Silver(I) complex with 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazin-3-one (Phx-1) ligand: crystal structure, vibrational spectra and?biological?studies

Article abstract of DOI:10.1080/00958972.2017.1384822

The first metal complex of Phx-1 ligand, bis(2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazin-3-one)nitratosilver(I), [Ag(Phx-1)₂NO₃], has been obtained and investigated by single crystal X-ray diffraction and vibrational spectroscopy methods. The Ag⁺ is bonded to heterocyclic nitrogen atoms of two organic ligands and one oxygen atom of a nitrate anion. The Phx-1 ligand coordination mode is supported by IR and Raman spectra, interpreted with the help of theoretical DFT studies. The antibacterial activity of the ligand and its Ag(I) complex as well as some reference compounds were screened against Gram-positive and Gram-negative bacteria, applying microdilution procedures. High sensitivity to the studied complex was found for Rhodococcus erythropolis and Bacillus licheniformis strains. Modified Phx-1 ligand preparation procedures are also presented.

Full text of DOI:10.1080/00958972.2017.1384822

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Silver(I) complex with 2-amino-4,4α-dihydro-4α,7-
dimethyl-3H-phenoxazin-3-one (Phx-1)
ligand: Crystal structure, vibrational spectra
and biological studies
K. Helios, H. Maniak, M. Sowa, W. Zierkiewicz, M. Wąsińska-Kałwa, M. Giurg,
P. Drożdżewski, A. Trusek-Hołownia, M. Malik & K. Krauze
To cite this article: K. Helios, H. Maniak, M. Sowa, W. Zierkiewicz, M. Wąsińska-Kałwa, M.
Giurg, P. Drożdżewski, A. Trusek-Hołownia, M. Malik & K. Krauze (2017): Silver(I) complex
with 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazin-3-one (Phx-1) ligand: Crystal
structure, vibrational spectra and biological studies, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2017.1384822
To link to this article: http://dx.doi.org/10.1080/00958972.2017.1384822
Accepted author version posted online: 26
Sep 2017.
Submit your article to this journal
Article views: 1
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gcoo20
Download by: [Australian Catholic University]
Date: 02 October 2017, At: 23:37

Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://doi.org/10.1080/00958972.2017.1384822
Silver(I) complex with 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-
phenoxazin-3-one (Phx-1) ligand: Crystal structure, vibrational spectra
and biological studies
K. HELIOS, H. MANIAK, M. SOWA, W. ZIERKIEWICZ, M. WĄSIŃSKA-KAŁWA, M. GIURG,
P. DROŻDŻEWSKI*, A. TRUSEK-HOŁOWNIA, M. MALIK and K. KRAUZE
Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
The first metal complex of Phx-1 ligand, bis(2-amino-4,4α-dihydro-4α,7-dimethyl-3H-
phenoxazin-3-one)nitratosilver(I), [Ag(Phx-1) NO ], has been obtained and investigated by
2
3
+
single crystal X-ray diffraction and vibrational spectroscopy methods. The Ag is bonded to
heterocyclic nitrogen atoms of two organic ligands and one oxygen atom of a nitrate anion. The
Phx-1 ligand coordination mode is supported by IR and Raman spectra, interpreted with the help
of theoretical DFT studies. The antibacterial activity of the ligand and its Ag(I) complex as well
as some reference compounds were screened against Gram-positive and Gram-negative bacteria,
applying microdilution procedures. High sensitivity to the studied complex was found for
R. erythropolis and B. licheniformis strains. Modified Phx-1 ligand preparation procedures are
also presented.
Keywords: Phenoxazine-3-one derivative; Ag Complex; Crystal and molecular structure;
Infrared and Raman spectra; Vibrational assignment; DFT
1
. Introduction
Recently, the aminophenoxazin-3-ones have attracted growing attention in medicine. The
simplest 2-aminophenoxazin-3-one (Questiomycin A, Phx-3) is a naturally occurring heterocycle
and it is the key part of Actinomycin D (figure 1), the first antibiotic with anticancer properties
[1]. Results obtained by Tomoda and coworkers [2] indicate that Phx-3 has the potential to
*
Corresponding author. Email: piotr.drozdzewski@pwr.edu.pl
1

promote apoptosis of cancer cells, and may be applied to treat gastric and colon cancer, which
are refractory to chemotherapy. The methyl derivative of Phx-3, 2-amino-4,4α-dihydro-4α,7-
dimethyl-3H-phenoxazin-3-one (Phx-1), used here gives in vitro and in vivo antitumor effects
against the human retinoblastoma cell line Y79 [3]. Phx-1 and Phx-3 exert anticancer activity
against human pancreatic cancer cell lines, KLM-1 and MIA-PaCa-2 [4]. Other studies have
shown that Phx-1, Phx-2 and Phx-3 may be useful as therapeutic agents against adult T-cell
leukemia (ATL), a malignant tumor of human CD4+ T cells infected with a human retrovirus,
T lymphotropic virus type-1 (HTLV-1) [5]. In vivo antiviral studies of activities against herpes
viruses (HSV) intravaginally inoculated into mice have been made and show promising data that
Phx-1, Phx-2 and Phx-3 are attractive candidates for agents to prevent replication of HSV and
aggravation of lesions caused by these viruses [6]. Also, the antibacterial activity of Phx-1,
Phx-2 and Phx-3 have been investigated. All presented derivatives show prominent antimicrobial
activity against non-tuberculosis mycobacterial strains, M. scrofulaceum, M. kansasii,
M. marinum and M. intracellurale [7].
In addition to the extensive studies of the biological activities of 2-aminophenoxazin-3-
ones, the properties of well-dispersed PtRu and Pd nanoparticles on Phx-3 functionalized multi-
walled carbon nanotubes (MWCNTs) have been reported and the results are very promising for
portable applications in direct methanol fuel cells (DMFCs) field [8, 9]. Until now only one
metal complex with Phx-3, ([Ag(Phx-3)NO ]) and its enhanced (in comparison to free ligand)
3
biological activity against S. aureus have been reported [10].
In this article we report the synthesis, crystal structure, Raman and IR spectra with band
assignments made on the basis of theoretical calculations of the first metal complex with Phx-1,
[
Ag(Phx-1) NO ]. Its biological activity has also been tested in order to validate the idea that
2 3
binding the metal by some ligands may enhance their biological activity.
Prior to the complex preparation, the Phx-1 ligand was obtained via two modified click
procedures using the corresponding 2-amino-5-methylphenol and hydrogen peroxide (H O ) in
2
2
the presence of ebselen or horseradish peroxidase (HRP).
2
2
. Experimental
.1. Materials and methods
All solvents were distilled before use. Commercially available reagents, 2-amino-5-
2

