Synthesis, characterization and SAR studies of bis(imino)pyridines as antioxidants, acetylcholinesterase inhibitors and antimicrobial agents

Article abstract of DOI:10.1016/j.bioorg.2020.104073

In this study we synthesized a series of sixteen bis(imino)pyridines (BIPs) starting from 2,6-diaminopyridine and various aromatic aldehydes, and evaluated their antioxidant, antibacterial, antifungal and acetylcholinesterase (AChE) inhibitory activity. T

Full text of DOI:10.1016/j.bioorg.2020.104073

Journal Pre-proofs
Synthesis, characterization and SAR studies of bis(imino)pyridines as antioxi-
dants, acetylcholinesterase inhibitors and antimicrobial agents
Milena D. Milošević, Aleksandar D. Marinković, Predrag Petrović, Anita
Klaus, Milica G. Nikolić, Nevena Ž. Prlainović, Ilija N. Cvijetić
PII:
S0045-2068(20)31370-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104073
YBIOO 104073
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Please cite this article as: M.D. Milošević, A.D. Marinković, P. Petrović, A. Klaus, M.G. Nikolić, N.Z.
Prlainović, I.N. Cvijetić, Synthesis, characterization and SAR studies of bis(imino)pyridines as antioxidants,
acetylcholinesterase inhibitors and antimicrobial agents, Bioorganic Chemistry (2020), doi: https://doi.org/
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Synthesis, characterization and SAR studies of bis(imino)pyridines as antioxidants,
acetylcholinesterase inhibitors and antimicrobial agents
Milena D. Miloševića, Aleksandar D. Marinkovićb, Predrag Petrovićc, Anita Klausd, Milica G.
Nikoliće, Nevena Ž. Prlainovićb, Ilija N. Cvijetićf,*
aSI Institute of Chemistry, Technology and Metallurgy, National Institute, Department of Ecology and
Techoeconomic, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia
bFaculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia
cInnovation Centre of Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000
Belgrade, Serbia
dDepartment for Industrial Microbiology, Institute for Food Technology and Biochemistry, University of
Belgrade-Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia
eFaculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, University of Niš
fInnovation Center of the Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade,
Serbia
Abstract
In this study we synthesized a series of sixteen bis(imino)pyridines (BIPs) starting from 2,6-
diaminopyridine and various aromatic aldehydes, and evaluated their antioxidant,
antibacterial, antifungal and acetylcholinesterase (AChE) inhibitory activity. The chemical
1
13
structures were elucidated by FTIR, elemental analysis, ESR and HRMS. H and C NMR
spectra couldn’t be acquired due to the formation of stable, carbon-centered radical cations in
a solution, as confirmed by ESR spectroscopy and DFT calculations. The in vitro antioxidant
potency was evaluated using four assays: free radical scavenging activity (DPPH and ABTS),
reducing power and total antioxidant capacity assay. BIPs demonstrated excellent antioxidant
properties, and two derivatives proved to be more potent than reference antioxidants (ascorbic
acid and Trolox) in all assays. DFT calculations on ωB97XD/6-311++g(d,p) level of theory
provided valuable insights into the radical scavenging mechanism of BIPs. For hydroxyl-
substituted BIPs, hydrogen atom transfer (HAT) is a predominant mechanism, while the
single electron transfer coupled with proton transfer (SET-PT) governs the antioxidant
activity of other derivatives. Intramolecular hydrogen bonding (IHB) plays an important role
in the mechanism of antioxidant activity as revealed by noncovalent interaction analysis and
rotational barrier calculations. The spin density of radical cations is localized on carbon atoms
* Corresponding author; e-mail: ilija@chem.bg.ac.rs, Phone: +381 11 3336788
1

of a pyridine ring, which corroborates with g-factors and multiplicity obtained from ESR
analysis. The most potent BIP exhibited moderate inhibitory activity toward AChE (IC50 =
20±4 μM), while molecular docking suggested binding at the peripheral anionic site of AChE
with the MMFF94 binding enthalpy of –43.4 kcal/mol. Moderate in vitro antimicrobial
activity of BIPs have been determined against several pathogenic bacterial strains:
Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus
and clinical isolate of methicillin resistant S. aureus (MRSA). The antifungal activity of BIPs
toward Candida albicans was also confirmed. The similarity ensemble approach combined
with molecular docking suggested leucyl aminopeptidase as the probable antimicrobial target
for the three most potent BIP derivatives.
Keywords: Bis(imino)pyridines; Antioxidant activity; Density functional theory; Electron
spin resonance; Antimicrobial activity; Acetylcholinesterase inhibition
1. Introduction
Currently, about 57% of approved drugs contain at least one heterocycle [1]. Among nitrogen-
containing heterocycles, pyridine and its derivatives are very interesting compounds for
biological, pharmacological and agricultural applications [2–5]. Particularly,
bis(imino)pyridine derivatives (BIPs) represent a versatile class of compounds with
antioxidant, antimicrobial, and antiproliferative activities [6,7]. BIPs as tridentate ligands are
often utilized for the synthesis of organometallic compounds with a broad range of
applications [7–9]. Schiff bases structurally similar to BIPs exhibited antimicrobial activity,
and were more potent upon complexation to transition metal ions [7,10,11]. The presence of
imino group appears to be essential for metal complexation and biological activity of BIPs.
Also, pyrrole and thiophene-substituted heterocyclic Schiff bases are reported as highly
potent, non-toxic antioxidants [12].
Aside from the broad spectrum of biological activities, BIPs are important compounds
for many technological applications. They are utilized as polymer materials, corrosion
inhibitors, catalysts for many organic reactions, analytical reagents and fluorescent sensors
[13–15].
For
example,
N,N'-(pyridine-2,6-diyl)bis[1-(2-hidroxyphenyl)]methanimine
(compound 6, Fig. 1) incorporated into PVC membrane acts as a sensor for selective
determination of Pb2+ ions. The same compound is a useful material for the extraction and
analytical determination of Cu2+ ions in a solution [15]. BIPs-based polymers were
successfully applied for the immobilization of glucose oxidase (GOx) [13].
2

Antioxidants are compounds that interact with harmful free radicals in the body and
prevent their deleterious effects on biomolecules, and the knowledge of their mechanism of
action is a prerequisite for the design of more potent compounds [16]. The radical scavenging
activity is one of the most important mechanisms of antioxidant activity of compounds. The
reactive free radical abstracts the H atom from the antioxidant via three mechanisms:
hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT),
and sequential proton loss electron transfer (SPLET). The inhibition of Fenton reaction
through metal chelation of Fe2+ is another important antioxidant mechanism [17].
In HAT, a hydrogen atom (which can be observed as proton + one electron) is
transferred from the antioxidant (Mol–H) to the free radical:
Mol–H
Mol· + H·
The HAT mechanism is quantified by the bond dissociation enthalpy, BDE:
BDE = H (Mol·) + H (H·) – H (Mol–H)
(1)
The stabilization of radical formed (Mol·) through the electronic and resonance effect
lowers BDE values and contributes to the increased antioxidant activity of a molecule.
The SET-PT mechanism is initiated by the electron transfer (ET) from the antioxidant
to the free radical, followed by deprotonation of radical-cation formed:
Mol–H
Mol–H·+ + e–
Mol–H·+
Mol·+ H+
The first step of this mechanism, SET-PT, determines the overall thermodynamic
feasibility of this process and can be quantified by ionization potential, IP:
IP = H (Mol–H·+) + H (e–) – H (Mol–H)
(2)
The higher is the stability of radical cation, the lower is IP values and SET-PT is a
more feasible process. The second step, proton-transfer step, is characterized by proton
dissociation enthalpy, PDE:
PDE = H (Mol·) + H (H+) – H (Mol–H·+)
(3)
The SPLET mechanism starts with the deprotonation of the group responsible for
antioxidant activity, followed by the ET from the anion:
Mol–H
Mol–
Mol– + H+
Mol· + e–
The first step of this mechanism is characterized by proton affinity, PA:
PA = H (Mol–) + H (H+) – H (Mol–H)
(4)
The ET step of this process is quantified by the electron transfer enthalpy, ETE:
ETE = H (Mol·) + H (e–) – H (Mol–)
(5)
3

