LH2
100
A solution of ZnCl2.2H2O (0.172 g, 1 mmol) in ethanol (20 mL) was added to a
101
solution of ligand (0.424 g (1 mmol) for LH2, dissolved in ethanol (20 mL) by stirring in a
102
water bath at 50 ◦C for 2 h in order to complete precipitation. The apparent pH of the solution
103
was adjusted to 5.0-5.5 by the addition of 1 M NaOH solution. The precipitate was filtered,
104
washed with water, and dried in vacuo at 60 ◦C. The complexes are very slightly soluble in
105
common organic solvents. The structures of the complexes are shown in Scheme 1.
106
2.3 Antimicrobial assays
107
Sixteen bacterial strains and four yeast strain were obtained from the American Type
108
Culture Collection (ATCC, Rockville, MD, USA). A strain (Candida trophicalis) was
109
obtained from Faculty of Medicine, Adnan Menderes University. The Gram-negative (G-)
5 110
were: Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Proteus
111
vulgaris ATCC 33420, Serratia marcescens ATCC 13880, Klebsiella pneumoniae ATCC
112
13882, Pseudomonas aeruginosa ATCC 35032, Enterobacter aerogenes ATCC 13048 and
113
the Gram-positive (G+) were: Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC
114
25923, Staphylococcus epidermidis ATCC 12228, Corynebacterium xerosis ATCC 373,
115
Bacilllus cereus ATCC 11778, Bacilllus subtilis ATCC 6633, Enterococcus faecalis ATCC
116
29212, and Listeria monocytogenes ATCC 19112 and acid-fast bacteria species was:
117
Mycobacterium smegmatis ATCC 607. The following five yeast strains, i.e. Candida utilis
118
ATCC 9950, C. albicans ATCC 10231, C. tropicalis, Kluyveromyces fragilis ATCC 8608 and
119
Saccharomyces cerevisiae ATCC 9763, were also tested using disc diffusion method [16,17]
120
and the minimum inhibitory concentration (MIC) was determined by broth dilution method
121
[18].
122
2.3.1 Disc diffusion method
123
Screening for antibacterial and antifungal activities were carried out using sterile
124
antibiotic discs (6 mm), following the standard procedure of Antimicrobial Disc Susceptibility
125
Tests outlined by the National Committee for Clinical Laboratory Standards- NCCLS [16,17].
126
Fresh stock solutions (1x10-4 M) of the compounds were prepared in DMSO according to the
127
needed concentrations for the experiments. The inoculum suspensions of the tested bacteria
128
and yeasts were prepared from the broth cultures (18–24 h) and the turbidity equivalent
129
adjusted to 0.5 McFarland standard tube to give a concentration of 1x10-8 bacterial cells and
130
1x10-6 yeast cells/mL. To test the antimicrobial activity of each hydrazone-oxime derivative
131
bearing egonol or its metal complexes, a Mueller Hinton agar plate was inoculated with 0.1
132
mL broth culture of bacteria or yeast. Then a hole of 6 mm in diameter and depth was made
133
on top with a sterile stick and filled with 50 µL of the hydrazone-oxime derivative bearing
134
egonol or its metal complexes.
6 135
Plates inoculated with E. coli ATCC 25922, S. typhimurium ATCC 14028, S. aureus
136
ATCC 25923, S. epidermidis ATCC 12228, E. faecalis ATCC 29212, L. monocytogenes
137
ATCC 19112, P. vulgaris ATCC 33420, Corynebacterium xerosis ATCC 373, Klebsiella
138
pneumoniae ATCC 13882, Mycobacterium smegmatis ATCC 607, Pseudomonas aeruginosa
139
ATCC 35032, Enterobacter aerogenes ATCC 13048 and S. marcescens ATCC 13880 were
140
incubated at 37 0C for 24 h and those inoculated with M. luteus ATCC 9341, B. cereus ATCC
141
11778, B.subtilis ATCC 6633, S. cerevisiae ATCC 9763, C. albicans ATCC 10231, C. utilis
142
ATCC 9950, C. tropicalis and Kluyveromyces fragilis ATCC 8608 were incubated at 30 0C
143
for 24 h. The diameter of the inhibition zone was then measured. Discs of Chloramphenicol
144
(C30, Oxoid), Gentamycin (GN10 Oxoid), Tetracycline (TE30), Erytromycine (E15),
145
Ampicillin (AMP10) and Nystatine (NS100) were used as positive controls. The inhibition
146
zones were compared with those of the reference discs.
147
2.3.2 Dilution method
148
Screening for antibacterial and antifungal activities were carried out by preparing a
149
microdilution broth, following the procedure outlined in Manual of Clinical Microbiology
150
[18]. All the bacteria were inoculated in the nutrient broth and incubated at 30–37 0C for 24 h
151
while the yeasts were inoculated in malt extract broth and incubated at 30 0C for 48 h. The
152
compounds were dissolved in DMSO (2 mg mL-1) and then diluted in Mueller Hinton broth.
153
Twofold serial dilution of the compounds were employed to determine the MIC ranging from
154
256 to 0.125 µgmL-1. Cultures were grown at 30–370C (18–20 h) and the final inoculum was
155
approximately 106 cfu mL-1. Test cultures were incubated at 37 0C (24 h). The lowest
156
concentration of antimicrobial agent that resulted in complete inhibition of the
157
microorganisms was represented as MIC (µgmL-1). As positive controls, streptomycin (I.E.
158
Ulagay) for bacteria and nystatin (NS100, Oxoid) for yeast were used in the dilution method.
159
7 160
3. Results and discussion
161
Egonol (Scheme1; 1a) isolated from the seeds of S. Officinalis L. was used as the main
162
starting material (Figures 1a-b). Egonol aldehyde (1b) was obtained from an oxidation reaction
163
of egonol using PCC in acetone–CH2Cl2 (Scheme 1).
164
Egonol hidrazon (L; 1c) were obtained from the condensation of egonol aldehyde with
165
hydrazine hyrate in the presence of alcohol. Subsequently, (1Z,2E)-N'-{(1E)-2-[2-(1,3-
166
benzodioxol-5-yl)-1-benzofuran-5-yl]ethylidene}-2(hydroxyimino)ethanehydroximo
167
hydrazide (1d, LH2) was prepared from 1c with anti-chloroglyoxime in presence of alcohol
168
(Figure 2).
169
The mononuclear Ni(II), Cu(II), Co(II) and Zn(II) complexes were prepared from the
170
egonol hydrazone glyoxime (1d) and a stoichiometric amount of NiCl2.6H2O, CuCl2.2H2O,
171
CoCl2.6H2O and ZnCl2.2H2O in ethanol (Scheme 1). The metal complexes were characterized
172
using, mass spectrometry, FT-IR, and magnetic susceptibility measurements. For all
173
compounds, melting points were given Table 1.
174
given in Table 2. 1H-NMR and 13C-NMR spectra of egonol and egonol aldehyde are given in
175
Table 3. 1H-NMR and 13C-NMR spectra of L, LH2 and metal complexes could not be taken
176
because of their very low solubility in organic solvents. Antimicrobial activities of
177
compounds were given in Tables 4 and 5.
FT-IR data of LH2 and its complexes are
178 179
3.1IR spectra, mass and magnetic susceptibility
180
The IR spectra of ligands exhibited –NH (3448 cm-1 for L and 3419 cm-1 for LH2), -OH
181
(3224 cm-1 for LH2), -C=Noxime (1630 cm-1 for LH2), and –NO (948 cm-1 for LH2) stretching
182
vibrations [19]. The C=N and OH stretching vibrations appear similar to other vic-dioxime
183
derivatives [20-21].
8 184
The FT-IR spectra of KBr pellets containing the egonol hydrazone glyoxime complexes of
185
the general formula M(LH)2, in which M is Ni(II), Cu(II), or Co(II).2H2O, exhibited C=Noxime
186
absorption bands at 1565-1515 cm-1. These bands suggest that the ligands are N,N΄
187 188 189
Figure 1a Flower of Styrax officinalis
Figure 1b Fruits of Styrax officinalis
(http://en.wikipedia.org/wiki/Styrax_officinalis) ( http://en.wikipedia.org/wiki/Styrax_officinalis)
190 191
coordinated with the metal ion in correspondence with the proposed structures[22-26].
192
Intramolecular hydrogen bonding was indicated by a peak appearing between 1845-1775 cm-1
193
[25, 26].