methylphenol, DMSO (Aldrich), AgNO and aqueous hydrogen peroxide (H O ) (Avantor
3
2
2
Performance Materials Poland S.A.), and Horseradish Peroxidase catalyst (HRP) (Sigma), were
used without purification. Water for enzymatic transformation was distilled twice.
Analytical TLC was performed on PET foils precoated with silica gel (Merck silica gel,
6
0 F254) and were made visual under UV light (λmax = 254 nm). Melting points were determined
on an Electrothermal IA 91100 digital melting point apparatus using the standard open capillary
method.
2
2
.2. Syntheses
.2.1. Preparation of the Phx-1 ligand. An exhaustive literature survey showed that the
preparation of Phx-1 required 2-amino-5-methylphenol [11, 12] and 2-nitro-5-methylphenol
13]. However, no multigram scale procedures were described to date. Prior to the complex
[
preparation, the Phx-1 ligand was obtained in gram scale via two modified click procedures
using the corresponding 2-amino-5-methylphenol and hydrogen peroxide (H O ) in the presence
2
2
of ebselen or horseradish peroxidase (HRP); see Supporting Information for details.
2
.2.2. Preparation of the silver complex - bis(2-amino-4,4α-dihydro-4α,7-dimethyl-3H-
phenoxazin-3-one)nitratosilver(I). For complex [Ag(Phx-1) NO ] preparation, an ethanolic
2
3
3
solution (5 cm ) of AgNO (0.2 mmol, 0.0339 g) was added to Phx-1 (0.2 mmol, 0.0454 g)
3
3
dissolved in 10 cm of ethanol (333 K). The reaction mixture was stirred and heated at 333 K for
a few minutes in a temperature-controlled bath. From the resulting orange solution, stored in a
beaker covered with parafilm, the orange crystals precipitated the next day. After a few days,
crystals were washed abundantly with ethanol and dried at room temperature. Yield: 0.13 g,
0
1
.20 mmol (36% based on AgNO ]). Anal. Calcd. for C H Ag N O (%): C, 51.39; H, 4.31; N,
3
28 28
1
5
7
0.70. Found: C, 52.84; H, 4.26; N, 10.76.
1
3
3
H NMR (DMSO-d , 600 MHz): 7.24 (d, J = 7.9 Hz, 1 H, H-9), 6.84 (dd, J = 7.9 Hz,
6
4
4
J = 1.1 Hz, 1 H, H-8), 6.80 (s, 2 H, NH ), 6.76 (d, J = 1.1 Hz, 1 H, H-6), 6.14 (s, 1 H, H-1),
2
2
2
3
.27 (d, J = 15.9 Hz, 1 H, H-4a), 3.05 (d, J = 15.9 Hz, 1 H, H-4b), 2.27 (s, 3 H, MeC-7), 1.14
1
3
(
s, 3 H, MeC-4α). C NMR (DMSO-d , 151 MHz): 191.20 (C-3), 162.46 (C-10α), 147.59 (C-2),
6
1
43.89 (C-5α), 137.03 (C-7), 131.91 (C-9α), 125.55 (C-9), 123.08 (C-8), 116.66 (C-6), 104.87
(C-1), 71.44 (C-4α), 48.99 (C-4), 22.08 (MeC-4α), 20.79 (MeC-7).
3

2
.3. X-ray analysis
Crystallographic measurement was performed on a Xcalibur R automated four-circle
diffractometer with graphite monochromated Mo Kα radiation at 100(2) K using an Oxford
Cryosystems cooler. Selected crystallographic data are provided in table 1. Data collection, cell
refinement, and data reduction and analysis were carried out with CRYSALISCCD and
CRYSALISRED, respectively [14]. Analytical absorption correction was applied with
CRYSALISRED [14]. The structure was solved by direct methods using SHELXS-97 [15] and
refined by full-matrix least squares with SHELXL-97 [15], with anisotropic thermal parameters
for all non-H atoms (unless otherwise stated below). N-bound H atoms were found in difference
Fourier maps, but in the final refinement cycles they were treated with DFIX and AFIX 3
instructions [15] and refined with U (H) = 1.2U (N). C-bound H atoms were placed in their
iso
eq
calculated positions and refined using a riding model, with C–H = 0.95–0.99 Å and U (H) =
iso
1
.5U (CH ) or 1.2U (CH , CH). During the refinement, the Ag ion and one of two ligand
eq 3 eq 2
molecules were disordered and each one was refined over two sites with 0.970(2)/0.030(2) and
.948(3)/0.052(3) occupancies, respectively. C and O atoms with minor occupancies were
0
refined isotropically and in the final refinement cycles constrained to have equal displacement
parameters. ORTEP [16] was used for the creation of figures.
2
.4. Spectroscopic measurements
The FT-Raman spectra were measured on a Bruker MultiRAM spectrometer equipped with a
Nd:YAG laser (1064 nm) and a liquid nitrogen cooled germanium detector. The spectra were
-1
recorded (128 scans) at a resolution of 2 cm and 250 mW laser power for Phx-1 and 50 mW for
-1
complex in the range 3500-50 cm . The middle- and far-infrared spectra were measured on a
Bruker VERTEX 70V vacuum spectrometer equipped with a diamond ATR accessory and air-
cooled DTGS detector. The instrument was kept under vacuum during the measurements, and
-1
the spectra were recorded (128 scans) at a resolution of 2 cm .
1
13
The H and C NMR spectra were recorded in DMSO-d or CH OH-d on a Bruker
6
3
4
1
13
Avance 600 spectrometer (600 for H and 151 for C) at 295 K. Chemical shifts (δ) are given in
parts per million (ppm) downfield relative to TMS, and coupling constants (J) are in Hz.
4