In the Equations (1)-(5), enthalpies (H) of the molecule, radical, H atom, radical
cation, anion, proton and electron are labeled as Mol–H, Mol·, H·, Mol–H·+, Mol–, H+, and e–,
respectively.
It should be noted that the net result of all three processes is the same, the transfer of H
atom from antioxidant to free radical. The SET-PT and SPLET mechanisms involve charged
intermediates which are better stabilized in polar media, so these are the preferred radical
scavenging mechanism in polar environments.
Previous DFT studies on the antioxidant activity of phenolic Schiff bases revealed the
importance of BDE for the rationalization of antioxidant mechanism but also showed that
SET-PT is the main antioxidant mechanism of this type of compounds [6].
Our previous work on bis(styryl)pyridines revealed their moderate antioxidant
capacities [18]. In the present study, we combined the benefits of pyridine moiety with two
imine fragments to develop more potent bis-substituted pyridine antioxidants. As current
literature lacks thorough studies on the antioxidant and antimicrobial activity of BIPs, herein
we report the synthesis of 16 BIPs and SAR study of their antioxidant and antimicrobial
activities. The structural characterization of BIPs using NMR spectroscopy was not possible
due to the formation of a stable, paramagnetic species in a solution that caused
broadening/disappearance of resonance signals. This difficulty is a possible reason for a
limited number of studies on BIPs in the literature. The antioxidant potential of BIPs was
mapped using different in vitro assays: DPPH, ABTS, CUPRAC and TAC
(phosphomolybdenum method), and the underlying mechanisms were rationalized with the
aid of DFT calculations. Antimicrobial activity of BIPs was determined toward three Gramm-
positive and two Gramm-negative bacteria, and the plausible molecular targets for the most
potent compounds were identified using molecular similarity approach and molecular
docking. Also, AChE inhibitory potential of BIPs was determined and the results were
supported by docking calculations.
2. Experimental
2.1.
Materials and characterization methods
Full details on materials and characterization methods are given in Electronic
Supplementary Information (ESI).
4

2.2. Synthesis and characterization of symmetrical N,N'-(pyridine-2,6-diyl)bis[1-(substituted
phenyl)]methanimine BIPs (1-16)
The solution of substituted aromatic aldehyde (21 mmol) in 50 mL ethanol (Procedure A,
applied for the synthesis of compounds 1, 2, 3, 6, 12 and 16, Fig. 1) or 20 mL DMSO
(Procedure B, applied for all other compounds) was added dropwise into a solution of 2,6-
diaminopyridine (10 mmol) in 100 mL ethanol. The mixture was heated at 80°C for 2h under
N2 atmosphere. The extent of reaction was monitored by TLC. Upon completion of a reaction,
the precipitate was separated from the hot solution by vacuum filtration, washed with cold
absolute ethanol and air-dried (Procedure A) or dried under N2 (Procedure B).
Fig. 1. General procedure for the synthesis of BIPs (1-16)
Compounds 1, 3 and 4 [19], 6 [20], 8 [19,21], 11 [19,22], 14 [13,19] have been
previously synthesized as precursors and used in situ for the synthesis of other compounds.
The detail characterization of previously reported BIPs using elemental analysis, melting
point determination, IR and HRMS is given in ESI. The results showing the structure and
purity of newly synthesized compounds are reported below. ESR spectra of compounds 8 and
5

14 are shown in ESI, Fig. S2. The atom numbering of synthesized compounds is outlined in
Fig. 1.
N,N'-(pyridine-2,6-diyl)bis[1-(phenyl)]methanimine (1)
Yellow solid, yield 57%, m.p. >250 °C; Elemental analysis: Calcd. for C19H15N3 (Mw =
285.35 g mol-1): C, 79.98; H, 5.30; N, 14.73%; Found: C, 79.91; H, 5.36; N, 14.68%; IR, cm-
1: νmax: 3377 brd (C-H stretching)Py, 3057w, 3025w (C-H stretching)Aryl, 2926s (C-H
stretching)Alkyl, 1663sshp, (C=N stretching)azomethine, 1578sshp (C=C stretching)Aryl, 1445m
(C=C asymmetric stretching)Aryl, 1224w (C=N stretching)Py, 1090m, 1027m (C-H in plane
bending, 700shp (C-H out of plane deformation of phenyl group)Aryl; HRMS: calculated for
C19H16N3 [M+H]+: 286.1347 observed: 286.1346.
N,N'-(pyridine-2,6-diyl)bis[1-(1-naphthyl)]methanimine (2)
Yellow solid, yield 55%, m.p. >270 °C, Elemental analysis: Calcd. for C27H19N3 (Mw =
385.47 g mol-1): C, 84.13; H, 4.97; N, 10.90%; Found: C, 84.05; H, 5.07; N, 11.00%; IR, cm-
1: νmax: 3378brd (C-H)Py, 3058w (C-H)Aryl, 2926s (C-H)Alkyl 1600sshp (C=N)azomethine
,
1584sshp (C=C)Aryl, 1477w (C=C)Aryl, 1216w (C=N)Py, 1022m (C-H)Aryl, 770 (C-H)Aryl;
HRMS: calculated for C27H20N3 [M+H]+: 386.1678 observed: 386.1680.
N,N'-(pyridine-2,6-diyl)bis[1-(4-methylphenyl)]methanimine (3)
Yellow solid, yield 42%, m.p. >280 °C, Elemental analysis: Calcd. for C21H19N3 (Mw =
313.40 g mol-1): C, 80.48; H, 6.11; N, 13.41%; Found: C, 80.35; H, 6.03; N, 13.52%; %; IR,
cm-1: νmax: 3379 brd (C-H)Py, 3020w (C-H)Aryl, 2920s (C-H)Alkyl, 2863shp (C-H stretching of
CH3)Alkyl, 1595sshp (C=N)azomethine, 1509w (C=C)Aryl, 1478w (C=C)Aryl, 1222w, 1170w
(C=N)Py, 1020m (C-H)Aryl 778sshp (C-H)Aryl; HRMS: calculated for C21H20N3 [M+H]+:
314.1657 observed: 314.1657.
N,N'-(pyridine-2,6-diyl)bis[1-(4-methoxyphenyl)]methanimine (4)
Yellow solid, yield 70%, m.p. >270 °C, Elemental analysis: Calcd. for C21H19N3O2 (Mw =
345.40 g mol-1): C, 73.03; H, 5.54; N, 12.17%; Found: C, 72.98; H, 5.58; N, 12.20%; IR, cm-
1: νmax: 3377brd (C-H)Py, 3065w, 3000w (C-H)Aryl, 2932s (C-H)Alkyl, 2834m (C-H stretching of
CH3-O)Alkyl, 1609sshp (C=N)azomethine, 1508m (C=C)Aryl, 1461m (C=C)Aryl, 1247s (Ph-O
stretching), 1172s (CH3-O stretching)Alkyl, 1110w (C=N)Py, 1031m (C-H)Aryl, 810 (C-H)Aryl;
HRMS: calculated for C21H20N3O2 [M+H]+: 346.1561 observed: 346.1559.
6