194
Egonol’s positive ion-mode LSMS/APCI showed molecular ion peaks at m/z: 327.12
195
[M]+ consistent with a molecular formula of C19H18O5. FAB mass spectral analysis indicated
196
m/z ratios of 324 [M]+ for egonol aldehyde (1b), 338 [M]+ for egonol hydrazone (L), 424
197
[M]+ for LH2 (Scheme 2), 904 [M-1]+ for complex [Ni(LH)2], 910 [M]+ for complex
198
[Cu(LH)2], 942 [M+1]+, 905 [M-2H2O]+ for complex [Co(LH)2(H2O)2] and 559 [M+1]+ for
199
complex [Zn(LH)Cl2]. The MS-determined metal:ligand ratio was 1:2 for Ni(II), Cu(II) and
200
Co(II) complexes, while metal:ligand ratio was 1:1 for Zn(II) complex.
201 202 203 204
9 205
Table 1. Melting point, yields, and values of UV-Vis. of egonol and its derivatives. Compounds
Melting point (0C)
Yield (%)
UV-Vis. (nm)
Egonol
119.2
-
317, 223
Egonol aldehyde L
112.1 145.5
26.02 74.09
317, 218 317, 214
LH2 [Ni(LH)2]
>300 >300
68.87 45.67
302, 272, 214 304, 268, 216
[Cu(LH)2] [Co(LH)2(H2O)2]
>300 >300
56.23 47.04
304, 278, 215 330, 276, 223
[Zn(LH)Cl2]
>300
60.10
302, 263, 225
206 207 208 209 OH O
O MeO
O
Egonol 1a
H
OH
NH2 H
H
N
N
+
O 1) NH2NH2.H2O/ EtOH
O
Cl
O
N
OMe O
O
reflux
O
HO 1c
OMe
O
OH
H
1b
NH H NH
N
O
O
H
O
N
O
Cl EtOH, NaOH
1d
O MCl2.XH2O EtOH, NaOH H O
O
NH
N
N
H
N
N
O
O
H N
O O
O OMe
H
M
H NH
O M: Ni(II), Cu(II), Co(II)
210 211 212
N
H OMe
N OH
OMe
OH
OMe
Cl ZnCl2 2H2O
Zn H
O
N
O
N
N
O O
Scheme 1. Synthesis of the ligands (L and LH2) and metal complexes [Ni(II), Cu(II), Co(II) and Zn(II)]
10 213
Based on IR spectral and magnetic susceptibility data, the complexes have a metal:ligand
214
ratio of 1:2 and square planar or octahedral structures for Ni(II), Cu(II) and Co(II) complexes
215
and 1:1 and tetraheral structure for Zn(II) complex (Scheme 1). The magnetic susceptibility
216
measurements of the Ni(II) and Zn(II) complexes indicate that these complexes are
217
diamagnetic. Room temperature magnetic moment measurements indicate that the Co(II)
218
complex ([Co(LH)2(H2O)2]) is paramagnetic with a magnetic susceptibility of 3.73 B.M for
219
LH2. The magnetic susceptibilities are within the range of high spin octahedral cobalt(II)
220
complex (the three-spin value is 3.87 B.M.) [23]. For [Co(LH)2(H2O)2], coordinated H2O
221
molecules were identified by a broad OH absorption band near 3220 cm−1 whose intensity
222
remained constant following heating to 110 ◦C for 24 h.
223
The Cu(II) complex was also paramagnetic with µ eff = 1.74 for LH2, a close fit for the spin
224
value of 1.73 B.M. Although the magnetic moment is somewhat low, there are likely some
225
diamagnetic contributions from the ligand that ultimately decrease the total paramagnetism of
226
the complexes [27].
227
Ligands form mononuclear complexes [(LH)2M] with a metal to ligand ratio of 1:2 with
228
M=Co(II)(H2O)2, Ni(II), and Cu(II). Zn(II) forms complexes [Zn(LH)Cl2] with a metal to
229
ligand ratio of 1:1 [28-29]. The Co(II) complex of the ligand is proposed to be octahedral with
230
water molecules as axial ligands, while the Ni(II) and Cu(II) complexes are proposed to be
231
square planar, and the complex of Zn(II) is tetrahedral. Two chloride ions are also coordinated
232
to the Zn(II) ion. The Zn(II) complex is tetrahedral with a 1:1 metal to ligand ratio, according
233
to the MS, and the ligand is coordinated only by the N, O atoms of the vic-dioxime [28-29].
234
The tetrahedral structure of the Zn(II) complex is based on the disappearance of the
235
band (O-H...O) of the complexes and appearance of the O-H bands of these compounds in the
236
FT-IR spectra[29].
237
11 238 239
3.2 NMR spectra The 1H and
13
C NMR spectra of egonol exhibited peaks at 3.73 ( t, 2H, J= 6.4, 6.4)
240
(H3") and 62.70 ppm (C3"). The corresponding signals for egonol aldehyde were at 9.85 (t,
241
1H, J= 0.8, 1.6) (H3"). The 1H NMR spectrum of egonol aldehyde indicated two methylenes
242
(δH 3.05, 2.85 instead of at δH 1.97, 2.85 in egonol), six aromatic methynes (δH 6.77, 6.96,
243
6.82, 7.32, 6.88, 7.41), one methylenedioxy (δH 6.00), one methoxy (δH 4.04) and one
244
aldehyde signal (δH 9.85). The main difference in the 1H NMR spectrum of egonol aldehyde
245
with that of egonol is the presence of the aldehyde proton signal at δH 9.85 (1H, t) and the
246
absence of a signal at δH 3.73 (2H, t) in egonol (Table 3). Therefore, the structure of egonol
247
aldehyde
248
yl]propanal [6a-b].
was
established
as
3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-
249 250 251
252 253 254
Table 2. Characteristic IR bands of the ligands and their metal complexes (cm-1, KBr) Compound
NH
1 2 3 4 5 6
3448 b 3419 b 3433 b 3414 b 3424 b 3415 b
-OH/ H2O 3224b 3220 b 3256 b
Aromatic
O-H..O
C=Na
C=Nb
N-O
3020 w 3028w 2982 w 2999 w 3021 w 2960 w
1845 w 1790 w 1775 w -
1653 s 1648 s 1648 s 1648 s 1648 s 1690 s
1630 s 1565 s 1572 s 1515 s 1551 s
948 m 984 m 960 m 960 m 948 m
_________________________________________________________________________________________________________ b: board s: strong, m: medium, w: weak, ν(C=N)a: hydrazone moiety, ν(C=N)b: oxime moiety.
(1) L, (2) LH2, (3)[Ni(LH)2], (4) [Cu(LH)2], (5) [Co(LH)2(H2O)2], (6) [Zn(LH)Cl2
12 +
H
O
O
N
N
H
OCH3 O
OH
NH
N
OH
m/z= 424 (-) OH H
O
N
O
N
H
OCH3 m/z= 411
O
H
(-) HCN
N +
H
N O
N O
+
(-)
N O
OH
NH
N
O
NH2
OH
NH
N +
OCH3 O
O
m/z= 380
H3CO O m/z= 338
HO-N=C(-) H
(-) O H NH N O
+
N NCH(-)
O
NH
N
+
O
O
OCH3 OCH3
O
O m/z= 364 m/z= 295 H
H N
H2N
NH
+
HN
+
NH2
+
O
O OCH 3
+
N O
255 256
m/z= 280
O
257 258 259 260 261 262 263
O OCH3
O
Scheme 2 The propesed fragmentation pattern of LH2
m/z= 295
NH
+
13 264 265 266
Table 3. 1H NMR of Egonol and egonol-aldehyde (CDCl3, 400 MHz) and 13 C NMR of compound of egonol (CDCl3, 100MHz) 267
Egonol
Egonol-aldehyde δ ppm
No
268 Egonol δ ppm
H3
6.81,s ,1H
6.77,s,1H
C2
156.51,q270
H4
6.99, s, 1H
6.96,s,1H
C3
100.76,d
H6
6.66, s, 1H
6.62,d,1H, J=1.6
C3a
H2'
7.34, d, 1H J=1.5
7.32,d,1H, J=4.0
C4
112.72,q
H5'
6.90,d,1H J=8.1
6.88,d,1H, J=8.0
C5
137.92,q
H6'
7.43,dd,1H J=1.6, 8.1
7.41,dd,H J=1.6, 8.0
C6
107.86,d
OCH2O
6.02, s, 2H,
6.00,s,2H
C7
145.20.q
OMe
4.05,s, 3H
4.04,s,3H
C7a
142.88,q
H1"
2.81, t, 2H J=7.5, 7.8
2.85,t,2H, J=1.2, 6.8
C1'
125.12,q277
H2"
1.97, p, 2H
3.05,t,2H, J=8.0, 7.2 9.85,t,1H, J=0.8, 1.6
C2'
105.95,d278
C3'
148.46,q279
C4'
148.39,q280
C5'
109.02,d
C6'
119.62,d
H3"
3.73, t,2H, J=6.4,6.4
269 271
131.46,q
272 273 274 275 276
281 282
283 284
4
3 '' 5
3
HO
285
A
286 287
O
7
O
2'
B
6
2
290 291 292
6'
4' 5'
Scheme 3 Suggested conformation of egonol
O
C
1
CH3
288 289
3'
1'
O
14 293
3.3. Antimicrobial Assays
294
A novel vic-dioxime-hydrazone derivative containing egonol group and its Ni(II),
295
Cu(II), Co(II) and Zn(II) complexes detemined remarkable antimicrobial activity (Tables 4
296
and 5).
