New water soluble bis-imidazolium salts with a saldach scaffold: Synthesis, characterization and in vitro cytotoxicity/bactericidal studies

Article abstract of DOI:10.1016/j.ica.2014.05.029

A series of water-soluble bis-imidazolium salts of the type H ₂(ⁱPr)₂saldach(1,2-Me₂Im ⁺-X^-)₂ (4) and their mononuclear complexes [M(III)Cl{(ⁱPr)₂saldach(1,2-Me₂Im ⁺-X^-)₂}] (M = Mn, 5; Fe, 6), (X = Cl, a; PF₆, b; BF₄, c), where saldach = N,N′- bis(salicylidene)-(±)-trans-1,2-diaminocyclohexane, have been synthesized and characterized using elemental analysis, electronic, spectral, magnetic as well as conductometric methods and MALDI-TOF-, ESI-MS. All complexes possess a distorted square pyramidal coordination geometry with MN₂O ₂Cl chromophore, as revealed by the elemental, spectral and literature data. These salts have been evaluated for in vitro cytotoxicity against HepG-2 and MCF-7 cell lines. Among them, 4c (IC₅₀ = 22.17 μM) exhibited potency against MCF-7. The bactericidal efficacy of 4a-c was screened against a panel of common pathogenic bacteria. Compound 4a was found to be the most potent antibacterial agent and could inhibit all the bacterial strains more effectively than standard antibiotics.

Full text of DOI:10.1016/j.ica.2014.05.029

Accepted Manuscript
New water soluble bis-imidazolium salts with a saldach scaffold: Synthesis,
characterization and in vitro cytotoxicity/ bactericidal studies
Reda F.M. Elshaarawy, Zeinab H. Kheiralla, Abeer A. Rushdy, Christoph Janiak
PII:
S0020-1693(14)00318-1
http://dx.doi.org/10.1016/j.ica.2014.05.029
ICA 16011
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 April 2014
21 May 2014
26 May 2014
Please cite this article as: R.F.M. Elshaarawy, Z.H. Kheiralla, A.A. Rushdy, C. Janiak, New water soluble bis-
imidazolium salts with a saldach scaffold: Synthesis, characterization and in vitro cytotoxicity/ bactericidal studies,
Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.2014.05.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New water soluble bis-imidazolium salts with a saldach scaffold: Synthesis,
characterization and in vitro cytotoxicity/ bactericidal studies.
Reda F.M. Elshaarawy,a,c,* Zeinab H. Kheiralla,b Abeer A. Rushdyb and Christoph Janiakc,*
a
Faculty
of
Science,
Suez
University,
Suez,
Egypt.
Email:
reda_elshaarawi@science.suez.edu.eg; Reda.El-Shaarawy@uni-duesseldorf.de
bBotany Department, University College for Women, Ain Shams University, Cairo, Egypt.
Email: kheiralla@hotmail.com
c
Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universität
Düsseldorf, 40204 Düsseldorf, Germany. Email: janiak@uni-duesseldorf.de
Abstract
A series of water-soluble bis-imidazolium salts of the type H2(iPr)2saldach(1,2-Me2Im+-X–)2
(4) and their mononuclear complexes [M(III)Cl{(iPr)2saldach(1,2-Me2Im+-X–)2}] (M = Mn, 5;
Fe, 6), (X = Cl, a; PF6, b; BF4, c), where saldach = N,N`-bis(salicylidene)-( )-trans-1,2-
diaminocyclohexane, have been synthesized and characterized using elemental analysis,
electronic, spectral, magnetic as well as conductometric methods and MALDI-TOF-, ESI-MS.
All complexes possess a distorted square pyramidal coordination geometry with MN2O2Cl
chromophore, as revealed by the elemental, spectral and literature data. These salts have been
evaluated for in vitro cytotoxicity against HepG-2 and MCF-7 cell lines. Among them, 4c
(IC50 = 22.17 µM) exhibited potency against MCF-7. The bactericidal efficacy of 4a-c was
screened against a panel of common pathogenic bacteria. Compound 4a was found to be the
most potent antibacterial agent and could inhibit all the bacterial strains more effectively than
standard antibiotics.
Keywords: Bis-imidazolium salts – Metallosaldach – cytotoxicity – antibacterial.
1. Introduction
One of the most chemotherapeutic problems we are facing today in the context of fighting
bacterial infectious diseases is the relentless increase and spread of multidrug-resistant (MDR)
[1,2]. Thus, studies for the identification of novel targets and drugs for the treatment of
infectious diseases are at the forefront. Several approaches to negating antibiotic resistance
are currently being investigated, including inactivation of enzymes in essential metabolic
pathways and inhibiting signal transduction systems [3,4]. These approaches involve the
1
2
3
4
1

1
2
development of new antimicrobial drugs with modes of action that circumvent current
resistance mechanisms [5,6].
3
Most of the platinum-based anticancer drugs [7] had an enormous impact on current
cancer chemotherapy. However, the spectrum of cancer that can be treated with platinum
agents is narrow and treatment efficacy suffers from side effects and resistance phenomena
[8,9]. In order to overcome clinical problems associated with the relatively limited activity of
platinum-based agents against the broad spectrum of human malignancies, acquired resistance
and side effects, novel non-platinum metal-based anticancer complexes have been developed
[9,10].
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Notably, metallo-saldach compounds are very well-studied class of chemical nucleases
that bind, cleave, and damage nucleic acids [11,12] via oxidative alkylation of nucleobases
[13]. Furthermore, various Fe(III)-saldach derivatives have been implicated in efficient
asymmetric catalysis and in catalyzing the hydrolytic cleavage of DNA and RNA [14]. Recent
studies have demonstrated that Fe(III)-saldach induces apoptosis in human embryonic kidney
cells [15]. Moreover, the in vitro DNA cleavage activity of Fe(III)-salen complexes is
inversely correlated with their apoptotic activities in cultured human cells [16]. Mn(III)-
saldach complexes were shown to exhibit superoxide dismutase (SOD), catalase activity and
are considered to be synthetic SOD mimics [17,18]. Also Mn(III)-saldach induces tumor
selective apoptosis in human cells [19].
In the race to synthesize new pharmaceutical drugs, ionic liquids (ILs) have attracted a
great deal of attention. IL strategies can take advantage of the dual nature (discrete ions) of
ILs to realize enhancements which may include controlled solubility, bioavailability or
bioactivity, stability, elimination of polymorphism, new delivery options (e.g., slow release or
the IL-API as ‘solvent’), or even customized pharmaceutical cocktails [20]. 1,3-
Dialkylimidazolium salts have become an attractive candidates for application in medicinal
chemistry due to their tunable properties and ability to generate biological responses upon
binding to several biological targets [21]. They have been recognized as bactericidal [22],
fungicidal [22], acetylcholinesterase (AChE) inhibitor [23], AMP deaminase inhibitor [24],
delivery of anti-inflammatory drugs [25], local anesthetic [22], anti-nociceptive [20],
anticholinergic [20], anticancer drugs [26] and in protein formulations [27]. Carson et al. have
reported the broad spectrum antibiofilm activity of 1-alkyl-3-methylimidazolium chloride ILs
against a panel of clinically important microbes [28]. Recently, the anti-microbial effect of a
series of imidazolium-based ionic liquids revealed broad-spectrum activities against cocci,
rods and fungi [29]. Also the length of N-3 alkyl substituent plays a significant role in the
2

≡N
anti-tumor activity. Noteworthy, imidazolium units can slowly interact with the C=N or C
1
2
module, through electrophilic-nucleophilic interaction, leading to the fragmentation of the
molecule [30].
3
4
In continuation of our ongoing programs directed toward the development of novel,
potent, selective and less toxic therapeutic agents [31], we now report a concise, practical
synthetic route and in vitro biological (antimicrobial and anticancer) evaluation of new
saldach-bis(imidazolium) salts and their metal complexes (Scheme 1) which may allow us to
develop a promising therapeutic strategy to combat antibiotic resistance and offer potent
cytotoxic agents.
5
6
7
8
9
10
(Scheme 1)
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
2. Materials and methods
Melting points were measured using a BÜCHI Melting point B-540 apparatus; all melting
points were measured in open glass capillaries and are uncorrected. Elemental analyses for C,
H, N, were performed with a Perkin–Elmer 263 elemental analyzer. FT-IR spectra were
recorded on a BRUKER Tensor-37 FT-IR spectrophotometer in the range 400–4000 cm–1 as
KBr disc in the 4000-550 cm–1 region with 2 cm–1 resolution or with an ATR (attenuated total
reflection) unit (Platinum ATR-QL, diamond). For signal intensities the following
abbreviations were used: br (broad), sh (sharp), w (weak), m (medium), s (strong), vs (very
strong). UV/Vis spectra were measured at 25 °C in ethanol (10–5 mol/L) on a Shimadzu UV-
2450 spectrophotometer using quartz cuvettes (1 cm). NMR-spectra were obtained with a
Bruker Avance DRX200 (200 MHz for 1H) or Bruker Avance DRX500 (125, 202 and
470 MHz for 13C, 31P and 19F respectively) spectrometer with calibration to the residual
proton solvent signal in DMSO-d6 (1H NMR: 2.52 ppm, 13C NMR: 39.5 ppm), CDCl3 (1H
NMR: 7.26 ppm, 13C NMR: 77.16 ppm) against TMS (δ = 0.00 ppm) for H and 13C,
1
85% phosphoric acid (δ = 0.00 ppm) for 31P and CFCl3 (δ = 0.00 ppm) for 19F NMR.
Multiplicities of the signals were specified s (singlet), d (doublet), t (triplet), q (quartet) or m
(multiplet). The mass spectra of the synthesized saldach-bis(imidazolium) salts and their
metal complexes were acquired in the linear mode for positive ions on a UHR-QTOF maXis
4G (Bruker Daltonics) and BRUKER Ultraflex MALDI-TOF instrument equipped with a 337
nm nitrogen laser pulsing at a repetition rate of 10 Hz. The 2+ charge assignment of ions in
HR-ESI-MS was confirmed by the m/z = 0.5 difference between the isotope peaks (x, x+1,
x+2). The MALDI matrix material (1,8-dihydroxy-9(10H)-anthracenone (dithranol, DIT,
12
C H10O3, M = 226.077 g/mol) was dissolved in chloroform at a concentration of 10
14
mg/mL. MALDI probes were prepared by mixing compound solution (1 mg/mL in CH2Cl2)
3