2
.5. Theoretical studies
The DFT calculations were performed for the Phx-1 ligand and its [Ag(Phx-1) NO ] complex
2
3
using the Gaussian09 program [17]. The respective crystallographic data was used as the starting
geometry in the optimization procedure. Full geometry optimizations were performed by means
of the three-parameter hybrid B3LYP-D3 [18, 19] method (B3LYP functional corrected with the
original D3 damping function [20]) combined with the D95V(d, p) [21] basis set for all ligand
atoms. For the Ag atom, the LanL2DZ effective core potential with the concomitant valence
basis set was applied [22].
The optimized structure corresponds very well to that measured, as results from table 2
show where selected structural parameters from the X-ray analysis are compared with
corresponding calculated values. The RMS difference between measured and calculated skeletal
bond lengths is about 0.04 Å whereas for the plane angles it equals 1.7. The noticeable
difference is in the mutual orientation of organic ligands. In the crystalline state, both ligands are
almost flat and are positioned close to the same plane. In the optimized structure, the aromatic
rings are slightly twisted in relation to the respective chelate rings, which in turn are more
twisted in relation to each other.
For the systems investigated, full geometry optimizations were followed by the
calculations of theoretical spectra based on vibrational frequencies and infrared intensities or
Raman activities. Figures S1 and S2 show the comparison of observed spectra with theoretical
–
1
ones, calculated for half-band-width equal 5 cm . The vibrational assignments of the
experimental ligand spectra were accomplished by the examination of the calculated potential
energy distribution, PED terms (ligand) and visualization of normal modes (ligand and complex).
For better correlation between observed and calculated wavenumbers, the latter were scaled by a
0
.97 factor, except for the NO group vibrations, for which the correlation was satisfactory.
3
2
.6. In vitro antibacterial assay
The antimicrobial activities of tested compounds were determined using a modified two-fold
microdilution procedure [23] and presented in Minimum Inhibitory Concentration values (MIC,
–
3
mg·dm ). The MIC was defined as the lowest concentration of tested compound that inhibited
the visible growth of microorganism after 24 h of incubation in defined cultivation conditions.
5

2
.6.1. Microorganisms and inocula preparation. The biological activity of each compound
was tested against Gram-positive and Gram-negative bacterial strains. The Gram-positive
microorganisms such as Staphylococcus aureus (PCM 2054), Bacillus licheniformis (PCM
1
847), Rhodococcus erythropolis (PCM 2149) and Gram-negative Escherichia coli (PCM 2057),
Pseudomonas fluorescens (PCM 2123), Alcaligenes faecalis (PCM 2223) originated from Polish
Collection of Microorganisms. The strains were cultivated in liquid Mueller-Hinton medium
–
3
(
MH) with the composition of g∙dm : beef extract, 2; casein hydrolysate, 17.5; starch, 1.5;
pH 7.2. All chemicals were of high purity grade used for microbiological procedures (Sigma-
3
Aldrich). The bacterial liquid cultures (inocula) were carried out in 250 cm glass flasks closed
3
with cotton plugs. The flasks contained 100 cm of the MH medium with appropriate
microorganism and were agitated on rotary-shaker with 120 rpm at 32 °C. Growth of the culture
was monitored by spectrophotometric measurements of an optical density at 600 nm until it
reached a logarithm phase growth which corresponded to OD600 between 0.4-0.6. Subsequently,
a microbial cell suspension was diluted to obtain the final optical density at 0.02 and used as an
inoculum to antimicrobial activity evaluation.
2
.6.2. MIC evaluation. The compounds in the crystalline form, [Ag(Phx-1) NO ] and AgNO ,
2 3 3
or as powders, 2-amino-4,4α-dihydro-4α,7-dimetylo-3H-phenoxazine-3-one (Phx-1) and
-amonophenoxazine-3-one (Questiomycin A), were dissolved in DMSO. Such prepared stock
2
solutions were further dissolved in the MH medium to obtain the 5.0% (v/v) concentration of the
DMSO. The serial microdilutions of tested compounds were performed in 96 microwell plates
with the same cultivation medium, in the range of concentrations varied from 0.183 to
–
3
–3
3
0
75 mg∙dm . Each well containing 0.1 cm of diluted compound was then inoculated with
–
3
.005 cm of previously prepared diluted bacterial cell suspension. Next, the 96-microwells
plates were incubated for 24 h in the dark at 32 °C, after which, the results were noted. The
plates were further incubated at the same condition for the next 24 h to confirm the former
results. Similar experiments were conducted for DMSO (positive control) at an initial
concentration of 5.0% (v/v) to exclude a toxic effect on microbial growth of the applied solvent.
Nifuroxazide and chloramphenicol were used as reference standards (negative controls) for
representative Gram-negative and Gram positive, E. coli and S. aureus strains, respectively. All
experiments were performed in duplicate. The assays performed in duplicate and resulting in the
6