297
Among the test compounds assayed, compounds L, LH2, [Ni(LH)2], [Cu(LH)2],
298
[Co(LH)2(H2O)2] and [Zn(LH)Cl2] showed activity against some bacteria and yeasts (Table
299
4). According to Table 4, compounds L, LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2] and
300
[Zn(LH)Cl2] demostrated stronger activity against using the some bacteria and yeasts C.
301
xerosis ATCC 373, M. smegmatis ATCC 607, Micrococcus luteus ATCC 9341,
302
Stapylococcus epidermidis ATCC 12228, Entereococcus faecalis ATCC 29212, Enterobacter
303
aerogenes ATCC 13048, Serratia marcescens ATCC 13880, B. subtilis ATCC 6633, C.
304
albicans ATCC 10231, C. utilis ATCC 9950.
305
The MIC values in Table 5 presented that some of the compounds tested demonstrated
306
the strongest activity against C. xerosis ATCC 373, M. smegmatis ATCC 607, M. luteus
307
ATCC 9341, S. epidermidis ATCC 12228, S. marcescens ATCC 13880, E. aerogenes ATCC
308
13048, B. subtilis ATCC 6633, C. trophicalis. For example, C. xerosis ATCC 373 (compound
309
[Zn(LH)Cl2]= 16 µgmL-1, LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2] and L= 32 µgmL-1),
310
M. smegmatis ATCC 607 (LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2], [Zn(LH)Cl2] and
311
L= 32 µgmL-1), M. luteus ATCC 9341 (LH2= 32 µgmL-1), S. epidermidis ATCC 12228 (L,
312
LH2,
313
([Ni(LH)2]= 32 µgmL-1), E. aerogenes ATCC 13048 ([Ni(LH)2] and [Zn(LH)Cl2] = 32
314
µgmL-1), B. subtilis ATCC 6633 (compound [Zn(LH)Cl2]= 32 µgmL-1), C. trophicalis
315
([Cu(LH)2] = 32 µgmL-1).
[Cu(LH)2],
[Co(LH)2(H2O)2] and [Zn(LH)Cl2]), S. marcescens ATCC 13880
15 316
In addition, tested compounds have indicated moderate effect against some bacteria and
317
yeasts (Table 5). For example, E. coli ATCC 25922 (L, [Cu(LH)2], [Co(LH)2(H2O)2] and
318
[Zn(LH)Cl2]), S. typhimirium ATCC 14028 (L, LH2, [Cu(LH)2] and [Co(LH)2(H2O)2]= 64
319
µgmL-1), K. pneumoniae ATCC 13882 (L, [Ni(LH)2]= 64 µgmL-1), P. vulgaris ATCC 33420
320
(L, LH2, [Co(LH)2(H2O)2] and [Zn(LH)Cl2]= 64 µgmL-1), E. faecalis ATCC 29212
321
([Cu(LH)2], [Co(LH)2(H2O)2] and [Zn(LH)Cl2]= 64 µgmL-1), B. subtilis ATCC 6633 (LH2,
322
[Ni(LH)2], [Cu(LH)2] and [Co(LH)2(H2O)2]= 64 µgmL-1), E. aerogenes ATCC (L,
323
[Cu(LH)2]= 64 µgmL-1), P. aeroginosa ATCC 35032 (L, [Zn(LH)Cl2]= 64 µgmL-1), S.
324
marcescens ATCC 13880 (L=64 µgmL-1), L. monocytogenes ATCC 19112(L=64 µgmL-1), B.
325
cereus ATCC 11778 (L=64 µgmL-1), C. albicans ATCC 10231 ([Ni(LH)2] and [Zn(LH)Cl2]=
326
64 µgmL-1), C. utilis ATCC 9950 ([Ni(LH)2] and [Zn(LH)Cl2]=64 µgmL-1), C. trophicalis
327
(L=64 µgmL-1), K. fragilis ATCC 8608 (LH2, [Ni(LH)2] and [Co(LH)2(H2O)2]= 64 µgmL-1),
328
S. cerevisiae ATCC 9763 (L, [Cu(LH)2]=64 µgmL-1).
329
Suggestions are made that the negative inductive effect plays a significant role.
330
Dimerisation of oxime involves the formation of a pair of H bonds [30]. This feature causes a
331
decrease in electronic density of oximes compared with phenylhydrazones, thereby
332
facilitating entry of the oxime into the cell. This is likely to increase the antibacterial potency
333
[30].
334 335 336 337 338 339 340 341 342
16 343
Table 4. Antimicrobial activities of compounds (Inhibition zone mm)
344 Compounds
Reference antibiotics
Test Microorganisms 1
2
3
4
5
6
C30
CN10
TE30
E15
AMP10
NS100
Escherichia coli ATCC 25922 Salmonella typhimirium ATCC 14028 Micrococcus luteus ATCC 9341 Stapylococcus aureus ATCC 25923 Stapylococcus epidermidis ATCC 12228 Klebsiella pneumoniae ATCC 13882 Pseudomonas aeruginosa ATCC 35032 Proteus vulgaris ATCC 33420 Entereococcus faecalis ATCC 29212 Corynebacterium xerosis ATCC 373 Mycobacterium smegmatis ATCC 607 Serratia marcescens ATCC 13880 Listeria monocytogenes ATCC 19112 Enterobacter aerogenes ATCC 13048 Bacilllus cereus ATCC 11778 Bacillus subtilis ATCC 6633 Candida albicans ATCC 10231 Candida utilis ATCC 9950
10
10
12
13
12
12
24
21
15
11
-
NT
12
10
12
12
11
13
17
16
15
8
8
NT
14
11
10
12
11
20
25
15
26
30
28
NT
8
-
8
9
9
11
23
20
22
23
20
NT
14
11
14
14
15
15
22
17
19
11
17
NT
-
13
10
10
9
13
21
19
20
14
-
NT
10
10
10
10
12
14
22
20
20
21
-
NT
13
11
11
11
12
13
17
24
16
20
-
NT
11
11
13
13
13
17
16
11
19
-
14
NT
16
16
16
16
17
20
20
17
25
26
27
NT
15
15
15
14
15
20
23
18
26
25
19
NT
11
15
11
11
11
13
23
19
13
-
19
NT
11
10
10
11
10
13
19
14
12
-
12
NT
11
15
12
10
14
13
19
20
14
-
-
NT
10
11
11
10
10
13
23
24
25
26
-
NT
12
13
13
12
14
18
22
20
12
25
-
NT
10
12
11
10
12
16
NT
NT
NT
NT
NT
22
11
13
9
9
13
15
NT
NT
NT
NT
NT
21
Candida trophicalis*
11
10
14
9
10
12
NT
NT
NT
NT
NT
12
12
10
12
10
14
NT
NT
NT
NT
NT
10
11
12
10
10
12
NT
NT
NT
NT
NT
Kluyveromyces fragilis ATCC 8608 Saccharomyces cerevisiae ATCC 9763
345 346 347 348 349 350 351 352
15 19 15
Note: 1 = LH2 2 = [Ni(LH)2], 3 = [Cu(LH)2], 4 = [Co(LH)2(H2O)2] 5 = [Zn(LH)Cl2] 6=L (–) = No zone, NT = Not tested. C30: Chloramphenicol (30 mg Oxoid); CN10: Gentamycin (10 mg Oxoid); TE30: Tetracycline (30 mg Oxoid); E15: Erytromycin (15mg Oxoid); AMP10: Ampicillin (10 mg Oxoid); NS: Nystatin (100 mg Oxoid); (-):Zone did not occur. NT: Not tested, *From Faculty of Medicine, Adnan Menderes University.
