Structure, CO-releasing property, electrochemistry, DFT calculation, and antioxidant activity of benzimidazole derivative substituted [Mn(CO)3(bpy)L]PF6 type novel manganese complexes

Article abstract of DOI:10.1016/j.ica.2016.05.027

Metal carbonyl complexes are prominent group organometallic compounds because of their applications in industrial and pharmaceutical chemistry. In recent years, metal carbonyl complexes have been accepted major for storage and transportation of carbon mon

Full text of DOI:10.1016/j.ica.2016.05.027

Accepted Manuscript
Research paper
Structure, CO-releasing property, electrochemistry, DFT calculation, and anti-
oxidant activity of benzimidazole derivative substituted [Mn(CO)3(bpy)L]PF6
type novel manganese complexes
Elvan Üstün, Melek Çol Ayvaz, Mutlu Sönmez Çelebi, Gizem Aşci, Serpil
Demir, İsmail Özdemir
PII:
S0020-1693(16)30269-9
http://dx.doi.org/10.1016/j.ica.2016.05.027
ICA 17063
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 February 2016
10 May 2016
12 May 2016
Please cite this article as: E. Üstün, M.C. Ayvaz, M.S. Çelebi, G. Aşci, S. Demir, I. Özdemir, Structure, CO-releasing
property, electrochemistry, DFT calculation, and antioxidant activity of benzimidazole derivative substituted
[Mn(CO)3(bpy)L]PF6 type novel manganese complexes, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/
10.1016/j.ica.2016.05.027
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Structure, CO-releasing property, electrochemistry, DFT calculation, and
antioxidant activity of benzimidazole derivative substituted
[Mn(CO)3(bpy)L]PF6 type novel manganese complexes
Elvan ÜSTÜNa, Melek ÇOL AYVAZa*, Mutlu SÖNMEZ ÇELEBİa, Gizem AŞCIa,
Serpil DEMİRb, İsmail ÖZDEMİRb,
a Department of Chemistry, Faculty of Science and Arts, Ordu University, 52200, Ordu, Turkey
b Department of Chemistry, Faculty of Science, İnönü University, 44280, Malatya, Turkey
Corresponding Author: mcol@odu.edu.tr
ABSTRACT
Metal carbonyl complexes are prominent group organometallic compounds because of their
applications in industrial and pharmaceutical chemistry. In recent years, metal carbonyl complexes
have been accepted major for storage and transportation of carbon monoxide which is an important
signaling molecule significantly in pathogenesis of some diseases. Thus, we designed and synthesized
novel manganese(I)carbonyl complexes with general formula [Mn(CO)3(bpy)L]PF6 (bpy=2,2-
bipyridyl, L= N-(2-chlorobenzyl)benzimidazole, N-(2-methoxybenzyl)benzimidazole, N-(2-
1
methylbenzyl)benzimidazole). The complex molecules were characterized by LC-MS, H-NMR, 13C-
NMR, IR spectroscopy methods and elemental analysis. The CO-releasing properties, antioxidant
activities and redox properties of these complexes were investigated. The DFT/TDDFT analyses were
made by ORCA package program.
INTRODUCTION
Since the discovery of nickel tetracarbonyl more than a century ago [1], metal carbonyls have played a
very important role in pharmaceutical chemistry. These complexes are now established as useful
biomolecules. The work on CO intensified, metal carbonyl complexes which have been accepted
major for storing and transporting CO are antiinflammatory, antiapoptotic, and antiproliferative,
protects tissues against hypoxia or ischemia-reperfusion injury, and causes vasodilatation [2]. The
studies show that endogenous increase of the CO amount in a tissue plays a role in eliminating the
problem and regulating the intracellular functions [3-8]. It has been also shown to have an appreciable
1