same MIC values were assumed as correct. Additional two assays were carried out in a case of
inconsistent results. In such cases, the mean values of all (four) tests were taken as final results
and standard deviations were calculated as the error analysis. Such procedures were carried out
for [Ag(Phx-1) NO ] tested towards A. faecalis, Phx-1 against B. licheniformis, AgNO towards
2
3
3
E. coli and questiomycin A against all Gram positive bacteria. For these experiments, table 3 lists
the mean MIC values and standard deviations. All results of MIC measurements are included in
table S1 (Supplementary Material).
3
3
. Results and discussion
.1. Molecular structure and crystal packing
The crystal structure of the investigated complex is slightly complicated by two effects, disorder
of the silver ions and the presence of two complex isomers, due to the chirality of the ligand.
Both effects are relatively small, which may be illustrated by respective occupancies. The minor
occupancy of Ag(I) site is 3.0(2)% whereas the second complex isomer contributes 5.2(3)%.
Both effects will be omitted in the figures and tables for better clarity. The refinement did not
reveal any disorder of the nitrate ion position. The fact seems surprising, yet a ~5% minor
occupancy would require refinement of less than half of an electron. The disordered position of
the nitrate ion might lie to close to the major occupancy to be detected and refined.
The structure of discrete [Ag(Phx-1) (NO )] entity is shown in figure 2. The central ion is
2
3
coordinated by two organic ligands acting in monodentate mode and one oxygen atom of the
nitrate counterion. The Ag(I) ion environment is close to planar with the metal ion being the
most separated from the best N10a-Ag-O1-N10b plane by 0.153 Å or by 0.213 Å from N10a-
O1-N10b plane. However, the mutual location of coordination centers is not symmetric, as seen
from the angles: N10a-Ag-N10b = 156.18(8), N10a-Ag-O1 = 83.20(8) and N10b-Ag-O1 =
1
16.64(8). The sum of these angles is 356.02, which again confirms the quasi-planarity of the
metal coordination vicinity.
The second Ag1 site is 0.58(2) Å from the original Ag(1) and located closer to the N10b
atom, which makes the Ag1-N bond lengths more different (Ag1-N10a 2.277(13) Å, Ag1-N10b
.060(11) Å).
The main part of the ligand skeleton is formed by three 6-membered rings, one of
aromatic character (R3 in figure 2). The two remaining rings lie very close to the plane of the
2
7

aromatic one. The largest displacement from the aromatic ring best plane is the C41a atom
0.498 Å). The next largest is the C3a atom with 0.214 Å displacement. Similar parameters for
(
second ligand are 0.728 Å (C41b) and 0.936 Å (C3b). Other small structural differences between
the ligand molecules can be deduced from table 2, where corresponding ligand parameters are
listed. In ligand skeletal bond lengths, the largest difference is 0.013 Å, found for the C41a–O5a
bond and its counterpart. The O5a and O5b atoms also form one of the most different angles
C41x–O5x–C51x (x = a or b) for which the difference is 2.3. The 29.4 difference between
C9x–C91x–N10x–Ag angles points out that two organic ligands present in the complex are
twisted relative to each other. This is also demonstrated by the dihedral angle of 46.2, measured
between the best planes composed for each ligand of all ring skeletal atoms, except C41a or
C41b.
Comparison with the structure of pure Phx-1 ligand [13] shows that formation of
Ag-N10a/b bonds caused very small changes in the vicinities of coordinating nitrogen atoms.
The C11a/b-N10a/b and N10a/b-C91a/b bonds enlarged less than 0.5%, whereas the C11a/b-
N10a/b-C91a/b angles changed less than 0.4%.
The orientation of the nitrate group, represented by the N1-O2-O3 plane, is fixed by five
hydrogen bonds with O2 and O3 atoms as acceptors. Among these is one intramolecular
C9b-H9b···O2 hydrogen bond of C9b···O2 distance = 3.414(4) Å. Probably by mere
coincidence, the nitrate plane lies very close to the plane of aromatic ring C51b–C91b (the
largest separation of 0.132 Å was found for the O2 atom).
The crystal packing is stabilized by nine intermolecular hydrogen bonds of N–H and C–H
donor types. The strongest connection between complex molecules is via N1a-H3a···O2i
i
ii
N1b-H3b···O1 and N1-bH2b···O2 hydrogen bonds (D···A 2.993(3), 2.928(3) and 3.200(3) Å,
respectively). Interactions are visualized in figure 3 (the caption of this figure contains the
corresponding symmetry codes). These bonds organize molecules related by the 2-fold screw
axis symmetry operation into chains extending along the b axis, as shown in figure 3. Additional
stabilization is provided by C–H···O interactions (not shown in figure 3). Weaker
iii
C4b-H4b2···O1a bonds (3.470(3) Å, symmetry code: (iii) x+1/2, 1/2-y, z-1/2) join the chains
into planes parallel to (1 0 1) plane. The planes are connected in a three-dimensional network by
iv
C13a-H13a···O1 bonds (3.451(3) Å; symmetry code (iv) x-1/2, 3/2-y, z-1/2).
8