1
2
with the matrix solution (1:10, v/v) in a 0.5 mL Eppendorf® micro tube. Finally 0.5 µL of this
mixture was deposited on the sample plate, dried at room temperature and then analyzed.
Peaks with chlorine showed the isotope ratio 35/37Cl = 75.8:24.2. Manganese (55Mn 54.938 Da,
100%) or iron (56Fe 55.934 Da, 91.2%) are either isotope pure or with a predominant isotope
(54Fe 53.939, 5.8%; 57Fe 56.935 Da, 2.1%). For the mass spectral assignment: Peaks are based
3
4
5
55
6
on 12C with 12.0000 Da,35Cl with 34.968 Da, Mn 54.938 Da, 56Fe 55.934. dithranol, DIT,
12
7
C H10O3, M = 226.077 g/mol C38H52N6O2 = [4 – 2 anions]2+ = 624.45 C38H50N6O2 = [4 –
14
8
2H+ – 2 anions]0 = 622.44 Me2Im = C5H8N2 = 96.078 Me2ImH = C5H9N2 = 97.086
C38H50N6O2FeCl = 713.3. The molar conductances of 10–3 mol/L solution of various salts
have been measured at ambient temperature with a digital conductivity meter (S30
SevenEasy™ conductivity, Mettler-Toledo Electronics, LLC, Polaris Parkway, Columbus).
The overall accuracy of the conductance measurements was found to be 0.2%. Magnetic
measurements of target complexes were carried out at room temperature using a Vibrating
Sample Magnetometer (VSM), (Model PAR 155).
9
10
11
12
13
14
15
16
17
18
19
Chemicals were obtained from the following suppliers and used without further
purification:
salicylaldehyde,
2-iso-propylphenol,
( )-trans-1,2-diaminocyclohexane,
·
anhydrous MgCl2 and Mn(CH3COO)2 4H2O (Sigma–Aldrich), paraformaldehyde (Roth), 1,2-
dimethylimidazole, 1-butylimidazole (Alfa Aesar), triethylamine (Et3N) and anhydrous ZnCl2
(GRÜSSING GmbH) and FeCl3 (Acros).
20
21
22
23
24
3. Experimental
The preparation details of the key starting materials 3-isopropylsalicylaldehyde (1), 3-iso-
propyl-5-chloromethyl-2-hydroxybenzaldehyde
(2),
3-(3-(iso-Propyl)-5-formyl-4-
hydroxybenzyl)-1,2-dimethylimidazol-3-ium chloride (3a) and anion metathesis products
(3b,c) can be seen in electronic supplementary information.
25
26
27
28
29
30
31
32
33
3.1 General procedure for the preparation of rac-trans-H2(iPr)2saldach(1,2-Me2Im+-X–)2
(4a-c):
A methanolic solution (10 mL) of ( )-trans-1,2-diaminocyclohexane (dach) (0.23 g, 2.0
mmol) in a Schlenk tube, was added dropwise to a methanolic solution (20 mL) of
salicylaldehyde-imidazolium salt H(iPr)sal(Me2Im+-X–) 3a-c (4.0 mmol) into a 100 mL
Schlenk flask under nitrogen atmosphere. The reaction mixture was stirred under N2 at 60 °C
for 3 h. Then the solvent was partially removed under reduced pressure, and the yellow
products of 4a-c were precipitated by the addition of ethyl acetate and kept in the refrigerator
overnight. Solvent was decanted off and the obtained crude product was sonicated for 15 min
4

1
2
3
4
5
in Et2O (3 x 25 mL). Et2O was also decanted off and the residual solid was washed
intensively with MeOH / Et2O mixture (1:2) to remove unreacted materials and then re-
dissolved in MeOH. EtOAc was added slowly (~15 min) to precipitate the products as pale
yellow-dark orange solids which were collected by filtration and dried under vacuum.
Samples of the isolated solids were characterized as follows.
6
7
8
9
N,N'-Bis[3-iso-propyl-5-((1,2-dimethylimidazol-3-ium)methylene)-salicylidene)-rac-trans-
1,2-cyclohexanediamine dichloride monohydrate (4a): Yellow-orange powder, (2.54 g, 89
%); mp 61-63 °C. FT-IR (KBr, cm-1): 3436 (m, br, ν(O-H)), 3131 (m, sh, νasym(C-H), Im and Ar),
3074 (m, sh, νsym(C-H), Im and Ar), 2931 (m, sh, ν(CH )) 2862 (m, sh, ν(C-H)), 1629 (vs, sh,
3
10
11
ν
(C=N)), 1536, 1460, 1384 (s, sh, ν(C=C
bend)), 1321 (m, sh, ν(C-H)), 1271 (s, sh, ν(Ar-O)),
+ C-H
Ar
1163 (s, sh, ν(H-C=C + H-C=N)bend, Im), 770 (m, sh), 645 (m, sh), 505 (m, br). 1H NMR (200 MHz,
12
13
14
15
16
17
18
19
20
21
22
23
24
25
CDCl3) δ (ppm): 13.98 (s, 2 H, 2 x Ar-OH), 8.55 (s, 2 H, 2 x H-C=N), 7.77 (d, J = 2.00 Hz, 2
H, 2 x Im-H), 7.72 (d, J = 1.97 Hz, 2 H, 2 x Im-H), 7.39 (d, J = 1.55 Hz, 2 H, 2 x Ar-H), 7.25
(d, J = 1.83 Hz, 2 H, 2 x Ar-H), 5.33 (s, 4 H, 2 x N(3)-CH2-Ar), 3.77 (s, 6 H, 2 x N(1)-CH3),
3.40 (m, 2 H, 2 x Cyhex-H), 3.22 (m(7), 2 H, 2 x CH(CH3)2), 2.64 (s, 6 H, 2 x C(2)-CH3), 1.80
(m, 4 H, 4 x Cyhex-H), 1.49 (m, 4 H, 4 x Cyhex-H), 1.16 (d, J = 7.89 Hz, 12 H, 2 x
CH(CH3)2). 13C NMR (125 MHz, CDCl3) δ (ppm): 165.23 (HC=N), 159.99 (C-OH), 144.58
(N(1)C(CH3)N(3)), 137.67 (C, Ar), 129.88 (CH, Ar), 123.79 (CH, Ar), 122.89
(N(1)CHCHN(3)), 121.16 (N(1)CHCHN(3)), 117.99 (C-C=N), 71.03 (CH, Cyhex), 50.57
(N(3)-CH2-Ar), 35.09 (N(1)-CH3), 34.83 (CH2, Cyhex),
32.87 (CH(CH3)2), 29.37
(CH(CH3)2), 24.12 (CH2, Cyhex), 9.79 (C(2)-CH3). ESI MS: m/z 624.4 (<5%, [C38H52N6O2]+•
= [M – 2Cl– + e–]+•), 431.3 (20%, [C38H52N6O2 – Me2ImH+ – Me2Im]+), 312.2 (50%,
[C38H52N6O2]2+), 264.2 (100%, [C38H52N6O2 – Me2Im]2+), 208.5 (45%, [C38H52N6O2 –
2+
·
Me2ImH – Me2ImCH2] ). Anal. Calcd. for C38H52Cl2N6O2 H2O (M = 713.80): C, 63.94; H,
7.63; N, 11.77; Found: C, 63.83; H, 7.83; N, 11.49.
26
27
28
29
N,N'-Bis-[3-iso-propyl-5-((1,2-dimethylimidazol-3-ium)methylene)-salicylidene)-rac-trans-
1,2-cyclohexanediamine bis-(hexafluorophosphate) monohydrate (4b): Yellow powder,
(3.39 g, 91 %); mp 84-85 °C. FT-IR (KBr, cm-1): 3438 (m, br, ν(O-H)), 3181 (m, sh, νasym(C-H)
Im and Ar), 3154 (m, sh, νsym(C-H), Im and Ar), 2938 (m, sh, ν(CH )) 2869 (m, sh, ν(C-H)), 1633
,
3
30
31
(vs, sh, ν(C=N)), 1539, 1466, 1389 (s, sh, ν(C=C
bend)), 1324 (m, sh, ν(C-H)), 1273 (s, sh, ν(Ar-
+ C-H
Ar
-
O)), 1160 (s, sh, ν(H-C=C + H-C=N) , Im), 838 (vs, sh, ν(PF )str), 774 (m, sh), 676 (m, sh), 557 (s,
bend
6
32
33
sh, δ(P-F)). 1H NMR (200 MHz, DMSO-d6) δ (ppm): 13.98 (s, 2 H, 2 x Ar-OH), 8.53 (s, 2 H, 2
x H-C=N), 7.66 (d, J = 2.12 Hz, 2 H, 2 x Im-H), 7.62 (d, J = 2.02 Hz,, 2 H, 2 x Im-H), 7.35
5

1
2
(d, J = 1.99 Hz, 2 H, 2 x Ar-H), 7.21 (d, J = 2.21 Hz, 2 H, 2 x Ar-H), 5.27 (s, 4 H, 2 x N(3)-
CH2-Ar), 3.75 (s, 6 H, 2 x N(1)-CH3), 3.51 (s, br, 2 H, 2 x Cyhex-H), 3.22 (m(7), 2 H, 2 x
CH(CH3)2), 2.61 (s, 6 H, 2 x C(2)-CH3), 1.83 (m, 4 H, 4 x Cyhex-H), 1.55 (m, 4 H, 4 x
3
13
4
Cyhex-H), 1.34 (dd, 12 H, J1 = 1.62 Hz, J2 = 6.93 Hz, 2 x CH(CH3)2). C NMR (125 MHz,
5
DMSO-d6) δ (ppm): 165.96 (HC=N), 160.11 (C-OH), 144.68 (N(1)C(CH3)N(3)), 137.68 (C,
Ar), 129.94 (CH, Ar), 123.90 (CH, Ar), 122.91 (N(1)CHCHN(3)), 121.19 (N(1)CHCHN(3)),
118.51 (C-C=N), 71.14 (CH, Cyhex), 50.64 (N(3)-CH2-Ar), 35.12 (N(1)-CH3), 32.91 (CH2,
Cyhex), 29.38 (CH(CH3)2), 26.73 (CH(CH3)2), 24.07 (CH2, Cyhex), 9.81 (C(2)-CH3) (see
Figure S1 in supplementary data). 31P NMR (202 MHz, DMSO-d6): –142.97 ppm (septet, 2JPF
= 711.24 Hz). 19F NMR (470 MHz, DMSO-d6): –70.58 ppm (doublet, 1JFP = 715.68 Hz) (see
Figure S2 in supplementary data). HR-ESI-MS: 527.3376 (<5%, [C38H52N6O2 – Me2ImH•]+),
431.2691 (25%, [C28H35N2O2]+ = [C38H52N6O2 – Me2ImH+ – Me2Im ]+), 312.2074 (100%,
6
7
8
9
10
11
12
13
14
15
16
17
18
[C38H52N6O2]2+), 264.1729 (60%, [C33H44N4O2]2+ = [C38H52N6O2 – Me2Im]2+), 208.47409
2+
+
(10%, [C38H52N6O2 – Me2ImH – Me2ImCH2] ). MALDI-TOF MS, m/z: 849.4 [M DIT –
– +
– +
H+ – 2 PF6 ] , 769.4 (<10%, [M – PF6 ] ), 657.3 (40%, [C38H52N6O2 + DIT – Me2ImH+ –
Me2Im]+), 431.2 (75%, [C38H52N6O2 – Me2ImH+ – Me2Im]+), 227.0 (100%, [DIT+H+]+).
·
Anal. Calcd. for C38H52F12N6O2P2 H2O (M = 932.80): C, 48.93; H, 5.83; N, 9.01; Found: C,
49.14; H, 5.75; N, 9.20.
19
20
21
22
Rac-trans-N,N'-Bis-[3-iso-propyl-5-((1,2-dimethylimidazol-3-ium)methylene)-salicylidene)-
rac-trans-1,2-cyclohexanediamine bis-(tetrafluoroborate) monohydrate (4c): Pale yellow
-1
−78 °C
powder, (3.04 g, 93 %); mp 76
. FT-IR (KBr, cm ): 3441 (m, br, ν(O-H)), 3183 (m, sh,
ν
asym(C-H), Im and Ar), 3151 (m, sh, νsym(C-H), Im and Ar), 2936 (m, sh, ν(CH )) 2867 (m, sh, ν(C-
3
23
24
H)), 1632 (vs, sh, ν(C=N)), 1539, 1466, 1388 (s, sh, ν(C=C
bend)), 1324 (m, sh, ν(C-H)), 1274
+ C-H
Ar
-
(s, sh, ν(Ar-O)), 1159 (s, sh, ν(H-C=C + H-C=N) , Im), 1060 (vs, sh, ν(BF )str), 865 (m, sh), 771 (m,
bend
4
25
26
27
28
29
30
31
32
33
sh), 678 (m, sh). 1H NMR (200 MHz, DMSO-d6) δ (ppm): 13.98 (s, 2 H, 2 x Ar-OH), 8.54 (s,
2 H, 2 x H-C=N), 7.66 (d, J = 2.08 Hz, 2 H, 2 x Im-H), 7.62 (d, J = 2.05 Hz, 2 H, 2 x Im-H),
7.36 (d, J = 1.96 Hz, 2 H, 2 x Ar-H), 7.22 (d, J = 2.08 Hz, 2 H, 2 x Ar-H), 5.27 (s, 4 H, 2 x
N(3)-CH2-Ar), 3.75 (s, 6 H, 2 x N(1)-CH3), 3.52 (s, br, 2 H, 2 x Cyhex-H), 3.22 (m(7), 2 H, 2
x CH(CH3)2), 2.61 (s, 6H, 2 x C(2)-CH3), 1.82 (m, 4 H, 4 x Cyhex-H), 1.50 (m, 4 H, 4 x
Cyhex-H), 1.17 (dd, J2 = 6.87 Hz, J1 = 1.32 Hz, 12 H, 2 x CH(CH3)2). 13C NMR (125 MHz,
DMSO-d6) δ (ppm): 165.59 (HC=N), 158.77 (C-OH), 144.70 (N(1)C(CH3)N(3)), 136.53 (C,
Ar), 129.36 (CH, Ar), 124.52 (CH, Ar), 122.91 (N(1)CHCHN(3)), 121.20 (N(1)CHCHN(3)),
118.07 (C-C=N), 71.44 (CH, Cyhex), 50.61 (N(3)-CH2-Ar), 35.13 (N(1)-CH3), 33.13 (CH2,
6

