Density functional theory molecular modeling and antimicrobial behaviour of selected 1,2,3,4,5,6,7,8-octahydroacridine-N(10)-oxides

Article abstract of DOI:10.1016/j.molstruc.2017.05.003

A series of 9-substituted 1,2,3,4,5,6,7,8-octahydroacridine-N(10)-oxides is evaluated against 12 bacterial and fungal strains, for their microbicidal and anti-pathogenic features. The largest spectrum of the antibacterial activity is evidenced for the nitro- (2b) and hydroxy- (5b) N-oxides, followed by the amino-N-oxide (3b). Density functional theory (DFT) modeling of the molecular structure and frontier molecular orbitals, i.e. highest occupied/lowest unoccupied molecular orbital (HOMO/LUMO), is accomplished by using the GAMESS 2012 software at M11/ktzvp level of theory in order to find their structural and electronic parameters. We show that the planarity of the molecules and the presence of the electron withdrawing group are advantages for its antimicrobial activity. Finally, we briefly present and discuss results on the processing of such compounds into thin films and hybrid structures by laser-assisted techniques, i.e. matrix-assisted pulsed laser evaporation (MAPLE) or laser-induced forward transfer (LIFT), to provide simple and environmental friendly, state-of-the-art solutions for antimicrobial/medical coatings and devices.

Full text of DOI:10.1016/j.molstruc.2017.05.003

Journal of Molecular Structure 1144 (2017) 14e23
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Density functional theory molecular modeling and antimicrobial
behaviour of selected 1,2,3,4,5,6,7,8-octahydroacridine-N(10)-oxides
,
,
Maria Marinescu a **, Ludmila Otilia Cinteza b ***, George Iuliu Marton c,
Luminita Gabriela Marutescu d e, Mariana-Carmen Chifiriuc d
,
,
, e
Catalin Constantinescu f
, g, *
a UB e University of Bucharest, Faculty of Chemistry, Department of Organic Chemistry, Biochemistry and Catalysis, 90-92 S¸ oseaua Panduri, Sector 5, RO-
050663 Bucharest, Romania
b UB e University of Bucharest, Faculty of Chemistry, Department of Physical Chemistry, 90-92 S¸ oseaua Panduri, Sector 5, RO-050663 Bucharest, Romania
c UPB e University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu bd., RO-011061 Bucharest, Romania
d UB e University of Bucharest, Faculty of Biology, Department of Botanic-Microbiology, 1-3 Aleea Portocalilor, RO-060101 Bucharest, Romania
e ICUB e Research Institute of the University of Bucharest, 91-95 Splaiul Independentei, RO-050095 Bucharest, Romania
f INFLPR e National Institute for Laser, Plasma and Radiation Physics, Department of Lasers, 409 Atomistilor St., RO-077125, Magurele, Bucharest, Romania
g University of Limoges, CNRS, ENSCI, SPCTS, UMR 7315, F-87000 Limoges, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 9-substituted 1,2,3,4,5,6,7,8-octahydroacridine-N(10)-oxides is evaluated against 12 bacterial
and fungal strains, for their microbicidal and anti-pathogenic features. The largest spectrum of the
antibacterial activity is evidenced for the nitro- (2b) and hydroxy- (5b) N-oxides, followed by the amino-
N-oxide (3b). Density functional theory (DFT) modeling of the molecular structure and frontier molecular
orbitals, i.e. highest occupied/lowest unoccupied molecular orbital (HOMO/LUMO), is accomplished by
using the GAMESS 2012 software at M11/ktzvp level of theory in order to find their structural and
electronic parameters. We show that the planarity of the molecules and the presence of the electron
withdrawing group are advantages for its antimicrobial activity. Finally, we briefly present and discuss
results on the processing of such compounds into thin films and hybrid structures by laser-assisted
techniques, i.e. matrix-assisted pulsed laser evaporation (MAPLE) or laser-induced forward transfer
(LIFT), to provide simple and environmental friendly, state-of-the-art solutions for antimicrobial/medical
coatings and devices.
Received 9 January 2017
Received in revised form
31 March 2017
Accepted 2 May 2017
Keywords:
1,2,3,4,5,6,7,8-Octahydroacridine-N(10)-
oxide
Antimicrobial activity
DFT study
Disc diffusion
Minimal inhibitory concentration (MIC)
Biofilm
© 2017 Elsevier B.V. All rights reserved.