role in preclinical animal models of cardiovascular disease, inflammatory disorders, and organ
transplantation [9-13].
Reactive oxygen species (ROS) are highly reactive molecules and can damage cell structures such as
carbohydrates, nucleic acids, lipids, and proteins and alter their functions. Antioxidant systems which
include enzymatic and nonenzymatic antioxidants are usually effective in blocking harmful effects of
ROS. The shift in the balance between oxidants and antioxidants in favor of oxidants is termed
“oxidative stress” and changes in oxidative stress are associated with reciprocal changes in antioxidant
capacity [14, 15]. Srisook et al. employed CORM-2 molecule to demonstrate that CO can inhibit ROS
-
production, especially O2 and NO, with murine macrophage cell line RAW 264.7 [16]. Backer et al.
studied with CORM-3 on surgical manipulated mice intestine about antioxidant effect of CO and
revealed that treatment with CORM-3 partially reduced oxidative stress in the muscularis externa and
confirmed a direct protective effect of CO in addition to the ability to induce HO-1 [17]. However, the
inhibition of the early “oxidative burst” in the mucosa fully depended on the induction of HO-1.
Similarly, Lamon et al. tried to enlighten with CORM-3 that CO acts whether as a vasodilator or
vasoconstrictor on vascular endothelial cells depending on ROS [18]. Although there are discrepancies
about the mechanism of action, it is apparent that CO and CORMs are really worth checking out in
point of antioxidant activity and an in-vitro molecular antioxidant activity study of novel CORM may
provide significant contribution while being directive for possible therapeutic applications.
Benzimidazole derivatives have been proven to function as antihypertensive [19], antiinflammatory
[20], antimicrobial [21], antiviral [22-24], antitumor [24-26], anticoagulants [27], antidiabetic [28]
agents. Benzimidazole derivatives are also useful antioxidants [29]. Transition metal complexes with
benzimidazoles can be tested in bioinorganic systems and may exhibit synergistic effect in possible
therapeutic applications.
Safe transmission of appropriate amount of CO to the tissue is crucial, and one of the most promising
candidates for this mission is metal carbonyl complexes. Many carbonyl complexes of transition
metals have been found to be photoreactive under irradiation into their lowest charge-transfer (CT)
absorption band, the observed primary reaction corresponding to a carbonyl loss [30-36]. One of
various ways to ensure CO-release is to irradiate the complexes with UV-light with certain
wavelength. These kinds of dark-stable, light-sensitive and potentially CO-releaser complexes are
called as photoCORMs (photo-activatable CO-releasing molecules) and manganese carbonyl
complexes are well-known photoCORMs [37-43].
Charge-transfer transitions {metal-to-ligand (MLCT), ligand-to-metal (LMCT)} of metal carbonyls
can lead to redox reactions and also result in reducing the metal center and generating radicals [44-46].
Density functional theory (DFT) and time-dependent density functional theory (TDDFT) are potent
and efficient tools for excited-state characterization and decomposition pathways of transition metal
2

complexes and often show how a broad absorption band can conceal beneath it several electronic
transitions. These are also essential for development of new molecules with suitable characteristics for
biological applications. Similar calculations have also been used to better explain the redox behavior
observed for carbonyls i.e., the specific location of the oxidizing or reducing equivalents in metal
carbonyls [47-50].
Studies about analyses of bioactivity of CO and synthesis of new CO-releasing molecules have
received considerable attention after the favorable results about functions of CO in tissue. It is clear
from the reference list that, although new CORMs having more advantageous half-life and more
totally released CO have been synthesized, research about biological function of CO has been focused
on initial CORMs (CORM-2, CORM-3 etc.) which could be synthesized easily or obtained
commercially [14]. We managed to give results in diverse fields for our novel molecules to provide
guidance and convenience for further research. In this study, novel [Mn(CO)3(bpy)L]PF6 [bpy=2,2-
bipyridyl, L= N-(2-chlorobenzyl)benzimidazole, N-(2-methoxybenzyl)benzimidazole, N-(2-
methylbenzyl)benzimidazole] type manganese complexes with benzimidazole derivative ligands have
been synthesized. The structures of the compounds were enlightened by 1H-NMR, 13C-NMR, IR, LC-
MS, and elemental analysis. Basic level in-vitro molecular antioxidant activity, redox properties and
CO-releasing properties have also been described hereunder. The excited-state characterization was
rationalized by DFT/TDDFT with ORCA package program [51-54].
DISCUSSION
Novel [Mn(CO)3(bpy)L]PF6 [bpy=2,2-bipyridyl, L= N-(2-chlorobenzyl)benzimidazole, N-(2-
methoxybenzyl)benzimidazole, N-(2-methylbenzyl)benzimidazole] type manganese complexes with
benzimidazole derivative ligands have been synthesized (Figure 1).The characterization of the ligands
and Mn(CO)3(bpy)Br were made by 1H-NMR, 13C-NMR and elemental analysis. The complexes 5-7
1
were characterized by H-NMR, 13C-NMR, IR and LC-MS and all characterization details could be
analyzed in Results. One broad band (379 nm for 5, 378 nm for 6, and 380 nm for 7) and a shoulder at
320 nm were observed for complexes 5-7 with lower intensity in UV-Vis Spectra. The absorbance
maximas and molar extinction coefficients of complexes 5-7 were determined in dimethyl sulfoxide
(Table 1).
Figure 1
CO-releasing properties of the complexes were identified by myoglobin-assay as detailed below. Since
the complexes are light sensitive but dark stable (photoCORMs), 366 nm UV lamp was used for
releasing (Figure S1 and Figure S2). Total released CO, CO equivalents, the percentage released CO
and half-life (t1/2) were determined with UV-Visible Spectrophotometer at 1 minute intervals (Figure
3

