Ag(I) camphor complexes: antimicrobial activity by design

Article abstract of DOI:10.1016/j.jinorgbio.2019.110791

Eleven new complexes of general formula [Ag(NO₃)(L-Y)₂] corresponding to Ag(I) camphorimine complexes [Ag(NO₃)(OC₁₀H₁₄NY)₂] (Y=NMe₂ (1); OH (2); C₆H₅ (3); 4

Full text of DOI:10.1016/j.jinorgbio.2019.110791

Accepted Manuscript
Ag(I) camphor complexes: Antimicrobial activity by design
M. Fernanda N.N. Carvalho, Silvestre Leite, Joana P. Costa,
Adelino M. Galvão, Jorge H. Leitão
PII:
S0162-0134(19)30380-0
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110791
Article Number:
Reference:
110791
JIB 110791
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
5 June 2019
19 July 2019
21 July 2019
Please cite this article as: M.F.N.N. Carvalho, S. Leite, J.P. Costa, et al., Ag(I) camphor
complexes: Antimicrobial activity by design, Journal of Inorganic Biochemistry,
https://doi.org/10.1016/j.jinorgbio.2019.110791
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ACCEPTED MANUSCRIPT
Ag(I) camphor complexes: antimicrobial activity by design
M. Fernanda N.N. Carvalho,*a Silvestre Leite, a,b Joana P. Costa, a Adelino M. Galvão, a Jorge H.
Leitão*,b
a Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais,
1049-001 Lisboa, Portugal. E-mail: fcarvalho@tecnico.ulisboa.pt
b Instituto de Bioengenharia e Biociências, Departamento de Bioengenharia, Instituto Superior Técnico
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
_________________
(*) Corresponding author:
M. Fernanda N.N. Carvalho , Centro de Química Estrutural, Instituto Superior Técnico, Universidade
de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. E-mail: fcarvalho@tecnico.ulisboa.pt
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Abstract
Eleven new complexes of general formula [Ag(NO3)(L)2] corresponding to Ag(I) camphorimine
complexes [Ag(NO3)(OC10H14NY)2] (Y=NMe2 (1); OH (2); C6H5 (3); 4-MeC6H4, (4); 3,5-(CH3)2C6H3) (5);
3-OHC6H4, (6); 3-ClC6H4 (7); 4-ClC6H4 (8); 4-FC6H4 (9); 4-CF3C6H4 (10)) and the camphor sulfonylimine
complex [Ag(NO3)(O2SNC10H14NY)2] (Y=NH2) were synthesized/characterized and their structural
properties and antibacterial activity studied to gain insights into the structure-antimicrobial activity
relationships. Five of the complexes were selected as representative examples and structures were
optimized by Density functional theory calculations. The results show that the imine substituents (Y)
at the camphor ligands drive the structure of the complexes from distorted octahedral to trigonal
prismatic or linear ionic while the effect of the sulfonylimine ring does not appreciably affects the
geometry of the complex. The lipophilicity and polarity which are important parameters concerning
the biological activity of the complexes are also high dependent of the characteristics of the camphor
ligands. The redox properties of the complexes studied by cyclic voltammetry showed that their
reduction potentials are essentially independent of their electronic and steric properties. The
antibacterial activity of all the complexes, against Gram-positive (S. aureus Newman) and Gram-
negative (Escherichia coli ATCC25922, Pseudomonas aeruginosa 477, Burkholderia contaminans
IST408) strains was evaluated through calculation of MIC values. Results show that complexes with
camphor imine ligands (1-10) that combine high lipophylicity with low dipolar moment (3-5) exhibit
enhanced antibacterial activity. The ability to establish hydrogen bonding emerged as an important
contribution to the antibacterial activity of the camphor sulphonylimine complex 11 (Y=NH2).
Keywords: Antibacterials, silver complexes, camphorimines, Gram-positive bacteria, Gram-negative
bacteria, structure-activity relationships.
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Introduction
Complexes are chemical entities where metal and ligands bind to afford compounds with properties
distinct from their precursors. Through the choice of the metal and the design of the ligands it is
possible to drive the properties, reactivity and applications of the complexes. The characteristics of
the metal are of utmost relevance concerning the biological interactions and toxicity of the complex.
Silver antiseptic properties are known since long ago. For instance, Hippocrates used silver in wound
care, and the ancient Egyptians used silver vessels to preserve the quality of the drinking water. Due
to its antimicrobial properties and general non-adverse effects for humans, silver and silver
derivatives are presently used mainly as antimicrobials.1 Examples of medical uses of silver
compounds include coating of bandages to protect burns and wounds, coating of catheters as well as
medical devices. 1,2,3 Non-medical uses may be found for example on coating of polyethylene fabrics
for food packing.4 A recent review reported that ca. fifty antimicrobial products based on silver were
industrially patented.5
Silver ions (Ag+) are highly toxic to microorganisms, while the reduced form (Ag0) is relatively inert.6
The broad spectrum of antimicrobial activities of silver ions is due to the non-specific mechanisms
that include the ability to bind to DNA, proteins and thiol groups, and generation of intracellular
reactive oxygen species. These non-specific mechanisms interfere with enzymes and membrane
proteins such as those involved in electron transport and energy generation and membrane ion
exchange systems.7,8 Recent studies highlighted that electron transfer processes9 and interaction
with efflux pumps10 are important aspects in the antimicrobial activity of silver compounds. Yan and
colleagues have recently reported the use of proteomics to investigate the cellular responses of
Pseudomonas aeruginosa to silver nanoparticles. These authors found a total of 59 proteins as being
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regulated by silver, 5 of them involved in silver binding. Furthermore, the main pathways found as
being involved in the antibacterial activity of silver nanoparticles were interference with the cell
membrane functions, and the generation of intracellular reactive oxygen species.7 In complexes, the
characteristics of the ligands and the silver ions (Ag+) combine, sometimes enhancing the
antimicrobial activity of the complex compared to the silver precursors (e.g. AgNO3), even when the
free ligand is not active as in the case of camphor derivatives.11 How the metal and the ligands
cooperate to enhance the antimicrobial activity of the complexes remains unclear, although there is
some consensus that the silver ion must be involved in the generation of reactive oxidative species,
binding to DNA, interaction with cell membranes or the thiol groups of vital enzymes.9 The ligands are
thus thought to provide the complexes with characteristics suitable to act as carriers to deliver the
silver ion into the cell. Therefore, complexes with weak binding ligands (nitrogen or oxygen) are
expected to perform better than those with ligands that bind tightly to silver (phosphorus or sulphur).