N,N'-(pyridine-2,6-diyl)bis[1-(3,4,5-methoxyphenyl)]methanimine (5)
Yellow solid; yield 48%, m.p. >250 °C, Elemental analysis: Calcd. for C25H27N3O6 (Mw =
465.51 g mol-1): C, 64.51; H, 5.85; N, 9.03%; Found: C, 64.48; H, 5.90; N, 9.10%; IR, cm-1:
νmax: 3371 brd (C-H)Py, 3060w, 3000w (C-H)Aryl, 2937s (C-H)Alkyl, 2834m (C-H of CH3-
O)Alkyl, 1599sshp (C=N)azomethine, 1502m (C=C)Aryl, 1462m (C=C)Aryl, 1232s (Ph-O stretching),
1183s (CH3-O stretching)Alkyl, 1125s (C=N)Py 1003m (C-H)Aryl, 776 (C-H)Aryl; HRMS:
calculated for C25H28N3O6 [M+H]+: 466.1899 observed: 466.1901.
N,N'-(pyridine-2,6-diyl)bis[1-(3-hidroxyphenyl)]methanimine (7)
Yellow solid, yield 45%, m.p. >250 °C, Elemental analysis: Calcd. for C19H15N3O2 (Mw =
317.35 g mol-1): C, 71.91; H, 4.76; N, 13.24%; Found: C,72.01; H, 4.68; N, 13.19%; IR, cm-1:
νmax: 3425sbrd (O-H), 3362 brd (C-H)Py, 3030w (C-H)Aryl, 2927s (C-H)Alkyl, 1595sshp
(C=N)azomethine, 1481m (C=C)Aryl, 1478w (C=C)Aryl, 1230s (C=N)Py, 1147 (Ph-O stretching),
1000m (C-H)Aryl, 781 (C-H)Aryl; HRMS: calculated for C19H16N3O2 [M+H]+: 318.1287
observed: 318.11280.
N,N'-(pyridine-2,6-diyl)bis[1-(2-quinolinyl)]methanimine (9)
Yellow solid, yield 71%, m.p. >280 °C, Elemental analysis: Calcd. for C25H17N5 (Mw =
387.45 g mol-1): C, 77.50; H, 4.42; N, 18.08%; Found: C, 77.45; H, 4.48; N, 18.12%; IR, cm-
1: νmax: 3379 brd (C-H)Py, 3055w (C-H)Aryl, 2927s (C-H)Alkyl, 1597sshp (C=N)azomethin, 1503m
(C=C)Aryl, 1478m (C=C)Aryl, 1306m (C=N streching)Aryl, 1233m (C=N)Py, 1043, 1023m (C-
H)Aryl, 780 (C-H)Aryl; HRMS: calculated for C25H18N5 [M+H]+: 388.1455 observed: 388.1456.
N,N'-(pyridine-2,6-diyl)bis[1-(8- hydroxy-2-quinolinyl)]methanimine (10)
Red solid, yield 68%, m.p. >280 °C, Elemental analysis: Calcd. for C25H17N5O2 (Mw =
419.14 g mol-1): C, 71.59; H, 4.09; N, 16.70; %; Found: C, 71.52; H, 4.05; N, 16.77 %; IR,
cm-1: νmax: 3425sbrd (O-H), 3387 brd (C-H)Py, 3032w (C-H)Aryl, 2927s (C-H)Alkyl, 1596sshp
(C=N)azomethine, 1504m (C=C)Aryl, 1478m (C=C)Aryl, 1326m (C=N)Aryl, 1238s, (C=N)Py,
1195wbrd (Ph-O), 1011m (C-H)Aryl, 781 (C-H); HRMS: calculated for C25H18N5O2 [M+H]+:
420.1402 observed: 420.1410.
N,N'-(pyridine-2,6-diyl)bis[1-(4-fluorophenyl)]methanimine (12)
Yellow solid, yield 78%, m.p. >275 °C, Elemental analysis: Calcd. for C19H13F2N3 (Mw =
321.33 g mol-1): C, 71.02; H, 4.08; N, 13.08%; Found: C,70.94; H, 4.12; N, 13.11%; IR, cm-1:
7

νmax: 3388 brd (C-H)Py, 3066w (C-H)Aryl, 2923s (C-H)Alkyl, 1600sshp (C=N)azomethine, 1506m
(C=C)Aryl, 1477m (C=C)Aryl, 1227s (C=N)Py, 1152s, (Ar-F stretching), 1014m (C-H)Aryl, 768
(C-H)Aryl; HRMS: calculated for C19H14F2N3 [M+H]+: 322.1156 observed: 322.1150.
N,N'-(pyridine-2,6-diyl)bis[1-(4-bromophenyl)]methanimine (13)
Yellow solid, yield 76%, m.p. >270 °C, Elemental analysis: Calcd. for C19H13Br2N3 (Mw =
443.14 g mol-1): C, 51.50; H, 2.96; N, 9.48%; Found: C, 51.58; H, 3.01; N, 9.42%; IR, cm-1:
νmax: 3378 brd (C-H)Py, 3047w (C-H)Aryl, 2924s (C-H)Alkyl, 1590sshp (C=N)azomethine, 1482m,
(C=C)Aryl, 1448w (C=C)Aryl, 1227w (C=N)Py, 1010m (C-H)Aryl, 768 (C-H)Aryl; HRMS:
calculated for C19H14Br2N3 [M+H]+: 444.7546 observed: 444.7545.
N,N'-(pyridine-2,6-diyl)bis[1-(4-nitrophenyl)]methanimine (15)
Orange solid, yield 49%, m.p. >275 °C, Elemental analysis: Calcd. for C19H13N5O2 (Mw =
375.34 g mol-1): C, 60.80; H, 3.49; N, 18.66%; Found: C, 60.76; H, 3.51; N, 18.73%; IR, cm-
1: νmax: 3362 brd (C-H)Py, 3030w (C-H)Aryl, 2927s (C-H)Alkyl, 1597sshp (C=N)azomethine, 1500m
(C=C)Aryl, 1478w (C=C)Aryl, 1350 (N=O stretching), 1228s (C=N)Py, 1023m (C-H)Aryl, 829s
(C-H)Aryl.. HRMS: calculated for C19H14N5O2 [M+H]+: 376.1019 observed: 376.1021.
N,N'-(pyridine-2,6-diyl)bis[1-(2-pyridinyl)]methanimine (16)
Yellow solid, yield 68%, m.p. >250 °C, Elemental analysis: Calcd. for C17H13N5 (Mw =
287.33 g mol-1): C, 71.06; H, 4.56; N, 24.37%; Found: C, 71.10; H, 4.61; N, 24.31%; IR, cm-
1: νmax: 3372 brd (C-H)Py, 3045w (C-H)Aryl, 2927s (C-H)Alkyl, 1614sshp (C=N)azomethine, 1500m
(C=C)Aryl, 1478w (C=C)Aryl, 1300m (C=N)Aryl, 1240s (C=N)Py, 1076, 1049m (C-H)Aryl 768s
(C-H)Aryl; HRMS: calculated for C17H14N5 [M+H]+: 288.1315 observed: 288.1321.
2.3. Synthesis of other 2,6-bis(imino)pyridine derivatives
To better understand the antioxidant activity of BIPs, structurally similar compounds 17
[23], 18 [24], 19 [24,25], 20 [7] and 21 [26] were synthesized and their characterization is
given in ESI (Fig. S3).
2.4. Computational methods
The previous study showed that BIPs predominantly exist in enolimine form in the solid
state and solution, so geometry optimization of BIPs started from bis(imino) structure [20,27].
The structures of the possible conformations of compound 1 are shown in Fig. S4. Concerning
8

the geometrical isomerism around imine bonds, s-trans/s-trans isomer was taken as an initial
geometry, as this isomer proved to be more stable than s-cis/s-cis in structurally related
bis(styryl)pyridines [18,28].
The initial 3D structures were generated using MMFF94 force field [29]. The
conformational search was done by systematic rotation of all flexible bonds with 15°
increment, using AMMP program implemented in Vega ZZ 3.1.0 [30]. The lowest-energy
conformer was submitted to full geometry optimization at ωB97XD/6-311++g(d,p) level in
the gas phase. The vibrational analysis confirmed that all structures correspond to a minimum
of PES (no imaginary frequency). The solvent effects were accounted using the SMD model
of water as a biologically relevant medium, methanol, and benzene whose low dielectric
constant imitates the inner part of phospholipid membrane [31]. The solution enthalpies were
obtained by adding the gas-phase thermal corrections to enthalpy (TCE) to the single-point
energies calculated with the SMD model. This approach is justified by the fact that TCE does
not significantly depend on a solvent model applied, while calculations of TCE with implicit
solvation models extremely increase the computational time without giving an additional
accuracy [32]. The geometry optimization of anions, radicals, and radical-cations was
performed using the same procedure, where unrestricted calculations were performed for
radicals and radical-cations. The solution enthalpies of H+ and e– were taken from [33], while
enthalpy of H atom was calculated at the same level of theory.
For studying the intramolecular hydrogen bonding, bond critical points (BCPs) were
calculated in Multiwfn 3.7 [34], starting from the geometries optimized at ωB97XD/6-
311++g(d,p) level of theory. The noncovalent interactions (NCI) were visualized according to
the method described in [35], implemented in Multiwfn program. NCI graphs were rendered
in VMD [36]. The rotational barrier for 6 was calculated by scanning the dihedral angle
between the phenol ring and azomethine moiety (C7-C8 bond). The reaction coordinate was
systematically modified by 20° while all other geometry parameters were reoptimized. All
calculations were performed using ωB97XD/6-311++g(d,p), in vaccum. Gaussian 16, revision
B.01 was used for all DFT computations [37].
The structure of compound 16 was docked into the active site of AChE (PDB code
1Q83 [38]) using AutoDock Vina [39]. This structure is a protein-ligand complex with a
large, bis-tacrine ligand which occupies both PAS and AS of AChE. Upon removal of ligand,
the binding pocket remains in „open“ conformation that allows BIPs to reach all parts of
AChE gorge. Exhaustiveness was set to 100 and 10 binding modes were calculated.
9