2). The t1/2 in this study is defined as the time taken for compounds 5-7 to release 50% of the total CO
ligands present per molecule.
Figure 2
In myoglobin assay, due to binding of released CO with myoglobin, the reaction is forced towards
product side. Carbonmonoxymyoglobin concentration [MbCO] and equivalence CO (eq. CO) which
become fixed after a while have been indicated total released CO. Molecule 5, the most efficient in
this study, released 2.19 of 3 carbon monoxide when 32.84 µM of carbonmonoxymyoglobin was
formed. Carbonmonoxymyoglobin concentration of 6 was 27.73 µM while the equivalence CO was
1.85. The amount of CO released as well as the release rate is obviously dependent on the nature of the
substituents on benzyl, the rest of the molecules are identical. Although it is not possible to have
common decision with three molecules, the results can be associated with mesomeric and inductive
effect of the substituents. Methoxy, more effective mesomeric substituent than chlorine and methyl,
induces the increasing of electron density on metal center and metal-CO back-bonding. Strong back-
bonding means difficult releasing of CO. On the other hand, chlorine has both inductive and
mesomeric effect while methyl has only inductive effect which is more dominant than the mesomeric
effect for chlorine. Half-lives of the molecules can be used for kinetic analysis. Most advantageous
molecule according to half-life is 7 which released half of its CO in 7.31 minute while the slowest one
is 6 with 11.07 minute. It is possible to pay attention that the more efficient CO-releaser is the slowest
one (Table 1).
Table 1
The released CO amount has been determined by myoglobin assay and also followed IR spectra.
Provided that the CO releasing occurs as observed in the myoglobin assay, the reactions would result
with the formation of di- or monocarbonyl complexes of manganese. Typical IR spectra for
manganese (I) fac-tricarbonyl complexes with a strong band 2030 cm-1 and two additional bands
around 1925 cm-1 were observed before illumination (Figure S3). During excitations, strong IR bands
of initial complexes disappeared indicating the dissociation of [MnI(CO)3]-units of complexes. On the
other hand new IR bands emerged in the region around 1975 cm-1 and 1860 cm-1. These bands could
be the indication of manganese (I) cis-dicarbonyl complexes [55, 56]. Ultimate IR bands which took
shape on 5th minute around 1850 cm-1 and 1875 cm-1 might be the sign for monocarbonyl manganese
(I) complexes [57].
Many transition metal complexes exhibit antioxidant activity [58]. Therefore, an in vitro investigation
was carried out to explore whether the compounds 5-7 have 2,2-diphenyl-1-picrylhydrazyl (DPPH),
−
superoxide (O2 ) and nitroxyl (NO·) radical scavenging activities.
4

As can be seen from the Table 2, DPPH scavenging ratios (%) of the 0.2 mM of all investigated
compounds were under 10% without UV exposure. But at the end of UV light exposure for 10
minutes, observed scavenging ratios were increased for each. Moreover, for compound 7, it is seen
that this increment continues at the end of the exposure time for 20 min. Even in this case, the highest
calculated inhibition percentage is lower than the calculated value for ascorbic acid (AA), which is
used as a standard antioxidant. 0.2 mM AA scavenged the 89.8 % of the DPPH radicals in the medium
under the same conditions.
Unlike DPPH radical scavenging activities, superoxide radical scavenging activities of the compounds
5-7 increased due to CO releasing and reached a high value comparable with the value calculated
using commercial SOD enzyme.
Table 2
Among these assays, the most interesting results were obtained for NO· radical scavenging abilities in
which CO releasing led to a decline in activity. Nitroxyl radical scavenging activities of these CORMs
were also higher than found for AA under the same conditions. The results are consistent with findings
in the literature recorded by Choi et al., 2015 [59]. They had proved that the CO-releasing molecule-3
suppressed Prevotella inter-media lipopolysaccharide-induced production of nitric oxide.
The results obtained by three different methods confirmed that the investigated compounds are more
effective to arrest superoxide radicals than the other radicals.
Figure 3
The electrochemical behaviors of the complexes were investigated by cyclic voltammetry on a
stationary glassy carbon electrode in the potential range 2.0 V to -2.0 V vs. Ag/AgCl. CVs of the
complexes are given in Figure 2 in acetonitrile containing 0.1 M TBAP at a scan rate of 50 mV s-1.
The nature of the voltammograms does not change with scan rate (50-200 mV s-1). All three
compounds show an irreversible oxidation peak around +1.4 V (1.40 V, 1.46 V and 1.48 V for
compounds 5, 6 and 7 respectively). As the potentials are quite close to each other, the peaks look
almost unaffected by differentiating the ligands and therefore should be attributed to the metal center
of the complexes rather than the ligand sites. These oxidation peaks are assigned to the monoelectronic
oxidation of Mn(I) to Mn(II) [60]. A less intensive oxidation process around 0.0 V was also
observed for all three compounds (-0.18 V, 0.27 V and 0.35 V for compounds 5, 6 and 7
respectively) which is also irreversible. Unfortunately we have not been able to identify the
species involved in the oxidation process. As seen from the voltammograms, all three compounds
feature complicated oxidation and reduction peaks at the negative potential region. This behavior is
assigned to ligand based oxidation/reduction of the complex molecules as the position of the peaks are
5