17 353 354
Table 5. Antimicrobial activities of compounds (MIC, µg.mL-1) Test Microorganisms
355 356 357 358 359 360 361 362
1
2
3
4
5
6
Str
NS 100
Escherichia coli ATCC 25922
128
128
64
64
64
64
64
-
Salmonella typhimirium ATCC 14028 Micrococcus luteus ATCC 9341 Stapylococcus aureus ATCC 25923 Stapylococcus epidermidis ATCC 12228 Klebsiella pneumoniae ATCC 13882 Pseudomonas aeruginosa ATCC 35032 Proteus vulgaris ATCC 33420 Entereococcus faecalis ATCC 29212 Corynebacterium xerosis ATCC 373
64
128
64
64
128
64
64
-
32
128
128
64
128
16
32
-
256
-
256
256
256
128
32
-
32
128
32
32
32
32
32
-
-
64
128
128
256
64
64
-
128
128
128
128
64
64
64
-
64
128
128
64
64
64
64
-
128
128
64
64
64
32
64
-
32
32
32
32
16
32
64
-
Mycobacterium smegmatis ATCC 607 Serratia marcescens ATCC 13880
32
32
32
32
32
32
128
-
128
32
128
128
128
64
64
-
Listeria monocytogenes ATCC 19112 Enterobacter aerogenes ATCC 13048 Bacilllus cereus ATCC 11778 Bacillus subtilis ATCC 6633 Candida albicans ATCC 10231 Candida utilis ATCC 9950 Candida trophicalis*
128
128
128
128
128
64
32
-
128
32
64
128
32
64
32
-
128
128
128
128
128
64
64
-
64
64
64
64
32
16
64
-
128
64
128
128
64
32
-
64
128
64
256
256
64
32
-
64
128
128
32
256
128
64
-
64
Kluyveromyces fragilis ATCC 8608
64
64
128
64
128
32
-
128
Saccharomyces cerevisiae 128 128 64 128 128 64 128 ATCC 9763 Note: 1 = LH2, 2 = [Ni(LH)2], 3 = [Cu(LH)2], 4 = [Co(LH)2(H2O)2], 5 = [Zn(LH)Cl2], 6=L Str= streptomycin, NS100= nystatin (–) = No effect * From Faculty of Medicine, Adnan Menderes University.
18 363
Conclusions
364
Novel vic-dioxime ligand (LH2) containing egonol hydrazone moiety and its mononuclear
365
Ni(II), Cu(II), Co(II) and Zn(II) complexes were synthesized and characterized. Reaction of
366
LH2 with metal(II) salts yielded mononuclear complexes corresponding to the general
367
formulas ([M(LH)2], M: Ni(II), Cu(II), Zn(II), or Co(II).2H2O).
368
The
antimicrobial activities
of
compounds
L,
LH2,
[Ni(LH)2],
[Cu(LH)2],
369
[Co(LH)2(H2O)2] and [Zn(LH)Cl2] were evaluated using disc diffusion method against 16
370
bacteria and 5 yeasts. Minimal inhibitory concentration (MIC) dilution against all tested
371
bacteria and yeasts were also determined. Among the test compounds attempted, compounds
372
L, LH2, [Ni(LH)2] and [Zn(LH)Cl2] showed the highest effects against certain Gram-positive,
373
Gram- negative bacteria and certain yeasts. These compounds were comparatively higher or
374
equipotent to the antibiotic and antifungal agents in the comparison tests. These compounds
375
appeared to have appreciable antibacterial and antifungal activity
376
References
377
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[17] C. H. Collins, P. M. Lyre, J. M. Grange, “Microbiological Methods”, 6th Edn., Butterworths Co. Ltd., London (1989). [18] R. N. Jones, A. L. Barry, T. L. Gaven, J. A. Washington, “Manual of Clinical
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433 434 435 436 437 438 439
Nano-Met. Chem. 32 (2002)1583-1610.
21 440 441 442 443 444
Highlights Preparation of novel ligands
445
Preparation of novel complexes
446
Characterization of ligands and complexes
447
1
448
Egonol derivatives
449
Antibacterial activity
450
H-NMR, 13C-NMR, MS, IR and, UV–VIS. spectroscopy
S1386-1425(14)01854-X http://dx.doi.org/10.1016/j.saa.2014.12.074 SAA 13124
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
19 August 2014 13 December 2014 17 December 2014
Please cite this article as: I. Babahan, S. Emirdağ-Öztürk, E. Poyrazoğlu-Çoban, Spectroscopic and biological studies of new mononuclear metal complexes of a bidentate NN and NO hydrazone-oxime ligand derived from egonol, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa. 2014.12.074
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1
4
Spectroscopic and biological studies of new mononuclear metal complexes of a bidentate NN and NO hydrazone-oxime ligand derived from egonol
5
Ilknur Babahan1,*, Safiye Emirdağ-Öztürk2, Esin Poyrazoğlu-Çoban3
1 2 3
6 7 8 9 10
1
11
Abstract
12
A novel ligand vicinal dioxime ligand (egonol hydrazone glyoxime (LH2) was synthesized
13
and characterized using 1H-NMR, 13C-NMR, MS, AAS, infrared spectroscopy, and magnetic
14
susceptibility measurements. Mononuclear nickel (II), copper (II) and cobalt (II) complexes
15
with a metal:ligand ratio of 1:2 for LH2 were also synthesized. Zn(II) forms complex
16
[Zn(LH)Cl2] with a metal to ligand ratio of 1:1. IR spectrum shows that the ligand act in a
17
bidentate manner and coordinates N4 donor groups of the ligands to NiII, CuII, CoII and ZnII
18
ions. The detection of H-bonding (O–H⋅⋅⋅O) in the [M(LH)2] metal complexes by IR spectra
19
supported the square-planar MN4 coordination of Ni(II), Cu(II) and Co(II) complexes.
20
The antimicrobial activities of compounds LH2 and their Ni(II), Cu(II), Co(II) and Zn(II)
21
complexes were evaluated using the disc diffusion method against 16 bacteria and 5 yeasts.
22
The minimal inhibitory concentrations (MICs) against all the bacteria and yeasts were also
23
determined. Among the test compounds attempted, it is showed that all the compounds (L,
24
LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2], [Zn(LH)Cl2]) were effect against used test
25
micrroorganisms.
Adnan Menderes University, Faculty of Science and Art, Department of Chemistry, 09010, Aydin, Turkey Ege University, Faculty of Sciences, Department of Chemistry, 35100, Bornova, İzmir, Turkey 3 Adnan Menderes University, Faculty of Science and Art, Department of Biology, 09010, Aydin, Turkey 2
__________________________________________________________________________________________
26 27
Key words: Styrax officinalis; vic-Dioxime; Hydrazone; Egonol; Transition metal complex;
28
Spectroscopy; Antimicrobial activity
29 30 31 32 33
___________________________________________________________________________ ___________ * Corresponding author: Tel.: 90-256-212-8498; fax: 90-256-213-5379 E-mail address:[email protected] (I. Babahan)
2 34
1. Introduction
35
Egonol, a natural 2-aryl benzofuran, is known to be an effective pyrethrum synergist
36
[1]. Egonol and its derivatives attracted the attention of synthetic chemists due to their
37
antibacterial and antifungal [2], anti-complement [3] activities besides their considerable
38
cytotoxic activities against human leukaemic HL-60 cells [4]. It was also reported that
39
significant activities were observed for egonol against C6 (rat glioma) and Hep-2 (larynx
40
epidermoid carcinoma) cell lines [5-6].
41
Hydrazones possessing an azomethine -NHN=CH- proton constitute an important class
42
of compounds for new drug development. Therefore, many researchers have synthesized these
43
compounds as well as their metal complexes as target structures and evaluated their biological
44
activities. These observations have been guiding for the development of new hydrazones that
45
possess varied biological activities [7]. Hydrazones have been demonstrated to possess,
46
among other, antimicrobial, anticonvulsant, analgesic, anti inflammatory, antiplatelet,
47
antitubercular and antitumoral activities [7].