1
2
3
4
5
6
7
Cyhex), 26.41 (CH(CH3)2), 24.41 (CH(CH3)2), 22.67 (CH2, Cyhex), 9.84 (C(2)-CH3) (see
19
Figure S4 in supplementary data). F NMR (470 MHz, DMSO-d6): -148.72 ppm (singlet)
+
+
(see Figure S5 in supplementary data). MALDI-TOF MS, m/z: 849.5 (10%, [M DIT – H –
– +
– +
2 BF4 ] ), 711.4 (25%, [M – BF4 ] ), 657.4 (60%, [C38H52N6O2 + DIT – Me2ImH+ –
Me2Im]+), 431.3 (100%, [C38H52N6O2 – Me2ImH+ – Me2Im]+), 227.0 (90%, [DIT+H+]+).
Anal. Calcd. for C38H52B2F8N6O2 H2O (M = 816.48): C, 55.90; H, 6.67; N, 10.29; Found: C,
56.29; H, 6.66; N, 10.35.
8
9
3.2 General procedure for the preparation of chlorido-metallosaldach-bis-imidazolium
complexes [M(III)Cl{(iPr)2saldach(Me2Im+-X–)2}] (M = Mn, Fe) (5a-c, 6a-c)
10
11
12
13
14
15
16
17
18
19
20
3.2.1. Synthesis of Mn(III) complexes (5a-c):
A yellow solution of the saldach-bis-imidazolium salts, H2(iPr)2saldach(Me2Im+-X–)2 4a-c,
(0.9 mmol) in ethanol (10 mL) was degassed for 15 minutes. An ethanolic solution (5 mL) of
·
Mn(OAc)2 4H2O (269 mg, 1.1 mmol) was then added with the yellow solution turning dark
brown immediately, and the reaction mixture was refluxed for 2 hours under N2. LiCl (69.9
mg, 1.65 mmol) was then added and the solution was refluxed for an additional 2 h under air
bubbling through the solution. After evaporating the solvent under reduced pressure, the
residue was re-dissolved in CH2Cl2 (3 ml), over-layered ethyl acetate (3 ml) and the mixture
kept in a refrigerator overnight. The precipitated solid was filtered off and washed with ethyl
acetate and diethyl ether. Recrystallization from CH2Cl2/n-hexane yielded pure
[Mn(III)Cl{(iPr)2saldach(Me2Im+-X–)2}] 5a-c.
21
22
23
24
Chlorido-trans-[[2,2`-][(1,2-cyclohexanediyl) bis(nitrilomethylidyne)] bis[4-((1,2-dimethyl-
imidazolium)methylene-6-(iPr-phenolato)]-[N,N`,O,O`]
manganese(III)
dichloride
-1
·
sesquihydrate (5a 1.5H2O): Dark-brown powder (679 mg, 93 %). FT-IR (KBr, cm ): 3434
(m, br, ν(O-H), lattice water), 3132 (m, sh, νasym(C-H), Im and Ar), 2948 (m, sh, ν(CH )) 2864 (m,
3
25
26
sh, ν(C-H)), 1620 (vs, sh, ν(C=N)), 1547, 1439, 1387 (s, sh, ν(C=C
bend)), 1332 (m, sh, ν(C-H)),
+ C-H
Ar
1280 (s, sh, ν(Ar-O)), 1179 (s, sh, ν(H-C=C + H-C=N)bend, Im), 832 (m, sh), 761 (m, sh), 678 (m, sh),
+
27
28
29
30
31
32
575 (m, sh, ν(Mn-N)), 474 (w, br, ν(Mn-O)). MALDI-TOF MS, m/z: 849.5 (10%, [ligand 4a DIT
– H+ – 2 Cl–]+)+, 710.3 (10%, [C38H50N6O2Mn + DIT – Me2ImH+ – Me2Im]+), 657.3 (30%,
[C38H52N6O2 + DIT – Me2ImH+ – Me2Im]+), 520.2 (20%, [C38H50N6O2MnCl – 2 Me2Im]+),
506.1 (5%, [Mn(DIT)2 – H+]+, 431.3 (20%, [C38H52N6O2 – Me2ImH+ – Me2Im]+), 227.0
+ +
·
(100%, [DIT+H ] ). Anal. Calcd. for C38H50Cl3MnN6O2 1.5H2O (M = 811.16): C, 56.27; H,
6.59; N, 10.36; Found: C, 56.16; H, 6.23; N, 10.29. µeff = 4.86 µB.
7

1
2
3
4
Chlorido-trans-[[2,2`-][(1,2-cyclohexanediyl) bis(nitrilomethylidyne)] bis[4-((1,2-dimethyl-
imidazolium)methylene-6-(iPr-phenolato)]-[N,N`,O,O`] manganese(III) bis-(hexafluoro-
-1
·
phosphate) monohydrate (5b H2O): Brown powder (835 mg, 91 %). FT-IR (KBr, cm ): 3436
(m, br, ν(O-H), lattice water), 3157 (m, sh, νasym(C-H), Im and Ar), 2956 (m, sh, ν(CH )) 2868 (m,
3
5
6
sh, ν(C-H)), 1619 (vs, sh, ν(C=N)), 1549, 1440, 1389 (s, sh, ν(C=C
bend)), 1332 (m, sh, ν(C-H)),
+ C-H
Ar
-
1279 (s, sh, ν(Ar-O)), 1179 (s, sh, ν(H-C=C + H-C=N) , Im), 840 (vs, sh, ν(PF )str), 840 (s, sh), 782
bend
6
7
8
(m, sh), 740 (m, sh), 678 (m, sh), 573 (m, sh, ν(Mn-N)), 559 (m, sh, δ(P-F)), 473 (w, br, ν(Mn-O)).
MALDI-TOF MS, m/z: ligand spectrum with 657.3 and 431.3; and 710.3 (5%,
[C38H50N6O2Mn + DIT – Me2ImH+ – Me2Im]+), 520.2 (10%, [C38H50N6O2MnCl – 2
Me2Im]+), 506.0 (<5%, [Mn(DIT)2 – H+]+ 227.0 (100%, [DIT+H+]+). Anal. Calcd. for
9
10
11
12
·
C38H50ClF12MnN6O2P2 H2O (M = 1021.18): C, 44.69; H, 5.13; N, 8.23; Found: C, 44.55; H,
5.19; N, 8.20. µeff = 4.96 µB.
13
14
15
16
Chlorido-trans-[[2,2'-][(1,2-cyclohexanediyl) bis(nitrilomethylidyne)] bis[4-((1,2-dimethyl-
imidazolium)methylene-6-(iPr-phenolato)]-[N,N`,O,O`] manganese(III) bis-(tetrafluoro-
-
·
borate) sesquihydrate (5c 1.5H2O): Reddish-brown powder (724 mg, 88 %). FT-IR (KBr, cm
1): 3437 (m, br, ν(O-H), lattice water), 3149 (m, sh, νasym(C-H), Im and Ar), 2958 (m, sh, ν(CH )
)
3
17
18
2867 (m, sh, ν(C-H)), 1620 (vs, sh, ν(C=N)), 1548, 1441, 1389 (s, sh, ν(C=C
bend)), 1334 (m,
+ C-H
Ar
-
sh, ν(C-H)), 1281 (s, sh, ν(Ar-O)), 1178 (s, sh, ν(H-C=C + H-C=N) , Im), 1058 (vs, sh, ν(BF )str), 830
bend
4
19
20
21
22
23
24
(m, sh), 779 (m, sh), 756 (m, sh), 679 (m, sh), 572 (m, sh, ν(Mn-N)), 471 (w, br, ν(Mn-O)).
MALDI-TOF MS, m/z:ligand spectrum with 849.5, 657.4 and 431.3, and 710.3 (15%,
[C38H50N6O2Mn + DIT – Me2ImH+ – Me2Im]+), 520.2 (20%, [C38H50N6O2MnCl – 2
Me2Im]+), 506.0 (<5%, [Mn(DIT)2 – H+]+, 227.0 (100%, [DIT+H+]+). Anal. Calcd. for
·
C38H50B2ClF8MnN6O2 1.5H2O (M = 913.33): C, 49.94; H, 5.85; N, 9.20; Found: C, 49.63; H,
5.91; N, 9.44. µeff = 4.91 µB.
25
26
27
28
29
30
31
32
3.2.2. Synthesis of Fe(III) complexes (6a-c):
A yellow solution of the saldach-bis(imidazolium) salts H2(iPr)2saldach(Me2Im+-X–)2 4a-c
(0.9 mmol) in ethanol (10 mL) was degassed for 15 minutes. An ethanolic solution (5 mL) of
FeCl3 (177 mg, 1.1 mmol) was then added with the yellow solution turning dark reddish-
brown immediately, and the reaction mixture was refluxed for 2 hours under N2. Then, the
solution was concentrated and the residue was kept in a refrigerator overnight. The
precipitated solid was filtered off and washed with cold ethanol (2 x 3mL) and diethyl ether (3
x 3mL) to yield [Fe(III)Cl{(iPr)2saldach(Me2Im+- X–)2}] 6a-c.
8