3
.2. Vibrational spectroscopy
In the spectroscopic part of the present work, the vibrational spectra of pure Phx-1 ligand and its
Ag(I) complex were compared for tracing the changes caused by metal coordination. Special
attention was focused on skeletal stretching vibrations absorbing mainly in the 1800 – 600 cm─1
region, shown in figure 4. The band assignment was made with the help of DFT calculated
–
1
frequencies, which show about 12 cm RMS observed – calculated differences in the mentioned
region.
The monodentate coordination mode of the Phx-1 ligand suggests that the vibrations of
the C11a=N10a-C91a subgroup (atom numbering as in figure 2) should be the most affected by
metal bonding. However, as results from the calculations indicate, it is difficult to find IR bands
characteristic for the stretching ν(C11a=N10a) or ν(C91a-N10a) vibrations. For a conjugated
system such as the Phx-1 skeleton, most stretching skeletal vibrations are strongly coupled and
involve simultaneous motion of many atoms. This is confirmed by PED analysis where the
largest contribution of particular internal stretching modes is typically less than 30% (except
ν(C=O) with 70% contribution). The largest contributions of the ν(C11a=N10a) internal mode
─1
have been found in normal vibrations of Phx-1 at 1565 and 1554 cm (for respective PED
values see table 4). Similar character show two normal vibrations calculated for
─1
─1
[
Ag(Phx-1) (NO )] complex at about 30 cm lower wavenumbers (1536 and 1523 cm ). In the
2 3
─1
─1
1
600 – 1500 cm region of the observed IR spectra the Phx-1 bands at 1587 and 1533 cm
─1
show the largest downshift upon coordination (1574 and 1528 cm in the complex spectrum)
and these bands have been assigned to vibrations with a substantial ν(C=N) contribution.
The stretching vibration of the neighboring C91a-N10a bond is more dispersed over
─1
several normal modes, with maximum contribution of 22% in 1217 cm vibration. For two
ligand molecules present in the complex, the similar ν(C-N) mode was calculated at 1222 and
─1
─1
1
218 cm . For these vibrations, the bands observed at 1229 and 1231 cm have been proposed.
Other normal vibrations with PED contributions of oxazine ring stretching modes exceeding 5%
can be found in table 4.
The most visible spectral change caused by metal coordination is the splitting of
characteristic ν(C=O) vibration. In Phx-1, this vibration generates the strong IR band at
─1
─1
1
692 cm , whereas in the complex spectrum, two bands at 1711 and 1696 cm are observed.
Similar splitting was predicted by calculations, pointing out that two ligand subunits present in
9

the complex are not structurally and spectrally equal. The first difference is confirmed by the
X-ray analysis showing that the C=O bond lengths are slightly different (table 2) and only one
C3a=O1a group is involved in hydrogen bonding, as an acceptor. The second difference is
monitored by calculated PED terms. In both ν(C=O) normal vibrations the C=O stretching mode
contributes in 70%, but remaining contributions are different, for example: 5% of bending
δ(C1b-C2b-C3b) for higher wavenumber vibration and 4% of ν(C3a-C4a) for lower one.
The new spectral features caused by metal coordination are the bands resulting from so
called "metal-ligand" vibrations. For the presented complex, these vibrations are expected at low
wavenumbers because of the Ag atomic mass and stiffened motion of coordinating nitrogen
atoms mounted into ring systems. Animation of calculated normal vibrations demonstrates that
the simultaneous stretching of both Ag-N bonds is connected with motions of whole Phx-1
─1
ligands. The largest amplitudes has been found for 74 and 167 cm normal vibrations
─1
(
symmetric ν (Ag-N)) and for 116 and 174 cm modes (asymmetric ν (Ag-N)). Such vibrations
s
a
─1
may contribute in respective bands at 76, 118 and 177 cm found in the FIR spectrum of the
Ag(Phx-1) (NO )] complex. Changes of Ag-N bond length have also been observed for oxazine
[
2
3
─1
ring deformations with large nitrogen atom displacements, calculated at 450 and 455 cm and
─1
attributed to 452 cm band. The Ag-O distance changes significantly when whole NO group is
3
─1
displaced, as in normal vibration at 155 cm , or during NO in-plane bending vibrations,
3
─1
─1
calculated at 712 and 695 cm . First vibration was assigned to 155 cm IR shoulder; two
remaining modes will be discussed latter.
The NO group vibrations have been localized by comparison of the literature data with
3
respective regions of complex and additionally the ligand spectra. In the former, additional IR
─1
bands were found at 1426, 1383, 1042, 823, 736 and 715 cm corresponding to ν , ν , ν , ν and
3
3
1
2
ν , ν , vibrations of simple nitrate salts [24]. The splitting of the ν and ν modes (doubly
4
4
3
4
-
degenerated in free NO ion) and IR activity of ν mode confirm the symmetry lowering of
3
1
nitrate ion upon metal coordination.
The foregoing discussion, and those bands listed in table 4, do not cover all vibrations of
Phx-1 and its silver complex. As predicted for Phx-1 by DFT calculations, about 40% of total 90
normal vibrations have weak IR intensity, being more than 50 times lower than the intensity of
–
1
strong ν(C=O) band at 1694 cm , taken for example as a reference. The wavenumbers of many
of these transitions are not available because of the overlap by stronger ones. In the case of the
1
0