48
Nowadays, vic-dioximes are appreciated as coordination compounds in lots of usage areas
49
such as analytical, biologically, pigment and medicinal chemistry. Many researchers have
50
studied vic-dioximes which have important role of the complexes especially 1,2-dioximes in
51
coordination chemistry [8-10]. Their complexes have been the source, through the decades, of
52
a never-ending series of interesting reports. Owing to their importance as stable MN4 core-
53
containing coordination compounds, vic-dioxime complexes have been much investigated
54
[11]. The exceptional stability and unique electronic properties of these complexes can be
55
attributed to their planar structure, stabilized by hydrogen bonding [12–15].
56 57 58
3 59
2. Experimental
60
2.1. Materials and measurements
61
All reagents used were purchased from Merck. 1H N.M.R-13C N.M.R spectra (Bruker
62
400 MHz), I.R spectra (Varian 900), melting points (Buchi SPM-20) and pH measurements
63
(Orion Expandable Ion Analyzer EA 940) were used to elucidate the structures of the
64
products. The magnetic moments of the complexes were measured by the Gouy method with
65
a Newport type D-104 instrument magnet power supply. Mass spectrometry (MS) spectra
66
were recorded on a Bruker LC/MS/MS-8030 Triple Quadrupole Mass Spectrometer.
67
The
starting
materials,
Egonol
(5-[300-(hydroxy)propyl]-7-methoxy-2-(30,40-
68
methylene dioxyphenyl)benzofuran; Scheme 1, 1a) was isolated from the seeds of S.
69
Officinalis and its isolation procedure and egonol aldehyde (3-[2-(1,3-Benzodioxol-5-yl)-7-
70
methoxy-1-benzofuran-5-yl]propanal; Scheme 1, 1b) were explained in our previous work
71
[6a-b].
72 73
2.2 Synthesis
74
2.2.1 Synthesis of (L)
75
A solution of egonol aldehyde [6a-b] (Scheme 1, 1b) (1 mmol) in absolute ethanol 30 mL was
76
added dropwise to a solution of hydrazine hydrate (1 mmol) with 1 mL AcOH in absolute
77
ethanol 10 mL for a 30 min period. The reaction mixture was stirred overnight at room
78
temparature. After the end of the period, yellow precipitated solid was filtered, washed
79
thoroughly with distilled water and dried. The chemical reaction and molecular structure are
80
shown in Scheme 1(1c).
81
2.2.1 Synthesis of (LH2)
82
A solution of egonol hydrazone (1c) (1 mmol) in absolute ethanol 30 mL was added
83
dropwise to a solution of anti-chloroglyoxime (1 mmol) in absolute ethanol 10 mL for a 30
84
min period. The reaction mixture was stirred overnight at room temparature. After cooling to
4 85
0 0C the pH of the mixture was raised to 5.0-5.5 with treatment with NaHCO3 dissolved in 5
86
mL distilled water, and stirring was continued for one hour. The solution was poured into 100
87
mL cold water with stirrring. After the end of the period, yellow precipitated solid was
88
filtered, washed thoroughly with distilled water and dried. The chemical reaction and
89
molecular structure are shown in Scheme 1(1d).
90
2.2.2 Synthesis of the Ni(II), Cu(II) and Co(II) Complexes of LH2
91
A solution of a metal salt (1 mmol of NiCl2.6H2O, CoCl2.6H2O and CuCl2.2H2O) in 20
92
mL of water were added to 2 mmol of the ligand solution (0.848 g LH2 in 30 mL of ethanol)
93
with stirring. An initial sharp decrease in the pH of the solution from 5.5 to about 3-3.5 is
94
observed. After raising the pH to 5.0-5.5 using 1% aqueous NaOH solution, the reaction
95
mixture was kept in a hot water bath (60 ◦C) for 2 h to complete the precipitation. Then the
96
precipitated complex compounds were filtered, washed with water and ethanol, and dried at
97
room temperature in a vacuum oven. The structure of the prepared complexes are shown in
98
Scheme 1.
99
2.2.3 Synthesis of the Zn(II) Complex of LH2
100
A solution of ZnCl2.2H2O (0.172 g, 1 mmol) in ethanol (20 mL) was added to a
101
solution of ligand (0.424 g (1 mmol) for LH2, dissolved in ethanol (20 mL) by stirring in a
102
water bath at 50 ◦C for 2 h in order to complete precipitation. The apparent pH of the solution
103
was adjusted to 5.0-5.5 by the addition of 1 M NaOH solution. The precipitate was filtered,
104
washed with water, and dried in vacuo at 60 ◦C. The complexes are very slightly soluble in
105
common organic solvents. The structures of the complexes are shown in Scheme 1.
106
2.3 Antimicrobial assays
107
Sixteen bacterial strains and four yeast strain were obtained from the American Type
108
Culture Collection (ATCC, Rockville, MD, USA). A strain (Candida trophicalis) was
109
obtained from Faculty of Medicine, Adnan Menderes University. The Gram-negative (G-)
5 110
were: Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Proteus
111
vulgaris ATCC 33420, Serratia marcescens ATCC 13880, Klebsiella pneumoniae ATCC
112
13882, Pseudomonas aeruginosa ATCC 35032, Enterobacter aerogenes ATCC 13048 and
113
the Gram-positive (G+) were: Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC
114
25923, Staphylococcus epidermidis ATCC 12228, Corynebacterium xerosis ATCC 373,
115
Bacilllus cereus ATCC 11778, Bacilllus subtilis ATCC 6633, Enterococcus faecalis ATCC
116
29212, and Listeria monocytogenes ATCC 19112 and acid-fast bacteria species was:
117
Mycobacterium smegmatis ATCC 607. The following five yeast strains, i.e. Candida utilis
118
ATCC 9950, C. albicans ATCC 10231, C. tropicalis, Kluyveromyces fragilis ATCC 8608 and
119
Saccharomyces cerevisiae ATCC 9763, were also tested using disc diffusion method [16,17]
120
and the minimum inhibitory concentration (MIC) was determined by broth dilution method
121
[18].
122
2.3.1 Disc diffusion method
123
Screening for antibacterial and antifungal activities were carried out using sterile
124
antibiotic discs (6 mm), following the standard procedure of Antimicrobial Disc Susceptibility
125
Tests outlined by the National Committee for Clinical Laboratory Standards- NCCLS [16,17].
126
Fresh stock solutions (1x10-4 M) of the compounds were prepared in DMSO according to the
127
needed concentrations for the experiments. The inoculum suspensions of the tested bacteria
128
and yeasts were prepared from the broth cultures (18–24 h) and the turbidity equivalent
129
adjusted to 0.5 McFarland standard tube to give a concentration of 1x10-8 bacterial cells and
130
1x10-6 yeast cells/mL. To test the antimicrobial activity of each hydrazone-oxime derivative
131
bearing egonol or its metal complexes, a Mueller Hinton agar plate was inoculated with 0.1
132
mL broth culture of bacteria or yeast. Then a hole of 6 mm in diameter and depth was made
133
on top with a sterile stick and filled with 50 µL of the hydrazone-oxime derivative bearing
134
egonol or its metal complexes.
6 135
Plates inoculated with E. coli ATCC 25922, S. typhimurium ATCC 14028, S. aureus
136
ATCC 25923, S. epidermidis ATCC 12228, E. faecalis ATCC 29212, L. monocytogenes
137
ATCC 19112, P. vulgaris ATCC 33420, Corynebacterium xerosis ATCC 373, Klebsiella
138
pneumoniae ATCC 13882, Mycobacterium smegmatis ATCC 607, Pseudomonas aeruginosa
139
ATCC 35032, Enterobacter aerogenes ATCC 13048 and S. marcescens ATCC 13880 were
140
incubated at 37 0C for 24 h and those inoculated with M. luteus ATCC 9341, B. cereus ATCC
141
11778, B.subtilis ATCC 6633, S. cerevisiae ATCC 9763, C. albicans ATCC 10231, C. utilis
142
ATCC 9950, C. tropicalis and Kluyveromyces fragilis ATCC 8608 were incubated at 30 0C
143
for 24 h. The diameter of the inhibition zone was then measured. Discs of Chloramphenicol
144
(C30, Oxoid), Gentamycin (GN10 Oxoid), Tetracycline (TE30), Erytromycine (E15),
145
Ampicillin (AMP10) and Nystatine (NS100) were used as positive controls. The inhibition
146
zones were compared with those of the reference discs.