Such assumption is supported by a considerable number of Ag(I) complexes with N or O binding
ligands reported as having antimicrobial properties.12 Additionally, the formation of strong Ag-S bonds
enabling the replacement of iron or zinc from proteins at the cell wall is one of the processes
considered as responsible for silver antimicrobial activity.13
Silver complexes with N-type ligands (camphor imines) fulfil the conditions to suitably deliver silver
ions to the microbial cell. In fact, these complexes display relevant antimicrobial properties as
corroborated by the high to moderate antibacterial or very high antifungal activities.14,15 The
characteristics of the camphor imine substituents (Y) in the delivery process of Ag(I) to the cells is still
to understand. The results obtained till now point to distinct mechanisms underlying the antimicrobial
activity of camphor imine silver complexes towards bacteria or fungi, since the complex with the
highest antibacterial activity is distinct from that with the highest antifungal activity. We have also
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concluded that a geometry-activity relationship may exist, since complexes that differ just in the meta
and orto position of the substituent at the aromatic ring in the camphor ligand, have considerably
different antimicrobial activities. However, parameters such as hydrogen bridging, electron
delocalization or the hydrophobic/hydrophilic character of the ligands are still to be evaluated. In the
present work, new complexes were synthesized and their antibacterial activity was evaluated. The
compounds were designed to get insights into the relationships between structural and electronic
properties (hydrogen bridging, electron delocalization, and hydrophobicity/hydrophilicity) and
antibacterial activity.
Results and discussion
Synthesis and characterization
A selection of camphor derivatives of the imine, hydrazone or sulphonyl imine types having distinct
structural and electronic properties were used as ligands (Figure 1) to prepare eleven new complexes
that differ in the mono camphor character from those whose antimicrobial properties were studied
before.14
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Figure 1 – Camphor imine (right) and camphor sulphonylimine (left) type-ligands used in this study
The structural characterization of the camphor imine (OC10H14NY) ligands Y=4-CH3C6H4 and Y=3-ClC6H4
was made by X-rays diffraction analysis and their ORTEP drawings and their relevant bond lengths and
angles are depicted in Figure 2 (cif file is available from CCDC).
(a)
(b)
Figure 2 - ORTEP drawings (ellipsoids with 25% probability) for camphor ligands OC10H14NY: a) Y=4-
CH3C6H4, [C=O, 120.5(3) pm; CN, 127.4(3) pm]; b) Y=3-ClC6H4. [C=O, 119.7(39) pm; CN 126.5(3) pm]
The new complexes fit in the formula [Ag(NO3)(OC10H14NY)2] (Y=NMe2, 1; OH, 2; C6H5, 3; 4-MeC6H4, 4;
3,5-(CH3)2C6H3), 5; 3-OHC6H4, 6; 3-ClC6H4, 7; 4-ClC6H4, 8; 4-FC6H4, 9; 4-CF3C6H4, 10) or
[Ag(NO3)(O2SNC10H14NNH2)2] (11) that essentially differ in the imine (1-10) or sulfonylimine (11)
character of the camphor ligand (Figure 2).
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The choice of the substituents (Y) at position 3 of the camphor skeleton (see number labelling at
Figure 1) was directed towards the synthesis of complexes where hydrogen (2, 6, 11) or halide (7-10)
could facilitate biological interactions due to the distinct electronic and/or geometric characteristics
of the complexes. Complexes 2-10 are of the imine type (-C=N-R) while complexes 1 and 11 are of the
hydrazone type (-C=N-NR2) and complex 11 is of the sulphonylimine type (Figure 1). Complex 12 was
also synthesized for comparative purposes since its structural characterization by X-rays11 was
obtained before. A relevant parameter concerning the interaction of compounds with the cells is their
lipophilic/hydrophilic character. Lipophilic compounds can diffuse through the cytoplasmic membrane
while the hydrophilic compounds do not. The characteristics of the camphor substituent (Y) may
prompt considerably different lipophilicities as predicted from log P = log [octanol]/[water] calculated
using ACDC software, thus tune the activity of the complex.
The complexes were characterized by elemental analysis (EA) and spectroscopic techniques (FTIR,
NMR). According to the elemental analysis all complexes fit in the 2:1 ligand to metal ratio. By FTIR
the camphor imine complexes (1-10) display bands in the ranges typical stretching frequencies for
C=O (νCO, 1707-1754 cm-1) and C=N (νCN, 1577-1654 cm-1). The camphor sulphonylimine complex (11)
displays two IR bands in the region of C=N stretching with values that differ considerably (C=NSO2,
1637 cm-1; C=N-N, 1575 cm-1) due to the distinct electronic properties of the sulphonylimine and the
-
13
hydrazone groups. Additional IR bands (1379-1385 cm-1) evidence the nitrate (NO3 ) group. By C
NMR the carbon atom of the keto (position 2, Figure 2) and the imine (position 3, Figure 2) groups of
the camphor ligands display chemical shifts in the narrow ranges of 204-207 ppm (1-10) and 173-175
ppm (2-9), respectively. The chemical shifts of the carbon atoms (C=N-N: 145.2 ppm, 1; 155.0 ppm,
10; 141.8 ppm, 11) of the hydrazone type complexes are lower than those of the imine groups
evidencing a higher shielding. The chemical shift of the sulphonylimine carbon atom (C=N-SO2: 186.4,
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11) is between the hydrazone and keto groups suggesting intermediate electron densities. The
analytical and spectroscopic characteristics of the camphor hydrazone complexes (1 and 11) fit well in
the chalice-type structural arrangement found for [Ag(NO3)(O2SNC10H13NNMe2)2] (12).11
The coordination of the camphor imine/hydrazone ligands to silver can involve the imine nitrogen
atom as found for [Ag(NO3)(OC10H13NC6H4NC10H13O)]16 (Figure 3a), combined imine/keto as found for
[(CuCl)2(Me2NNC10H14O)]n17 or the keto group may enable coordination to the same (Figure 3b) or
sequential (Figure 3c) silver atoms as found for metals of group 10.18
(a)
(b)
(c)
Figure 3- Types of coordination of camphor imines: a)- mono-hapto; b)- di-hapto; c)- bridging
The characteristics of the imine substituent (Y), tunes the structural arrangement and may play a role
in the interactions of the complex with the biological cell. Computational calculations were conducted
to optimize the geometries of a selection of complexes in order to ascertain on the effect of different
Y groups in the biological activity, if any.
Calculations
Compounds 1- 4, 8 and 12 were selected as representative of the different types of the Ag(I) camphor
complexes under study and their geometries were optimized by DFT.
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In the case of complexes with aromatic substituents (3, 4 and 8) the aim was the study of the effect of
the electron donor or donor characteristics of the substituent on the structural/antibacterial
properties of the complexes.
The calculated and experimental data (obtained by X-Ray diffraction analysis)10 for complex
[Ag(NO3)(O2SNC10H13NNMe2)2] (12) are in good agreement (Table 1). The optimized relevant distances
and angles for the selected complexes are displayed in Table 1.