To identify the potential antimicrobial targets for BIPs, SMILES strings of all
compounds were submitted to SEA Search Server [40]. 3D protein structure of leucyl-
aminopeptidase identified in the previous step was retrieved from PDB code 1CP6 [41]. The
ligand and water molecules were removed, hydrogen atoms were added to resemble
protonation state at pH = 7.40 [42]. The protein structure was minimized using 100ps
conjugate gradient minimization in NAMD 2.12 program [43] and CHARMM force field.
The protein backbone was kept fixed to preserve the experimental structure, so only steric
bumps were minimized. The active site was defined as all residues within a 12 Å radius from
Zn2+ ion. AutoDock4Zn [44] parameters were used for docking in AutoDock 4.2, and
VegaZZ 3.1.0 [30] and AutoDockTools 1.5.6 [45,46] were used for the preparation of input
files. One pseudo atom was placed in tetrahedral orientation to Zn2+ ion where inhibitor
should be coordinated. MMFF94 binding enthalpy calculation and binding mode visualization
were done in LigandScout 4.2.6 [47].
2.5. Antioxidant capacity methods
All details about the methods for the determination of antioxidant capacity of BIPs are
given in ESI.
2.6. AChE inhibition activity testing
The experimental procedure for the determination of AChE inhibition is given in ESI.
2.7. Antimicrobial activity testing
A detailed description of the methods for antimicrobial activity testing of BIPs is
given in ESI.
3. Results and discussion
3.1. Synthesis and characterization
The compounds were synthesized by the condensation of 2,6-diaminopyridine with
different phenyl-substituted aromatic aldehydes with the yields from 42 to 78% (Fig. 1).
During the routine 1H NMR analysis of the first three synthesized derivatives (4, 14, and 15),
we were surprised by the absence of sharp resonance peaks. Even after the prolonged signal
acquisition, only low-intensity, broad signals were obtained. Furthermore, we attempted to
acquire NMR spectra of all other derivatives in three deuterated solvents (CDCl3, DMSO-d6,
10

and acetone-d6) and using elevated temperature (50 ºC). Although the complete solubility of
compounds (>10mg/0.4mL) was ensured, no peaks were obtained in any case (examples of
NMR spectra of compounds 11 and 14 are given in ESI, Figs. S5 and S6). Nevertheless, there
are studies reporting the successful characterization of BIPs using the NMR spectroscopy
[48].
Therefore, we used different techniques to check the purity of the samples. According
to elemental analysis, samples with a good purity were obtained directly from the reaction
mixture by careful crystallization, washing with cold absolute ethanol and drying under
nitrogen atmosphere. HRMS data further confirmed the purity of synthesized compounds
(examples given in ESI, Figs. S7 and S8). Besides the molecular ion, M+1 and M+2 ions were
observed. These ions can be attributed to partial or full reduction of azomethine group [49].
Interestingly, previously reported compound 10 was also obtained with high yield and purity
without any purification [50]. Additionally, the characteristic IR absorption bands confirmed
the presence of azomethine group, pyridine and aromatic rings.
Therefore, the confirmed BIPs purity and the observed absence of NMR signals
suggest the presence of paramagnetic species in a solution. Although BIPs act as tridentate
ligands, the synthetic method didn’t include any metal ions as it was confirmed by analysis of
the acidic extract from synthesized BIPs using inductively coupled plasma mass spectrometry
(ICP-MS). Thus, the possibility for the formation of paramagnetic organometallic compounds
in the course of BIPs synthesis was excluded. Searching for the possible explanations, we
found one study reporting the inability to record NMR spectra of structurally similar BIPs
[50]. Using the electron spin resonance (ESR) spectroscopy, the authors explained NMR-
silence of BIPs through the formation of a stable, carbon-centered radical on a pyridine ring.
To test if this effect might explain the observed NMR silence, we recorded ESR
spectra for all compounds. All tested compounds showed the same ESR pattern but with
different intensities. The value of g-factor was about 2.006, indicating the formation of
carbon-centered, not N-centered radicals for all compounds [50]. As a representative, the ESR
spectra of 8 and 14 are given in ESI, Fig. S2. No hyperfine structure was observed in the ESR
spectrum of any compound, indicating the absence of nuclei with nuclear spin 1 or ½ in the
proximity of radical species. The results suggest the formation of a stable radical cation
through the ionization of BIPs in a solution.
3.2. Antioxidant capacity of BIPs
11

The antioxidant capacity (AOC) of natural antioxidants is mainly associated with low-
energy phenolic –OH bond(s), where highly stabilized radical species are formed after ET or
HAT. Apart from four derivatives with –OH bonds, the structure of studied
bis(imino)pyridines is very different from the structure of natural antioxidants. The AOC of
sixteen BIPs was investigated using four in vitro assays mapping different antioxidant
characteristics of a molecule: DPPH and ABTS as free radical methods, CUPRAC test as a
measure of reducing capacity, and phosphomolybdenum assay characterizing the total
antioxidant capacity of DIPs. The results obtained were compared with the AOC of two well-
known standards, ascorbic acid and Trolox. Furthermore, DFT calculations were performed in
order to gain more insights into the antioxidant mechanism of BIPs.
3.2.1. Free-radical methods
DPPH and ABTS tests have been widely used for screening the free radical
scavenging ability of novel synthetic or natural products antioxidants. These measurements
are based on the reduction of radical species through the electron- and/or hydrogen-atom
transfer from antioxidants to the free radical which is accompanied by the change in
absorbance.
It was previously shown that the heterocyclic imine-based Schiff bases possess a good
antioxidant activity in DPPH and ABTS assays, where the presence of an electron-donating
group on the phenolic ring improves the activity [12].
All sixteen BIPs showed good antiradical potency toward DPPH•, with IC50 values
between 0.027 and 0.375 mM (Table 1). Compounds 8 and 10 were the most potent in DPPH
assay, while 8, 10 and 14 were the most potent antioxidants in ABTS test. The most potent
compound 10 contains quinoline structure and hydroxyl group on heterocyclic moiety which
might contribute to the extended π-electron delocalization and stabilization of radical species.
The 8-hydroxyquinoline is recognized in the literature as a moiety with positive contribution
to the antioxidant activity [51].
Additionally, within the subset of phenolic BIPs, the AOC is correlated with the
position of the –OH group, with the following order of activity: 10>>8>7>6. The presence of
strong IHB in compound 6 is previously confirmed by crystal structure analysis [20], and this
interaction makes hydrogen atom transfer to reactive radical species more difficult.
Table 1. The IC50 values for BIPs (1-16) determined in DPPH and ABTS free radical assays
12

DPPH
IC50 , mM
ABTS
IC50 , mM
Compound
*
*
1
2
3
4
5
6
7
8
9
0.280±0.051
0.289±0.040
0.153±0.022
0.168±0.025
0.184±0.033
0.175±0.021
0.109±0.005
0.084±0.045
0.215±0.015
0.027±0.010
0.184±0.022
0.197±0.014
0.162±0.016
0.188±0.031
0.375±0.020
0.226±0.037
0.039±0.019
0.120±0.024
0.111±0.007
0.178±0.011
0.084±0.035
0.140±0.057
0.195±0.009
0.088±0.018
0.034±0.014
0.117±0.023
0.031±0.011
0.218±0.012
0.191±0.022
0.209±0.016
0.030±0.011
0.247±0.051
0.205±0.022
0.052±0.034
10
11
12
13
14
15
16
Ascorbic acid
*IC50 values are expressed as mean ± standard deviation (SD) of three independent measurements
Compared to DPPH assay BIPs proved to be even more potent in ABTS test with the
IC50 values range from 0.030 to 0.218 mM (Table 1). The results indicate that the positive
contribution of electron-donating substituents is more pronounced than in DPPH test. For
example, dimethylamino group as the strongest electron-donating substituent within this
series yielded the most potent BIP derivative when introduced in para-position. The methoxy
group, as another strong electron-donating substituent yielded a potent para-substituted
derivative. The trend in the activity of hydroxyl derivatives is the same as for DPPH assay.
Again, the least potent compound was para-nitro derivative (15), the strongest electron
acceptor within this series.
3.2.2. Reducing power assay, total antioxidant capacity
The antioxidant activity of compounds is also attributed to the chelation of transition
metal ions such as iron (Fe2+) and copper (Cu+) which have been proposed as catalysts for the
in vivo formation of reactive oxygen species. Therefore, in addition to typical radical
scavenging assays (DPPH and ABTS), reducing power method using CUPRAC have been
applied to provide additional details about the AOC of BIPs. The good metal-chelating ability
13