147
2.3.2 Dilution method
148
Screening for antibacterial and antifungal activities were carried out by preparing a
149
microdilution broth, following the procedure outlined in Manual of Clinical Microbiology
150
[18]. All the bacteria were inoculated in the nutrient broth and incubated at 30–37 0C for 24 h
151
while the yeasts were inoculated in malt extract broth and incubated at 30 0C for 48 h. The
152
compounds were dissolved in DMSO (2 mg mL-1) and then diluted in Mueller Hinton broth.
153
Twofold serial dilution of the compounds were employed to determine the MIC ranging from
154
256 to 0.125 µgmL-1. Cultures were grown at 30–370C (18–20 h) and the final inoculum was
155
approximately 106 cfu mL-1. Test cultures were incubated at 37 0C (24 h). The lowest
156
concentration of antimicrobial agent that resulted in complete inhibition of the
157
microorganisms was represented as MIC (µgmL-1). As positive controls, streptomycin (I.E.
158
Ulagay) for bacteria and nystatin (NS100, Oxoid) for yeast were used in the dilution method.
159
7 160
3. Results and discussion
161
Egonol (Scheme1; 1a) isolated from the seeds of S. Officinalis L. was used as the main
162
starting material (Figures 1a-b). Egonol aldehyde (1b) was obtained from an oxidation reaction
163
of egonol using PCC in acetone–CH2Cl2 (Scheme 1).
164
Egonol hidrazon (L; 1c) were obtained from the condensation of egonol aldehyde with
165
hydrazine hyrate in the presence of alcohol. Subsequently, (1Z,2E)-N'-{(1E)-2-[2-(1,3-
166
benzodioxol-5-yl)-1-benzofuran-5-yl]ethylidene}-2(hydroxyimino)ethanehydroximo
167
hydrazide (1d, LH2) was prepared from 1c with anti-chloroglyoxime in presence of alcohol
168
(Figure 2).
169
The mononuclear Ni(II), Cu(II), Co(II) and Zn(II) complexes were prepared from the
170
egonol hydrazone glyoxime (1d) and a stoichiometric amount of NiCl2.6H2O, CuCl2.2H2O,
171
CoCl2.6H2O and ZnCl2.2H2O in ethanol (Scheme 1). The metal complexes were characterized
172
using, mass spectrometry, FT-IR, and magnetic susceptibility measurements. For all
173
compounds, melting points were given Table 1.
174
given in Table 2. 1H-NMR and 13C-NMR spectra of egonol and egonol aldehyde are given in
175
Table 3. 1H-NMR and 13C-NMR spectra of L, LH2 and metal complexes could not be taken
176
because of their very low solubility in organic solvents. Antimicrobial activities of
177
compounds were given in Tables 4 and 5.
FT-IR data of LH2 and its complexes are
178 179
3.1IR spectra, mass and magnetic susceptibility
180
The IR spectra of ligands exhibited –NH (3448 cm-1 for L and 3419 cm-1 for LH2), -OH
181
(3224 cm-1 for LH2), -C=Noxime (1630 cm-1 for LH2), and –NO (948 cm-1 for LH2) stretching
182
vibrations [19]. The C=N and OH stretching vibrations appear similar to other vic-dioxime
183
derivatives [20-21].
8 184
The FT-IR spectra of KBr pellets containing the egonol hydrazone glyoxime complexes of
185
the general formula M(LH)2, in which M is Ni(II), Cu(II), or Co(II).2H2O, exhibited C=Noxime
186
absorption bands at 1565-1515 cm-1. These bands suggest that the ligands are N,N΄
187 188 189
Figure 1a Flower of Styrax officinalis
Figure 1b Fruits of Styrax officinalis
(http://en.wikipedia.org/wiki/Styrax_officinalis) ( http://en.wikipedia.org/wiki/Styrax_officinalis)
190 191
coordinated with the metal ion in correspondence with the proposed structures[22-26].
192
Intramolecular hydrogen bonding was indicated by a peak appearing between 1845-1775 cm-1
193
[25, 26].
194
Egonol’s positive ion-mode LSMS/APCI showed molecular ion peaks at m/z: 327.12
195
[M]+ consistent with a molecular formula of C19H18O5. FAB mass spectral analysis indicated
196
m/z ratios of 324 [M]+ for egonol aldehyde (1b), 338 [M]+ for egonol hydrazone (L), 424
197
[M]+ for LH2 (Scheme 2), 904 [M-1]+ for complex [Ni(LH)2], 910 [M]+ for complex
198
[Cu(LH)2], 942 [M+1]+, 905 [M-2H2O]+ for complex [Co(LH)2(H2O)2] and 559 [M+1]+ for
199
complex [Zn(LH)Cl2]. The MS-determined metal:ligand ratio was 1:2 for Ni(II), Cu(II) and
200
Co(II) complexes, while metal:ligand ratio was 1:1 for Zn(II) complex.
201 202 203 204
9 205
Table 1. Melting point, yields, and values of UV-Vis. of egonol and its derivatives. Compounds
Melting point (0C)
Yield (%)
UV-Vis. (nm)
Egonol
119.2
-
317, 223
Egonol aldehyde L
112.1 145.5
26.02 74.09
317, 218 317, 214
LH2 [Ni(LH)2]
>300 >300
68.87 45.67
302, 272, 214 304, 268, 216
[Cu(LH)2] [Co(LH)2(H2O)2]
>300 >300
56.23 47.04
304, 278, 215 330, 276, 223
[Zn(LH)Cl2]
>300
60.10
302, 263, 225
206 207 208 209 OH O
O MeO
O
Egonol 1a
H
OH
NH2 H
H
N
N
+
O 1) NH2NH2.H2O/ EtOH
O
Cl
O
N
OMe O
O
reflux
O
HO 1c
OMe
O
OH
H
1b
NH H NH
N
O
O
H
O
N
O
Cl EtOH, NaOH
1d
O MCl2.XH2O EtOH, NaOH H O
O
NH
N
N
H
N
N
O
O
H N
O O
O OMe
H
M
H NH
O M: Ni(II), Cu(II), Co(II)
210 211 212
N
H OMe
N OH
OMe
OH
OMe
Cl ZnCl2 2H2O
Zn H
O
N
O
N
N
O O
Scheme 1. Synthesis of the ligands (L and LH2) and metal complexes [Ni(II), Cu(II), Co(II) and Zn(II)]
10 213
Based on IR spectral and magnetic susceptibility data, the complexes have a metal:ligand
214
ratio of 1:2 and square planar or octahedral structures for Ni(II), Cu(II) and Co(II) complexes
215
and 1:1 and tetraheral structure for Zn(II) complex (Scheme 1). The magnetic susceptibility
216
measurements of the Ni(II) and Zn(II) complexes indicate that these complexes are
217
diamagnetic. Room temperature magnetic moment measurements indicate that the Co(II)
218
complex ([Co(LH)2(H2O)2]) is paramagnetic with a magnetic susceptibility of 3.73 B.M for
219
LH2. The magnetic susceptibilities are within the range of high spin octahedral cobalt(II)
220
complex (the three-spin value is 3.87 B.M.) [23]. For [Co(LH)2(H2O)2], coordinated H2O
221
molecules were identified by a broad OH absorption band near 3220 cm−1 whose intensity
222
remained constant following heating to 110 ◦C for 24 h.
223
The Cu(II) complex was also paramagnetic with µ eff = 1.74 for LH2, a close fit for the spin
224
value of 1.73 B.M. Although the magnetic moment is somewhat low, there are likely some
225
diamagnetic contributions from the ligand that ultimately decrease the total paramagnetism of
226
the complexes [27].
227
Ligands form mononuclear complexes [(LH)2M] with a metal to ligand ratio of 1:2 with
228
M=Co(II)(H2O)2, Ni(II), and Cu(II). Zn(II) forms complexes [Zn(LH)Cl2] with a metal to
229
ligand ratio of 1:1 [28-29]. The Co(II) complex of the ligand is proposed to be octahedral with
230
water molecules as axial ligands, while the Ni(II) and Cu(II) complexes are proposed to be
231
square planar, and the complex of Zn(II) is tetrahedral. Two chloride ions are also coordinated
232
to the Zn(II) ion. The Zn(II) complex is tetrahedral with a 1:1 metal to ligand ratio, according
233
to the MS, and the ligand is coordinated only by the N, O atoms of the vic-dioxime [28-29].