obviously affected by altering the substituents on the ligands coordinating the metal. The reduction
process assigned to the ligand sites of the molecules may be referred to electron accommodation of the
*
π MO of the ligand (the LUMO of the compound) which is clearly affected by the substituents (i.e. –
Cl, – OCH3, and – CH3 for compounds 5,6 and 7 respectively). For example, when we compare the
first reduction peaks of compounds 5 and 7 (-1.43 V and -1.76 V respectively), reduction of the latter
is observed at a more negative potential. As the substituent becomes more electron withdrawing (as in
*
the case of compound 5 relative to compound 7), the relative energy of the π MO becomes lower
which causes the compound to be reduced easier leading to a more positive reduction potential [61,
62]. As expected according to the electron withdrawing moieties of the substituents of the ligands, the
first reduction peak of the compound 7 is observed at a more positive potential than compound 5.
An understanding of the photochemistry of transition metal compounds requires knowledge of the
properties of molecular orbitals and appropriate excited states. Frontier orbitals play a relevant role in
such systems, because they rule the electronic excitations and the transition characters. With the aid of
TDDFT calculations, it is possible to have comments about the contributions of the ligand and metal
orbitals to molecular orbitals. It is not practical to analyze all the electronic transitions and the orbitals;
therefore some restrictions were used. Only strong transitions with an oscillator strength >0.01 in the
300−600 nm range were reported and only contributions >10% were listed. The ground-state
electronic structures were calculated accounting for the solvent effect of water in COSMO model.
For all complexes, the highest occupied molecular orbital (HOMO) is basically manganese-centered
with limited contribution from benzimidazole and CO moieties like HOMO-1 and HOMO-2. In 5,
HOMO-4 and HOMO-6 are completely benzimidazole-based while HOMO-7 consists of bpy. The
lowest unoccupied molecular orbital (LUMO) is localized on the bpy’s for all complexes. LUMO+1
and the other unoccupied virtual orbitals, up to LUMO+3, are all ligand based but LUMO+5 and
LUMO+6 have little manganese contributions. All molecular orbitals which have contribution to
singlet electronic states can be seen in Table 3, Figure S3-S5, and Table S1-S3.
Table 3
One broad band (379 nm for 5, 378 nm for 6 and 380 nm for 7) and a shoulder at 320 nm were
observed in the experimental spectra of complexes 5-7. These broad energy bands which calculated
→ bpy; MLCT =
377 nm for all molecules are attributed MLCT states (Mn
metal-to-ligand charge
transfer) with highest oscillator strength (fosc) in TDDFT results. But the shoulders which occur 320
nm in experimental UV spectra for all complexes could not be confirmed with TDDFT calculations.
The single calculated result at 321.2 nm with 0.0138 oscillator strength for 5 has contributions of
HOMO-1→LUMO+5 (46.0%, MLCT Mn→imid), HOMO-1→LUMO+6 (37.6%, MLCT Mn→imid)
→LUMO+2 (10.2%, LLCT imid→bpy)
and HOMO-6
transitions. Complex 6 and 7 haven’t got a
6