of BIPs is already reported, acting as a good tridentate ligand forming pincer complexes with
metal ions [52]. The results of CUPRAC assay were compared with Trolox as a reference
antioxidant (Table 2) and determined from the ratio of molar absorptivity (ESI, Fig. S9, and
Table S1). The compounds 10 and 14 bearing strong electron-donating groups had the highest
AOC, while electron-withdrawing substituents (11-13, 15) decreased the AOC.
Table 2. The results of CUPRAC assay, given relative to Trolox, and the results of total
antioxidant capacity (TAC) assay using the phosphomolybdenum method, expressed as the
equivalent mass of ascorbic acid.
Reducing power assay
CUPRAC
TEAC
0.273
Total antioxidant capacity (TAC)
Compound
Phosphomolybdenum assay
1
2
3
4
5
6
7
8
9
0.165
0.132
0.133
0.156
0.183
0.178
0.119
0.146
0.536
0.209
0.163
0.148
0.139
0.306
0.146
0.211
-
0.292
0.663
0.350
0.839
0.143
0.198
0.451
0.453
1.104
0.193
0.256
0.171
0.977
0.202
0.533
1.000
10
11
12
13
14
15
16
Trolox
Total antioxidant capacity (TAC) was performed by phosphomolybdenum test, which
is based on the reduction Mo(VI) to Mo(V) and monitoring of corresponding change in the
absorbance [53]. The obtained results are presented as mass equivalents of ascorbic acid
(Table 2). The most potent compound 9 bears 2-quinoline substituents, indicating the
importance of additional coordinating N-atoms in the complexation of Mo(VI).
14

3.3. DFT studies of structure-antioxidant capacity relationships
Antioxidant activity of the molecule is a direct consequence of its structure,
conformation and solvent properties. The structural features which stabilize the radical
species formed upon electron or hydrogen atom transfer improve the antioxidant capacity of a
molecule. Therefore, not only the presence of particular functional groups but also the overall
molecular geometry of antioxidant should be considered when deriving structure-antioxidant
relationships within the congeneric series of compounds.
The geometry optimization of a ground state of BIPs provided the important structural
parameters for further structure-antioxidant capacity relationship studies. The optimized
structures of 1-16 are presented in Fig. 2 and geometry data are given in ESI, Table S2. The
shortest bonds between central pyridine ring and imino groups are found for the strong
electron-donating substituents like N,N-dimethyl (compound 14), indicating that the electronic
effects can be transferred from substituents to the central pyridine moiety.
Considering the symmetry of compounds, a large degree of planarity could be
expected. Therefore, the values of torsional angles between the central pyridine ring and
imino “arm” groups are studied (Fig. 2). This torsion will strongly influence the degree of
conjugation through the molecule, which might have implications for the antioxidant activity.
The lowest torsion angle of 10.64o and the most planar structure is for compound 6, due to
presence of OH group in –ortho position to imino group. Such an arrangement enables
intramolecular hydrogen bonding (IHB) between hydroxyl hydrogen and imino nitrogen thus
forming six-membered rings and stabilizing almost planar structure. Somewhat higher values
of torsional angle, around 16o for the compounds 2, 9 and 10 are probably due to the presence
of rigid fused rings. For all other compounds, the values range between 23 and 26o.
15

Fig. 2. Optimized geometries of BIPs with the torsional angles indicated
Molecules exhibit their antioxidant activity and diminish the oxidative stress by
adopting one of the previously described mechanisms (HAT, SET-PT or SPLET), or their
combination. According to the experimental data (Tables 1 and 2), it can be concluded that
the electron-donating substituents improve, while electron-withdrawing substituents decrease
the AOC of BIPs. To search for further details about the antioxidant mechanism of BIPs, we
computed several molecular descriptors of the antioxidant activity using the DFT method in
gas phase and simulating the solvation effects of water, benzene and methanol (Table 3).
16

Table 3. BDE, IP and PA values of BIPs computed at ωB97XD/6-311++g(d,p) level in the gas phase and using SMD solvation model of
benzene, water, and methanol.
BDE (kcal/mol)
C6H6 H2O MeOH
IP (kcal/mol)
C6H6 H2O MeOH
PA (kcal/mol)
C6H6 H2O
Reactive
group
Comp.
Gas
Gas
Gas
MeOH
Imino
97.89
98.11 100.53 99.98 174.13 153.15 138.81 139.46 399.38 156.22 92.19 93.19
1
Py 4-H 111.16
Py 3-H 114.30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
3
4
5
Imino
Imino
Imino
Imino
Imino
OH
Imino
OH
Imino
OH
Imino
Imino
OH
Imino
Imino
Imino
Imino
Imino
Imino
94.23
97.27
99.25 101.12 102.62 172.79 152.74 141.21 144.34 389.94 148.80 85.73 85.88
97.40 99.66 99.14 169.03 149.27 136.11 136.71 400.74 156.62 90.98 92.33
100.02 100.43 102.65 102.33 165.12 146.59 135.12 135.46 394.49 150.66 85.44 86.69
97.21 97.33 99.54 99.01 168.30 149.90 138.49 138.84 400.02 157.07 91.74 92.79
99.69 100.05 102.02 101.74 177.66 156.12 141.07 141.55 390.64 150.42 89.92 90.94
96.70 95.25 91.46 92.43 141.55 349.77 110.10 43.80 45.83
100.25 100.66 102.90 102.58 174.16 153.36 138.75 139.30 391.34 148.38 84.33 85.55
91.46
97.65
81.75
93.07
94.43
88.27
97.62
97.49
97.65
97.47
95.50
94.93
6
7
-
-
-
91.30 92.94 93.84
97.84 100.23 99.69 168.18 148.72 135.97 136.25 400.99 157.61 93.04 94.08
81.76 84.64 85.00 136.25 335.33 96.80 38.04 39.42
-
-
-
139.30 348.71 107.18 38.59 40.79
8
9
-
-
-
94.18 98.19 97.68 171.59 152.48 139.97 140.45 381.91 143.15 87.76 88.32
95.28 98.42 97.99 177.12 156.69 141.88 142.56 378.29 141.34 87.09 87.60
10
85.98 81.23 82.37
-
-
-
142.56 345.80 105.99 40.08 42.25
11
12
13
14
15
16
97.89 100.31 99.80 177.17 155.51 139.48 140.27 393.26 152.58 91.11 92.00
97.72 100.06 99.55 176.28 154.29 138.64 139.39 387.84 146.22 84.20 85.29
97.92 100.34 99.83 177.15 155.88 139.68 140.48 392.81 152.42 91.02 91.91
97.53 99.74 99.18 152.77 136.17 127.07 127.09 407.21 162.03 94.19 95.40
95.14 95.49 95.12 189.02 163.29 143.13 144.16 366.96 131.19 73.43 73.91
96.15 99.81 99.39 173.61 153.38 140.05 140.58 386.46 145.93 87.41 88.22
17