234
The tetrahedral structure of the Zn(II) complex is based on the disappearance of the
235
band (O-H...O) of the complexes and appearance of the O-H bands of these compounds in the
236
FT-IR spectra[29].
237
11 238 239
3.2 NMR spectra The 1H and
13
C NMR spectra of egonol exhibited peaks at 3.73 ( t, 2H, J= 6.4, 6.4)
240
(H3") and 62.70 ppm (C3"). The corresponding signals for egonol aldehyde were at 9.85 (t,
241
1H, J= 0.8, 1.6) (H3"). The 1H NMR spectrum of egonol aldehyde indicated two methylenes
242
(δH 3.05, 2.85 instead of at δH 1.97, 2.85 in egonol), six aromatic methynes (δH 6.77, 6.96,
243
6.82, 7.32, 6.88, 7.41), one methylenedioxy (δH 6.00), one methoxy (δH 4.04) and one
244
aldehyde signal (δH 9.85). The main difference in the 1H NMR spectrum of egonol aldehyde
245
with that of egonol is the presence of the aldehyde proton signal at δH 9.85 (1H, t) and the
246
absence of a signal at δH 3.73 (2H, t) in egonol (Table 3). Therefore, the structure of egonol
247
aldehyde
248
yl]propanal [6a-b].
was
established
as
3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-
249 250 251
252 253 254
Table 2. Characteristic IR bands of the ligands and their metal complexes (cm-1, KBr) Compound
NH
1 2 3 4 5 6
3448 b 3419 b 3433 b 3414 b 3424 b 3415 b
-OH/ H2O 3224b 3220 b 3256 b
Aromatic
O-H..O
C=Na
C=Nb
N-O
3020 w 3028w 2982 w 2999 w 3021 w 2960 w
1845 w 1790 w 1775 w -
1653 s 1648 s 1648 s 1648 s 1648 s 1690 s
1630 s 1565 s 1572 s 1515 s 1551 s
948 m 984 m 960 m 960 m 948 m
_________________________________________________________________________________________________________ b: board s: strong, m: medium, w: weak, ν(C=N)a: hydrazone moiety, ν(C=N)b: oxime moiety.
(1) L, (2) LH2, (3)[Ni(LH)2], (4) [Cu(LH)2], (5) [Co(LH)2(H2O)2], (6) [Zn(LH)Cl2
12 +
H
O
O
N
N
H
OCH3 O
OH
NH
N
OH
m/z= 424 (-) OH H
O
N
O
N
H
OCH3 m/z= 411
O
H
(-) HCN
N +
H
N O
N O
+
(-)
N O
OH
NH
N
O
NH2
OH
NH
N +
OCH3 O
O
m/z= 380
H3CO O m/z= 338
HO-N=C(-) H
(-) O H NH N O
+
N NCH(-)
O
NH
N
+
O
O
OCH3 OCH3
O
O m/z= 364 m/z= 295 H
H N
H2N
NH
+
HN
+
NH2
+
O
O OCH 3
+
N O
255 256
m/z= 280
O
257 258 259 260 261 262 263
O OCH3
O
Scheme 2 The propesed fragmentation pattern of LH2
m/z= 295
NH
+
13 264 265 266
Table 3. 1H NMR of Egonol and egonol-aldehyde (CDCl3, 400 MHz) and 13 C NMR of compound of egonol (CDCl3, 100MHz) 267
Egonol
Egonol-aldehyde δ ppm
No
268 Egonol δ ppm
H3
6.81,s ,1H
6.77,s,1H
C2
156.51,q270
H4
6.99, s, 1H
6.96,s,1H
C3
100.76,d
H6
6.66, s, 1H
6.62,d,1H, J=1.6
C3a
H2'
7.34, d, 1H J=1.5
7.32,d,1H, J=4.0
C4
112.72,q
H5'
6.90,d,1H J=8.1
6.88,d,1H, J=8.0
C5
137.92,q
H6'
7.43,dd,1H J=1.6, 8.1
7.41,dd,H J=1.6, 8.0
C6
107.86,d
OCH2O
6.02, s, 2H,
6.00,s,2H
C7
145.20.q
OMe
4.05,s, 3H
4.04,s,3H
C7a
142.88,q
H1"
2.81, t, 2H J=7.5, 7.8
2.85,t,2H, J=1.2, 6.8
C1'
125.12,q277
H2"
1.97, p, 2H
3.05,t,2H, J=8.0, 7.2 9.85,t,1H, J=0.8, 1.6
C2'
105.95,d278
C3'
148.46,q279
C4'
148.39,q280
C5'
109.02,d
C6'
119.62,d
H3"
3.73, t,2H, J=6.4,6.4
269 271
131.46,q
272 273 274 275 276
281 282
283 284
4
3 '' 5
3
HO
285
A
286 287
O
7
O
2'
B
6
2
290 291 292
6'
4' 5'
Scheme 3 Suggested conformation of egonol
O
C
1
CH3
288 289
3'
1'
O
14 293
3.3. Antimicrobial Assays
294
A novel vic-dioxime-hydrazone derivative containing egonol group and its Ni(II),
295
Cu(II), Co(II) and Zn(II) complexes detemined remarkable antimicrobial activity (Tables 4
296
and 5).
297
Among the test compounds assayed, compounds L, LH2, [Ni(LH)2], [Cu(LH)2],
298
[Co(LH)2(H2O)2] and [Zn(LH)Cl2] showed activity against some bacteria and yeasts (Table
299
4). According to Table 4, compounds L, LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2] and
300
[Zn(LH)Cl2] demostrated stronger activity against using the some bacteria and yeasts C.
301
xerosis ATCC 373, M. smegmatis ATCC 607, Micrococcus luteus ATCC 9341,
302
Stapylococcus epidermidis ATCC 12228, Entereococcus faecalis ATCC 29212, Enterobacter
303
aerogenes ATCC 13048, Serratia marcescens ATCC 13880, B. subtilis ATCC 6633, C.
304
albicans ATCC 10231, C. utilis ATCC 9950.
305
The MIC values in Table 5 presented that some of the compounds tested demonstrated
306
the strongest activity against C. xerosis ATCC 373, M. smegmatis ATCC 607, M. luteus
307
ATCC 9341, S. epidermidis ATCC 12228, S. marcescens ATCC 13880, E. aerogenes ATCC
308
13048, B. subtilis ATCC 6633, C. trophicalis. For example, C. xerosis ATCC 373 (compound
309
[Zn(LH)Cl2]= 16 µgmL-1, LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2] and L= 32 µgmL-1),
310
M. smegmatis ATCC 607 (LH2, [Ni(LH)2], [Cu(LH)2], [Co(LH)2(H2O)2], [Zn(LH)Cl2] and
311
L= 32 µgmL-1), M. luteus ATCC 9341 (LH2= 32 µgmL-1), S. epidermidis ATCC 12228 (L,
312
LH2,
313
([Ni(LH)2]= 32 µgmL-1), E. aerogenes ATCC 13048 ([Ni(LH)2] and [Zn(LH)Cl2] = 32
314
µgmL-1), B. subtilis ATCC 6633 (compound [Zn(LH)Cl2]= 32 µgmL-1), C. trophicalis
315
([Cu(LH)2] = 32 µgmL-1).
[Cu(LH)2],
[Co(LH)2(H2O)2] and [Zn(LH)Cl2]), S. marcescens ATCC 13880
15 316
In addition, tested compounds have indicated moderate effect against some bacteria and
317
yeasts (Table 5). For example, E. coli ATCC 25922 (L, [Cu(LH)2], [Co(LH)2(H2O)2] and
318
[Zn(LH)Cl2]), S. typhimirium ATCC 14028 (L, LH2, [Cu(LH)2] and [Co(LH)2(H2O)2]= 64
319
µgmL-1), K. pneumoniae ATCC 13882 (L, [Ni(LH)2]= 64 µgmL-1), P. vulgaris ATCC 33420
320
(L, LH2, [Co(LH)2(H2O)2] and [Zn(LH)Cl2]= 64 µgmL-1), E. faecalis ATCC 29212
321
([Cu(LH)2], [Co(LH)2(H2O)2] and [Zn(LH)Cl2]= 64 µgmL-1), B. subtilis ATCC 6633 (LH2,
322
[Ni(LH)2], [Cu(LH)2] and [Co(LH)2(H2O)2]= 64 µgmL-1), E. aerogenes ATCC (L,
323
[Cu(LH)2]= 64 µgmL-1), P. aeroginosa ATCC 35032 (L, [Zn(LH)Cl2]= 64 µgmL-1), S.