Table 1 – Calculated distances / angles and dipole moment for complexes [Ag(NO3)(OC10H14NY)2] (1-4,
8) and [Ag(NO3)(O2SNC10H13NNMe2)2] (12)
Y
Distance (Å) /
Angles (°)
NMe2 (1)
OH (2)
C6H5 (3)
4-MeC6H5 (4) 4-ClC6H5 (8)
NMe2 (12)
Ag-N(Y)
Ag-Za
Ag-O1(NO3)
Ag-O2(NO3)
C=N
2.39/2.47
2.59/2.66
2.40
2.23
2.75/2.85
2.70
2.28/2.33
2.84/2.92
2.41
2.28/2.34
2.84/2.89
2.42
2.28/2.34
2.84/2.91
2.42
2.83
2.32 (2.40)b
2.45
2.67
131
2.74
129
2.69
129
2.69
129
2.68
129
2.45
129
C=O
126
125
125
125
125
̶
N-Ag-N
Z-Ag-Za
Z-Ag-O
144.8
82.3
160.3
77.8
144.4
68.2
143.8
70.1
144.6
68.4
136.1 (138.2)
91.8
128.7/127.7
15.40
167.9/136.5 139.4/132.7 162.5/144.2 158.4/148.8 162.2/144.2
10.00 10.03 11.37 11.37 14.11
Dipole (D)
b
a Z=O or N. Experimental data obtained by X-rays diffraction analysis.11
According to the calculated bond lengths and angles, the geometry of the Ag(I) complexes vary from
octahedral (1, 12) to trigonal prismatic (3, 4, 8) or linear ionic (2). At complexes 1 and 12 (hydrazone
type) the camphor ligands act as bidentate through the nitrogen of the imine and the oxygen atoms
of the ketone groups (1) or through the nitrogen atoms of the imine and sulphonylimine groups (12)
-
within a chalice-type arrangement (Figure 2). The coordination sphere is completed by the NO3 group
that acts as a bidentate O-O ligand such as in the high distorted octahedral silver-nitrate complex
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19
[Ag(NO3)L2] (L=2-(bis(methylthio)methylene)-1-phenylbutane-1,3-dione.
Distortion from the
octahedral geometry is lower in 1 (imine type) than in 12 (sulphonylimine type) (Figure 4).
1
12
Figure 4 – Calculated structures for [Ag(NO3)(OC10H13NY)2] (1) and [Ag(NO3)(O2SNC10H13NY)2] (12)
(Y=NMe2)
At complexes 3, 4 and 8 of the camphor imine ligands that differ in the characteristics of the
substituent (Y, aromatic) at the camphor skeleton, the ligands behave as mono dentate binding to
silver through the imine nitrogen atoms with bond lengths that do not differ in the three complexes
(Table 1). The oxygen atom of the camphor ketone group occupies a close non-binding distance such
as in group 10 (Pd, Pt) complexes.20 The geometries tend to trigonal prismatic (Figure 5).
3
4
8
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Figure 5 – Calculated structures for [Ag(NO3)(OC10H13NY)2] (Y=C6H5, 3; 4-MeC6H4, 4; 4-ClC6H4, 8)
According to calculations, complex 2 (Y=OH) shows a considerably high cationic character (Q=0.67).
Therefore, the interaction of the nitrate group with the metal has an essentially outer sphere (non-
bonding) character (Figure 6).
Figure 6 – Calculated structure for [Ag(NO3)(OC10H13NOH)2] (2)
Results from calculations clearly evidence that the characteristics of the camphor substituents (Y)
drive the geometry and the coordination numbers (NC) of the complexes as well as the dipole
moment (Table 1). The coordination number varies from six (octahedral) to three (trigonal prismatic)
or two (linear) fostering a variable number of coordinative vacant positions and interactions with the
microbial cell wall. The dipole moment drives the polarity of the complexes which is of utmost
relevance to biological interactions.
Structure - antibacterial activity relationships
Bacteria are commonly divided in Gram-positive and Gram-negative categories according to the
characteristics of their cell wall. Both are composed of a cell membrane or cytoplasmitc membrane,
which is surrounded by a thick layer of peptidoglycan and teichoic acids in the case of Gram-positive
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bacteria, while in the case of Gram-negative bacteria, a thin layer of peptidoglycan is contained in the
periplasmic space that is surrounded by an outer membrane to which lipopolysaccharides are
anchored. Gram-negative therefore have an additional barrier to antimicrobials which provides an
extra protective layer keeping the vital role of material exchanges with the exterior of the cell. In
order to gain insights into the relationships between structure of the silver camphor imine complexes
and their antibacterial activity both Gram-negative and Gram positive bacteria were included in this
study.
A relevant parameter concerning the interaction of compounds with the bacterial cell membrane is
their lipophilic/hydrophilic character. Lipophilic compounds can cross the cytoplasmic membrane by
diffusion while hydrophilic compounds need to be transported through the membrane to reach
intracellular targets. At complexes, the substituent (Y) at the camphor imine ligand (Figure 1) may
prompt considerably different lipophilicities, as predicted from log P = log [octanol]/[water] (using
ACDC software) , thus tuning the antibacterial activity of the complex.
The antibacterial properties of the complexes [Ag(NO3)(OC10H14NY)2] (1-10) and
[Ag(NO3)(O2SNC10H13NY)2] (11,12) towards the Gram-positive Staphylococcus aureus Newman, and
the Gram-negative Pseudomonas aeruginosa 477, Escherichia coli ATCC25922, and Burkholderia
contaminans IST408, were assessed by determining the MIC values by microplate dilution assay and
by fitting the data with a modified Gompertz equation as previously described.16 All complexes display
antibacterial activities (Tables 2 and 3).
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Table 2 – Data for complexes [Ag(NO3)(OC10H14NY)2]
MIC (μg/mL)
E. coli
ATCC25922
P. aeruginosa B. contaminans
S. aureus
Newman
LIGAND
퐸푝푟푒푑
Volta
Log Pb
477
IST408
Y
1
NMe2
58
51
59
37
54
41
134
119
54
0.97 ± 0.60
1.06 ± 0.59
2.94 ± 0.58
3.40 ± 0.58
2
3
4
0.11
0.12
OH
C6H5
52
55
53
59
91
66
4-MeC6H4
55
5
6
0.11
3,5-Me2C6H3
3-OHC6H4
40
52
53
52
53
53
105
75
47
3.86 ± 0.59
2.36 ± 0.59
3.65 ± 0.59
0.074
0.044
113
110
7
8
3-ClC6H4
4-ClC6H4
44
0.12
52
59
52
55
53
60
57
77
55
151
93
3.78 ± 0.59
3.24 ± 0.62
3.91 ± 0.61
9
0.097
4-FC6H4
10
4-CF3C6H4
0.11
105
a Potential in Volt (±10 mV) vs sce. b Partition coefficient in octanol/water predicted using ACDC software.
Table 3 - Data for complexes [Ag(NO3)(O2SNC10H13NY)2]
MIC (μg/mL)
LIGAND
Log Pb
푬풓풑풆풅
Volta
E. coli
ATCC25922
P. aeruginosa B. contaminans
S. aureus
Newman
477
IST408
Y
11
12
NH2
0.10
0.17
0.18
52
107
47
36
38
114
257
73
-0.99 ± 0.69
NMe2
108
56
-0.69 ± 0.70
[Ag(NO3)]
39
74
̶
a Potencial in Volt (±10 mV) vs sce. b Predicted partition coefficient in octanol/water using ACDC software.