Virulence
1. Introduction
therefore provides selective pressure, which makes the surviving
bacteria more likely to be resistant. Other factors contributing to-
Widespread, repeated and improper use of antibiotics, i.e.
chemicals, compounds, or drugs designed to cure or prevent in-
fections, are the primary causes of the increase in antibiotic-
resistant bacteria. Sensitive bacteria are killed, but resistant
germs may be left to grow and multiply. Exposure to antibiotics
wards resistance include incorrect diagnosis, unnecessary pre-
scriptions, improper use of antibiotics by patients, and the use of
antibiotics as livestock food additives for growth promotion. This
problem is amplified by the ability of bacteria to grow in a sessile,
adherent state, developing microbial communities called biofilms,
1000-fold more resistant to antibiotic treatments with respect to
their planktonic counterparts [1e4]. To illustrate, although the
treatment of choice for Staphylococcus aureus (S. aureus) infection is
penicillin, more than half (52%) of all current cases are resistant to
penicillin, methicillin, tetracycline and erythromycin [5]. Thus, with
the occurrence of multi-drug, extended-drug, and pan-drug resis-
tance phenotypes, the necessity of identifying innovative, radical
antimicrobial agents is of paramount importance. Smart use of
* Corresponding author. Current address: University of Limoges, CNRS, ENSCI,
SPCTS, UMR 7315, F-87000 Limoges, France.
** Corresponding author.
*** Corresponding author.
E-mail addresses: maria.marinescu@chimie.unibuc.ro (M. Marinescu), ocinteza@
gw-chimie.math.unibuc.ro
(C. Constantinescu).
(L.O.
Cinteza),
catalin.constantinescu@unilim.fr
http://dx.doi.org/10.1016/j.molstruc.2017.05.003
0022-2860/© 2017 Elsevier B.V. All rights reserved.

M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
15
antibiotics is therefore the key to controlling the spread of resis-
tance [6e8].
according to the reaction pathways presented in Fig. 1 [46]. sym-
OHA (1a) resulted together with trans-syn-trans-perhydroacridine
and trans-anti-cis-perhydroacridine by the reaction of 2,2'-methy-
lene-bis-cyclohexanone with formamide. 9-Nitro-sym-OHA (1b)
was synthesized in two steps from sym-OHA. Amino-N-oxide (3b)
was obtained by reducing nitro-N-oxide (2b) with hydrogen over
Pd catalyst and methoxy-N-oxide (4b), water-soluble, resulted also
from (2b), by the reaction with methanol on potassium carbonate
as catalyst. Hydrolysis with 48% HBr of (4b) gives hydroxy-N-oxide
(5b). The 1H, 13C, 15N NMR spectra of the compounds, and the
thermal analysis data, are all in accordance with those previously
reported [58e63]. Finally, the (2b-5b) N-oxides have been studied
for their qualitative and quantitative in vitro properties to evaluate
their biologic activity.
There are many commercial products claimed to be microbicidal.
Recently, we have shown that biologically active 2-substituted
benzimidazoles can be used as antimicrobial coatings [9]. Herein,
we present and discuss results on 1,2,3,4,5,6,7,8-octahydroacridine
(sym-octahydroacridine, or sym-OHA), exposing the relationship
between the structure vs. their microbicidal and anti-pathogenic
features. Pyridines, quinolines, acridines and their N-oxides are
important not only as structural units in natural compounds and
synthetic pharmaceuticals, but have also elicited considerable
theoretical interest; sym-OHA and its substituted derivatives gained
much attention as starting materials in the syntheses of hydrogen-
bonding receptors for biomolecules [10e15], of metal-bonding
polyaza torands [13e15] and of other polyaza cavity-shaped li-
gands [16e22]. Organic N-oxides are also an important structural
class in many pharmaceutical and industrial chemicals, with
numerous pyridine and pyridine-1-oxide derivatives exhibiting a
broad range of biological activities, such as antimicrobial [23e29],
antiviral [30e32], antioxidative [29e33], antidiabetic [34], fungi-
static activity [35,36], vasorelaxant [37,38], antiviral [39,40], anti-
malarial [41,42], antitumoral [43,44]. Some are also known for their
central nervous system depressant activity [45]. The use of 1,2,3,4-
tetrahydroacridines in treating Alzheimer's disease [46,47] and as
AchE inhibitors [48] is also noteworthy.
In this work, we present and discuss on the electronic
and structural parameters of four 9-substituted 1,2,3,4,5,6,7,8-
octahydroacridin-N(10)-oxides (sym-OHA-N(10)-oxides), calcu-
lated by using the density functional theory (DFT) at M11/ktzvp
level of theory, and further correlate the results to their biologic
activity. The largest antibacterial activity spectrum is evidenced for
the nitro- (2b) and hydroxy- (5b) N-oxides, followed by the amino-
(3b) N-oxide. Finally, we briefly present and discuss results on the
processing of such compounds into thin films and hybrid structures
by laser-assisted techniques, i.e. matrix-assisted pulsed laser
evaporation (MAPLE) or laser-induced forward transfer (LIFT), to
provide simple and environmental friendly, state-of-the-art solu-
tions for antimicrobial/medical coatings and devices.
2.3. Microbial strains
The four chemical compounds have been tested for their anti-
microbial activity using the following microbial strains: Gram
negative (Salmonella sp. 9246, extended spectrum beta-lactamase
(ESBL) Klebsiella pneumoniae, Escherichia coli 1576 ESBL þ, Serratia
marcescens 0804, Citrobacter freundii 1748, Providencia stuartii 1116,
Pseudomonas aeruginosa 846), Gram positive (Staphylococcus aureus
ATCC 25923, Staphylococcus epidermidis 1736, Enterococcus faecalis
ATCC 29212, Bacillus subtilis 6688) bacterial strains and the fungal
strain Candida albicans ATCC 10231, by using qualitative and
quantitative screening assays (Table 1).