1
2
3
4
Chlorido-trans-[[2,2`-][(1,2-cyclohexanediyl) bis(nitrilomethylidyne)] bis[4-((1,2-dimethyl-
imidazolium)methylene-6-(iPr-phenolato)]-[N,N`,O,O`] iron(III) dichloride sesquihydrate
-1
·
(6a 1.5H2O): reddish-brown powder (687 mg, 94 %). FT-IR (KBr, cm ): 3429 (m, br, ν(O-H)
,
lattice water), 3137 (m, sh, νasym(C-H), Im and Ar), 2935 (m, sh, ν(CH )) 2865 (m, sh, ν(C-H)),
3
5
6
1613 (vs, sh, ν(C=N)), 1549, 1449, 1388 (s, sh, ν(C=C
bend)), 1334 (m, sh, ν(C-H)), 1278 (s,
+ C-H
Ar
sh, ν(Ar-O)), 1177 (s, sh, ν(H-C=C + H-C=N)bend, Im), 834 (m, sh), 745 (m, sh), 678 (m, sh), 570 (m,
7
8
sh, ν(Fe-N)), 498 (w, br), 467 (m, sh, ν(Fe-O)). MALDI-TOF MS, m/z: 746.3 (10%,
[C38H50N6O2FeCl + DIT – Me2ImH+ – Me2Im]+), 711.3 (60%, [C38H50N6O2Fe + DIT –
Me2ImH+ – Me2Im]+), 552.2 (30%, [C38H50N6O2FeCl(OMe) – 2 Me2Im]+, 521.2 (80%,
[C38H50N6O2FeCl – 2 Me2Im]+), 507.1 (20%, [Fe(DIT)2 – H+]+, 226.9 (100%, [DIT+H+]+).
9
10
11
12
·
Anal. Calcd. for C38H50Cl3FeN6O2 1.5H2O (M = 812.07): C, 56.20; H, 6.58; N, 10.35; Found:
C, 56.14; H, 6.42; N, 10.72. µeff = 5.63 µB.
13
14
15
16
17
Chlorido-trans-[[2,2`-][(1,2-cyclohexanediyl) bis(nitrilomethylidyne)] bis[4-((1,2-dimethyl-
imidazolium)methylene-6-(iPr-phenolato)]-[N,N`,O,O`] iron(III) bis-(hexafluorophosphate)
-1
·~
monohydrate (6b 2H2O): Brown powder (842 mg, 90 %). FT-IR (KBr, cm ): Dark-brown
powder (556 mg, 93 %). FT-IR (KBr, cm-1): 3430 (m, br, ν(O-H), lattice water), 3145 (m, sh,
ν
asym(C-H), Im and Ar), 2954 (m, sh, ν(CH )) 2867 (m, sh, ν(C-H)), 1615 (vs, sh, ν(C=N)), 1549,
3
18
19
1442, 1391 (s, sh, ν(C=C
bend)), 1328 (m, sh, ν(C-H)), 1279 (s, sh, ν(Ar-O)), 1178 (s, sh, ν(H-
+ C-H
Ar
-
bend, Im), 842 (vs, sh, ν(PF )str), 780 (m, sh), 740 (m, sh), 680 (m, sh), 557 (m, sh,
C=C + H-C=N)
6
20
21
22
23
24
δ(P-F)), 572 (w, br, ν(Fe-N)), 471 (m, br, ν(Fe-O)). MALDI-TOF MS, m/z: 746.3 (<5%,
[C38H50N6O2FeCl + DIT – Me2ImH+ – Me2Im]+), 711.3 (45%, [C38H50N6O2Fe + DIT –
Me2ImH+ – Me2Im]+), 521.2 (40%, [C38H50N6O2FeCl – 2 Me2Im]+), 507.1 (20%, [Fe(DIT)2 –
+ +
+ +
·~
H ] , 227.0 (100%, [DIT+H ] ). Anal. Calcd. for C38H50ClF12FeN6O2P2 2H2O (M =
1040.10): C, 43.88; H, 5.23; N, 8.08; Found: C, 44.01; H, 5.20; N, 8.11. µeff = 5.86 µB.
25
26
27
28
Chlorido-trans-[[2,2'-][(1,2-cyclohexanediyl) bis(nitrilomethylidyne)] bis[4-((1,2-dimethyl-
imidazolium)methylene-6-(iPr-phenolato)]-[N,N`,O,O`] iron(III) bis-(tetrafluoroborate)
-1
·
monohydrate (6c H2O): Dark-red powder (816 mg, 89 %). FT-IR (KBr, cm ): 3442 (m, br,
ν
(O-H), lattice water), 3150 (m, sh, νasym(C-H), Im and Ar), 2938 (m, sh, ν(CH )) 2868 (m, sh, ν(C-
3
29
30
H)), 1616 (vs, sh, ν(C=N)), 1551, 1453, 1391 (s, sh, ν(C=C
bend)), 1329 (m, sh, ν(C-H)), 1282
+ C-H
Ar
-
(s, sh, ν(Ar-O)), 1179 (s, sh, ν(H-C=C + H-C=N) , Im), 1060 (vs, sh, ν(BF )str), 834 (m, sh), 748 (m,
bend
4
31
32
sh), 678 (m, sh), 573 (m, sh, ν(Fe-N)), 497 (w, br), 467 (m, sh, ν(Fe-O)), 443 (w, br). MALDI-
TOF MS, m/z: 711.3 (20%, [C38H50N6O2Fe + DIT – Me2ImH+ – Me2Im]+), 521.2 (15%,
9

1
2
3
[C38H50N6O2FeCl – 2 Me2Im]+), 507.1 (5%, [Fe(DIT)2 – H+]+, 227.0 (100%, [DIT+H+]+).
·
Anal. Calcd. for C38H50B2ClF8FeN6O2 H2O (M = 905.77): C, 50.39; H, 5.79; N, 9.28; Found:
C, 50.54; H, 5.78; N, 9.33. µeff = 5.75 µB.
4
5
3.3 In vitro anticancer (cytotoxicity) activity
3.3.1 Reagents: Dimethylsulphoxide (DMSO), crystal violet and Trypan blue dye were
purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Dulbecco’s modified Eagle’s
medium (DMEM), Roswell Park Memorial Institute medium (RPMI-1640), Fetal Bovine
Serum (FBS), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer solution,
L-glutamine, gentamycin and 0.25% Trypsin- EDTA were obtained from Lonza.
Crystal violet stain: prepared as 0.5% crystal violet solution by dissolving 0.5 g of crystal
violet stain in 50 mL methanol, then the solution was completed to 100 mL with deionized
water, filtered through Whatmann No.1 and stored at room temperature.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
3.3.2 Cell cultures: Two human tumor cell lines, MCF-7 (breast adenocarcinoma) and Hepg2
(liver hepatocellular carcinoma) were obtained from the VACSERA Tissue Culture Unit and
cultured in either RPMI-1640 or DMEM media supplemented with 10% heat-inactivated
FBS, 1% L-glutamine, HEPES buffer and 50 µg/mL gentamycin. All cells were maintained at
37 °C in a humidified atmosphere with 5% CO2 and sub-cultured two times a week. Cell
toxicity was monitored by determining the effect of the tested samples on cell morphology
and cell viability. The effect of the vehicle solvent (DMSO) on the growth of these cell lines
was evaluated in all the experiments by exposing untreated control cells to the maximum
concentration (0.5%) of DMSO used in each assay.
22
23
24
25
26
27
28
29
30
31
32
33
3.3.3 Cytotoxicity assay: The in vitro cytotoxicity of the saldach-bis-imidazolium salts and
their chlorido M(III) complexes were measured by the cytotoxic effect assay according to the
procedure adopted by the Regional Center for Mycology & Biotechnology, Egypt. Briefly, the
cells were seeded in 96-well plates at a cell concentration of 1×104 cells per well in 100 µL of
growth medium. Fresh medium containing different concentrations of the tested sample was
added after 24 h of seeding. Serial two-fold dilutions of the tested compound were added to
confluent cell mono layers dispensed into 96-well, flat-bottomed micro titer plates (Falcon,
NJ, USA) using a multichannel pipette. The micro-titer plates were incubated at 37 ºC in a
humidified incubator with 5% CO2 for a period of 48 h. Three wells were used for each
sample concentration. Control cells were incubated without tested sample and with or without
DMSO. The little percentage of DMSO present in the wells (maximal 0.1%) is not affecting
the experiment. After incubation of the cells for 24 h at 37 °C, various concentrations of
10

1
2
sample (1.50-70.04 µM) were added, and the incubation was continued for 48 h then the
viable cells yield was determined by a colorimetric method. After the end of the incubation
period, media were aspirated and the crystal violet solution (1%) was added to each well for at
least 30 minutes. The stain was removed and the plates were rinsed using tap water until all
excess stain was removed. Glacial acetic acid (30%) was then added to all wells and mixed
thoroughly, and then the absorbance of the plates were measured after gently shaken on Micro
plate reader (TECAN, Inc.), using a wavelength of 490 nm. All results were corrected for
background absorbance detected in wells without stain. Treated samples were compared with
the cell control in absence of the tested compounds. All experiments were carried out in
triplicate and the average values were calculated. The cell cytotoxic effect of each tested
compound was calculated [32].
3
4
5
6
7
8
9
10
11
12
3.4 Antibacterial survey
13
14
3.4.1 Reagents: Dimethylsulphoxide (DMSO) and Ampicillin antibiotic were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
15
16
17
18
19
20
21
22
23
24
25
26
3.4.2 Bacterial cultures: Multi-drug resistant (MDR) strains used in this study from National
Organization for Drug Control and Research (NODCAR), Cairo, Egypt. The different strains
are Staphylococcus aureus (S. aureus, ATCC-29737), Staphylococcus epidermidis (S.
epidermidis, ATCC-12228), Streptococcus pneumoniae and (S. pneumoniae, ATCC-49619)
as representatives for the Gram-positive bacteria and Escherichia coli (E. coli, ATCC-10536),
Pseudomonas aeruginosa (P. aeruginosa, ATCC-27853), Shigella flexneri (S. flexneri,
ATCC-12022), Klebsiella pneumonia (K. pneumonia, ATCC-13883) and Neisseria
meningitidis (N. meningitidis, ATCC-13090) as the most important Gram-negative pathogenic
bacteria. Stock cultures grown aerobically on nutrient broth (NB) agar slants (Hi-Media) at
37°C were maintained at 4°C. Pre-cultures containing 105 CFU/ml, grown aerobically in
Mueller Hinton (MH) liquid medium (Hi-Media) at 37°C for 5 h, were used as inoculum for
all experiments.
27
28
29
30
31
32
3.4.3 Bactericidal assay (antibacterial susceptibility testing): Antibacterial susceptibility of
the bacterial strains was carried out by agar well diffusion method [33] towards the most
potent cytotoxic compounds (4a-c). As an indicator of antibacterial activity, the clear zone
around the wells was measured as inhibition zones and the diameter of these zones of
inhibition (ZOI, mm) were measured accurately using a transparent meter rule and recorded if
the zone of inhibition was ≥10 mm [34]. Duplicates were maintained in each extract and the
11