[
Ag(Phx-1) (NO )] complex, which molecule has 201 normal vibrations, the number of observed
2 3
bands is not significantly increased because two coordinating ligand molecules have very close
wavenumbers of corresponding vibrations.
3
.3. In vitro antibacterial assay
The comparative biological studies of the [Ag(Phx-1) NO ] complex as well as remaining
2
3
reference compounds such as AgNO and Phx-1, and Questiomycin A, both prepared at
3
enzymatic procedures and other antibiotics were performed against Gram-positive and Gram-
negative bacteria, applying commonly known microdilution procedure. The antibacterial
activities, presented in MIC values, are included in table 3.
The MIC values of the various organic ligands, the silver(I) salt and its coordination
compound can vary significantly between applied procedures [25, 26], bacteria species or even
between the strains, as found in the literature [27-29]. Nevertheless, an effort to perform a
comparable qualitative analysis of the obtained data has been made.
Results from table 3 show the Phx-1 ligand exhibited lack of activity against Gram-
negative representatives and only very weak action towards some Gram-positive bacteria.
Comparing these results to the Phx-1 analog, Questiomycin A, the latter presented weak
antibacterial action towards Gram-positive strains, especially for S. aureus and R. erythropolis.
Similar results were obtained by Shimizu and co-workers [30] who investigated the antibacterial
activity of these two phenoxazine derivatives on mycobacteria and other bacteria strains,
applying similar microdilution method. In their research both compounds did not present
biological action against E. coli and S. aureus, in tested range of concentrations with the
–
3
maximum saturation at 45 mg·dm . The lack of antimicrobial activity for both organic
molecules could be attributed to a phenoxazine system, which probably easily undergoes
inactivation during the metabolic pathways. The hydroxylation of heterocyclic aromatic rings is
a typical and essential step in carbon cycle [31] and defense mechanism of the microorganisms
when exposed on unfriendly environment [32, 33].
Further comparison of the results obtained for tested compounds showed that the
complexation of silver nitrate lead to appreciably improved activities of the organic ligand
Phx-1. For selected Gram-positive and Gram-negative representatives the antibacterial activity of
the complex was significantly enhanced in comparison to pure ligand, respectively. Thus, the
1
1

[
Ag(Phx-1) NO ] compound presents a wide spectrum of the biological activity against both
2 3
Gram-positive and Gram-negative bacteria in the investigated range of concentrations (0.183-
–
3
3
75 mg·dm ). The minimum inhibitory concentrations corresponded to E. coli, P. fluorescens
–
3
–3
(
23 mg·dm ) and S. aureus, B. licheniformis strains (47 mg·dm ) are of the same order of
magnitude, indicating comparable antibacterial activity. However, in these two groups of
microorganisms, there are strains which do not follow the tendency being more resistant
(A. faecalis) or more sensitive (R. erythropolis) to tested coordination compound. Different
results were obtained [28], where the authors investigated the beneficial effect of
Questiomycin A complexation with silver(I) nitrate. The resulted coordination compound
[
Ag(2-aminophenoxazine-3-one)(NO )] presents enhanced biological activity only against
3
S. aureus in comparison to pure ligand and inorganic salt.
Figure 5 clearly shows that the biological activity of [Ag(Phx-1) NO ] is predominantly
2
3
an effect of the metal cation action. For all tested microorganisms, the AgNO solution exhibited
3
very strong toxic effect, which remains in agreement with the literature data [34-36]. Silver is an
antimicrobial agent of medical application due to its non-toxicity for human cells. An excellent
example of its usage is silver(I)-sulfadiazine, a polymeric drug commonly used on a wound
surface of scalded skin to accelerate healing and simultaneously prevent bacterial infections [37].
The inhibitory action of silver and its compound on microorganisms is poorly understood.
Nevertheless, recent research provides some possible explanations of antibiological mechanisms.
In the mechanisms mentioned, the following are frequently listed: damage of cell membrane and
interaction with protein having thiol groups or with active side of enzymes. Jung and co-workers
[34] performed a detailed study on antibacterial effect of silver(I) solution on E. coli and
S. aureus by analyzing the growth and structure of bacterial cell. They concluded that silver
cation exhibited better activity towards gram-negative rather than gram-positive bacteria due to
the differences in cell membrane morphology. The exposition of cell on silver solution caused
cell wall damage and separation of cellular components from microbial membrane. The
additional studies were conducted on possible interactions of Ag(I) ion with sulfhydryl (thiol) or
imidazole groups present in various organic compounds [36, 38] as well as in amino acid
polymeric chains in enzymes and other proteins [29, 37, 39]. Above examples showed that
antibacterial properties of silver cation can affect various processes inside the microbial cell.
In summary, based on the obtained MICs, the antibacterial activity should be classified
1
2

rather as moderate in comparison to values of tested antibiotics such as chloramphenicol (2.9 mg
-3
-3
dm ) and nifuroxazide (0.73 mg dm ). Nevertheless, among tested microorganisms were strains
R. erythropolis, B. licheniformis) of better antibacterial activity than for AgNO and are
(
3
comparable to chloramphenicol. Thus, the group of Gram-positive bacteria should be further
tested as potential group sensitive for [Ag(Phx-1) NO ] complex.
2
3
4
. Conclusion
Silver(I) complex with phenoxazine derivative (Phx-1) has been synthesized and characterized
by X-ray, IR and Raman and biological methods. The ligand acts in monodentate mode, binding
the silver(I) ion via nitrogen atom of phenoxazine ring. Due to ligand chirality, the obtained
[
Ag(Phx-1) NO ] crystals are composed mainly of complex molecules with R and one S
2 3
enantiomers; only about 5% of molecules are built of the same enantiomers.
Binding the Ag(I) ion causes very small changes (less than 0.5%) in lengths of bonds
around coordinating nitrogen atom. Nevertheless, the respective vibrations changed significantly
shifting to lower wavenumbers, which suggests that this is mostly due to the "mass effect" of
heavy metal attachment to the nitrogen atom. The [Ag(Phx-1) NO ] complex shows moderate
2
3
antibacterial activity in comparison with some commercial antibiotics, but exhibits better
antibacterial activity than for Phx-1 or AgNO for selected Gram-positive bacteria. This
3
observation confirms the hypothesis that metal complexation may enhance the biological activity
of the used ligand or metal salt.
Supplementary material
CCDC 1525422 contains the supplementary crystallographic data for this paper. The data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/structures.
Acknowledgements
This work was financed in part by a statutory activity subsidy from the Polish Ministry of
Science and Higher Education for the Faculty of Chemistry of Wroclaw University of
Technology. A generous computer time from the Wroclaw Supercomputer and Networking
Center as well as the Poznan Supercomputer and Networking Center is acknowledged.
1
3