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A first insight into table 2 shows that the antimicrobial activity of the complexes (1-10) towards P.
aeruginosa is almost independent of the characteristics of the camphor ligands and the structure of
the complexes, in contrast to the other bacterial strains. Such behaviour points to the mechanisms
underlying the antimicrobial activity of the complexes towards P. aeruginosa as being essentially
distinct from those towards E. coli, S. aureus or B. contaminans (Tables 2). A Pseudomonas-specific
process apparently overwhelms other mechanisms involved in the antimicrobial activity of the silver
camphorimine complexes and drives a structure-independent process while for the other strains the
characteristics of the complexes modulated their antibacterial activity. A trait specific of P. aeruginosa
strains is their ability to secrete pyocyanin, a redox active compound involved in the activation of
molecular oxygen and formation of hydrogen peroxide. A recent study by Muller6 showed that the
silver ion (Ag+) competes with molecular oxygen by electron transfer from pyocyanin and
concomitantly decreases the production of hydrogen peroxide from molecular oxygen. A redox
process mediated by pyocyanin leading to Ag+Ag0 reduction, decomposition of the camphorimine
complex and generation of a naked silver species (conceivably of the nano size) can explain the
complexes-independent anti-P. aeruginosa activity. To support such proposal the redox properties of
a selection of the complexes were studied by cyclic voltammetry. The results show that the potentials
at which the silver metal sites reduce (Tables 2 and 3) are slightly higher than the potential at which
pyocyanin oxidizes (E=-0.34 V)6 thus accounting for a favourable redox process between them. Such
process conceivably buffers the activity of the complexes due to less Ag+ ion being available to
promote electron capture and cell wall impairment which is one of the mechanisms responsible for
the antimicrobial action of silver compounds.21 That is not the case of the camphor sulphonylimine
complexes 11 and 12 that reveal structural dependent activity towards P. aeruginosa. Complex 11
exhibits MIC values almost 3 times lower than complex 12 (36 and 108 μg/mL, respectively, Table 3).
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The main structural difference between 11 and 12 refers to the Y group at the amine substituent
(Figure 1) that at complex 11 (Y=NH2) is prone to establish hydrogen bonding with carboxyl or amino
groups of proteins or other molecular components of the bacterial cells enabling the anti-bacterial
activity of the complex against P. aeruginosa. Non-covalent bonding is less feasible in the case of 12
(Y=NMe2) thus the antibacterial activity is much lower. Further support to hydrogen bonding
relevance towards the antibacterial activity of the complexes is obtained from the MIC values of
complexes 11 and 12 (Table 3) where 11 performs better than complex 12 for the Gram-positive and
Gram-negative strains studied. From the set of complexes under study, complexes 2 (Y=OH) and 6
(C6H4OH) could in principle establish hydrogen bridges with the cell components. However, the
cationic character / linear geometry of 2 and the low acidity of 6 (due to electron delocalization
through the aromatic ring) decrease hydrogen bonding establishment.
Complexes 3-5 display the lowest MIC values towards the Gram-positive S. aureus which are ca. half
of the values calculated for complexes 6-10 (Table 2). Since, the geometry of complexes 3-10 is
trigonal prismatic according to calculations (Figure 5) their distinct activities must rely on the
characteristics of the aromatic imine group (Figure 1) that is essentially non-polar [C6H4R: R=4-H (3),
R=4-Me (4), R=3,5-Me2 (5)] or polar [R=3-OH (6), R=3-Cl (7), R=4-Cl (8), R=4-F (9), R=4-CF3 (10)].
Complexes 1-10 perform better towards Gram-negative than Gram-positive bacteria, except
complexes 3-5 that display similar activities towards S. aureus, E. coli and P. aeruginosa (Table 2). In
fact, complexes 4 and 5 display the lowest MIC values towards Gram-negative E. coli (37 and 40
μg/mL, respectively) and additionally 5 displays the lowest value against S. aureus (47 μg/mL). These
data points to high lipophilicity (estimatedError! Bookmark not defined. through calculation of logP values,
Table 2 and 3) associated with low polarity as structural features favouring the antibacterial activity of
the silver complexes. High lipophilicity enables molecules to cross the membrane by diffusion to reach
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the bacterial cytoplasm 22 while hydrophilic compounds such as β-lactams need to use the outer
membrane porins or other transport systems to gain access to the interior of the bacterial cell.
The activity of the silver camphor imine complexes (1-12) towards B. contaminans contrasts with the
above mentioned, since low lipophilicity irrespectively of the polarity, tends to favours the
antimicrobial activity of the complexes (1, 2, 11, 12) with hydrogen bridging also playing a role in the
process, as observed for complex 11 that displays the lowest MIC values towards B. contaminans
(Table 3). This strain belongs to a large group of species known as the Burkholderia cepacia complex,
characterized by possessing large genomes encoding more than 7500 genes and encompassing
numerous membrane transport systems which might be “highjacked” by hydrophilic camphor
complexes.23
The results presented herein show that bacterial resistance to antimicrobials is multifactorial and
often strain-specific including intrinsic resistance as a result of structural features such as poor
membrane permeability, constitutive expression of efflux pumps, high resistance due to the
acquisition of resistance genes or genetic modifications and active efflux mechanisms.24
The antimicrobial activities of complexes 1-12 reflect the differences in the mechanisms used by each
strain to resist to the antimicrobials and additionally show that the relationships between the
structures of the complexes and their activity are highly dependent of the strain. Therefore,
generalization is not possible since a wide variety of mechanisms can be used by each bacterial stain,
the compounds they secret, electron transfer processes between the cell and the complexes and
many other parameters. However, it is evident from the herein results that the activity of the Ag(I)
camphor imine can be tuned towards Gram-positive or Gram-negative bacteria through design of the
camphor ligands.
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Conclusions
Two sets of new silver camphor complexes [Ag(NO3)(L)2], that essentially differ on the ligands
[camphor imine (OC10H14NY); camphor sulphonylimine (SO2NC10H14NY)] were designed,
synthesized/characterized and their antibacterial properties evaluated through calculation of the MIC
values. All complexes display antimicrobial activity towards Gram-positive (Staphylococcus aureus
Newman) and Gram-negative bacteria (Pseudomonas aeruginosa 477, Escherichia coli ATCC 25922,
Burkholderia contaminans IST408) with magnitudes that vary according to the characteristics of the
substituent (Y) of the camphor imine ligand and the bacteria strain.
The structures of a selection of complexes (1-4, 8 and 12) were optimized by Density Functional
Theory. Data revealed that the geometries considerably vary according to the substituent (Y) at the
imine group (NY). Hydrazone type substituents (Y=NR2) prompt complexes with octahedral distorted
geometries (1, 12) and coordination number equal to six due to cooperative binding of the nitrogen
and oxygen atoms of the imine ligand (1) or the two nitrogen atoms of the sulphonylimine ligand (12),
-
while nitrate (NO3 ) binds through two oxygen atoms. Complex 2 (Y=OH) has a cationic character with
-
a linear geometry, the ligand binding the silver atom through the imine nitrogen atom, the NO3 ion
occupying the outer sphere of the complex. Complexes 3, 4 and 8 display trigonal prismatic
geometries (Y=C6H4R) with the ligands (L) binding the metal through the nitrogen atom and the
nitrate through one of the oxygen atoms.
As a general trend, the Ag(I) complexes (1-12) perform better towards Gram-negative than Gram-
positive bacteria. Exceptions are complexes 3-5 that display similar MIC values for P. aeruginosa, E.
coli and S. aureus. The combined high lipophilicity and low polarity of complexes 3-5 is considered to
enhance their activity towards S. aureus. These parameters also seem relevant for the lowest MIC
17

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values displayed by complexes 4 and 5 against E. coli. Hydrogen bonding in 11 is considered to
account for the enhanced activity against P. aeruginosa (MIC, 36 μg/mL) and B. contaminans (MIC, 38
μg/mL) compared to the related complex 12.