As a molecular descriptor of HAT, BDE was calculated for all compounds. Taking
compound 1 as a model, we initially screened several hydrogen atoms (imino group and
pyridine H-atoms) to find the lowest-energy bond. According to the results given in Table 3,
the imino C–H atom is the most reactive site. Therefore, for all other BIPs the imino moiety
was considered as HAT site. For hydroxyl-substituted derivatives 6, 8 and 10, the BDE of
phenolic –OH group was also considered.
The thermodynamically feasible HAT reaction between DPPH• and BIPs might be
expected only for hydroxyl-substituted derivatives 8 and 10, as BDE for DPPH•–H is 82.6
kcal/mol [54], so for all other compounds the equilibrium: BIP–H + DPPH•
BIP· +
DPPH2 will be highly shifted toward reactants. The higher BDE values for 6 might be
explained with the formation of IHB between the –OH group and imino N atom which
stabilizes the ground state. The HAT from –OH group disrupts the IHB, leading to the larger
energy difference between the ground state and O radical, and higher BDE according to Eq.
(1).
To test the hypothesis that IHB plays an important role in the reaction of BIPs and
DPPH•, we calculated the strength of IHB for two derivatives containing –OH group (6 and
10) using Atoms in Molecules (AIM) analysis. The positions of BCPs for IHB of 6 and 10 are
indicated via NCI analysis, Fig. S10. Topology analysis provided several parameters such as
electron density (ρ), Laplacian of electron density (∇2ρ) and potential energy density (V(r))
which are correlated with the H-bond binding energy. According to Espinosa et. al [55], the
strength of hydrogen bond (EHB) can be approximated by 1/2 of V(r). The results are given in
Table 4.
Table 4. Electron density, Laplacian of electron density, potential energy density, H-bond
….
binding energy and donor-acceptor distance for O–H N intramolecular hydrogen bonds of 6
and 10.
Comp. ρ, a.u. ∇2ρ, a.u. D-A distance, Å V(r), a.u. EHB, kcal/mol
6
10
0.04772 0.1167
0.02466 0.0930
1.748
2.069
-0.04450
-0.01934
13.96
6.07
The nature of IHB in compound 6 is additionally studied by calculating the rotational
barrier around the C7-C8 bond (Fig. S11). The calculated rotational barrier (15.52 kcal/mol)
originates from strong IHB interaction with a small contribution of steric effect.
The exceptionally strong IHB of 6 rationalize its low antioxidant activity. On the other
hand, compound 10 is a highly potent antioxidant in all assays (Tables 1 and 2) although IHB
18

might be expected in this structure as well. From Table 4 we can see that the strength of
corresponding IHB is significantly lower compared to 6, so hydroxy-group at position 8 is
more available for the interaction with free radicals. Compared with the computed interaction
energy for phenol-water pair (7.7 kcal/mol) [56], we conclude that –OH group of compound 6
remains „locked“ in pseudo 6-membered ring when solvated by explicit water molecules,
while 10 prefers intermolecular H-bonding with solvent molecules, releasing the –OH group
from intramolecular 5-membered ring.
The spin density (SD) distribution is a useful tool to visualize the degree of spin
delocalization in the radical species formed upon HAT or SET-PT. Higher delocalization
indicates better stabilization of radicals/radical cations, and lower BDE and IP values (Eqs.
(1) and (2)). SD distribution maps can also provide information about the reactivity of various
active sites in the molecule [57]. The SD distribution maps of two potent compounds 8 and 14
are shown in Fig. 3. Hydroxyl radicals (Fig. 3b) have much more pronounced spin
delocalization compared to imino radicals (Fig. 3a). The spin density of radical cations (Figs.
3c and 3d) is delocalized over the entire molecule, where the majority of spin density is
located at carbon atoms of the pyridine ring. The schematic representation of SD distribution
for all 16 BIPs in the gas phase and water solvation model is given in ESI, Figs. S12-S15.
Fig. 3. Spin density distribution mapped for: (a) imino radical of 14; (b) O radical of 8; (c)
radical cation of 14; (d) radical cation of 8, at the isovalue 0.0004 a.u.
19

Highly localized SD of imino radicals explains the weak influence of substituents on
BDE values. On the other hand, pronounced delocalization of radical cation’s SD indicate its
stability and supports the presence of paramagnetic species in a solution. Overall results
reveal weak dependence of BDEs on substituent’s nature and the medium tested, so additional
descriptors are necessary to fully rationalize the experimental results.
The suggested formation of stable radical cation in a solution indicates the facile
ionization of BIPs, which should favor the SET-PT mechanism. The lowest value of IP in all
environments is found for compound 14, and the highest for compound 15. As those
compounds have strong electron-donating or electron-accepting group attached in p-position
of benzene rings, respectively, this implies that the IP is strongly influenced by the position
and nature of substituents. Furthermore, statistically significant correlations between IP and
Hammett substituents constants (Fig. S16) suggest that the ionization potential of BIPs might
be successfully predicted using LFER principles, thus saving computational time for the
design of novel BIP-based antioxidants. On the other hand, the low statistical validity of BDE
versus σ constant correlations (r = 0.02 (for gas phase) to 0.08 (for water)) was observed,
which further confirms the weak influence of substituents on BDE of BIPs.
Accordingly, high BDE of compound 14 suggests that HAT is not a thermodynamically
favorable process, so SET-PT mechanism must be an important contributor to the excellent
potency of 14 observed in DPPH and ABTS tests.
From the thermodynamic point of view, the preferred antioxidant mechanism is
determined by the absolute values of BDE, IP, and PA as the first step of HAT, SET-PT and
SPLET mechanisms, respectively. From Table 3 it is clear that HAT is a favorable radical
scavenging mechanism in the gas phase and benzene, while SPLET is feasible only in water.
It should be noted that the overall antioxidant performance of a compound also depends on
the nature of scavenged free radical, the explicit interactions with the environment, as well as
the kinetics of the process.
There is another common procedure for the elucidation of a preferable antioxidant
mechanism from the DFT-computed descriptors. According to Wright et al. [58], two main
radical scavenging mechanisms, HAT and SET-PT, occur simultaneously but with different
rates, and the dominant process can be elucidated from the difference in gas-phase BDE and
IP between phenol and antioxidant. They concluded that the HAT dominates for antioxidants
with ΔBDE ≈ –10 kcal/mol and ΔIP up to –36 kcal/mol, while SET-PT is a dominant
mechanism for antioxidants with ΔIP above –45 kcal/mol. Taking into account the
20

experimental data for BDE and IP of phenol (88.0 kcal/mol and 196.2 kcal/mol, respectively)
[58,59] and the data from Table 3, we conclude that HAT is a dominant mechanism for
hydroxyl-substituted derivatives 8 and 10, while SET-PT is a dominant mechanism for
compound 14.
The main mechanism for compound 8 (4-OH derivative) can be also deduced by the
comparison with experimental data of 4-OCH3 derivative (compound 4). Compound 4 lacks
the reactive –OH group and cannot react via HAT mechanism, while stronger electron-
donating groups should additionally stabilize radical cation, favoring the SET-PT mechanism.
Substitution of –OH with –OCH3 group increases the IC50 values 2-3 times (Table 1),
indicating that HAT is a dominant mechanism for compound 8.
3.5. Structure-antioxidant capacity relationships - Interpretation of mechanisms by
correlation methods
The antioxidant mechanism of the BIPs was also analyzed using one- and two-
parameter linear regression analysis, i.e. linear free energy relationships (LFER) principles.
Correlation analysis of DFT descriptors such as HOMO/LUMO energies, energy gap (Egap)
values (ESI, Table S3), BDE, and IP (Table 3), along with Hammett constants (ESI, Table S4)
with the antioxidant activities further help in the interpretation of the antioxidant mechanisms.
The BIPs radical scavenging ability versus HOMO energy value (Table S3) was
considered in the first step. The exponential and linear relations of DPPH and ABTS activity
versus HOMO energy are given in Figs. S17 and S18, respectively. From Fig. S18, increased
activity versus increasing HOMO energy was observed. Generally the electron-donating
groups increase the energy of HOMO orbitals, while opposite trend is for electron-accepting
groups. Also, Egap values generally follow a similar trend (ESI, Fig. S19 and Table S3). For
compound 10, the appropriate stabilization of HOMO/LUMO orbital leads to the lowest Egap.
In this compound, both HOMO and LUMO orbitals are delocalized symmetrically over 8-
hydroxyquinolin-2-yl moiety in former and imino moieties in later orbital. Also, IHB and
electron-donating character of dimethylamino and hydroxy groups in compounds 6, 8 and 14,
respectively, cause a higher extent of delocalization (Fig. S19), leading to the increased
activity. The linear dependence of ABTS activity versus HOMO energy (Fig. S18) is in
accordance with the proposed antioxidative mechanism (Eq. (2)).
The results obtained from preliminary correlations between IC50 and BDE, IP and PA,
using all synthesized compounds, showed low statistical validity. Acceptable and similar
correlation results of IC50 (DPPH and ABTS) and TEAC (CUPRAC) versus BDE were
21