1
2
average values were calculated for the eventual antibacterial activity. Ampicillin,
Antibacterial, was employed as standard drugs
3
4
Determination of MIC and MBC; As a parameter of the antibacterial efficacy, the minimal
inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of new
compounds (4a-c) against Gram-positive and Gram-negative isolates were determined using
the macro-dilution broth susceptibility test. Freshly prepared MH broth was used as diluents
in the macro-dilution method. A serial dilution of each target compound was prepared within
a desired range (0.12 mM to 28.02 mM). One milliliter of the Stock cultures was then
inoculated and tubes were incubated at 37 °C for 24 h, control tubes without any addition
were assayed simultaneously. MIC was examined visually, by checking the turbidity of the
tubes. Furthermore the tubes having lesser concentration than MIC level were inoculated on
MHA plate for MBC determination.
5
6
7
8
9
10
11
12
13
14
15
4. Results and Discussion
4.1 Chemistry
4.1.1 Synthesis of rac-trans-(H2(iPr)2saldach(Me2Im+-X–)2) (X = Cl, 4a; PF6, 4b; BF4,4 c)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
The synthesis of saldach-scaffold bearing bis-imidazolium terminal compartments 4a-c is
depicted in Scheme 2. The key starting materials salicylaldehyde-imidazolium salts 3a-c were
synthesized starting from 2-iso-propylphenol following modified literature procedures [35].
Under extra dry conditions and using anhydrous magnesium dichloride as O-magnesiation
reagent, 2-iso-propylphenol was ortho-formylated by paraformaldehyde in the presence of
catalytic amounts of triethylamine. 3-iso-Propylsalicylaldehyde was then chloromethylated to
yield 5-chloromethyl-3-iso-propylsalicylaldehyde 2 in high purity (see Scheme 2A).
Compound 2 allowed the preparation of a common precursor, 3a, through the quarternization
reaction of 1,2-dimethylimidazole with 2 to afford the salicylaldehyde-imidazolium chloride
(3a). This salt was then readily metathesized into the corresponding hexafluorophosphate and
tetrafluoroborate salts (3b,c) via reaction with aqueous hexafluorophosphoric acid, HPF6(aq),
and sodium tetrafluoroborate, NaBF4, respectively. The ligands, rac-trans-
H2(iPr)2saldach(Me2Im+-X–)2 (4a-c), were synthesized by Schiff-base condensation reaction
between compounds 3a-c and ( )-trans-1,2-diaminocyclohexane (rac-trans-dach) in
methanolic solution (see Scheme 2B). The rac-trans-configuration of the N,N′-
bis(salicylidene)-1,2-cyclohexanediamine (saldach) backbone was selected, because metal-
ligand binding will force the ligand to get a flat chair (planar) conformation, similar to the
12

1
2
3
4
5
6
7
1,2-cyclohexanediamine moiety present in oxaliplatin, a very promising metal-based drug
[36]. Furthermore, the imine bond and salicyl effect lead to a very rigid configuration with
only possible rotation around the N-C axis of diaminocyclohexane group. The saldach-
bis(imidazolium) salts 4a-c were isolated in high yields and characterized by FTIR, UV/Vis,
1H NMR, 13C NMR, 19F NMR, 31P NMR, ESI-MS, MALDI-TOF MS and conductivity
measurements.
(Scheme 2)
8
9
4.1.1 Synthesis of the metallosaldach-imidazolium salts [M(III)Cl{(iPr)2saldach(Me2Im+-
X–)2}] (M = Mn, 5; Fe, 6), (X = Cl, a; PF6, b; BF4, c)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
A two-step one-pot preparative route (see Scheme 3) was used to prepare the chlorido
Mn(III) complexes, [Mn(III)Cl{(iPr)2saldach(Me2Im+-X–)2}] (X = Cl, PF6, BF4) (5a-c). This
synthetic route involve exchange of acetate ligands in Mn(OAc)2·4H2O by
(iPr)2saldach(Me2Im+-X–)2 under anaerobic conditions (N2-atmosphere). Then spontaneous
oxidation of Mn(II) center into Mn(III) species was allowed by molecular oxygen [37],
followed by coordination of a chloride ion from lithium chloride (LiCl).
(Scheme 3)
The Fe(III) complexes, [Fe(III)Cl{(iPr)2saldach(Me2Im+-X–)2}] (X = Cl, PF6, BF4) (6a-c),
were prepared by refluxing a solution of the corresponding saldach ligands,
(H2(iPr)2saldach(Me2Im+-X–)2), with anhydrous Fe(III) chloride in methanol (UV-
spectroscopy grade) under aerobic conditions (cf. Scheme 3).
Unfortunately all attempts to obtain X-ray diffraction quality single crystals of the
Mn(III)- and Fe(III)-saldach-imidazolium chlorides ((5,6)a-c) were unsuccessful. Yet, the
metal-ligand structures suggested in this work based upon elemental and spectral analysis
(FTIR, UV-Vis, MALDI-TOF [38] conductivity as well as magnetic measurements and match
with the structures of reported metal-saldach analogues (Table S1, supplementary data).
26
27
28
29
30
31
32
33
4.2 Characterizations of the saldach-bis(imidazolium) salts and their complexes
4.2.1 Microanalytical data, conductivity and mass spectrometry
Saldach-bis(imidazolium) salts (4a-c) and their complexes were prepared in high yields,
gave satisfactory C, H, and N elemental analyses, which are consistent with the proposed
formula for the ligands and their chelate complexes (see the Experimental section).
Molar conductance values of all the saldach-bis(imidazolium) salts and their complexes in
EtOH (1 × 10−3 M) at 25 °C are in the region of 49.1-84.5 and 51.3-95.0 µS/cm, respectively,
in accordance with their ionic nature. The molar conductivities of the saldach-
13

1
2
bis(imidazolium) salts H2(iPr)2saldach(Me2Im+-X-)2 decrease in the following order: 4c (X =
BF4) > 4a (X =Cl) > 4b (X = PF6).
3
The HR-ESI+ mass spectra of 4a and 4b show dominant signals for the doubly-charged
cation [C38H52N6O2]2+ and species assuming both imidazolium groups have been lost,
[C38H52N6O2 – Me2ImH+ – Me2Im ]+ and [C38H52N6O2 – Me2Im]2+.
4
5
6
The MALDI-TOF spectra of 4b and 4c display signals corresponding to the cation-(DIT–
+
+
– +
– +
+
7
H )-matrix adduct [M DIT – H – 2 X6 ] , the cation with one anion [M – X ] , the cation
where both imidazolium groups have been lost (with and without DIT) [C38H52N6O2 + DIT –
Me2ImH+ – Me2Im]+ and [C38H52N6O2 – Me2ImH+ – Me2Im]+. The protonated DIT+H+
matrix signal is usually the base peak. The DIT-matrix adduct ion is formed as a result of the
intermolecular hydrogen bonding with the phenolic oxygen and π-π stacking of the aromatic
rings [39].
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
In the metal complexes 5,6a-c the metal-containing ions consist of metal-ligand species
where both imidazolium groups have been lost, [C38H50N6O2M + DIT – Me2ImH+ – Me2Im]+
and [C38H50N6O2MCl – 2 Me2Im]+. Either the chlorido ligand is retained or replaced by a DIT
ligand. Additionally, a signal for a metal-bis(DIT) complex, [M(DIT)2 – H+]+, is also
observed. In the iron complexes 6a,b also a signal for [C38H50N6O2FeCl + DIT – Me2ImH+ –
Me2Im]+ was detected. The protonated DIT+H+ matrix signal is also the base peak in the
MALDI-TOF-MS of the metal complexes.
4.2.2 IR spectroscopic data
The most informative evidence confirming the anchoring of central saldach backbone to
the terminal imidazolium groups was obtained by FT-IR spectra. All saldach-
bis(imidazolium) salts show a broad band at the range of 3436-3441 cm–1 attributed to the
stretching vibration of the intramolecular hydrogen bonded phenolic OH group. All saldach-
bis(imidazolium) salts contain two O–H⋯N intramolecular hydrogen bonds within the 2-
hydroxybenzylidene-imine moieties (Scheme 4) which is further confirmed by the
displacement of the azomethene (-HC=N-) stretching band of 4a-c to lower wavenumber, ca
1631 cm–1, compared with that of the free azomethene moiety [40,41]. Moreover, an
additional band around 1271 was observed for the samples, which was assigned to the
stretching vibration of Ar-O. The vibrational bands at ~865 and ~770 cm–1 are due to the in-
plane and out-of plane flexible vibrations of the imidazolium ring.
(Scheme 4)
14

1
2
The presence of the saldach-bis(imidazolium) salts as well as its binding mode within the
complexes was assigned on the basis of vibrational spectroscopy. The general displacement of
the ν(C=N) bands to lower frequencies compared to that of the free ligands (Figure 1) (see Table
S2, supplementary data) confirms the coordination of the azomethene nitrogen atom to the
metal center [42].
3
4
5
6
(Figure 1)
7
The appearance of a new band in the spectra of complexes around 570 cm–1, assigned to
νM(III)–N vibrations (M= Mn, Fe) [43], is further support for this coordination. Strong bands
attributable to phenolic oxygen ν(Ar–O) undergo positive frequency shift, which is evidence for
the ligation of the phenolate oxygen to metal ion, this is further confirmed by the appearance
of a new band at 467-474 cm-1 which is assigned to the νM–O vibration. The presence of ν(O-H)
bands around 3430 cm-1 confirms the hydrate nature suggested by the analytical data for new
complexes.
8
9
10
11
12
13
14
15
The IR spectral studies reveal that, the imine nitrogen atoms and phenolate oxygen atoms
coordinate to a metal atom.
16
4.2.3 NMR studies and tautomerism scenario
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
We could identify the possible tautomeric equilibria with their relative tautomer
population, in the saldach-bis-imdazolium salts 4a-c by comparing with the NMR studies of
the related salen and salophen Schiff base-based ligands (Scheme S1, supplementary data)
isolated in the former work [44,45] and salophen ligands [46]. These studies reveal that the
central salen/ salophen/ saldach backbone is in the expected O-protonated (enol-imine)
1
tautomeric form (cf. Scheme S1, supplementary data). However, at first glance in the H
NMR spectrum of the ligand 4a (Figure 2) appear to be quite complicated. It exhibits two
groups of signals due to a pair of tautomers with different populations, the neutral bis-
(enolimine) with some contribution of the ionic phenolate-iminium resonance structure (cf.
Scheme 4). Two singlets at 13.98 ppm, 2H, of phenolic O-H typical for bis-(enolimine)
tautomer and at 13.56 ppm, 1H, for O-H of enolimine/ phenolate-iminium tautomer. The NH
resonance (8.55 ppm) is a singlet which is consistent with the structure of the bis-(enolimine)
tautomer and a singlet/doublet set (8.62/ 8.43 ppm), this is agrees with enolimine/ phenolate-
1
iminium tautomer. Thus, H NMR spectrum demonstrates that, in 4a the central saldach
backbone is in the enolimine form with some contribution of the enolimine/ phenolate-
iminium form in the solution (cf. Scheme 4).
(Figure 2)
15