In contrast to the sensitivity of the geometry and the antibacterial activity of the complexes to the
electronic and steric characteristics of the imine substituents (Y), Y-independent antibacterial activity
of complexes 1-10 was found towards P. aeruginosa. Such behaviour was attributed to reduction of
the silver site by the redox active pyocyanin secreted by P. aeruginosa. Such electron transfer process
is feasible according to the redox potentials of the complexes measured by cyclic voltammetry.
In what concerns the geometry and the antimicrobial activity of the complexes no direct relationship
was found, however, there is a relationship between the characteristics of the camphor imine
substituents (Y) the geometry and the antibacterial activity of the complexes (1-10). Complexes with
camphor imine ligands combining high lipophilicity with low dipolar moment (3-5) exhibit low MIC
values, thus high activity towards Gram-positive S. aureus, while camphor imine complexes with high
polarity (6-10) perform better towards Gram-negative bacteria. The high polarity and low lipophilicity
of the camphor sulphonylimine complexes (11, 12) apparently favour the activity against Gram-
negative bacteria with further enhancement due hydrogen bonding in the case of complex 11.
Therefore, the rational design of complexes driving their activity towards Gam-positive or Gram-
negative is feasible through choice of the suitable substituent (Y) of the camphor imine or camphor
sulphonylimine ligand.
Experimental
The complexes were synthesized under nitrogen using Schlenk and vacuum techniques. Camphor
ligands (OC10H14NY: Y=OH, C6H5, NH2, NMe2 and O2SNC10H14NNH2) were prepared according to
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reported procedures.25 Silver nitrate, (1R)-(+)-camphor, (1S)-(+)-10-sulfonic acid and amines were
purchased from Sigma Aldrich. Acetonitrile (PA grade) was purchased from Carlo Erba purified by
conventional techniques26 and distilled before use. The FTIR spectra were obtained from KBr pellets
19
using a JASCO FT/IR 4100 spectrometer. The NMR spectra (1H, 13C, F, DEPT, HSQC and HMBC) were
obtained from CD2Cl2, CD3CN, Acetone-d6 or CDCl3 solutions using a Bruker Avance II+ (300 or 400
MHz) spectrometers. NMR chemical shifts are referred to TMS (δ = 0 ppm). The redox properties were
studied by cyclic voltammetry using a three compartments cell equipped with a Pt wire electrode and
interfaced with a VoltaLab PST050 equipment. The cyclic voltammograms were obtained using
NBu4BF4 as electrolyte (0.10 M) in CH3CN. The potentials were measured in Volts (± 10 mV) versus SCE
푟푒푑
at 200 mV/s using (Fe(η5-C5H5)2]0/+ (퐸 =0.382 V; CH3CN) as internal reference.
1/2
Synthesis
Camphor derivatives
The new camphor imines were obtained from camphorquinone by reaction with the appropriate
amine or hydrazine in ethanol, using Forster procedures27 and improvements.11,28,29
Characterization details:
OC10H14NC6H4CH3-4
Elem. Anal. (%) for C17H21NO: Found.: C, 80.4; N, 5.5 H, 8.3. Calc.: C, 80.0; N, 5.5; H, 8.2.IR (cm-1): 1748
1
(C=O), 1652 (C=N). H NMR (CDCl3, δppm): 7.15 (d, J=8.0 Hz, 2H); 6.84 (d, J=8.0 Hz, 2H); 2.84 (d, J= 4.7
Hz, 1H); 2.33 (s, 3H); 2.17-2.01 (m, 1H); 1.91-1.77 (m, 1H); 1.72-1.55 (m, 2H) 1.08 (s, 3H); 0.96 (s, 3H);
13
0.87 (s, 3H). C NMR (CDCl3, δppm): 206.3 (C=O); 171.4 (C=N); 146.6, 135.4 (CPhipso); 129.6, 120.9
(CPh); 58.0 (C1); 50.2 (C4); 44.8 (C7); 30.2 (C6); 24.4 (C5); 21.0, 17.6 (C9, C10); 9.1 (C8).
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OC10H14NC6H3(CH3)2 -3,5
Elem. Anal. (%) for C18H23ON: Found.: C, 80.3; N, 5.5; H, 9.0. Calc.: C, 80.3; N, 5.2; H, 8.6; IR (cm-1):
1
1748 (C=O); 1663 (C=N); 1601 (Ph); H NMR (CDCl3, δppm): 6.79 (s, 1H); 6.50 (s, 2H); 2.80 (d, J=4.7 Hz,
1H); 2.29 (s, 6H); 2.10-2.01 (m, 1H); 1.88-1.78 (m, 1H); 1.68-1.56 (m, 2H); 1.08 (s, 3H); 0.96 (s, 3H);
13
0.88 (s, 3H); C NMR (CDCl3, δppm): 206.6 (C=O); 171.4 (C=N); 149.6, 138.6 (CPhipso); 126.9, 117.9
(CPh); 58.1 (C1); 50.1 (C4); 44.5 (C7); 30.1, 24.4 (C5, C6); 21.4 ((CH3)2); 21.0, 17.5 (C9, C10); 9.1 (C8).
OC10H14NC6H4OH-3
Elem. Anal. (%) for C16H19O2N∙1/5H2O: Found.: C, 73.6; N, 5.7; H, 7.6. Calc.: C, 73.7; N, 5.4; H, 7.4. IR
1
(cm-1): 3361 (OH); 1748 (C=O); 1666 (C=N); 1580 (Ph). H NMR (CDCl3, δppm): 7.17 (t, J=8.0 Hz, 1H);
6.64 (d, J=8.0 Hz, 1H); 6.47-6.38 (m, 2H); 2.82 (d, J= 4.7 Hz, 1H); 2.11-2.01 (m, 1H); 1.88-1.77 (m, 1H);
13
1.68-1.53 (m, 2H); 1.07 (s, 3H); 0.96 (s, 3H); 0.87 (s, 3H). C NMR (CDCl3, δppm): 206.5 (C=O); 172.2
(C=N); 156.5, 150.7 (CPhipso); 130.0, 112.6, 112.3, 107.8 (CPh); 58.2 (C1); 50.2 (C4); 44.5 (C7); 30.1,
24.4 (C5, C6); 21.0, 17.5 (C9, C10); 9.0 (C8).
OC10H14NC6H4CF3-4
Elem. Anal. (%) for C17H18F3ON∙¼H2O: Found.: C, 64.8; N, 4.6; H, 5.5. Calc.: C, 65.1; N, 4.5; H, 5.9;IR
1
(cm-1): 1752 (C=O); 1672 (C=N); 1609 (Ph); 1325, 1112 (CF3); H NMR (CDCl3, δppm): 7.60 (d, J=8.3 Hz,
2H); 6.94 (d, J=8.3 Hz, 2H); 2.70 (d, J=4.6 Hz, 1H); 2.13-2.02 (m, 1H); 1.93-1.80 (m, 1H); 1.73-1.51 (m,
13
2H); 1.10 (s, 3H); 0.97 (s, 3H); 0.91 (s, 3H); C NMR (CDCl3, δppm): 205.8 (C=O); 173.2 (C=N); 152.9,
126.9 (CPhipso); 126.3 (q, J=3.8 Hz) (CF3); 120.0 (CPh); 58.2 (C1); 50.1 (C4); 44.5 (C7); 30.0, 24.4 (C5,
C6); 21.1, 17.4 (C9, C10); 9.0 (C8).