2.3.1. Qualitative screening of the antimicrobial properties of the
tested compounds
The four compounds have been solubilized in water or methanol
(10 mg/ml) and the antimicrobial activity was performed by spot-
ting 10 ml of the compound solutions on the solid medium, previ-
ously inoculated with bacterial suspension. The Petri dishes were
left at the room temperature to ensure the equal diffusion of the
compounds in the medium or to allow the drop of the solution to be
absorbed into the medium, and thereafter, the dishes were incu-
bated at 37 ꢁC for 24 h [64,65].
2. Experimental details
2.3.2. Quantitative assay of the antimicrobial activity
The quantitative assay concerning the antimicrobial activity of
the compounds was performed by using the two fold microdilution
method in liquid broth distributed in 96-multi well plates, in order
to establish the minimal inhibitory concentration (MIC). Over
Naturally occurring in coal tar together with its derivatives,
acridine (C13H9N) is a stable nitrogen heterocycle molecule that
exhibits weakly basic properties, structurally related to that of
anthracene (C14H10), with one of the central (CH) groups replaced
by nitrogen [49,50]. These molecules can also be produced through
many synthetic processes, e.g. the Bernthsen synthesis [51e54], the
Lehmstedt-Tanasescu reaction [55,56], etc.
150
pound stock solution were added, performing thereafter serial two
fold dilutions from 5000 to 9.7 g/ml. Finally, in each well, 30 ml
ml liquid medium distributed in each well, 150 ml of the com-
m
microbial suspension of 0.5 McFarland density were added. The
plates were incubated for 24 h at 37 ꢁC and MICs were determined
as the last concentration of the respective tested compound [60,61].
2.1. Fourier transform infrared spectroscopy
The Fourier transform infrared spectroscopy (FTIR) spectra have
been recorded at a resolution of 4 cmꢀ1 and a spectral range of
4000e400 cmꢀ1, by using a Vertex 70-Bruker spectrophotometer,
as presented elsewhere [57]. The KBr pellet technique is used: 1 mg
of each sample was dispersed in about 300 mg of anhydrous KBr,
and pressed under vacuum at 20 tons/cm2. Finally, all spectra were
processed by means of the OPUS 6.5 software. Atmospheric
compensation, vector normalization, baseline correction, and one
additional concave rubber band correction, have all been applied,
too.
2.3.3. Investigation of the compounds' influence upon the
expression of virulence factors
In order to investigate the influence of the new chemical com-
pounds upon the expression of different virulence factors, the mi-
crobial strains were cultivated for 24 h in the presence of sub
inhibitory concentrations of the tested compounds; thereafter, the
respective bacterial cultures were investigated for the production
of the following virulence factors: i) the bacterial ability to colonize
the abiotic surface: the liquid cultures from the MIC plates were
removed and the wells were washed three times with phosphate
buffer saline (PBS), the microbial cells adhered to the plastic wells
were fixed with methanol, stained with 1% crystal violet, washed
with distilled water and solubilized in 33% acetic acid, the absor-
bance of the colored suspension bring read to an Elisa reader at the
2.2. Synthesis of sym-OHA-N(10)-oxides
Four 9-substituted N-oxides (2b-5b) have been synthesized

16
M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
O
HCONH2
167-170oC
O
+
+
N
N
N
H
H
1a
NO2
HNO3 / H2SO4
H2O2 / AcOH
N
+
N
N
+
_
_
O
O
2b
1a
1b
OCH3
OH
NH2
MeOH / K2CO3
H2 / Pd-C
HBr
48%
2b
N
+
N
+
N
+
_
_
_
O
O
O
5b
3b
4b
Fig. 1. The synthesis route for the sym-octahydroacridine-N(10)-oxides 1b-5b.
Table 1
determined using pig stomach mucin (to a final concentration of
1%) incorporated in brain heart agar supplemented with 2% NaCl.
The strains were spotted and incubated at 35 ꢁC for 48 h; the
enzyme activity was noticed by the presence of a clear area around
the culture spot. The clear area became more evident when some
Lugol's drops were poured upon; viii) the amylase production was
tested on 10% starch supplemented agar medium. The strains were
stubbed and incubated at 37 ꢁC for 24 h, starch hydrolysis being
registered by the presence of a clear area around the culture spot;
ix) esculin hydrolysis with the formation of esculethol was inves-
tigated after stubbing the bacterial culture on solid medium con-
taining 1% esculin and iron citrate. After incubation at 37 ꢁC for 24 h,
the positive cultures induced the occurrence of a black area around
the culture spot, due to the reaction of the resulted esculethol with
the iron salt [66e70].
Qualitative screening of the antimicrobial activity of the tested compounds.