324
marcescens ATCC 13880 (L=64 µgmL-1), L. monocytogenes ATCC 19112(L=64 µgmL-1), B.
325
cereus ATCC 11778 (L=64 µgmL-1), C. albicans ATCC 10231 ([Ni(LH)2] and [Zn(LH)Cl2]=
326
64 µgmL-1), C. utilis ATCC 9950 ([Ni(LH)2] and [Zn(LH)Cl2]=64 µgmL-1), C. trophicalis
327
(L=64 µgmL-1), K. fragilis ATCC 8608 (LH2, [Ni(LH)2] and [Co(LH)2(H2O)2]= 64 µgmL-1),
328
S. cerevisiae ATCC 9763 (L, [Cu(LH)2]=64 µgmL-1).
329
Suggestions are made that the negative inductive effect plays a significant role.
330
Dimerisation of oxime involves the formation of a pair of H bonds [30]. This feature causes a
331
decrease in electronic density of oximes compared with phenylhydrazones, thereby
332
facilitating entry of the oxime into the cell. This is likely to increase the antibacterial potency
333
[30].
334 335 336 337 338 339 340 341 342
16 343
Table 4. Antimicrobial activities of compounds (Inhibition zone mm)
344 Compounds
Reference antibiotics
Test Microorganisms 1
2
3
4
5
6
C30
CN10
TE30
E15
AMP10
NS100
Escherichia coli ATCC 25922 Salmonella typhimirium ATCC 14028 Micrococcus luteus ATCC 9341 Stapylococcus aureus ATCC 25923 Stapylococcus epidermidis ATCC 12228 Klebsiella pneumoniae ATCC 13882 Pseudomonas aeruginosa ATCC 35032 Proteus vulgaris ATCC 33420 Entereococcus faecalis ATCC 29212 Corynebacterium xerosis ATCC 373 Mycobacterium smegmatis ATCC 607 Serratia marcescens ATCC 13880 Listeria monocytogenes ATCC 19112 Enterobacter aerogenes ATCC 13048 Bacilllus cereus ATCC 11778 Bacillus subtilis ATCC 6633 Candida albicans ATCC 10231 Candida utilis ATCC 9950
10
10
12
13
12
12
24
21
15
11
-
NT
12
10
12
12
11
13
17
16
15
8
8
NT
14
11
10
12
11
20
25
15
26
30
28
NT
8
-
8
9
9
11
23
20
22
23
20
NT
14
11
14
14
15
15
22
17
19
11
17
NT
-
13
10
10
9
13
21
19
20
14
-
NT
10
10
10
10
12
14
22
20
20
21
-
NT
13
11
11
11
12
13
17
24
16
20
-
NT
11
11
13
13
13
17
16
11
19
-
14
NT
16
16
16
16
17
20
20
17
25
26
27
NT
15
15
15
14
15
20
23
18
26
25
19
NT
11
15
11
11
11
13
23
19
13
-
19
NT
11
10
10
11
10
13
19
14
12
-
12
NT
11
15
12
10
14
13
19
20
14
-
-
NT
10
11
11
10
10
13
23
24
25
26
-
NT
12
13
13
12
14
18
22
20
12
25
-
NT
10
12
11
10
12
16
NT
NT
NT
NT
NT
22
11
13
9
9
13
15
NT
NT
NT
NT
NT
21
Candida trophicalis*
11
10
14
9
10
12
NT
NT
NT
NT
NT
12
12
10
12
10
14
NT
NT
NT
NT
NT
10
11
12
10
10
12
NT
NT
NT
NT
NT
Kluyveromyces fragilis ATCC 8608 Saccharomyces cerevisiae ATCC 9763
345 346 347 348 349 350 351 352
15 19 15
Note: 1 = LH2 2 = [Ni(LH)2], 3 = [Cu(LH)2], 4 = [Co(LH)2(H2O)2] 5 = [Zn(LH)Cl2] 6=L (–) = No zone, NT = Not tested. C30: Chloramphenicol (30 mg Oxoid); CN10: Gentamycin (10 mg Oxoid); TE30: Tetracycline (30 mg Oxoid); E15: Erytromycin (15mg Oxoid); AMP10: Ampicillin (10 mg Oxoid); NS: Nystatin (100 mg Oxoid); (-):Zone did not occur. NT: Not tested, *From Faculty of Medicine, Adnan Menderes University.
17 353 354
Table 5. Antimicrobial activities of compounds (MIC, µg.mL-1) Test Microorganisms
355 356 357 358 359 360 361 362
1
2
3
4
5
6
Str
NS 100
Escherichia coli ATCC 25922
128
128
64
64
64
64
64
-
Salmonella typhimirium ATCC 14028 Micrococcus luteus ATCC 9341 Stapylococcus aureus ATCC 25923 Stapylococcus epidermidis ATCC 12228 Klebsiella pneumoniae ATCC 13882 Pseudomonas aeruginosa ATCC 35032 Proteus vulgaris ATCC 33420 Entereococcus faecalis ATCC 29212 Corynebacterium xerosis ATCC 373
64
128
64
64
128
64
64
-
32
128
128
64
128
16
32
-
256
-
256
256
256
128
32
-
32
128
32
32
32
32
32
-
-
64
128
128
256
64
64
-
128
128
128
128
64
64
64
-
64
128
128
64
64
64
64
-
128
128
64
64
64
32
64
-
32
32
32
32
16
32
64
-
Mycobacterium smegmatis ATCC 607 Serratia marcescens ATCC 13880
32
32
32
32
32
32
128
-
128
32
128
128
128
64
64
-
Listeria monocytogenes ATCC 19112 Enterobacter aerogenes ATCC 13048 Bacilllus cereus ATCC 11778 Bacillus subtilis ATCC 6633 Candida albicans ATCC 10231 Candida utilis ATCC 9950 Candida trophicalis*
128
128
128
128
128
64
32
-
128
32
64
128
32
64
32
-
128
128
128
128
128
64
64
-
64
64
64
64
32
16
64
-
128
64
128
128
64
32
-
64
128
64
256
256
64
32
-
64
128
128
32
256
128
64
-
64
Kluyveromyces fragilis ATCC 8608
64
64
128
64
128
32
-
128
Saccharomyces cerevisiae 128 128 64 128 128 64 128 ATCC 9763 Note: 1 = LH2, 2 = [Ni(LH)2], 3 = [Cu(LH)2], 4 = [Co(LH)2(H2O)2], 5 = [Zn(LH)Cl2], 6=L Str= streptomycin, NS100= nystatin (–) = No effect * From Faculty of Medicine, Adnan Menderes University.
18 363
Conclusions
364
Novel vic-dioxime ligand (LH2) containing egonol hydrazone moiety and its mononuclear
365
Ni(II), Cu(II), Co(II) and Zn(II) complexes were synthesized and characterized. Reaction of
366
LH2 with metal(II) salts yielded mononuclear complexes corresponding to the general
367
formulas ([M(LH)2], M: Ni(II), Cu(II), Zn(II), or Co(II).2H2O).
368
The
antimicrobial activities
of
compounds
L,
LH2,
[Ni(LH)2],
[Cu(LH)2],
369
[Co(LH)2(H2O)2] and [Zn(LH)Cl2] were evaluated using disc diffusion method against 16
370
bacteria and 5 yeasts. Minimal inhibitory concentration (MIC) dilution against all tested
371
bacteria and yeasts were also determined. Among the test compounds attempted, compounds
372
L, LH2, [Ni(LH)2] and [Zn(LH)Cl2] showed the highest effects against certain Gram-positive,
373
Gram- negative bacteria and certain yeasts. These compounds were comparatively higher or
374
equipotent to the antibiotic and antifungal agents in the comparison tests. These compounds
375
appeared to have appreciable antibacterial and antifungal activity
376
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377
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21 440 441 442 443 444
Highlights Preparation of novel ligands
445
Preparation of novel complexes
446
Characterization of ligands and complexes
447
1
448
Egonol derivatives
449
Antibacterial activity
450
H-NMR, 13C-NMR, MS, IR and, UV–VIS. spectroscopy