OC10H14NC6H4F-4
Elem. Anal. (%) for C16H18ONF∙½H2O: Found.: C, 71.4; N, 5.7; H, 7.0. Calc.: C, 71.6; N, 5.2; H, 7.1; IR
1
(cm-1): 1747 (C=O); 1655 (C=N); 1591 (Ph); H NMR (CDCl3, δppm): 7.05 (t, J=8.3 Hz, 2H); 6.94-6.89 (m,
20

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2H); 2.82 (d, J=4.6 Hz, 1H); 2.15-2.06 (m, 1H); 1.91-1.82 (m, 1H); 1.71-1.56 (m, 2H); 1.09 (s, 3H); 0.98
13
(s, 3H); 0.88 (s, 3H); C NMR (CDCl3, δppm): 206.2 (C=O); 172.1 (C=N); 145.3, 126.2 (CPhipso); 122.5,
122.4, 115.9, 115.7 (CPh); 58.0 (C1); 50.1 (C4); 44.7 (C7); 30.2, 24.3 (C5, C6); 21.0, 17.5 (C9, C10); 9.1
(C8)
OC10H14NC6H4Cl-4
Elem. Anal. (%) for C16H18ONCl: Found: C, 69.5; 5.0; 6.5; Calc.: C, 69.7; N, 5.1; H, 6.5; IR (cm-1): 1747
1
(C=O); 1673 (C=N); 1662 (C=N); H NMR (Acetone-d6, δppm): 7.41 (d, J=8.6 Hz, 2H); 6.91 (d, J=8.6 Hz,
2H); 2.82 (sbr, 1H); 2.17-2.09 (m, 1H); 1.96-1.88 (m, 1H); 1.68-1.54 (m, 2H); 1.03 (s, 3H); 1.01 (s, 3H);
0.87 (s, 3H); 13C NMR (Acetone-d6, δppm): 205.8 (C=O); 173.6 (C=N); 150.0, 130.7 (CPhipso); 130.0, 122.6
(CPh); 58.6 (C1); 51.0 (C4); 45.0 (C7); 30.7, 24.7 (C5, C6); 21.0, 17.6 (C9, C10); 9.3 (C8).
OC10H14NC6H4Cl-3
Elem. Anal. (%) for C16H18ONCl∙1/20 CHCl3: Found.: C, 68.7; N, 5.1; H, 6.6. Calc.: C, 68.4; N, 5.0; H, 6.4.
1
IR (cm-1): 1754 (C=O); 1681 (C=N); 1589 (Ph); H NMR (CDCl3, δppm): 7.20 (t, J=8 Hz, 1H); 7.13 (d, J=8
Hz, 1H); 6.88 (d, J=1.8 Hz, 1H); 6.76 (d, J=7.6 Hz, 1H); 2.77 (d, J=4.8 Hz, 1H); 2.14-2.03 (m, 1H); 1.91-
13
1.81 (m, 1H); 1.70-1.53 (m, 2H); 1.09 (s, 3H); 0.98 (s, 3H); 0.90 (s, 3H); C NMR (CDCl3, δppm): 206.0
(C=O); 172.9 (C=N); 151.0, 134.7 (CPhipso); 130.1, 125.2, 120.3, 118.4 (CPh); 58.2 (C1); 50.1 (C4); 44.5
(C7); 30.1, 24.4 (C5, C6); 21.0, 17.5 (C9, C10); 9.0 (C8).
Complexes
The complexes were synthesized using the following typical procedure: AgNO3 (0.50 mol) was added
to the solution of the suitable ligand (1.0 mol) in acetonitrile (ca. 5 ml). Then the mixture was
21

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protected from light, stirred for ca. 2h at RT and filtered (if necessary) to remove silver particles. The
solvent was then evaporated till precipitation starts. Cooling in the fridge for ca. 1day favours
precipitation of the yellowish complexes that may then be obtained by filtration (Yield =50-65%).
Characterization details:
[Ag(NO3)(OC10H14NNMe2)2] (1)
Elem. Anal. (%) for AgC20H40N5O5 ∙ 2H2O: Found.: C, 46.1; N, 11.1; H, 6.6; Calc.: C, 46.3; N, 11.3; H, 7.1;
IR (cm-1): 1707 (C=O); 1566 (C=N); 1385 (NO3); 1H NMR (CD3CN, δppm): 3.22 (d, J=4.3, 1H); 3.12 (s, 6H);
2.14-2.02 (m, 1H); 1.83-1.71 (m, 1H); 1.56-1.46 (m, 1H); 1.42-1.31 (m, 1H); 0.95 (s, 3H); 0.94 (s, 3H);
0.81 (s, 3H); 13C NMR (CD3CN, δppm): 206.3 (C=O); 145.2 (C=N); 57.5 (C1); 51.7 (C4); 46.9 ((CH3)2); 46.7
(C7); 30.8 (C6); 26.4 (C5); 21.0, 18.1 (C9, C10); 9.6 (C8).
[Ag(NO3)(OC10H14NOH)2] (2)
Elem. Anal. (%) for AgC20H30N3O7: Found.: C, 45.1; N, 7.3; H, 5.7; Calc.: C, 45.1; N, 7.9; H, 5.6. IR (cm-1):
3433 (OH); 1743 (C=O); 1643(C=N); 1379 (NO3); 1H NMR (CD3CN, δppm): 10.33 (s, 1H); 3.19 (d, J=4.3 Hz,
1H); 2.22 (s, 1H); 2.14-1.73 (3); 1.50-1.37 (1); 0.99 (s, 3H); 0.96 (s, 3H); 0.82 (s, 3H); 13C NMR (CD3CN,
δppm): 205.3 (C=O); 160.6 (C=N); 59.2 (C1); 47.5 (C4); 45.5 (C7); 31.4 (C6); 24.4 (C5); 20.8, 17.7 (C9,
C10); 9.3 (C8).
[Ag(NO3)(OC10H14NC6H5)2] (3)
Elem. Anal. (%) for AgC32H38N3O5: Found.: C, 58.7; N, 6.7; H, 6.1. Calc.: C, 58.9; N, 6.4; H, 5.8; IR (cm-1):
1746 (C=O); 1659 (C=N); 1592 (Ph); 1379 (NO3). 1H NMR (CD2Cl2, δppm): 7.38 (d, J=7.8 Hz, 2H); 7.25 (t,
J=7.3 Hz, 1H); 7.03 (d, J=8 Hz, 2H); 2.885 (d, J=4.8 Hz, 1H); 2.17-2.07 (m, 1H); 1.95-1.85 (m, 1H); 1.69-
13
1.53 (m, 2H); 1.07 (s, 3H); 0.97 (s, 3H); 0.82 (s, 3H); C NMR (CD2Cl2, δppm): 205.6 (C=O); 174.4 (C=N);
22

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148.1 (CPhipso); 129.7, 127.3, 121.9 (CPh); 58.5 (C1); 51.2 (C4); 45.2 (C7); 30.5 (C6); 24.6 (C5); 21.4,
17.4 (C9, C10); 9.1 (C8).