1
1
2
Contrary, examination of the H NMR spectra of H2(iPr)2saldach(1,2-Me2Im+-X-)2 (4b,c)
(see supplementary data) leads us to highlight the following aspects: (i) The singlets
corresponding to the phenol OH and azomethine (H-C=N) protons are observed at 13.98 ppm
(2H) and round 8.54 (2H), respectively. (ii) The central saldach backbone is in the O-
protonated tautomeric form not in the N-protonated tautomeric form and the downfield shift
of phenolic proton signal in all ligands as a result of the intramolecular H-bond to the imine
nitrogen (cf Scheme 4). (iii) The phenyl, imidazolium, methylene, cyclohexyl, methyl and
isopropyl protons are slight affected by anion metathesis, depending on the different
arrangement adopted by the protons.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Further evidence for the O-protonated tautomer in both compartments is provided by 13C
NMR spectroscopy (see supplementary data). Claramunt et al. remarked that the 13C
resonance varies from ∼160 ppm for an enol-imine to ∼180 ppm for a pure keto-enamine
tautomer [47], making the 13C resonance a powerful parameter to discern the relative
tautomeric population. The 13C NMR spectra of H2(iPr)2saldach(1,2-Me2Im+-X-)2 exhibits
resonance at ca 160.0 ppm typical for enol-imines tautomer (160.9 ppm) [48]. Furthermore,
signal around 166 ppm is observed, which can be assigned to the carbons of the aldimine (H-
C=N) moieties [49]. Thus, NMR studies of 4b,c reveal that, the acidic protons of the central
saldach backbone are bond to O and not bond to N.
1H-13C COSY NMR (HMQC) experiments (Figure S4, S8, supplementary data) prove
correlations between with the “imine” proton (H-C=N-Cyhex), which appears as a singlet
(8.53 ppm for X = PF6; 8.54 ppm for X = PF6), and aldimine carbon (H-C=N-Cyhex), which
appears at (165.96 ppm for X = PF6; 166.00 ppm for X = PF6). This correlation pattern has
already been observed for the O-protonated ligands H2(iPr)2saldach(1,2-Me2Im+-X-)2 and was
proposed for the pure O-protonated tautomer. These results demonstrate that the acidic
protons are bond to oxygen only in solution.
26
27
28
29
30
31
32
33
34
4.2.4 Electronic Absorption Spectroscopy and magnetic susceptibility
In H2(iPr)2saldach(1,2-Me2Im+-X–)2 (4a-c) the absorption centered at ca 222 and 259 nm
originate from the π→π* and n→π* transitions associated with the phenolic chromophor [50],
while the peak at 325 nm can be assigned to the π→π* transition involving the imine group
[50], (see Figure S16 in supplementary data).
By going from the free saldach-bis-imdazolium salts to their mononuclear complexes
[M(III)Cl{(iPr)2saldach(Me2Im+-X-)2}] ((5,6)a-c), the UV/Vis spectra give further evidence
for complexation where the characteristic bands of the neat saldach-bis-imdazolium salts are
red-shifted (Table S3, Figure S7-S10, supplementary data). The most important feature in the
16

1
2
3
4
5
6
7
8
9
near-UV region of the M(III)-saldach-imidazolium salts is the shift of the imine π→π*
transition band from 324 nm (ε x 103 = 0.48-1.10 M-1cm-1) to higher wavelengths over 400 nm (ε
x 103 = 0.46-0.77 M-1cm-1) which indicates the coordination of metal ion with ligands [51]. In
addition, the low intensity broad absorption round 500 nm (ε x 103 = 0.09-0.84 M-1cm-1) which
can be assigned to the three allowed d–d transitions expected for complexes with a square
pyramidal geometry, (dxz→dx2-y2), (dxy→ dx2-y2, dyz) and (dz2→dx2-y2) [52].
The magnetic moment values for the complexes (4.85-4.96 µB for Mn(III) saldach and
5.63-5.86 µB Fe(III) saldach) are also supportive of square pyramidal geometry with high spin
metal centers [53].
10
11
12
13
14
15
16
17
4.3 Pharmacology
Many clinical trials of new active pharmaceutical ingredients (API) end in failure due to
the low efficacy of the drug due to a limited bioavailability or solubility. With respect
amphiphilicity, lipophilicity and solubility of imidazolium units, the anchoring of
imidazolium compartments to the H2(iPr)2saldach provides a synergetic effect: it increases
water solubility and at the same time enhances the pharmacological effect. Also the
symmetric compounds, with two imidazolium units, are easier to synthesize than the
unsymmetric ones, with one imidazolium unit
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
4.3.1 In vitro cytotoxicity
The in vitro cytotoxicity of the saldach-bis(imdazolium) salts, H2(iPr)2saldach(1,2-
Me2Im+-X–)2, 4a-c as well as their metal complexes was evaluated in relation to the
anticancer drug doxorubicin (C27H29NO11, 543.52 g/mol) against human breast carcinoma
(MCF-7) and human hepatocellular carcinoma (HepG2) cell lines (Tables S5, S6
supplementary data). IC50 values of the target compounds are in the range of 18.1->50.9 µg
(Figure 3, Table 1). The cell viability assays revealed that, all compounds had inhibitory effects on
the growth of MCF-7 cell line more effectively than HepG2 cell line. Also H2(iPr)2saldach-
(Me2Im+)2X- (4a-c) are more effective in inducing cell death (cytotoxic) than their M(III)-
complexes [M(III)Cl{(iPr)2saldach(Me2Im+-X-)2}] ((5,6)a-c).
(Figure 3)
(Table 1)
Noteworthy, the saldach-bis-imidazolium chloride, hexafluorophosphate and
tetrafluoroborate (4a-c) showed different levels of cytotoxicity against MCF-7 cells where the
tetrafluoroborate salt (4c) (IC50 = 22.17 µM) is the most effective in inducing cell death and
the chloride derivative (4a) (IC50 = 33.34 µM) affect MCF-7 cell viability more than the
17

1
2
hexafluorophosphate one (4b) (IC50 = 40.41 µM). Lipophilicity and/or vulnerability to
hydrolytic cleavage seem to be the key structural features leading to the observed anion-
dependent cytotoxicity. Interestingly, the antitumor activity of imidazolium salts may
be due to their amphiphilic structure, in which the hydrophilic cationic segments, 1,2-
dimethylimidazolium, could have strong electrostatic interactions with the phosphate groups
3
4
5
-
-
6
of DNA and hydrogen-bonding association between the anions, BF4 and PF6 , with the DNA
bases [54]. Tetrafluoroborate salt, 4c, is more cytotoxic than hexafluorophosphate analog, 4b,
because of [BF4]− anions have higher tendency to establish, on average, more hydrogen bonds
with the DNA bases than the [PF6]− anion.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Notably, Fe(III)-saldach-bis-imidazolium (6a-c) complexes appeared to be slightly more
cytotoxic than Mn(III) analogs (5a-c). The IC50 values for the parent ligands and their metal
complexes suggest that, chelation of the saldach-bis-imdazolium salts to the Mn(III)/Fe(III)
ions significantly decreased their cytotoxicity. This is probably because the hydrogen bonding
interactions between two phenolic hydroxyl groups of the free ligands and the functional
groups positioned to the edges of DNA bases are no longer possible [55]. Notably, a
hydrophobic isopropyl substituents at the ortho-position of the phenolic moieties increase the
lipophilicity of the molecule and affect the propensity of a ligand to bind to DNA, contributed to
the enhanced cytotoxicity [56].
The lower cytotoxic activities of these metal complexes compared to the parent ligands may be
assigned to either difference in their mechanism of action, electronic effects and/or the
complex geometry. Where coordination metal ion to the ligand compel the cyclohexane ring to lie
perpendicular to the plan of complex [57] and may hinder or even prevent thier intercalation
with DNA nucleobases (Scheme 5). Consequently, Fe(III) and Mn(III) saldach complexes
may induce apoptosis via the mitochondrial pathway [58]. Furthermore, Fe(III) complexes are
more cytotoxic than Mn(III) complexes due to the ability of iron chelates to generate reactive
oxygen species (ROS) [59] that damage-induced signaling cascades in the tumor cells.
(Scheme 5)
28
29
30
31
32
33
34
4.3.2 Antibacterial Screening
The saldach-bis-imidazolium compounds 4a-c and standards drugs Gentamycin
(C21H43N5O7, 477.596 g/mol ), Tetracycline (C22H24N2O8 , 444.435 g/mol ), Amoxicillin
(C16H19N3O5S, 365.400 g/mol ) were in vitro assessed separately for their capacity to inhibit
the growth of the pathogenic bacterial strains by observing the inhibitory zone. From the
bactericidal activity data, ZOIs (Figure 4), Table S6 (see supplementary data) it has been
observed that, the selected compounds showed good to moderate activity against bacteria as
18

1
2
compared to known standard drugs. All compounds inhibited the growth of Gram-positive
strains slightly more effective than Gram-negative strains. This could be ascribed to their cell
walls structural differences, where the outer walls of Gram-negative species are more
3
4
complex
than
those
of
Gram-positive
organisms,
so
all
tested
5
compounds diffuse easily through the loose outer wall of Gram-positive bacteria. The antibacterial
activities of the target compounds decrease in the following order: chloride salt of saldach-
bis(imidazolium) chloride (4a) > hexafluorophosphate (4c) > tetrafluoroborate (4b). The
imidazolium units with chloride anions demonstrate weak antibacterial activity as revealed by
higher MIC/ MBC values. Exchange of the halide by other anions resulted in an increase of
bactericidal and bacteriostatic activities of imidazolium-supported saldach salts. For example,
the counterion-dependent antibacterial activity of H2(iPr)2saldach(1,2-Me2Im+-X–)2 against S.
aureus and E. coli follows the trend below:
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
X (MIC/ MBC mM)S. aureus: BF4 (1.33/ 4.99) > PF6 (1.37/ 10.11) > Cl (14.30/ 14.47)
X (MIC/ MBC mM)E. coli: BF4 (1.35/ 5.05) > PF6 (1.39/ 2.58) > Cl (3.50/ 3.46)
Similar results were obtained by Pernak et al. for imidazolium ionic liquids with an
appended alkoxy functional group and anions ([Cl], [BF4] and [PF6]) [60].
Also 4a showed greater antibacterial effect against different bacterial types while 4b and
4c exhibited high resistance against S. pneumoniea (ATCC 49619) and P. aeruginosa (ATCC
27853), respectively.
(Figure 4)
To investigate growth-inhibitory effects of 4a-c against S. aureus and E. coli, the minimum
inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were
determined by broth dilution method, from the percentages of inhibition at five different
concentration levels, 0.12 mM-28.02 mM, and the obtained results are recorded in Table 2.
The bacterial growth inhibition is susceptible to the concentration of the compound and the
activity is greatly enhanced at the higher concentration. The observed MIC and MBC values
for 4a, 4b and 4c against S. aureus and E. coli revealed that, although 4a has the greatest
antibacterial effect, ZOIs, compared to 4b and 4c it needs a high concentration to become
bactericidal and can be classified as a new good candidate in the fight against resistant
antibiotic bacteria.
The nature of the cell wall, geometry of molecule, positive charge density, hydrophilicity,
lipophilicity, pharmacokinetic factors, etc. play decisive roles in determining an antibacterial
activity of a Schiff-base and its metal complexes [61]. These factors may lead to enhanced
antibacterial activity in two different ways: (i) by interactions of the cationic imidazolium
19