obtained for the compounds 6 - 8 and 10 in water and methanol (ESI, Figs. S20, S22, and S24,
respectively). Different type of correlation line was obtained: linear for DPPH assay (Fig
S20), and parabolic for ABTS and CUPRAC (Figs. S22 and S24). Such results indicate that
HAT is a favorable mechanism for hydroxy-substituted compounds 6, 8 and 10 in DPPH
assay, while in the ABTS and CUPRAC assays other processes also participate, with the main
contribution of HAT process. Also, the different trends of the correlation lines of DPPH,
ABTS and CUPRAC scavenging data versus IP for the other compounds, given in Figs. S21,
S23 and S25, indicate a complex mechanism of antioxidative activity. In line with this, the
opposite slopes of linear correlations, obtained for IC50 versus IP for ABTS (Fig. S23) and
CUPRAC assay (Fig. S25) in two solvents, corroborate with the complex influences of
different parameters on obtained AOC values.
The AOC of BIPs is also correlated with Hammett σ constants (Figures S26-S29).
Different types of correlations are observed for DPPH, ABTS, CUPRAC and TAC activity vs.
Hammett σ constants, suggesting that electronic effects of substituents are not solely
responsible for the antioxidant mechanism of BIPs.
The LFER multi-parameter approach was valuable in an estimation of the
contributions of thermodynamic values BDE and IP to the measured antioxidant activity.
Thus, the obtained correlation results, with the assumption that correlation coefficients relate
to contributions of HAT and SET-PT mechanism, were used in an analysis of the overall
antioxidant mechanism. The two-parameter correlation approach using a set of obtained
activities (Table 1) versus BDE and IP descriptors (Table 3) yielded the correlation results
(Table S5) used for calculation of the percentage contribution of HAT and SET-PT
mechanism (Table S5). The correlation results were used for the analysis of the nature of
antioxidant mechanism for BIPs. Results from two-parameter correlations of IC50 versus BDE
and IP, including all compounds, indicated a somewhat higher BDE contribution, 52%, over
IP, 48%, in DPPH assay (Table S5). Exclusion of compound 15 from correlation increased the
BDE contribution to 69%, while the opposite is true if dimethylamino is excluded (46%). This
result indicates that dimethylamino group contributes to the significant stabilization of imino
radical formed, i.e. stabilization of the benzylidene radical is strongly favored by the extended
π-delocalization. Exclusion of all hydroxyl-substituted BIPs did not provide satisfactory
correlations, while the exclusion of 4-OH substituted BIP decreased HAT contribution to 38%
(Table S5). Different correlation results were obtained for ABTS test considering all
compounds studied. A higher IP contribution of 65% suggests that the molecule reacts
primarily with the ABTS•+ via SET-PT mechanism. Exclusion of compounds 1, 2, 4 and 9
22

improved the statistical validity of correlation with 40% and 60% of HAT and SET-PT
participation, respectively. The opposite correlation obtained by the exclusion of compounds
2, 4, 6-8 and 10, indicates the prevalence of SET-PT mechanism in water and methanol.
Ortho-hydroxy substituted compound 6 behaves as SET-PT promoting compound due to π-
delocalization within pseudocyclic structure which prevents an efficient H atom abstraction
(Fig. S10). In general, the obtained correlation results, summarized in Table S5, indicate that
the radical scavenging mechanism is composed of the appropriate contribution of the electron
(IP) and H atom (BDE) transfer steps, depending on electronic properties and the nature of
substituents.
To find the additional evidence on the antioxidant mechanism of BIPs, compounds 17-
21 (Fig. S3) were synthesized and tested in DPPH and ABTS assays. Consideration of the
structural analogy and antioxidant activities helped in the understanding of active sites taking
place in a concomitant process of electron and proton abstraction. Structural isomerism of
compounds 1 and 17 and similar activity (12% and 20% higher activity of compound 17)
indicate on similar effect on stabilization of either radical or radical cation formed in DPPH
and ABTS test, respectively. It could be supposed that participation of both HAT and SET-PT
takes place in a DPPH assay, while SET-PT is a major process in ABTS assay. Also, the
significant increase of DPPH activity for compound 18 (48% higher than for 6) indicates the
existence of a free hydroxyl group able to participate in the HAT process. This is opposite to
the structurally arranged hydrogen bonding in compound 6 which decreases the effectiveness
of hydrogen transfer. Fairly similar activity of compounds 8 and 19 (less than 10% difference)
confirmed this conclusion. Additionally, increased and decreased DPPH activity of
compounds 20 and 21 for 22% and 46%, respectively, compared to compound 1, indicate that
the HAT process participates at approximately 46% considering imino radical formation
(Table S5). Otherwise, a similar activity of compounds 1 and 21 in ABTS test is additional
evidence of the derived conclusions.
In summary, these results showed complex influences of structural and electronic
effects of substituents on the HAT and SET-PT processes, which participate in the overall
antioxidant mechanism in different extents depending on the assay performed.
3.6. AChE inhibitory activity of BIPs
All tested compounds showed poor to moderate inhibitory activity towards AChE
(ESI, Fig. S30 and Table S6). The most potent derivative was compound 16 (IC50 = 21±4 μM)
bearing two o-pyridyl substituents. The low potency of BIPs bearing various substituents with
23

different electronic and steric effects indicates that further modification of scaffold is
necessary to achieve inhibitory activity in nanomolar range. To elucidate the binding mode of
16 into the active site of AChE, molecular docking was performed. The tentative binding
mode and ligand interaction diagram are shown in Fig. 4.
Fig. 4 a) Binding mode of 16 into the active site of AChE obtained by docking
calculations. The hydrophobic pharmacophores are shown as a yellow sphere, and hydrogen-
bond acceptor pharmacophores are shown as red spheres. Hydrogen bonding interactions
between protein and ligand are depicted with red arrows while stacking interactions between
16 and Trp 286 and Tyr 72 are shown as blue rings on the upper pyridine ring of a ligand; b)
Ligand interaction diagram showing a 2D depiction of stabilizing interactions between 16 and
AChE; c) The orientation of 16 (light blue sphere) inside AChE binding pocket. Amino acid
residues comprising peripheral anionic site are colored green, the acyl-binding pocket is in
red, deep anionic site is in yellow, and catalytic triad (Glu 334, His 447, Ser 203) is shown as
blue sticks.
As can be seen from Fig. 4c, compound 16 occupies mainly peripheral anionic site
(PAS) located at the rim of AChE binding pocket [60]. One pyridyl substituent forms π- π
stacking interactions with Trp 286 and Tyr 72, and central pyridine ring forms hydrogen bond
with Tyr 124. The pyridine central ring also forms hydrophobic interactions with Tyr 341.
The only interacting residue outside PAS is Tyr 337, a part of anionic site (AS) located deeper
24

in the AChE gorge. The calculated binding affinity was -8.9 kcal/mol, and MMFF94 binding
enthalpy was -43.43 kcal/mol. An increase in the molecular size to allow more interactions
inside AS, particularly with Trp 86, appears as a rational structural modification to improve
the potency of BIPs toward AChE.
3.7. Antibacterial and antifungal activity of BIPs
BIPs and particularly their metal complexes are often studied as antimicrobial
agents [7,24]. To evaluate the antimicrobial potential of 16 BIPs and derive preliminary SAR
information, we tested all compounds toward several Gramm + and Gramm – strains that are
common human pathogens, as well as one fungus (C. Albicans). Screening the several
bacterial and one fungal strain we aimed to gain more general insights into the antimicrobial
potential of BIPs.
The tested compounds showed bacteriostatic potential toward all studied bacteria (Table 5).
The compounds were very effective against E. coli (MIC 0.31 - 5.00 mmol/L), E. faecalis
(MIC 0.16 - 5.00 mmol/L) and S. aureus (MIC 0.16 - 5.00 mmol/L). More importantly, the
compounds were active against highly resistant S. aureus MRSA (MIC 0.08 - 5.00 mmol/L).
Along with this, almost half of the analyzed compounds exert a fungistatic effect toward C.
albicans (MIC 0.16 - 5.00 mmol/L). The compound 16 showed the ability to destroy the
bacteria E. faecalis and P. aeruginosa (MBC 5.00 mmol/L).
Previous studies have shown that compounds 6, 8 and 16 exert moderate antimicrobial
activity while metal complexation significantly improves the activity [7]. Two compounds, 16
and 6, exerted a potent fungicidal effect against C. albicans comparable with fluconasole
(MBC 0.31 mmol/L for 16 vs. 0.16 mmol/L for fluconasole). The overall assessment
indicated that among sixteen studied BIPs, compound 16 (bis-2-pyridyl derivative) was the
most potent antimicrobial agent, followed by 2-OH derivative (compound 6).
25