[Ag(NO3)(OC10H14NC6H4CH3-4)2] (4)
Elem. Anal. (%) for AgC34H42N3O5∙⅓HNO3: Found.: C, 58.5; N, 7.1; H, 6.4. Calc.: C, 58.2; N, 6.7; H, 6.0. IR
1
(cm-1): 1747 (C=O); 1654 (C=N); 1384 (NO3). H NMR (CD2Cl2, δppm): 7.17 (d, J=8.0 Hz, 2H); 6.96 (d,
J=8.0 Hz, 2H); 2.91 (d, J=4.8 Hz, 1H); 2.33 (s, 3H); 2.21-2.05 (m, 1H); 1.93-1.80 (m, 1H); 1.65-1.50 (m,
13
2H); 1.05 (s, 3H); 0.97 (s, 3H); 0.78 (s, 3H). C NMR (CD2Cl2, δppm): 205.5 (C=O); 173.3 (C=N); 145.2,
137.8 (CPhipso); 130.2, 122.4 (CPh); 58.4 (C1); 51.3 (C4); 45.3 (C7); 30.5 (C6); 24.4 (C5); 21.3, 21.1, 17.4
(CH3, C9, C10); 9.0 (C8).
[Ag(NO3)(OC10H14NC6H3(CH3)2-3,5)2] (5)
Elem. Anal. (%) for AgC36H46N3O5 2H2O: Found.: C, 57.9; N, 5.9; H, 6.4; Calc.: C, 58.1; N, 5.6; H, 6.7;
IR (cm-1): 1754 (C=O); 1680 (C=N); 1592 (Ph); 1383 (NO3); 1H NMR (CD3CN, δppm): 6.88 (s, 1H); 6.54 (s,
2H); 2.80 (d, J=4.4 Hz, 1H); 2.30 (s, 6H); 2.12-2.04 (m, 1H); 1.92-1.86 (m, 1H); 1.63-1.54 (m, 2H); 1.05
13
(s, 3H); 0.98 (s, 3H); 0.84 (s, 3H); C NMR (CD3CN, δppm): 207.3 (C=O); 173.4 (C=N); 150.3, 140.0
(CPhipso); 128.0, 118.9 (CPh); 58.9 (C1); 51.2 (C4); 45.2 (C7); 30.8 (C6); 24.8 (C5); 21.3 ((CH3)2); 21.2,
17.4 (C9, C10); 9.3 (C8).
[Ag(NO3)(OC10H14NC6H4OH-3)2] (6)
Elem. Anal. (%) for AgC32H38N3O7 ∙2H2O: Exp.: C, 53.0; N, 6.1; H, 5.1. Calc.: C, 53.3; N, 5.8; H, 5.8.
IR (cm-1): 3375 (OH); 1750 (C=O); 1664 (C=N); 1588 (Ph); 1384 (NO3).
1H NMR (CD2Cl2, δppm): 7.22 (t, J=7.8 Hz, 1H); 6.89 (s, 1H) 6.81 (s, 1H); 6.47 (d, J=7.3 Hz, 1H); 3.01 (d,
J=4.6 Hz, 1H); 2.25-2.14(m, 1H); 1.99-1.88 (m, 1H); 1.71-1.58 (m, 2H); 1.07 (s, 3H); 1.01 (s, 3H); 0.78 (s,
13
3H); C NMR (CD2Cl2, δppm): 205.6 (C=O); 174.4 (C=N); 158.8, 148.5 (CPhipso); 130.5, 115.5, 112.5,
109.9 (CPh); 58.5 (C1); 51.6 (C4); 45.3 (C7); 30.6 (C6); 24.4 (C5); 21.3, 17.2 (C9, C10); 9.0 (C8).
23

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[Ag(NO3)(OC10H14NC6H4Cl-3)2] (7)
Elem. Anal. (%) for AgC32H36N3O5Cl2 ∙2H2O: Found.: C, 50.7; N, 5.8; H, 5.1; Calc.: C, 50.7; N, 5.5; H, 5.3;
IR (cm-1): 1755 (C=O); 1684 (C=N); 1590 (Ph); 1381 (NO3); 1H NMR (CD3CN, δppm): 7.38 (t, J=8.0 Hz, 1H);
7.22 (d, J=8.0 Hz, 1H); 6.95 (s, 1H); 6.83 (d, J=7.9, 1H); 2.75 (d, J=4.7 Hz, 1H); 2.09-2.03 (m, 1H); 1.92-
13
1.87 (m, 1H); 1.65-1.56 (m, 2H); 1.05 (s, 3H); 0.98 (s, 3H); 0.86 (s, 3H); C NMR (CD3CN, δppm): 206.9
(C=O); 174.7 (C=N); 152.2, 135.3 (CPhipso);131.6, 125.9, 120.8, 119.3 (CPh); 58.9 (C1); 51.1 (C4); 45.1
(C7); 30.7 (C6); 24.6 (C5); 21.2, 17.4 (C9, C10); 9.3 (C8).
[Ag(NO3)(OC10H14NC6H4Cl-4)2] (8)
Elem. Anal. (%) for AgC32H36N3O5Cl2 ∙H2O: Found.: C, 52.1; N, 5.9; H, 4.9; Calc.: C, 52.2; N, 5.7; H, 5.2.
1
IR (cm-1): 1745 (C=O); 1663 (C=N); 1383 (NO3); H NMR (CD3CN, δppm): 7.41 (d, J=8.7 Hz, 2H); 6.92 (d,
J=8.7 Hz, 2H); 2.78 (d, J=4.8 Hz, 1H); 2.12-2.03 (m, 1H); 1.92-1.87 (m, 1H); 1.64-1.55 (m, 2H); 1.04 (s,
13
3H); 0.98 (s, 3H); 0.84 (s, 3H); C NMR (CD3CN, δppm): 206.9 (C=O); 174.4 (C=N); 149.3; 130.0; 123.1;
122.9; 58.9 (C1); 51.1 (C4); 45.2 (C8); 30.7 (C6); 24.6 (C5); 21.1, 17.4 (C9, C10); 9.2 (C8).