suitable transition among calculated wavelength and oscillator strength in TDDFT results for 320 nm
shoulder. Residual transitions of 5 which have contribution more than %10 but not given in Table S1
and all electronic transitions of 6 and 7 were given in Table S2 and Table S3. With the aid of
DFT/TDDFT calculation, we had knowledge about frontier molecular orbitals, electronic transitions,
and singlet excitation density differences of compounds.
RESULTS
N-(3-chlorobenzyl)benzimidazole (1)
1
Yield: 2.09 g (86.0%). H NMR (300 MHz, CDCl3): δ (ppm)= .26 (s, 1H, NCHN), 7.78-7.75 (m, 1H,
NC6H4N), 7.48-7.37 (m, 2H, NC6H4N), 7.26-7.00 (m, 4H, CH2C6H4(Cl)-3), 6.99-6.98 (m, 1H,
NC6H4N), 5.87 (s, 2H, CH2C6H4(Cl)-3). 13C NMR (300 MHz, CDCl3): δ (ppm) =141.9 (NCHN),
136.4, 133.6, 122.7, 121.9, 112.7, and 109.2 (NC6H4N), 129.7, 127.6, 127.1, 126.2, 125.5, and 124.1
(CH2C6H4(Cl)-3), 49.9 (CH2C6H4(Cl)-3), Anal. Calc. for C14H11N2Cl (242.70): C, 69.28; H, 4.57; N,
11.54. Found: C, 69.25; H, 4.61; N, 11.50%.
N-(3-methoxybenzyl)benzimidazole (2)
1
Yield: 3.12 g (87.2%). H NMR (300 MHz, CDCl3): δ (ppm)= 8.04 (s, 1H, NCHN), 7.87-7.84 (m, 1H,
NC6H4N), 7.35-7.25 (m, 4H, NC6H4N and CH2C6H4(OCH3)-3), 6.88-6.74 (m, 3H, CH2C6H4(OCH3)-
3), 5.35 (s, 2H, CH2C6H4(OCH3)-3), 3.76 (s, 3H, CH2C6H4(OCH3)-3). 13C NMR (300 MHz, CDCl3):
δ (ppm) =143.5 (NCHN), 160.1, 143.1, 136.9, 133.9, 130.2, 123.2, 122.4, 120.3, 119.4, 113.4, 113.0
and 110.1(NC6H4N and CH2C6H4(OCH3)-3), 55.3 (CH2C6H4(OCH3)-3), 48.7 (CH2C6H4(OCH3)-3).
Calc. for C15H14N2O(238.3): C, 75.61; H, 5.92; N, 11.76. Found: C, 75.59; H, 5.93; N, 11.73%.
N-(3-methylbenzyl)benzimidazole (3)
1
Yield: 5.82 g (95.4%). H NMR (300 MHz, CDCl3): δ (ppm)= 2.33 (s, 3H, NCH2C6H4(CH3)-3), 5.33
(s, 2H, NCH2C6H4(CH3)-3), 7.00-7.86 (m, 8H, NC6H4N, NCH2C6H4(CH3)-3), 7.98 (s, 1H, NCHN).
13C NMR (300 MHz, CDCl3): δ (ppm) =21.4 (NCH2C6H4(CH3)-3), 48.9 (NCH2C6H4(CH3)-3), 110.1,
120.2, 122.4, 123.2, 124.2, 127.8, 128.9, 138.9 (NC6H4N, NCH2C6H4(CH3)-3), 143.8 (NCHN). Calc.
for C15H14N2 (222.3): C, 81.05; H, 6.35; N, 12.60. Found: C, 80.90; H, 6.39; N, 12.71%.
Mn(CO)3(bpy)Br (4)
Yield: 580 mg (85%). 1H NMR (500 MHz, DMSO-D6): δ (ppm) = 7.72 (s, 2H, 5/5’), 8.23 (s, 2H,
4/4’), 8.65 (s, 2H, 3/3’), 9.19 (s, 2H, 6/6’). 13C NMR (500 MHz, DMSO-D6): δ (ppm) = 123.1 (5/5’),
126.7 (2/2’), 139.1 (4/4’), 153.3 (3/3’), 154.9 (6/6’), 181.3, 186.6, 192.1 (CO). Anal. Calc. for
7