References
[1]
[2]
[3]
U. Hollstein. Chem. Rev., 74, 625 (1974).
A. Tomoda, K. Miyazawa, T. Tabuchi. Int. J. Mol. Sci., 14, 17573 (2013).
K. Kimura, Y. Usui, T. Hattori, N. Yamakawa, H. Goto, M. Usui, S. Okada, K. Shirato,
A. Tomoda. Oncol. Rep., 19, 3 (2008).
[4]
[5]
S. Kato, K. Shirato, K. Imaizumi, H. Toyota, J. Mizuguchi, M. Odawara, X.-F. Che, S.
Akiyama, A. Abe, A. Tomoda. Oncol. Rep., 15, 843 (2006).
N. Miyano-Kurosaki, K. Ikegami, K. Kurosaki, T. Endo, H. Aoyagi, M. Hanami, J.
Yasumoto, A. Tomoda. J. Pharmacol. Sci., 110, 87 (2009).
[
6]
7]
K. Hayashi, T. Hayashi, K. Miyazawa, A. Tomoda. J. Pharmacol. Sci., 114, 85 (2010).
S. Shimizu, M. Suzuki, A. Tomoda, S. Arai, H. Taguchi, T. Hanawa, S. Kamiya. Tohoku
J. Exp. Med., 203, 47 (2004).
[
[
[
[
8]
Y. Zhao, X. Yang, J. Tian. Electrochim. Acta, 54, 7114 (2009).
Y. Zhao, X. Yang, J. Tian, F. Wang, L. Zhan. Mater. Sci. Eng., B, 171, 109 (2010).
9]
10] K. Pandurangan, S. Gallagher, G.G. Morgan, H. Müller-Bunz. F. Paradisi. Metallomics,
, 530 (2010).
2
[
[
[
[
11] M. Giurg, K. Piekielska, M. Gębala, B. Ditkowski, M. Wolański, W. Peczyńska–Czoch,
J. Młochowski. Synth. Commun., 37, 1779 (2007).
12] J.M. Granda, K. Piekielska, M. Wąsińska, N. Kawecka, M. Giurg. Synthesis, 47, 3321
(
2015).
13] S.M. Jansze, V. Saggiomo, A.T.M. Marcelis, M. Lutz, A.H. Velders. Tetrahedron Lett.,
6, 1060 (2015).
5
14] Oxford Diffraction, CrysAlis CCD and CrysAlis RED in Xcalibur PX and Kuma KM-4-
CCD Software, Oxford Diffraction Ltd., Yarnton, Oxfordshire, England (2009).
15] G.M. Sheldrick. Acta Crystallogr., A64, 112 (2008).
[
[
[
16] L.J. Farrugia. J. Appl. Cryst., 30, 565 (1997).
17] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
1
4

H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J.
Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K.
Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M.
Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J.
Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J.
Cioslowski, D.J. Fox, Gaussian, Inc., Wallingford, CT (2009).
[
[
[
[
18] A.D. Becke. J. Chem. Phys., 98, 5648 (1993).
19] C. Lee, W. Yang, R.G. Parr. Phys. Rev. B, 37, 785 (1988).
20] S. Grimme, J. Antony, S. Ehrlich, H. Krieg. J. Chem. Phys., 132, 154104 (2010).
21] T.H. Dunning Jr., P.J. Hay. In Modern Theoretical Chemistry, H.F. Schaefer (Ed.),
Vol. 3, pp. 1-28. Plenum Press, New York (1976).
[
[
[
22] P.J. Hay, W.R. Wadt. J. Chem. Phys., 82, 299 (1985).
23] EUCAST discussion document. Clin. Microbiol. Infect., 9, 1 (2003).
24] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds,
Part A, p. 183, John Wiley & Sons, Hoboken (2009).
[25] I. Chopra. J. Antimicrob. Chemother., 59, 587 (2007).
[26] S. Zhang, L. Liu, V. Pareek, T. Becker, J. Liang, S. Liu. J. Microbiol. Methods, 105, 42
(2014).
[27] M. Cavicchioli, A.C. Massabni, T.A. Heinrich, C.M. Costa-neto, E.P. Abrão, B.A.L.
Fonseca, E.E. Castellano, P.P. Corbi, W.R. Lustri, C.Q.F. Leite. J. Inorg. Biochem., 104,
5
33 (2010).
[
28] K. Pandurangan, S. Gallagher, G.G. Morgan, H. Mu. Metallomics, 5901, 4 (2010).
29] U. Kalinowska-Lis, A. Felczak, L. Che, K. Lisowska, J. Ochocki. J. Organomet. Chem.,
[
7
49, 394 (2014).
[30] S. Shimizu, M. Suzuki, A. Tomoda, S. Arai, H. Taguchi, T. Hanawa, S. Kamiya. Tohoku
J. Exp. Med., 203, 47 (2004).
[
[
[
31] W.C. Evans. J. Gen. Microbiol., 32, 177 (1963).
32] R. Ullrich, M. Hofrichter. Cell. Mol. Life Sci., 64, 271 (2007).
33] P. Di Gennaro, A. Bargna, G. Sello. Appl. Microbiol. Biotechnol., 90, 1817 (2011).
1
5

[
[
[
34] W.K. Jung, H.C. Koo, K.W. Kim, S. Shin, S.H. Kim, Y.H. Park. Appl. Environ.
Microbiol., 74, 2171 (2008).
35] C.P. Randall, L.B. Oyama, J.M. Bostock, I. Chopra, A.J.O. Neill. J. Antimicrob.
Chemother., 68, 131 (2013).
36] K. Nomiya, Y. Kondoh, K. Onoue, N.C. Kasuga, H. Nagano, M. Oda, T. Sudoh, S.
Sakuma. J. Inorg. Biochem., 58, 255 (1995).
[
[
[
37] C.G. De Gracia. Burns, 27, 67 (2001).
38] K. Nomiya, K. Tsuda, T. Sudoh, M. Oda. J. Inorg. Biochem., 68, 39 (1997).
39] R.B. Wakshlak, R. Pedahzur, D. Avnir. Sci. Rep., 5, 1 (2015).
1
6