[Ag(NO3)(OC10H14NC6H4F-4)2] (9)
Elem. Anal. (%) for AgC32H36N3O5F2 ∙ 1.5H2O: Exp.: C, 53.5; N, 6.0; H, 5.0; Calc.: C, 53.7; N, 5.9; H, 5.4;
IR (cm-1): 1747 (C=O); 1651 (C=N); 1499 (Ph); 1384 (NO3);
1H NMR (CD3CN, δppm): 7.16 (t, J=8.5 Hz, 2H); 6.99-6.96 (m, 2H); 2.805 (d, J=4.6 Hz, 1H); 2.11-2.04 (m,
1H); 1.91-1.86 (m, 1H); 1.66-1.55 (m, 2H); 1.05 (s, 3H); 0.98 (s, 3H); 0.84 (s, 3H);
13C NMR (CD3CN, δppm): 207.1 (C=O); 173.9 (C=N); 162.8, 160.4, 146.6 (CPhipso); 123.5, 123.4, 116.8,
116.6 (CPh); 58.8 (C1); 51.1 (C4); 45.3 (C7); 30.8 (C6); 24.6 (C5); 21.1, 17.5 (C9, C10); 9.3 (C8);
[Ag(NO3)(OC10H14NC6H4CF3-4)2] (10)
Elem. Anal. (%) for AgC34H36N3O5F6: Found.: C, 51.8; N, 5.3; H, 4.5; Calc.: C, 51.8; N, 5.3; H, 4.6;
24

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1
IR (cm-1): 1751 (C=O); 1671 (C=N); 1609 (Ph); 1383 (NO3); 1325, 1113 (CF3); H NMR (CD2Cl2, δppm):
7.635 (d, J=8.3 Hz, 2H); 7.10 (d, J=8.3 Hz, 2H); 2.78 (d, J=4.7 Hz, 1H); 2.19-2.04 (m, 1H); 1.97-1.82 (m,
13
1H); 1.72-1.48 (m, 2H); 1.08 (s, 3H); 0.98 (s, 3H); 0.85 (s, 3H); C NMR (CD2Cl2, δppm): 205.2 (C=O);
175.6 (C=N); 127.0 (q, J=3.8 Hz); 121.7 (CPh); 58.6 (C1); 51.2 (C4); 45.1 (C7); 30.4, 24.6 (C5, C6); 21.5,
-
17.3 (C9, C10); 9.0 (C8); 19F NMR (CD3CN, δppm): 62.54 (CF3), using BF4 (Br4NBF4) as reference
(δppm=-151.9 ppm).
[Ag(NO3)(O2SNC10H13NNH2)2] (11)
Elem. Anal. (%) for AgC20H30N7S2O7: Found.: C, 37.3; N, 14.6; S, 9.7; H, 4.9; Calc.: C, 36.8; N, 15.0; S,
9.8; H, 4.6; IR (cm-1): 3401, 3280, 3200 (N-H); 1637, 1575 (C=N); 1383 (NO3); 1330 (SO2 asym); 1158
1
(SO2 sym). H NMR (CD3CN, δppm): 7.92 (s, 0.4H (Z-isomer)); 7.21 (s, 1.6H (E-isomer)); 3.30, 3.10 (2d,
J=13.7 Hz, 2H); 3.045 (d, J=4.1 Hz, 1H); 2.89 (s, 1H); 2.77 2.89 (s, 1H); 2.19-2.00 (m, 2H); 1.71-1.46 (m,
2H); 1.05 (s, 3H); 0.82 (s, 3H); 13C NMR (CD3CN, δppm): 186.4 (C2); 141.8 (C3); 64.5 (C1); 50.0 (C8); 48.5
(C7); 47.4 (C4); 30.4, 23.9 (C5, C6); 19.5, 18.6 (C9, C10).
Antibacterial activity determinations
The Gram-positive Staphylococcus aureus Newman, and the Gram-negative Pseudomonas aeruginosa
477, Escherichia coli ATCC 25922, and Burkholderia contaminans IST408 were used in this work. When
in use, bacteria were maintained in Lennox Broth (LB) solid medium (Sigma-Aldrich, St. Louis, USA),
otherwise cultures were kept frozen at -80°C. Minimum Inhibitory Concentration (MIC) of
camphorimine Ag(I) complexes were assessed by microdilution assays in Muller-Hinton broth (MH)
(Sigma-Aldrich, St. Louis, USA) using at least 3 independent experiments carried out in duplicate, as
previously described.14,16 For these purposes, overnight bacterial cultures grown at 37 ˚C in MH with
orbital shaking (250 rev.min-1) were diluted with fresh MH medium to a final optical density,
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measured at 640 nm (OD640) of 0.02, in a Hitachi U-2000 UV/Vis spectrophotometer. 100 μL aliquots
of cell suspensions were mixed in 96-wells polystyrene plates with 100 μL of fresh MH containing
previously serially diluted (1:2) camphorimine Ag(I) complexes from stock solutions. The OD640 of the
cultures were measured after 24 h of incubation at 37°C using a SpectrostarNano microplate reader
(BMG Labtech, Germany). The MIC values were estimated by fitting the OD640 mean values with a
Gompertz modified equation.14
Computational details
The optimization of the structures and the molecular geometry of the complexes were carried out by
DFT calculations using GAMESS-US,30 version R3 with a CAMB3LYP functional,31 and 65% HF exact
exchange at long range and 19% at short range, using a SBKJC basis set.
X-ray diffraction
X-ray data for camphor imine ligands OC10H14NY (Y=4-CH3C6H4 and Y=3-ClC6H4 ) was collected at room
temperature using a Bruker AXS-KAPPA APEX II area detector apparatus equipped with a graphite-
monochromated Mo K (=0.71073 Å) and were corrected for Lorentz polarization and, empirically,
for absorption effects. The structures were solved by direct methods using SHELX9732 and refined by
full matrix least squares against F2 using SHELX97 all included in the suite of programs WinGX v1.70.01
for Windows.33 The non-hydrogen atoms were refined anisotropically and the H atoms were inserted
in idealized positions and allowed to refine riding on the parent atom. Crystal data and refinement
parameters are summarized in Table 4. Illustrations of the molecular structures were made with
ORTEP3.34
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Table 4 - Crystallographic data for camphor imines (OC10H14NY, figure 2)
Y
4-CH3C6H4
3-ClC6H4
Empirical formula
Formula weight
Crystal system
Space group
C17H21NO
255.36
Orthorhombic
P212121
C16H18ClNO
275.78
Orthorhombic
P212121
Unit cell dimensions
a/ Å
9.991(1)
10.386(2)
20.214(5)
90
7.212(2)
10.921(3)
18.562(5)
90
b/ Å
c/ Å
α/ deg
β/ deg
90
90
γ/ deg
90
90
Volume (Å-3)
1467.6(5)
4, 1.156
0.071
1461.9(7)
4, 1.253
0.253
Z, Dcal (g/cm3)
Absorption coefficient (mm-1)
F(000)
552
584
Crystal size (mm3)
θ range for data collection (deg)
Index ranges
0.3 x 0.2 x 0.3
2.01 to 30.56
0.3 x 0.3 x 0.4
2.16 to 30.78
-9<=h<=9, -13<=k<=14, -24<=l<=28 -10<=h<=9, -15<=k<=12, -26<=l<=25
Reflections collected / unique
Data/restraints/parameters
Final R (observed)
9955 / 4447 [R(int)=0.050]
4447 / 0 / 176
R1 = 0.055, wR2 = 0.127
10378 / 4497 [R(int) = 0.041]
4497 / 0 / 175
R1 = 0.0485, wR2 = 0.136
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Acknowledgements: Financial support by FCT-Fundação para a Ciência e Tecnologia through projects
ID/QUI/00100/2013, UID/BIO/04565/2013, Grant BL-CQE/2018-013 and the NMR Network (IST-UTL
Node) for facilities are gratefully acknowledged.
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