C13H8N2O3BrMn (375.06): C, 41.63; H, 2.15; N, 7.47. Found: C, 41.57; H, 2.05; N, 7.51%. IR (cm-1,
ATR): ν= 1471.8, 1442.3 (s, C-H), 1604.5 (s, C-N), 2019.6, 1938.3, 1912.4 (s, CO).
[Mn(CO)3(bpy){N-(3-chlorobenzyl)benzimidazole}]PF6 (5)
Yield: 135.00 mg (69.6%). 1H NMR (300 MHz, DMSO-D6): δ (ppm)= 5.36 (s, 2H, NCH2C6H4Cl),
6.91 (s, 1H, NCHN), 8.04 (s, 1H, NCH2C6H4Cl), 7.88-7.65 (m, 5H, NCH2C6H4Cl, NC6H4N), 7.40-
7.19 (m, 3H, NCH2C6H4Cl, NC6H4N ), 7.55 (t, J=3.9, 1H, NCH2C6H4Cl), 8.31 (d, J=12.3, 2H,
NC10H8N), 8.62 (d, J=6.0, 2H, NC10H8N), 9.48 (d, J=3.6, 2H, NC10H8N). 13C NMR (300 MHz,
DMSO-D6): δ (ppm) = 47.16 (NCH2C6H4Cl), 137.78 (NCHN), 145.77, 144.38, 140.92, 132.84,
130.54, 127.064, 125.87, 123.30, 117.12, 116.68, 113.42, 112.32 (NCH2C6H4Cl, NC6H4N), 154.96,
154.63, 140.29, 128.03, 124.00 (NC10H8N). LCMS: m/z 571.034 [M-bpy]+. IR (cm-1, ATR): ν=
1446.6.7, 1473.6 (s, C-H), 1604.8 (s, C-N), 1925.0, 2033.0 (s, CO).
[Mn(CO)3(bpy){N-(3-methoxybenzyl)benzimidazole}]PF6 (6)
1
Yield: 163.02 mg (84.6%). H NMR (300 MHz, DMSO-D6): δ (ppm)= 3.67 (s, 3H, NCH2C6H4OCH3),
5.30 (s, 2H, NCH2C6H4OCH3), 9.48 (s, 1H, NCHN), 9.70-6.43 (m, 16H, NCH2C6H4OCH3, NC10H8N,
NC6H4N). 13C NMR (300 MHz, DMSO-D6): δ (ppm) = 48.43 (NCH2C6H4OCH3), 55.55
(NCH2C6H4OCH3), 137.38 (NCHN), 159.82, 155.51, 146.31, 141.87, 139.0, 133.54, 119.70, 117.63,
112.94 (NCH2C6H4CH3, NC6H4N), 155.23, 140.88, 130.31, 124.54, 113.78 (NC10H8N). LCMS: m/z
576.15 [M-PF6]+. IR (cm-1, ATR): ν= 1442.7, 1492.9, 1516.1 (s, C-H), 1604.8 (s, C-N), 2033.0,
1928.8 (s, CO).
[Mn(CO)3(bpy){N-(3-methylbenzyl)benzimidazole}]PF6 (7)
1
Yield: 167.30 mg (88.8%). H NMR (300 MHz, DMSO-D6): δ (ppm)= 2.19 (s, 3H, NCH2C6H4CH3),
9.47 (s, 2H, NC10H8N), 5.30 (s, 2H, NC10H8N), 8.51-6.72 (m, 15H, NC10H8N, NCH2C6H4CH3,
NC6H4N). 13C NMR (300 MHz, DMSO-D6):
δ (ppm) =20.85 (NCH2C6H4CH3), 47.86
(NCH2C6H4CH3), 136.34 (NCHN), 145.67, 144.84, 141.28, 135.18, 132.88, 128.45, 127.66, 124.54,
124.27, 123.77, 123.02, 117.05 (NCH2C6H4CH3, NC6H4N), 154.95, 154.69, 140.30, 137.88, 123.97
(NC10H8N). LCMS: m/z 551.13 [M-bpy]+. IR (cm-1, ATR): ν= 1442.7, 1516.0 (s, C-H), 1604.8 (s, C-
N), 2036.8, 1944.5 (sh) 1928.8, 1925.0 (s, CO).
EXPERIMENTAL and COMPUTATIONAL SECTION
General Remarks:
All chemicals were purchased from Sigma Aldrich and used without further purification. All reactions
were carried out under an atmosphere of pure argon by using standard Schlenk and vacuum
8

techniques. Solvents were freshly distilled after refluxing over metallic sodium or phosphorous
pentoxide for 3-4 days. IR spectra were recorded on pure solid samples with a Shimadzu IRAffinity-1
ATR spectrometer. Intensities of stretching vibrations were marked as strong(s), medium (m), weak
(w), or shoulder (sh). NMR spectra were recorded on a Bruker Ultra Shield 300 MHz spectrometer.
Chemical shifts δ in parts per million indicate a downfield shift relative to tetramethylsilane (TMS)
and were referenced relative to the signal of the solvents. Coupling constants J are given in hertz.
Individual peaks are marked as singlet (s), doublet (d), triplet (t), or multiplet (m). Absorption spectra
were measured using a Shimadzu UV-1800 in quartz cuvettes (d=1 cm). Elemental analysis (C, H, and
N) of ligands was carried out using a CHNS-932 (LECO) and liquid chromatography-mass
spectrometry (LC-MS) of complexes was carried out on an Agilent 1100 Series. The complexes were
precipitated as PF6 salt by anion exchange procedure.
Synthesis of Ligands (1-3):
Compound 1-3 were prepared by similar methods. Small pieces of lithium (45 mmol, 312 mg) were
added slowly to ethylene diamine at 110 oC. The solution was allowed to reach room temperature after
stirring for 1 hour and n-alkylbenzylchloride (50 mmol) and toluene (40 mL) were added. Precipitated
lithium chloride was filtered and the N-(n-alkyl benzyl)ethylene diamine was isolated by distillation
o
from the oily mixture (120 C/0.01 mmHg) after solvents were removed under vacuum. N-(n-
alkylbenzyl)ethylenediamine (35 mmol) and N,N-dimethylformamide dimethylacetal (40 mmol) were
stirred for 2 hours at 100 oC and methanol and dimethyl amine were separated at 120 oC by distillation.
o
The last product was isolated from yellow oily residual by distillation under vacuum (124 C/0.01
mmHg).
Synthesis of Mn(CO)3(bpy)Br (4) :
Mn(CO)5Br (500 mg, 1.82 mmol) and 2,2’-bipyridyl (312.51 mg, 2.00 mmol) were refluxed in diethyl
ether for 3 hours under argon atmosphere. Precipitated orange product was filtered and washed with
10 mL cold diethyl ether.
Synthesis of manganese complexes (5-7):
Compounds 5-7 were prepared by similar methods. Mn(CO)3(bpy)Br (100 mg, 0.267 mmol) were
added into the solution of AgOTf (82.2 mg, 0.320 mmol) in acetone (10 mL). Participated AgBr was
filtrated by Celite and the ligand was added after stirring for a day at room temperature. Acetone was
evaporated under vacuum and KPF6/methanol solution was added for anion exchange. Precipitated
orange product was filtered and washed with 5 mL cold methanol and 10 mL cold diethyl ether.
Myoglobin Assay:
9