Tested microbial strains
Diameter of the growth inhibition zone (mm)
2b
3b
4b
5b
Control
S. epidermidis 1736
S. aureus ATCC 25923
E. faecalis ATCC 29212
B. subtilis 6688
P. stuartii 1116
P. aeruginosa 846
K. pneumoniae ESBLþ
S. marcenscens 0804
C. freundii 1748
E. coli 1576, ESBL þ
Salmonella sp. 9246
C. albicans ATCC 10231
5
8
6
11
6
9
5
6
6
5
9
9
e
e
e
e
5
e
e
e
e
e
4.5
e
e
7
5
8
e
6.5
8
7.5
5
8
5
8
11
5
7.5
5.5
e
2.5
8
7.5
6
6
5
5
8.5
6
5
6.5
5.5
5.5
11
5.5
5.5
10
5.5
10
2.4. Computational analysis
wavelength of 490 nm; ii) the plate hemolysis was evidenced by
streaking the tested strains on blood agar plates containing 5% (v/v)
sheep blood in order to obtain isolated colonies. After incubation at
37 ꢁC for 24 h, the clear areas (total lysis of the red blood cells)
around the colonies were registered as positive reactions; iii) the
lipase production was investigated after spotting the microbial
strains on. Twin 80 agar as a substrate at a final concentration of 1%
and the plates were incubated at 37 ꢁC until 7 days. A precipitation
zone around the spot was registered as positive reaction; iv) for the
lecithinase production, the cultures were spotted into 2.5% yolk
agar and were incubated at 37 ꢁC for 7 days. An opaque halo around
the spot indicated the lecithinase production; v) the DN-ase pro-
duction was studied on DNA supplemented agar. The strains were
spotted and after incubation at 37 ꢁC for 24 h, a drop of HCl 1 N
solution was added upon the spotted cultures; a clearing zone
around the culture was interpreted as positive reaction; vi)
caseinase activity was determined using 15% soluble casein agar as
substrate. The strains were spotted and after incubation at 37 ꢁC for
24 h, a precipitation zone surrounding the bacteria growth indi-
cated the casein production; vii) the mucinase production was
A quantum mechanical modeling was performed on each 9-
substituted sym-OHA-N-oxide, using the GAMESS 2012 [71] soft-
ware in order to find theirs structural parameters. The modeling
was performed on a computer cluster consisting of 12 nodes and 96
cores running on Linux CeontOS. The results were visualized using
wxMacMolPlt [72]. The molecular geometries of the compounds
were optimized using DFT at M11/ktzvp level of theory. Tuhlar's
M11 [73] is a modern functional that provides better results than
classical B3LYP. Also basis set used is a more recent one (Karlsruhe
valence triple zeta basis with a set of single polarization) intro-
duced by Prof. Ahlrichs [74,75]. The parameters used for geometry
optimization are the default ones used in GAMESS. The geometry
used during calculations is described by “natural internal co-
ordinates” generated by GAMESS.
3. Results and discussion
The electronic and structural properties of all the sym-OHA-N-
oxides have been investigated and further correlated to their

M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
17
microbicidal and anti-pathogenic activity. We first present and
discuss the results regarding their in vitro properties in order to
evaluate their biologic activity, compare and discuss with results on
the frontier molecular orbital (FMOs) analysis performed on all
sym-OHA-N-oxides by using the DFT theory, followed by calcula-
tions of the atomic charges and geometric parameters of these
molecules.
S. epidermidis 1576 (Table 3). The expression of soluble virulence
factors has not been influenced in the presence of subinhibitory
concentrations of the tested compounds, excepting the compound
2b which inhibited the expression of lipases and siderophores like
production by S. marcescens 0804 strain.