Table 5. Antimicrobial activity of BIPs, amoxicillin (AMX, used only for bacterial strains) and fluconazole (FLU, used only for fungal strain),
expressed as MIC (mmol/L) and MBC (mmol/L), determined by the broth microdilution method
Pseudomonas
aeruginosa
Enterococcus
Staphylococcus
aureus
Staphylococcus
aureus MRSA
clinical isolate
Candida albicans
Escherichia coli
faecalis
Compound
ATCC 10231
ATCC 27853
ATCC 25922
ATCC 29212
ATCC 25923
MIC
-
-
-
MBC
MIC
MBC
MIC
5.00a,A
5.00a,A
-
5.00a,A
5.00a,A
2.50b,B
MBC
MIC
5.00a,A
2.50b,B
5.00a,A
5.00a,A
-
MBC
MIC
MBC
MIC
5.00a,A
2.50b,B
-
5.00a,A
5.00a,A
0.08d,E
MBC
1
1
2
3
4
5
6
7
8
9
-
-
-
-
-
5.00a,A2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5.00a,A
0.62d,C
5.00a,A
5.00a,A
5.00a,A
1.25c,C
-
-
-
-
-
-
-
-
-
-
5.00a
-
-
-
-
-
1.25c,B
0.16d,D
0.31a,B
5.00a,A
2.50b,B
2.50b,A
-
-
-
-
-
-
-
-
5.00a,A
-
-
-
-
-
-
-
5.00a,A
5.00a,A
5.00a,A
5.00a,A
5.00a,A
5.00a,A
5.00a,A
-
-
-
-
2.50b,B
5.00a,A
5.00a,A
5.00a,A
5.00a,A
2.50b,B
2.50b,B
-
-
-
-
-
-
-
1.25c,C
-
5.00a,A
2.50b,B
2.50b,B
1.25c,C
2.50b,B
5.00a
2.50b,B
5.00a,A
5.00a,A
5.00a,A
-
-
-
-
-
10 5.00a,A
11 5.00a,A
12
13 2.50b,B
14
15 1.25c,C
5.00a,A
-
-
-
-
-
5.00a
-
5.00a,A
-
-
5.00a,A
-
-
-
-
-
5.00a,A
-
-
1.25c,C
-
16 0.16d,B 0.31a,C 0.31b,A 5.00a,A 0.31c,A 5.00a,A 0.16c,B 5.00a,A 0.16e,B 2.50b,B 0.08d,C 2.50b,B
Amoxycillin
Fluconazole
0.07c,A
0.14b,A
0.02d,B
0.07b,B 0.001d,C 0.01b,C 0.001f,C 0.004c,D
0.04e
0.16b
As standard deviations do not exist, they are not presented in the table.
1 Not achieved
2 Values in the same column with different lowercase letters, as well as values in the same row with different capital letters are significantly different at α = 0.05 (ANOVA, Tukey's HSD test).
26

3.8. Target fishing and molecular docking indicate possible antibacterial targets for BIPs
To indicate a possible mechanism of antimicrobial action of BIPs, similarity ensemble
approach was applied [40]. In this method, the proteins are clustered according to the chemical
similarity of their ligands, so chemical similarity of novel molecules with the ensemble of
molecules from database gives a rational guess of potential cellular targets and possible
pharmacology pathways. The three highest-ranked molecular targets for each of the most active
ligands (compounds 6, 10 and 16) are shown in Table 6, while suggested molecular targets for
other ligands are listed in Table S7.
Table 6. The three best-ranked molecular targets suggested by similarity ensemble search for
compounds 6, 10 and 16. Overall rank is a combination of several parameters such as distance
from the mean (Z-score) and Tanimoto similarity index (Max TC).
Compound
Suggested target
Endothelin-converting enzyme 2
Cysteine protease falcipain-3
Bacterial leucyl aminopeptidase
Gag-Pro-Pol polyprotein
Z-score Max TC
61.9499 0.3590
35.7526 0.4286
17.6778 0.3235
76.2021 0.3000
56.1682 0.3256
21.9179 0.4000
6
10
16
Endothelin-converting enzyme 2
Bacterial leucyl aminopeptidase
Amiloride-sensitive amine oxidase [copper-
containing]
Bacterial leucyl aminopeptidase
5'-AMP-activated protein kinase subunit
gamma-1
17.6168 0.2955
17.1653 0.3143
15.7955 0.3171
As seen from Table 6, bacterial leucyl aminopeptidase is recognized as the best-ranked
target for compounds 6, 10 and 16. It is a Zn2+-dependent protease that selectively cleaves
amino-terminal Leu residue from polypeptides. These compounds showed notable activity
against a panel of bacteria (Table 5), so it is reasonable to hypothesize this enzyme as a
molecular target of BIPs in bacterial cells.
To characterize the possible molecular interactions between the most potent compound
16 and bacterial aminopeptidase we performed two molecular docking simulations. In the first
one, no energy minimization/MD was performed upon removal of ligand so aminopeptidase
binding pocket was in the exact conformation as in complex with the inhibitor 1-butaneboronic
acid. In the second one, the protein side chains were minimized to remove steric bumps.
27

According to the lowest estimated free energy of binding (-7.71 vs. -7.42 kcal/mol), the complex
of 16 in non-minimized protein structure (Fig. 5) is more stable compared to minimized one (Fig.
S31). One pyridyl moiety resides deep into the binding pocket, forming the hydrophobic
interactions with Met 180, Met 242, Phe 244, and Phe 248. Second pyridyl moiety is located at
the entrance of binding pocket, forming hydrophobic interaction with Leu 155 and hydrogen
bond with Gly 154 backbone. Two active site Zn2+ ions are coordinated to central pyridine N
atom that occupies tetrahedral position where co-crystalized inhibitor was bound. The bridging
Asp 117 is hydrogen bonded to azomethine N atom (Fig. 5).
Fig. 5. a) Possible binding mode of 16 into the active site of Leu aminopeptidase (PDB code
1CP6); b) Ligand interaction diagram showing the most important interactions. The binding
pocket of a receptor is shown as a transparent surface. The hydrophobic pharmacophores are
shown as a yellow sphere, and hydrogen-bond acceptor pharmacophores are shown as red
spheres. Hydrogen bonding interactions are depicted with red arrows, and zinc binding
interactions are indicated with blue cones.
The qualitative SAR analysis reveals that the compounds having an additional
coordinating atom near tridentate bis(imino)pyridine moiety are the most potent antibacterial
BIPs. As target fishing analysis indicated Zn2+-dependent Leu aminopeptidase as the most
probable molecular target for the most potent BIPs, it is reasonable to hypothesize the metal
chelating ability of BIPs as the main characteristics responsible for antibacterial activity.
4. Conclusion
In the present study, we reported synthesis, structural and biological characterization of a
series of bis(imino)pyridines (BIPs). The formation of a stable, carbon-centered radical on a
28

pyridine ring is suggested by EPR spectroscopy and spin density derived from DFT calculations.
Excellent antioxidant activity is found for several derivatives, while DFT calculations at
ωB97XD/6-311++g(d,p) level aided the elucidation of the antioxidant mechanism. HAT is the
main antioxidant mechanism for hydroxyl-substituted BIPs, while SET-PT governs the activity
of other derivatives. The moderate inhibitory activity of BIPs toward AChE was found. The most
potent compound 16 exert inhibition in the low micromolar range (IC50 = 20±4 μM), and binds
to the peripheral anionic site of AChE as suggested by molecular docking. Moderate activity
toward several bacterial and fungal strains is also confirmed. Similarity ensemble search
combined with molecular docking suggested leucyl aminopeptidase as the plausible bacterial
target for the most potent derivatives 6, 10 and 16.
Acknowledgements
The authors acknowledge financial support from Ministry of Education, Science and
Technological development of Serbia, Ministry of Education, Science and Technological
Development of Republic of Serbia, Contract number: 451-03-68/2020-14/200288, 451-03-
68/2020-14/200026, and 451-03-68/2020-14/200135.
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