Horse muscle myoglobin solution which prepared by dissolving in PBS (0.1 M, pH=7.4) was reduced
to deoxymyoglobin by addition of sodium dithionite solution in PBS (0.1 M, pH=7.4). Stock solutions
of complexes 5-7 that measured CO-releasing properties by myoglobin assay were prepared in DMSO.
PBS (0.1 M, pH=7.4), 100 mM sodium dithionite (100 µL), 15 µM metal carbonyl complex and 60
µM myoglobin were combined in a cuvette to give a total volume of 1000 µL. It was degassed by
bubbling with argon in each step of the procedure. Final solution was put perpendicularly in front of
the UV lamp far from 5 cm and all the spectra were collected by Shimadzu UV 1800 UV-Vis
Spectrophotometer. Irradiation was made by 366 nm CAMAG UV Lamp in 1 minute intervals during
the initial 20 minute and gone on 5 minutes intervals until no more differences in MbCO
concentration. All irradiation experiments were carried out in triplicate. Solutions were freshly
prepared for the dark stability and photo-activation experiments. Dark stability spectra were collected
automatically for established period of time by spectrometer software.
Antioxidant Activity:
DPPH is a stable free radical. Because of having a single electron, DPPH solution in methanol gives
strong absorbance at 517 nm. If DPPH abstracts a hydrogen radical from an external source, the
absorption decreases stoichiometrically depending on the number of electrons or hydrogen atoms [63].
An established change in absorbance because of the interaction between DPPH and investigated
compound could be evaluated as DPPH radical scavenging capacity. 0.2 mM compound in DMSO
was mixed with DPPH solution. The mixtures were shaken and incubated at room temperature for 30
min in dark. The decrease in absorbance of DPPH was measured at 517 nm. Inhibition percent was
calculated by comparing the absorbance values of control (DPPH + DMSO) and compounds.
Superoxide dismutase (SOD) activities of the compounds were determined by the method based on the
photo-reduction of nitroblue tetrazolium (NBT) of Beauchamp and Fridovich [64]. The reaction
mixtures were prepared so as to contain 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM
methionine, 75 µM NBT, 2 µM riboflavin and 0.2 mM of each compound. Riboflavin was added as
the last component and the reactions were performed for 10 min by placing the tubes under the
fluorescent source (24 W). The reaction in each tube was terminated by removing the tubes from the
light source. The photo-reduction of NBT was recorded spectrophotometrically at 560 nm and
compared with blank sample which had buffer instead of compound.
Nitric oxide scavenging capacity of the compounds was exhibited by Griess reagent method [65, 66].
0.2 mM compound was mixed with 5 mM sodium nitroprusside in phosphate buffered saline (pH 7.4)
to a final volume of 1 mL. All the mixtures were incubated at 25°C for 150 min. During this period,
sodium nitroprusside generates nitric oxide, which interacts with oxygen to produce nitrite ions.
Compounds which have nitric oxide scavenging ability leads to reduced nitrite ions
10