3.2. Electronic and structural properties
3.1. Microbicidal and anti-pathogenic activity
3.2.1. Electronic parameters of the 9-substituted sym-OHA-N-
oxides
The qualitative screening of the tested compounds evidenced
the occurrence of a bactericidal effect of at least one of the tested
compounds, superior to that of the methanol solvent, against all the
tested bacterial strains. None of the tested compounds did exhibit
antifungal activity superior to that of the solvent represented by
methanol (Table 2). It is to be noticed that the water soluble com-
pound 4b was the less active, exhibiting growth inhibition effect
only against two of the tested strains, i.e. Bacillus subtilis and
Escherichia coli. The 2b and 5b compounds proved to be more active
than 3b, against six of the tested strains. The active compounds
selected during the qualitative screening were further tested in the
quantitative assay for establishing the MIC value. The spectropho-
FMO analysis was performed on all sym-OHA-N-oxides by using
the DFT theory at M11/ktzvp level. The contour plots of the frontier
orbitals e i.e. the highest occupied molecular orbital (HOMO), and
the lowest unoccupied molecular orbital (LUMO), together with the
energies of the HOMO and LUMO orbitals EHOMO, ELUMO, Egap
(EHOMO e ELUMO), dipole moment (DM), obtained using DFT at M11/
ktzvp level of theory, and combined with the hydratation energy
(HE), versus the total antimicrobial activity for 2b-5b, are all shown
in Table 3. Molecular dynamics simulation has been employed in
the computation of the free energy of solvation for a large number
of solute molecules with different chemical functionalities in the
solvent. Furthermore, the outermost orbital occupied with elec-
trons, i.e. HOMO, works as an electron donor because it tends to
give these electrons, while LUMO, the innermost orbital containing
free orbitals accept electrons from HOMO [76]. FMOs have an
important role in intermolecular interactions through the interac-
tion between the HOMO of the drug with the LUMO of the receptor
and vice versa [77]. Therefore, a good interaction has been expected
in case of low Egaps. The following observations can be made,
studying the dates from Table 3: i.) the maximum Egap for 4b, which
means a minimum reactivity of all compounds, correlates with the
worst antimicrobial activity; ii.) the minimum Egap for 2b, therefore
a better reactivity for it, concretizes, in good qualitative antimi-
crobial activity, and also the best minimum inhibitory concentra-
tions (Tables 1 and 2); iii.) a very good correlation between the
energy of hydration of the studied compounds and their reactivity
can be made from Table 3. The order of increasing energy of hy-
dration is proportional with the decreasing antimicrobial activity of
N-oxides 2b-5b; iv.) compounds 5b and 3b have higher dipole
moments (5.682 and respectively 4.442 Debye), which increases
their ability to interact with the surroundings. Although 2b has a
very low dipole moment, of 0.674 D, it has a good capacity to
interact, so the other parameters must be studied to correlate its
good activity; v.) antimicrobial activities don't correlate very good
with all calculated electronic parameters from Table 3; we will try
tometric measurements have evidenced MIC values
(mg/ml)
ranging from 10 g/ml to 250 g/ml (Table 3). However, only in few
m
m
cases the MIC values were superior to that of the solvent, i.e. 3b
against S. aureus and E. faecalis, 4b against B. subtilis, and 5b against
S. epidermidis (Table 3). The alarming rates of microbial resistance
to antibiotics led to the search of new antimicrobial strategies,
based on the inhibition of the coordinated expression of virulence
factors implicated in different steps of the infectious process, i.e.
initial adherence to a certain substratum followed by the produc-
tion of soluble virulence factors. The anti-pathogenic strategy is not
selecting resistance because the anti-pathogenic drugs do not kill
the bacteria, they just turn them harmless. The inert substrata
including the prosthetic medical devices represent risk factors for
the occurrence of biofilm associated infections. Taking into account
the differences in the physiology and susceptibility to antibiotics of
biofilm embedded cells, the obtained compounds have been tested
also against biofilms developed on the plastic substratum. They
exhibited different influences on this process, depending on the
compound and also, on the tested strains (Table 3). Compound 2b
inhibited the biofilm formation by the Gram-negative P. stuartii
1116 strain, 3b inhibited the biofilm formation by K. pneumoniae
ESBL þ, 4b inhibited the adherence ability of the B. subtilis strain,
while the compound 5b inhibited only the biofilm formed by
Table 2
Minimal inhibitory concentration values and effects on biofilm development on the inert substratum of the tested compounds. Grayscaled data highlight the best minimum
inhibitory concentrations.
Compound
Microbial strain
MIC (mg/mL)
Methanol
The effect on the biofilm development
2b
S. aureus ATCC 25923
E. faecalis ATCC 29212
B. subtilis 6688
P. aeruginosa 846
S. marcescens 0804
P. stuartii 1116
K. pneumoniae, ESBLþ
E. faecalis ATCC 29212
S. aureus ATCC 25923
B. subtilis
0.025
0.025
0.83
0.05
0.025
2.5
0.039
0.15
0.15
0.15
0.01
0.2
0.2
0.2
0.2
0.025
0.01
0.02
0.15
0.31
0.31
e
N
N
N
No effect (N)
Inhibitory (I)
I
N
3b
4b
5b
I
C. freundii 1748
0.01
0.41
0.83
0.2
0.2
0.1
Stimulatory (S)
N
I
N
N
N
E. faecalis ATCC 29212
S. epidermidis 1736
Salmonella sp. 9246
K. pneumoniae, ESBLþ
P. stuartii 1116
0.1
0.2
0.2
0.1

18
M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
Table 3
The contour plots of the frontier orbitals HOMO, LUMO, the energies of the HOMO, LUMO orbitals, EGAP (eV), dipole moments, hydratation energies, and antimicrobial activities
for N-oxides 2b-5b.
Properties/Compound
HOMO/eV
sym-Octahydroacridine-N-oxide
2b
3b
4b
5b
LUMO/eV
Etotal (kcal/Mol)
ꢀ839.941
ꢀ8.870
ꢀ0.489
ꢀ8.381
0.674
ꢀ690.785
ꢀ7.428
1.659
ꢀ749.940
ꢀ8.054
1.224
ꢀ710.661
ꢀ7.809
1.442
E
E
E
HOMO/eV
LUMO/eV
GAP/eV
ꢀ9.087
¡9.278
ꢀ9.251
DM (D)
5.682
4.223
4.442
HE (Kcal/Mol)
Antimicrobial activity
ꢀ3.14
ꢀ1.72
ꢀ0.59
ꢀ3.76
50%
41.6%
33.3%
Weak
to provide an explanation, by calculating atomic charges and geo-
metric parameters of the N-oxides.
increasing of nucleophilicity due to participation of these two
heteroatoms is reflected in an increasing of interaction with re-
ceptor, and as a consequence, in an increasing of biological activity.