1
2
group with the negatively charged microbial cell wall to enhance the bactericidal activity [61],
(ii) by the ability of the hydrophobic isopropyl substituents to help to penetrate the lipophilic
cell walls and enhance the bactericidal activity [62].
3
4
(Table 2)
5
6
5. Conclusion
7
In conclusion, a series of water-soluble bis-imidazolium salts, H2(iPr)2saldach(1,2-
Me2Im+-X–)2, and their Mn(III)/ Fe(III) complexes have been prepared. The isolated
compounds have been structurally and biologically characterized. NMR and IR spectroscopies
affords signatures of the central saldach in the saldach-bis-imidazolium ligands 4. Ligand 4a,
is in the O-protonated tautomeric form with strong contributions of the ionic, phenolate-
iminium tautomer in contrast to the literature on salen and saldach ligands. The aim of this
protocol to develop new promising candidates for antibacterial and anticancer chemotherapy.
Structure-activity relationships for new compounds against human breast carcinoma (MCF-7)
and human hepatocellular carcinoma (HepG2) cell lines revealed a correlation between the
hydrophilicity of target compound as tuned by the substituents on the phenolate ligand, and its
cytotoxicity and antibacterial activity. The parent ligands (4a-c) act as better anticancer and
bactericidal agents rather than their complexes ((5,6)a-c). 4c (IC50 = 19.40 µM) exhibited
remarkable results against breast cancer. Also antimicrobial screening showed significant and
broad spectrum potency against pathogenic bacterial strains as compared to control drugs.
Hence, more distant chemical refinements of the target compounds may serve as a platform
towards discovery of exceptionally active antibacterial drug. Noably, exchange of the phenol
in the free ligand, by the metal-phenolate bond in the complexes led to an unexpected
dramatic decrease in cytotoxic properties. This effect might be a consequence of a reduced
degree of the interaction with the specific DNA nucleobase targets though H-bonding.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Acknowledgment.
This work was in part supported by DFG grant Ja466/24-1 (initiation of bilateral cooperation
RFME-CJ) and DAAD.
Appendix A. Supplementary data
Supplementary data (experimental, spectral and biological data) associated with this article
can be found, in the online version, at doi:
20

1
References
[1] A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, K. Suzuki, T. Ueda, H. Terauchi, M.
Kawasaki, K. Nagai, M. Wachi, J. Yamagishi, Bioorg. Med. Chem. 12 (2004) 5515-
5524.
[2] D.T. Chu, J.J. Plattner, L. Katz, J. Med. Chem. 39 (1996) 3853-3874.
[3] L. Ballell, R.A. Field, K. Duncan, R.J. Young, Antimicrob. Agents Chemother. 49 (2005)
2153-2163.
[4] J.F. Barrett, J.A. Hoch, Antimicrob. Agents Chemother. 42 (1998) 1529-1536.
[5] T. Miyamoto, J. Matsumoto, K. Chiba, H. Egawa, K. Shibamori, A. Minamida, Y.
Nishimura, H. Okada, M. Kataoka, M. Fujita, T. Hirose, J. Nakano, J. Med. Chem. 33
(1990) 1645-1656.
[6] N. Nakada, H. Shimada, T. Hirata, Y. Aoki, T. Kamiyama, J. Watanabe, M. Arisawa,
Antimicrob. Agents Chemother. 37 (1993) 2656-2661.
[7] (a) K.R. Barnes, S.J. Lippard, Met. Ions Biol. Syst. 42 (2004) 143-177; (b) L. Zhang, Y.
Zhang, P.Y. Huang, F. Xu, P.J. Peng, Z.Z. Guan, Cancer Chemother. Pharmacol. 61
(2008) 33-38; (c) L.J.K. Boerner, J.M. Zaleski, Curr. Opin. Chem. Biol. 9 (2005)135-
144. (d) K. I. Ansari, B.P. Mishra, S. S. Mandal, Biochim. Biophys. Acta 1779 (2008)
66-73; (e) B. Stordal, N. Pavlakis, R. Davey, Cancer Treat. Rev. 33 (2007) 347-357.
[8] J. L. Czlapinski, T. L. Sheppard, J. Am. Chem. Soc. 123 (2001) 8618-8619.
[9] I. Ott, R. Gust, Arch. Pharm. (Weinheim) 340 (2007) 117-126.
[10] (a) G. Sava, S. Zorzet, C. Turrin, E. Vita, M. Soranzo, G. Zabucchi, M. Cocchietto, A.
Bergamo, S. DiGiovine, G. Pezzoni, L. Sartor, S. Garbisa, Clin. Cancer Res. 9 (2003)
1898-1905; (b) P. Köpf-Maier, Eur. J. Clin. Pharmacol. 47 (1994) 1-16; (c) P. Köpf-
Maier, Anticancer Res. 19 (1999) 493-504; (d) C.G. Hartinger, M.A. Jakupec, S.
Zorbas-Seifried, M. Groessl, A. Egger, W. Berger, H. Zorbas, P.J. Dyson, B.K. Keppler,
Chem. Biodivers. 5 (2008) 2140-2155; (e) R. Gust, I. Ott, D. Posselt, K. Sommer, J.
Med. Chem. 47 (2004) 5837-5846; (f) B. K. Keppler, M.R. Berger, M.E. Heim, Cancer
Treat. Rev. 17 (1990) 261-277.
[11] (a) S. Bhattacharya, S.S. Mandal, Chem. Commun. (1996) 1515-1516; (b) C.J. Burrows,
R.P. Hickerson, J.G. Muller, B. Felden, S.E. Rokita, Biophysical J. 76 (1999) A5-a5; (c)
J.G. Muller, S.J. Paikoff, S.E. Rokita, C.J. Burrows, J. Inorg. Biochem. 54 (1994) 199-
206; (d) S. Routier, J.L. Bernier, M.J. Waring, P. Colson, C. Houssier, C. Bailly, J. Org.
Chem. 61 (1996) 2326-2331; (e) D.J. Gravert, J.H. Griffin, J. Org. Chem. 58 (1993)
21

820-822; (f) H.Y. Shrivastava, S.N. Devaraj, B.U. Nair, J. Inorg. Biochem. 98 (2004)
387-392.
[12] S.E. Rokita, C.J. Burrows, Salen-metal Complexes, Small Molecule DNA and RNA
Binders, vol. 1 (2003) Wiley-VCH.
[13] J.G. Muller, L.A. Kayser, S.J. Paikoff, V. Duarte, N. Tang, R.J. Perez, S.E. Rokita, C.J.
Burrows, Coord. Chem. Rev. 185-186 (1999) 761-774.
[14] (a) M. Irisawa, N. Takeda, M. Komiyama, J. Chem. Soc. Chem. Commun. (1995) 1221-
1222; (b) M. Komiyama, N. Takeda, H. Shigekawa, Chem. Commun. (1999) 1443-
1452; (c) D. Prema, A.V. Wiznycia, B.M.T. Scott, J. Hilborn, J. Desper, C.J. Levy,
Dalton Trans. 42 (2007) 4788-4496; (d) A.V. Wiznycia, J. Desper, C.J. Levy, Chem.
Commun. 37 (2005) 4693-4695.
[15] G.A. Woldemariam, S.S. Mandal, J. Inorg. Biochem. 102 (2008) 740-747.
[16] K.I. Ansari, J.D. Grant, G. Woldemariam, S. Kasiri, S.S. Mandal, Org. Biomol. Chem. 7
(2009) 926-932.
[17] S.R. Doctrow, K. Huffman, C.B. Marcus, G. Tocco, E. Malfroy, C.A. Adinolfi, H. Kruk,
K. Baker, N. Lazarowych, J. Mascarenhas, B. Malfroy, J. Med. Chem. 45 (2002) 4549-
4558.
[18] Y. Rong, S.R. Doctrow, G. Tocco, M. Baudry, Proc. Natl. Acad. Sci. USA 96 (1999)
9897-9902.
[19] K. I. Ansari, J. D. Grant, S. Kasiri, G. Woldemariam, B. Shrestha, S. S. Mandal, J. Inorg.
Biochem. 103 (2009) 818-826.
[20] W.L. Hough, M. Smiglak, H. Rodrıguez, R.P. Swatloski, S.K. Spear, D.T. Daly, J.
Pernak, J.E. Grisel, R.D. Carliss, M.D. Soutullo, New J. Chem. 31 (2007) 1429-1436.
[21] K.M. Hindi, M.J. Panzner, C.A. Tessier, C.L. Cannon, W.J. Youngs, Chem. Rev. 109
(2009) 3859-3884.
[22] (a) J. Pernak, I. Goc, I. Mirska, Green Chem. 6 (2004) 323-329; (b) D. Demberelnyamba,
K.-S. Kim, S. Choi, S.Y. Park, H. Lee, C.J. Kim, I.D. Yoo, Bioorg. Med. Chem. 12
(2004) 853-857; (c) J. Pernak, K. Sobaszkiewicz, I. Mirska, Green Chem. 5 (2003) 52-
56; (d) J. Pernak, K. Sobaszkiewicz, J. Foksowicz-Flaczyk, Chem. Eur. J. 10 (2004)
3479-3485.
[23] F. Stock, J. Hoffman, J. Ranke, B. Ondruschka, B. Jastorff, Green Chem. 6 (2004) 286-
290.
[24] A.C. Skladanowski, P. Stepnowski, K. Kleszczynski, B. Dmochowska, Environ. Toxicol.
Pharmacol. 19 (2005) 291-296.
22

[25] F. Hosseinzadeh, M. Mahkam, M. Galehassadi, Des. Monomers Polym. 15 (2012) 379-
388.
[26] (a) S. Budagumpi, R.A. Haque, S. Endud, G. Ur Rehman, A.W. Salman, Eur. J. Inorg.
Chem. (2013) 4367-4388; (b) A. Gautier, F. Cisnetti Metallomics 4 (2012) 23-32; (c)
S.V. Malhotra, V. Kumar, Bioorg. Med. Chem. Lett. 20 (2010) 581-585.
[27] (a) K. Fujita, D.R. MacFarlane, M. Forsyth, Chem. Commun. (2005) 4804-4806; (b) J.
Von Hagen, U. Michelsen, U.S. Patent US7470778B2 (2008).
[28] L. Carson, P.K.W. Chau, M.J. Earle, M.A. Gilea, B.F. Gilmore, S.P. Gorman, M.T.
McCann, K.R. Seddon, Green Chem. 11 (2009) 492-497.
[29] (a) J. Pernak, K. Sobaszkiewicz, I. Mirska, Green Chem. 5 (2003) 52-56; (b) J. Pernak, I.
Goc, I. Mirska, Green Chem. 6 (2004) 323-329; (c) K.M. Docherty, C.F. Kulpa, Green
Chem. 7 (2005) 185-189; (d) M.T. Garcia, N. Gathergood, P.J. Scammells, Green
Chem. 7 (2005) 9-14; (e) P.J. Scammels, J.L. Scott, R.D. Singer, Aust. J. Chem. 58
(2005) 155-169; (f) N. Gathergood, P.J. Scammels, Aust. J. Chem. 55 (2002) 557-560;
(g) J. Łuczak, C. Jungnickel, I. Łacka, S. Stolte, J. Hupka, Green Chem. 12 (2010) 593-
601.
[30] (a) S. Budagumpi, R. A. Haque, A.W. Salman, M.Z. Ghdhayeb, Inorg. Chim. Acta 392
(2012) 61–72; (b) A.W. Salman, R.A. Haque, S. Budagumpi, H.Z. Zulikha, Polyhedron
49 (2013) 200–206.
[31] (a) R.F.M Elshaarawy, Synthesis and reactions of some pyridoyl derivatives with
anticipated biological activities, M.Sc. thesis, Suez Canal university, Egypt, (2001); (b)
B. Wisser, C. Janiak, Z. Anorg. Allg. Chem. 633 (2007) 1796-1800; (c) R.F.M
Elshaarawy, H. K. Ibrahim, E. Eltamany, I. Mohy-Eldeen, Maced. J. Chem. Chem. Eng.
27(1) (2008) 65-79; (d) A.-C. Chamayou, M. A. Neelakantan, S. Thalamuthu, C. Janiak,
Inorg. Chim. Acta 365 (2011) 447-450.
[32] (a) Mosmann, T., 1983 J. Immunol. Methods, 65: 55-63; (b) Vijayan, P., C. Raghu, G.
Ashok, S.A. Dhanaraj, Indian J. Med. Res. 120 (2004) 24-29.
[33] C. Perez, P.M. Bazerque, Acta Biol. Med. Exp. 15 (1990) 113-115.
[34] A. J. Vlietink, L. Van Hoof, J. Totte, H. Laure, D. V. Berhe, P. C. Rwangabo, J.
Mrukiyunmwami, J. Ethnopharm. 46 (1995) 31-47.
[35] R.F.M. Elshaarawy, C. Janiak, Eur. J. Med. Chem. (2013), in press.
[36] A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E. Lederer, S. Wölfl, N.
Metzler-Nolte, S. Schäfer, W.S. Sheldrick, C. Bischof, U. Schatzschneider, R. Gust J.
Biol. Inorg. Chem. 14 (2009) 711-725.
23