production by competing with oxygen. At the end of this period 0.5 mL of 1X Griess reagent
(Modified, Sigma) was added. The absorbance was measured at 540 nm.
In order to exhibit the effect of CO releasing on antioxidative activity, the experiments were
performed on compounds which exposed to certain wavelength UV-light (366 nm) for 0, 10 and 20
minutes.
For all scavenging activity assays, % inhibition (I%) values caused by the 0.2 mM of compounds were
calculated by using the following equation:
I% = [(Acontrol - Asample)/ Ablank ] x 100
Electrochemistry:
Cyclic voltammograms (CVs) of the compounds were recorded using a CHI Model 600E Potentiostat
with 3-electrode configuration. The working electrode was a glassy carbon electrode (GCE) with a
diameter of 3 mm. A Pt wire was used as the counter electrode and a Ag/AgCl electrode was used as
the reference electrode. The complex molecules were dissolved in acetonitrile and tetra-n-
butylammonium perchlorate (TBAP) was used as the supporting electrolyte. Analytical concentration
of the solutions was 4.0 mM for each molecule. All the electrochemical experiments were performed
after purging a sufficient amount of pure nitrogen gas to the solutions in order to remove the dissolved
oxygen.
Theory/Calculation:
DFT calculations were carried out with ORCA version 2.8 using the BP86 functional with the
resolution-of-the-identity (RI) approximation, a def2-TZVP/ def2-TZVP/J basis set, the tightscf and
grid4 options, and the COSMO solvation model with water as the solvent for geometry optimizations
[51-54].
ACKNOWLEDGEMENTS:
Support of Scientific and Technological Research Council of Turkey (TÜBİTAK, Project No:
112T320) is gratefully acknowledged.
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15

5
6
7
Figure 1. fac-Manganese(I) tricarbonyl bipyridyl complexes with benzimidazole derivatives
Figure 2. Change of absorption of myoglobin with the irridation at 366 nm UV
light for a solution of complex 1 (15 µM) in 0.1 M PBS (pH=7.4) in the
presence of myoglobin (60 µM) and sodium dithionite (10 mM) under Argon
atmosphere as monitored by UV/Vis spectroscopy
16

Figure 3. CVs of 4.0 mM solutions of (a) compound 5, (b) compound 6, and (c) compound 7 recorded
in acetonitrile containing 0.1 M TBAP on GCE. Scan rate = 50 mV s-1
17

Table 1. CO-Release Data and Absorption Maxima and Extinction Coefficient for Complexes 5-7
Concentration of Half-life Equivalent of
MbCO [µM] t1/2 [min] CO Released
Percentage CO Wavelength
ε
Complex
Released [%]
[nm]
379
378
380
[M-1cm-1]
5
6
7
32.84
27.73
29.45
9.37
11.07
7.31
2.19
1.85
1.96
73.0
3698.8
3465.0
2604.2
61.7
65.3
Table 2. DPPH, SOD and NO radical scavenging ratio as % of 0.2 mM concentrations of some
CORMs due to carbon monoxide emissions by exposure to UV light (366 nm) at different time
intervals (0, 10 and 20 min)
Complex
DPPH
t=10
23.4
10.2
14.0
SOD
t=10
48.5
54.0
61.4
NO
t=10
ND
ND
ND
t=0
2.25
1.53
7.9
t=20
20.9
5.94
43.9
t=0
39.2
38.3
43.8
t=20
51.1
60.0
64.2
t=0
4.96
17.0
11.2
t=20
ND
3.0
5
6
7
ND
ND: Not determined.
18

Table 3. Energies (in nm), Oscillator Strength ( fosc), Main Orbital Contributions, and Type
of Transition Involved in the Most Important Singlet Excitations for 5 Calculated with
TDDFT/BP86
state
molecular orbitals
main transitions
585.1 nm
1
(%82.3)
HOMO
HOMO-1
HOMO-1
LUMO
MLCT (fosc:0.0171)
550.1 nm
2
(%82.0)
MLCT (fosc:0.0187)
LUMO
377.1 nm
11
18
21
(%59.0)
LUMO+2
MLCT (fosc:0.0620)
338.0 nm
(%49.4)
MLCT (fosc:0.0332)
HOMO-1
LUMO+3
352.0 nm
(%68.9)
HOMO-5
LUMO+1
LLCT (fosc:0.0182)
19

22
(%44.9)
329.3 nm
HOMO-5
LUMO+2
LLCT (fosc:0.0393)
304.7 nm
26
(%67.8)
LUMO+6
HOMO
MLCT (fosc:0.0148)
321.2 nm
31
(%46.0)
MLCT (fosc:0.0138)
LUMO+5
HOMO-1
32
326.1 nm
(%84.1)
HOMO-6
LUMO+2
LLCT (fosc:0.0250)
20

21

•
•
Synthesis of dark-stable and light-sensitive novel manganese complexes with benzimidazole
derivative ligands
Analytical, spectroscopic and electrochemical characterization of novel benzimidazole
substituted manganese complexes
•
•
CO-releasing properties and antioxidant activities of photoCORMs
DFT/TDDFT analysis of novel manganese complexes with ORCA package program
22