However, inferior charges of 2b presented superior antimicrobial
activities and superior charges on 3b provided inferior results. To
find an explanation for these results, the structural characteristics
(bonds and angles) of the compounds will be examined.
3.2.2. Atomic charges and geometric parameters of the sym-OHA-
N-oxides
Mulliken charges arise from the Mulliken population analysis,
and provide a means of estimating partial atomic charges from
calculations. Carried out by the methods of computational chem-
istry, and particularly by those based on the linear combination of
atomic orbitals molecular orbital method, such investigations are
routinely used as variables in linear regression procedures. The
Mulliken charge on the atoms in structures 2b-5b, that have been
calculated by using the DFT at M11/ktzvp level of theory (Table 4),
have been further correlated with the nucleophilicity and electro-
philicity concepts. The numbering of the atoms for 2b-5b com-
pounds is provided in Fig. 2. The calculated charges on the aromatic
atoms are very different, depending on, the electronic effects of the
heteroatoms in pyridine moiety, substituent and the presence of
the N-oxide oxygen. The Mulliken charge data showed that the
simultaneously presence of the charge on the oxygen from N-oxide
and on the heteroatom from the substituent are prerequisites for
the high antimicrobial activity on sym-OHA-N-oxides. So, the
Structural analysis of the target compounds 2b-5b has been
performed using DFT theory at M11/ktzvp level. The calculated
geometric parameters for all compounds are summarized in
Tables 5 and 6. To the best of our knowledge, there is no experi-
mental data on the geometric structure of these sym-OHA-N-oxides
in literature. Calculated lengths of the NeO bonds (from the N-
oxide group) are between 1.271 Å (2b) and 1.294 Å (3b), so the
mode of interaction of N-oxide with the receptor doesn't have an
influence, or has a smaller contribution with respect to this
parameter. As expected, a bigger length C4eN2 for 2b give a better
biological activity compared with 3b. This observation is not valid
for the C4O2 and C4eO distances from 3b and 4b. So, there is
another parameter more important in these cases. The most
important bond angles and dihedral angles calculated for the
structures 2b-5b are shown in Table 6. The dihedral angle C5C4O2H
in hydroxy-N-oxide 5b is 6.579ꢁ, which means a small deviation
from planarity, therefore the molecule is almost plane, the Mullikan
charges of the substituent (oxygen) and oxygen (from NO) act with
the great intensity. Therefore, the best biological activity is the one
of compound 5b. The nitro substituent on the 2b molecule is dis-
torted with almost 50ꢁ (49.969ꢁ) from the plane of the pyridine
ring, and exhibits a lower charge on the substituent and oxygen
(NO). However, the presence of nitro substituent is an advantage
[78] to its biological activity, an important aspect to be noted. The
very poor antimicrobial activity of 4b N-oxide seems to be very
much related with the most distorted dihedral angle from
planarity, of 100.815ꢁ (C5C4O2C7). This large deviation leads to poor
conjugation between substituent and the aromatic ring, and the
molecule will interact weaker with the receptor [77], therefore, a
poor antimicrobial activity. Compound 3b has an electron-donor
substituent (eNH2), which is deviated with 24.379ꢁ from the pyr-
idine ring, and, as a consequence, an antibacterial activity smaller
than the nitro-N-oxide 2b, with an electron-acceptor group and a
Table 4
Mulliken atomic charges of the 2b-5b N-oxides.
Atom
Mulliken charge
2b
3b
4b
5b
N-1 (from pyridine ring)
C-2 (from pyridine ring)
C-3 (from pyridine ring)
C-4 (from pyridine ring)
C-5 (from pyridine ring)
C-6 (from pyridine ring)
O-7 (from NO)
N-2 (from NO2 or NH2)
O-1eO-2 (from NO2)
H-1 (from NH2)
0.039
0.201
0.088
ꢀ0.198
0.061
0.210
¡0.490
0.138
¡0.206
e
0.008
0.251
ꢀ0.008
0.188
ꢀ0.059
0.259
¡0.558
¡0.644
e
0.021
0.296
ꢀ0.070
0.032
ꢀ0.096
0.294
¡0.530
e
0.011
0.258
ꢀ0.095
0.180
ꢀ0.151
0.286
¡0.544
e
e
e
0.275
e
e
e
O-3 (from OCH3, OH)
C-7 (from OCH3)
H-2 (from OCH3, OH)
e
¡0.362
ꢀ0.260
0.154
¡0.408
e
e
e
e
e
0.315

M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
19
R
4
5
3
2
6
1
N
O7
H2
H2
H2
1
O
2
O
H2
H1
H1
C
2
N
2
N
3
O
3
O
R:
2b
3b
4b
5b
Fig. 2. Numbering of atoms in the sym-octahydroacridine-N-oxides.