[37] M.J. Sabater, A. Corma, J.V. Folgado, H. Garcia, J. Phys. Org. Chem. 13 (2000) 57-62.
[38] C. Núňez, R. Bastida, A. Macías, L. Valencia, J. Ribas, J.L. Capelo, C. Lodeiro, Dalton
Trans. 39 (2010) 7673-7683.
[39] M.M. Stone, A.H. Franz, C.B. Lebrilla, J. Am. Soc. Mass Spectrom. 13 (2002) 964-974.
[40] M. Azam, Z. Hussain, I. Warad, S.I. Al-Resayes, M.S. Khan, M. Shakir, A. Trzesowska-
Kruszynskae, R. Kruszynski Dalton Trans. 41 (2012) 10854-10864.
[41] M.R. Mauryaa, A.K. Chandrakar, S. Chand, J. Mol. Cat. A 270 (2007) 225-235.
[42] (a) Webster F.X., Silverstein R.M., “Spectrophotometer Identification of Organic
Compounds”, 6th Ed., John Wiley, New York, 87 (1992); (b) O. Pouralimardan, A.-C.
Chamayou, C. Janiak, H.H.-Monfared, Inorg. Chimica Acta 360 (2007) 1599-1608; (c)
R. I. Kureshy, K. J. Prathap, T. Roy, N.C. Maity, N.H. Khan, S.H.R. Abdi, H.C. Bajaj,
Adv. Synth. Catal. 352 (2010) 3053-3060; (d) T. Chang, L.Jin, H. Jing, ChemCatChem
1 (2009) 379-383.
[43] (a) K. Nakomoto, “Infrared and Raman Spectroscopy of Inorganic and Coordination
Compounds”, 3rd Ed., Wiley Interscience, New York (1978); (b) R.F.M. Elshaarawy, Y.
Lan, C. Janiak Inorg. Chim. Acta 401 (2013) 85-94; (c) G. Karimipour, M.
Montazerozohori, N.H. Naeini, Iran. J. Chem. Chem. Eng. 30 (2011) 13-18; (d) X.
Guojin, W. Saili, F. Yingguo, Z. Libo, T. Yuhai, Z. Yuansuo, Chin. J. Catal. 33 (2012)
473-477.
[44] (a) K. Kurzak, I. Kuzniarska-Biernacka, B. Zurowska, J. Solution Chem. 28 (1999) 133-
151; (b) O.T. Benjelloun, M. Akkurt, S.Ö. Yıldırım, M. Daoudi, N. Ben Larbi, A.
Kerbal, B. Bennani, O. Büyükgüngör, A.F. Jalbout, T. Ben Hadda, ARKIVOC xi (2008)
56-63; (c) B.M. Drašković, G.A. Bogdanović, M.A. Neelakantan, A.-C. Chamayou, S.
Thalamuthu, Y.S. Avadhut, J. Schmedt auf der Günne, S. Banerjee, C. Janiak, Cryst.
Growth Des. 10 (2010) 1665-1676; (d) A. Blagu, D. Cinčić, T. Friščić, B. Kaitner, V.
Stilinović, Maced. J. Chem. Chem. Eng. 29 (2010) 117-138.
[45] (a) Ĕ. Tozzo, S. Romera, M.P. dos Santos, M. Muraro, R.H. de A. Santos, L.M. Lião, L.
Vizotto, E.R. Dockal, J. Mol. Struct. 876 (2008) 110-120; (b) P.J.K. Inba, B. Annaraj,
S. Thalamuthu, M.A. Neelakantan Spectrochim. Acta A 104 (2013) 300-309.
[46] (a) N. B. Pahor, M. Calligaris, P. Delise, G. Dodic, G. Nardin, L. Randaccio, Dalton
Trans. (1976) 2478-2483; (b) K. Lippe, D. Gerlach, E. Kroke, J.r. Wagler,
Organometallics 28 (2008) 621-629; (c) A. Dalla Cort, F. Gasparrini, L. Lunazzi, L.
Mandolini, A. Mazzanti, C. Pasquini, M. Pierini, R. Rompietti, L. Schiaffino, J. Org.
24

Chem. 70 (2005) 8877-8883; (d) C.-G.F. von Richthofen, A. Stammler, H. Bögge, T.
Glaser, J. Org. Chem. 77 (2012) 1435-1448.
[47] R.M. Claramunt, C. López, M.D. Santa María, D. Sanz, J. Prog. Nucl. Magn. Reson.
Spectrosc. 49 (2006) 169-206.
[48] S.H. Alarcon, A.C. Olivieri, M. Gonzalez-Sierra, J. Chem. Soc., Perkin Trans. (1994)
1067-1070.
[49] (a) A. Dalla Cort, F. Gasparrini, L. Lunazzi, L. Mandolini, A. Mazzanti, C. Pasquini, M.
Pierini, R. Rompietti, L. Schiaffino, J. Org. Chem. 70 (2005) 8877-8883; (b) D. M.
Boghaei, S. Mohebi, J. Chem. Res., Synop. (2002) 72-75.
[50] (a) S. M. Crawford, Spectrochim. Acta 19 (1963) 255-270; (b) B. Bosnich, J. Am. Chem.
Soc. 90 (1968) 627-632; (c) S. Di Bella, I. Fragala, I. Ledoux, M. A. Diaz-Garcia, T. J.
Marks, J. Am. Chem. Soc. 119 (1997) 9550-9557.
[51] L.J. Boucher, M.O. Farrell, J. Inorg. Nucl. Chem. 35 (1973) 3731-3738.
[52] B.P. Gaber, V. Miskowski, T.G. Spiro, J. Am. Chem. Soc. 96 (1974) 6868-6873.
[53] O. Kahn, Molecular Magnetism; VCH, New York, (1993).
[54] L. Cardoso, N.M. Micaelo, ChemPhysChem 12 (2011) 275- 277.
[55] S. Tabassum, M. Zaki, M. Afzal, F. Arjmand, Dalton Trans. 42 (2013) 10029–10041.
[56] N. Berthet, V. Martel-Frachet, F. Michel, C. Philouze, S. Hamman, X. Ronotb, F.
Thomas, Dalton Trans 42 .(2013) 8468–8483
[57] A. Hille, R. Gust, Arch. Pharm. Chem. Life Sci. 342 (2009) 625 – 631.
[58] (a) G. A. Woldemariam, S. S. Mandal, J. Inorg. Biochem. 102 (2008) 740–747; (b) K. I.
Ansari, J. D. Grant, G. A. Woldemariam, S. Kasiri, S. S. Mandal, Org. Biomol. Chem. 7
(2009) 926–932; (c) K. I. Ansari, S. Kasiri, J. D. Grant, S. S. Mandal, Dalton Trans.
(2009) 8525–8532; (d) K. I. Ansari, J. D. Grant, S. Kasiri, G. A. Woldemariam, B.
Shrestha, S. S. Mandal, J. Inorg. Biochem. 103 (2009) 818–826.
[59] A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E. Lederer, S. W_lfl, N.
Metzler-Nolte, S. Sch_fer, W. Sheldrick, C. Bischof, U. Schatzschneider, R. Gust, J.
Biol. Inorg. Chem. 14 (2009) 711–725.
[60] J. Pernak, K. Sobaszkiewicz, I. Mirska, Green Chem. 5 (2003} 52–56.
[61] (a) M. Kong, X.G. Chen, K. Xing, H.J. Park, Int. J. Food Microbiol. (2010) 51-63. (b)
V.P. Daniel, B. Murukan, B.S. Kumari, K. Mohanan, Spectrochim Acta A: Mol.
Biomol. Spectrosc. 70 (2008) 403-410.
[62] E.I. Rabea, M.E.I Badawy, T.M. Rogge, C.V. Stevens, M. Höfte, W. Steurbaut, G.
Smagghe, Pest. Manag. Sci. 61 (2005) 951-960.
25

Tables Captions
Table 1 IC50 values of saldach-bis(imidazolium) salts (4a-c) and their M(III)-complexes (5,6a-c)
derivatives towards MCF-7 and HepG-2 cell lines
Table 2 MIC (mg/mL) and MBC (mg/mL) assay results for the saldach-bis-(imdidazolium) salts (4a-
c) and standard drugs
Table 1 IC50 values of saldach-bis(imidazolium) salts (4a-c) and their M(III)-complexes (5,6a-c)
derivatives towards MCF-7 and HepG-2 cell lines
IC50 (µM) for the tested
compounds
Compound
MCF-7
HepG-2
>50
33.34 2.41
>50
4a
>50
5a
46.30 3.15
40.41 2.89
48.96 3.54
45.86 3.11
22.17 1.33
>50
>50
6a
>50
4b
>50
5b
6b
>50
4c
>50
5c
>50
6c
>50
>50
Doxorubicin
>50
2.21 1.17
26

Table 2 MIC (mM) and MBC (mM) assay results for the saldach-bis-(imdidazolium) salts (4a-c) and
standard drugs
MIC (mM)
MBC (mM)
Bacterium
Compounds
4a
14.30 1.88 14.47 2.03
4b
1.37 0.31
1.33 0.27
NA
10.11 0.76
4.99 0.55
NA
S. aureus
4c
(ATCC 29737)
Gentamycin
Tetracycline
NA
7.20 0.66
3.50 0.49
1.39 0.30
1.35 0.28
6.70 0.63
NA
4a
3.46 0.47
2.58 0.38
5.06 0.61
NA
4b
E. coli
4c
(ATCC 10536)
Gentamycin
Tetracycline
NA
NA= not assigned
27

Figures Captions
Figure 1 Selected IR region, for comparison of the azomethine and phenolate stretching vibrations
and their splitting patterns in 4b, 5b and 6b.
Figure 2 1H NMR spectra of 4a (200 MHz, DMSO-d6).
Figure 3 Concentration dependent analysis of cytotoxic effects of the bis-(Me2Im+X–)saldach salts
(4a-c) and their metal complexes (5,6a-c) on the human MCF-7 and HepG-2 cell lines.
Figure 4 Graph of zone of inhibition/mm for target compounds against different bacterial species.
Figure 1 Selected IR region, for comparison of the azomethine and phenolate stretching vibrations
and their splitting patterns in 4b, 5b and 6b.
28

Figure 2 1H NMR spectra of 4a (200 MHz, DMSO-d6).
29