Table 5
Bond lengths values of the 2b-5b N-oxides.
groups of the sym-OHA-N-oxides, are in accordance with their
antimicrobial activity.
Accurate processing of such compounds into thin films and
hybrid structures, e.g. by laser ablation matrix-assisted pulsed laser
evaporation (MAPLE) and/or laser induced forward transfer (LIFT)
[79e84] provide simple and environmental friendly, state-of-the
art solutions for antimicrobial applications. It was shown that the
antimicrobial activity of lysozyme [82e85] and lactoferrin [86,87]
is preserved after the laser processing as thin films. Therefore,
embedded and/or multi-layered structures [87], patterned coatings
and blends [88,89] can be further imagined in which novel struc-
tures can be designed and produced to further enhance, adjust and
control such microbicidal/anti-biofilm properties. There are
numerous potential markets for microbicidal coatings, including
consumer and household goods, building interiors and ventilation
ducts, hospital and medical supplies, etc. Much additional work is
therefore required to confirm stability and safety of such coatings.
Chemical bond
Length (Å)
2b
3b
4b
5b
N1]C2
1.368
1.384
1.387
1.387
1.384
1.368
1.271
1.473
1.212
e
1.359
1.383
1.398
1.398
1.382
1.360
1.294
1.385
e
1.363
1.384
1.389
1.390
1.385
1.363
1.284
e
1.359
1.385
1.390
1.389
1.382
1.363
1.289
e
C2]C3
C3]C4
C4]C5
C5]C6
C6]N1
N]O (N-oxide)
C4eN2
N]O (NO2)
NeH (NH2)
C4eO2 (OCH3)
O2eC7 (OCH3)
C4eO (from OH)
OeH
e
e
1.006
e
e
e
e
1.376
1.425
e
e
e
e
e
e
e
1.364
0.960
3.2.3. UVevisible absorption spectra of the sym-OHA-N-oxides
The recorded UVevisible spectra of sym-octahydroacridine-N-
oxides 2b-5b are given in Fig. 3. Except the nitro-N-oxide 2b, all
UVevisible spectra of the compounds are characterized by the
three bands structure. One can notice that the characteristic bands
of sym-octahydroacridines 2b-5b are in the regions around
bigger deviation from planarity than amino-N-oxide 3b. It can be
concluded that the presence of a particular group, namely an
electron acceptor, is more important for the antibacterial activity
than the planarity of the whole molecule. It can be also concluded
that the electronic parameters, the spatial geometry and charges on
196e265 nm and 242e333 nm. These values are common to
band, which is represented as HOMO and LUMO, due to the
p
/
p
*
*
p/
p
Table 6
transition of the aromatic pyridine ring or pring
/poxygen of aro-
Bond and dihedral angles of the 2b-5b N-oxides.
matic amine oxides therefore have to be attributed of the two
conjugated groups NeO and C]C from aromatic ring [90]. It is
obviously that the UVevisible spectrum of the nitro-N-oxide 2b is
quite different of the others in respect of numbers of bands and the
redshifted values of these. These redshifts are consistent with the
lower Egap (ꢀ8.381 eV) calculated for 2b.
Parameter
The angle value
2b
3b
4b
5b
C5C4N2O1
C5C4N2H1
C5C4O2C7
C5C4O2H
C2N1O7
49.969
e
e
e
e
24.379
e
e
e
e
100.815
e
e
e
e
6.579
119.552
122.050
117.098
e
119.120
118.209
118.224
124.878
e
119.703
120.705
119.949
e
119.388
118.744
120.292
e
3.2.4. Infrared analysis of the sym-OHA-N-oxides
C3C4N2
The FTIR spectra for all sym-octahydroacridine-N-oxides 2b-5b
are presented in Fig. 4. The main bands that appear for each com-
pound are shown in Table 7. The vibrations can be grouped as fol-
lows: OHA specific (a); N-oxide (b); 9-substituent vibrations. (a)
OHA characteristic vibration resulted from combined bands of
C5C4N2
O]N]O
HeNeH
O2C7H
114.600
e
e
e
e
110.570
e
e
C4O2H
e
e
110.600

20
M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
Fig. 3. Experimental UVeVisible spectra of the sym-octahydroacridine-N-oxides 2b-5b.
3500
3000
2500
2000
1500
1000
500
Wavenumber cm-1
Fig. 4. Experimental FTIR spectra of the sym-octahydroacridine-N-oxides 2b-3b (red: 2b; blue: 3b; green: 4b; 5b: pink). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
pyridine ring and aliphatic cycle [80]. The pyridine stretching
characteristic vibrations e
C]N, are between 1539 cmꢀ1 and
1569 cmꢀ1 and in domain 1261-1268 cmꢀ1 for
C-N. For the
For 9-substituent vibrations we found: two specific vibrations for
nitro-N-oxide: 1520 cmꢀ1 very strong attributed to asymmetric
stretching of nitro group and a weak one at 1343 cmꢀ1 for a sym-
metric vibration [91]. A very strong band at 3190 cmꢀ1 and a me-
dium one at 3452 cmꢀ1 are attributed to the amino group in the N-
oxide, 3b. The two strong peaks at 1154 cmꢀ1 and 1136 cmꢀ1 in the
case of 4b are attributed to OeCH3 stretching vibrations [92].
Finally, the broad band at about 3550 cmꢀ1 is attributed to the
hydroxyl group in compound 5b [93].
n
n
saturated rings asymmetric stretching vibrations are at about
2940 cmꢀ1 and symmetric stretching, between 2860 and
2870 cmꢀ1. Also, bending CH2 vibrations are present at approxi-
mately 1423e1460 cmꢀ1 for the ‘in plane’ bending, and in two in-
tervals: 1359-1397 cmꢀ1, and 739-757 cmꢀ1 for wagging vibrations.
For the carbon-carbon saturated bonds, the skeletal deformation
vibrations are identified at 1068-1082 cmꢀ1. (b) N-oxide specific
vibrations are observed in two regions: (1) a strong band attributed
4. Conclusion
to
1293 cmꢀ1 (2b) [91] and in general a medium band due to
plane deformation between 843 cmꢀ1 (4b) and 858 cmꢀ1 (2b) [92]. (c)
n
N-O(oxide) stretching vibration between 1283 cmꢀ1 (5b) and
N-Oin
n
Four 9-substituted-1,2,3,4,5,6,7,8-OHA-N(10)-oxides (with:
eNO2, eNH2, eOCH3, eOH) are screened for their microbicidal and

M. Marinescu et al. / Journal of Molecular Structure 1144 (2017) 14e23
21
Table 7
Vibrational assignments (wavenumber, cmꢀ1) of sym-octahydroacridine-N-oxides 2b-5b FTIR spectra.
IR Assignments
sym-Octahydroacridine-N-oxide
2b
3b
4b
5b
n
n
n
n
n
n
OH
NH
e
e
e
3550 b
e
e
3452 m; 3306 sh; 3190 vs
e
CHasym
CHsym
C]Nring stretch
NO2 asym
2939 w
2870 w
e
2945 w
2862 m
1569 w
e
2935 s
2942 s
2860 m
1543 m; 1539 m
e
2861 m
1558 m
e
1520 vs; 1481 sh
1460 m
1377 m
1343 w
1293s; 1319 sh
1268 m
e
d
d
CH2 in plane
CH2 wagging
1431 m
1387 m
e
1464 m
1359 m
e
1447 m; 1423 sh
1397 m
e
n
n
n
n
NO2 sym
N-O(oxide)
CeN(aromatic)
OeCH3
1289 s
1267 m; 1178 vs
e
1292 s; 1272 sh
1261 m; 1194 s
1154 s; 1136 s
1068 s; 980 m
843 m
1283 s
1243 m
e
d
n
d
CeC
NeOin plane deformation
CH2 wagging
1082 m
858 w
751 m
1076 m; 1049 m
855 m
757 m
1076 m
850 s
757 w
739 w
Infections Globally: Final Report and Recommendations, commissioned
study by the UK Government (UK Department of Health) and the Wellcome
Trust, May 19, 2016, https://amr-review.org/.
anti-pathogenic properties. The qualitative screening of the four
tested N-oxides evidenced a better bactericidal effect than the
antifungal one. The nitro- (2b) and hydroxy- (5b) N-oxides proved
to be more active than amino-N-oxide (3b), being active against six
of the tested strains. The spectrophotometric measurements evi-
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denced MIC values (mg/ml) ranging from 10 mg/ml to 250 mg/ml. The
OH substituted derivative proved to be very active also in the
quantitative assay, exhibiting very low MIC values. Some com-
pounds (5b, 3b, 4b) also inhibited the ability of the bacterial strains
to form biofilm on the inert substratum. The presence of the elec-
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good antibacterial activity, against 41.6% of tested strains. The
antimicrobial activity is consistent with the variation of the hy-
dration energy. Antimicrobial activity is increased in the order:
4b < 3b < 2b < 5b, simultaneously with the decrease of the hy-
dration energy from ꢀ0.59, to ꢀ1.72, to ꢀ3.14 and respectively
to ꢀ3.76 kcal/Mol. The maximum Egap for 4b N-oxide correlates
with the worst antimicrobial activity, the simultaneous presence of
the charge on the oxygen from N-oxide and on the heteroatom from
the substituent are prerequisites for the high antimicrobial activity
on sym-OHA-N-oxides, while the planarity of the molecule is an
advantage for its antimicrobial activity. Finally, we briefly present
and discuss on the processing of such compounds into thin films
and hybrid structures by laser-assisted techniques, i.e. matrix-
assisted pulsed laser evaporation or laser-induced forward trans-
fer, to provide simple and environmental friendly, state-of-the-art
solutions for antimicrobial/medical coatings and devices.
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