Table 1. Crystal data, data collection and refinement details for [Ag(Phx-1) NO ].
2
3
Compound
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
[Ag(Phx-1) NO ]
2
3
C H AgN O
7
2
8
28
5
654.42
Monoclinic
P2 /n
1
15.258(5)
10.895(3)
17.338(5)
111.73(5)
2677.4(16)
4
b (Å)
c (Å)
β (°)
Volume (Å3)
Z (molecule/cell)
-3
Dcalc (Mg m )
F(000)
1.624
1336
Crystal size (mm)
Wavelength (Å)
Temperature (K)
θ range (°)
0.15 × 0.12 × 0.06
0.71073
100(2)
2.874–30.796
0.810
-1
Absorption coefficient (mm )
Index ranges (limiting indices)
–21 ≤ h ≤ 13
–
–
15 ≤ k ≤ 13
24 ≤ l ≤ 24
Reflection collected / unique (Rint)
Goodness-of-fit
18407 / 7577 [R = 0.040]
int
1.039
Final R indices [I>2σ(I)]]
R =0.0433,
1
wR =0.0716
2
R indices (all data)
R =0.0679,
1
wR =0.0790
2
-3
Largest diff. peak and hole (e Å )
0.59 and -0.42
1
7

Table 2. Selected bond lengths [Å] and angles [] of [Ag(Phx-1)
2
NO
3
] with e.s.d.s. in parentheses.
X-ray
Aromatic ring parameters are omitted as being close to typical.
Parameter
X-ray
DFT
Parameter
DFT
Ag–N10a
2.193(2)
2.573(2)
1.256(2)
2.363
2.501
1.278
Ag–N10b
N1–O1
2.161(2)
1.257(2)
1.239(2)
2.304
1.275
1.254
Ag–O1
N1–O2
N1–O3
Ligand “a”
Ligand “b”
C1b–C2b
C1b–C11b
C2b–N1b
C2b–C3b
C3b–O1b
C3b–C4b
C4b–C41b
C41b–O5b
C1a–C2a
1.358(3)
1.416(3)
1.344(3)
1.491(3)
1.213(3)
1.501(3)
1.518(3)
1.447(3)
1.516(3)
1.521(3)
1.375(3)
1.386(3)
1.498(3)
1.413(3)
1.303(3)
119.9(2)
125.6(2)
116.3(2)
120.8(2)
112.4(2)
103.8(2)
112.3(2)
114.0(2)
118.5(2)
120.6(2)
120.7(2)
117.8(2)
117.62(15)
121.1(2)
123.5(2)
14.0(4)
1.382
1.418
1.349
1.504
1.224
1.517
1.532
1.449
1.528
1.539
1.368
1.417
1.513
1.402
1.320
119.6
123.6
115.7
120.9
112.7
105.1
111.4
113.9
119.2
120.4
121.6
117.4
123.2
120.9
122.8
6.0
1.368(3)
1.417(3)
1.336(3)
1.498(3)
1.208(3)
1.498(3)
1.519(4)
1.434(3)
1.375
1.433
1.363
1.501
1.225
1.514
1.532
1.442
1.528
1.540
1.372
1.411
1.512
1.407
1.311
120.2
124.6
116.7
120.4
114.6
104.4
111.9
114.5
119.1
120.8
121.1
118.1
C1a–C11a
C2a–N1a
C2a–C3a
C3a–O1a
C3a–C4a
C4a–C41a
C41a–O5a
C41a–C11a
C41b–C11b
1.518(3)
1.526(4)
1.377(3)
1.395(3)
1.506(4)
1.407(3)
1.306(3)
119.3(2)
125.5(2)
115.5(2)
121.0(2)
111.6(2)
105.3(2)
111.5(2)
111.7(2)
119.7(2)
120.8(3)
121.3(2)
117.2(2)
C41a–C12a
C41b–C12b
O5a–C51a
O5b–C51b
C51a–C91a
C51b–C91b
C7a–C13a
C7b–C13b
C91a–N10a
C91b–N10b
N10a–C11a
N10b–C11b
C1a–C2a–C3a
C1a–C2a–N1a
C2a–C3a–C4a
C2a–C3a–O1a
C3a–C4a–C41a
C4a–C41a–O5a
C4a–C41a–C12a
C41a–O5a–C51a
O5a–C51a–C6a
C6a–C7a–C13a
C9a–C91a–N10a
C91a–N10a–C11a
C91a–N10a–Ag
N10a–C11a–C1a
C11a–C1a–C2a
N1a–C2a–C3a–O1a
C2a–C3a–C4a–C41a
C3a–C4a–C41a–C12a
C51a–C6a–C7a–C13a
C1b–C2b–C3b
C1b–C2b–N1b
C2b–C3b–C4b
C2b–C3b–O1b
C3b–C4b–C41b
C4b–C41b–O5b
C4b–C41b–C12b
C41b–O5C–C51b
O5b–C51b–C6b
C6b–C7b–C13b
C9b–C91b–N10b
C91b–N10b–C11b
C91b–N10b–Ag
N10b–C11b–C1b
C2b–C1b–C11b
N1b–C2b–C3b–O1b
C2b–C3b–C4b–C41b
C12b–C41b–C4b–C3b
C51b–C6b–C7b–C13b
114.94(15) 120.2
121.0(2)
122.8(2)
-15.3(4)
46.3(3)
120.7
123.3
-3.8
-40.1(3)
-73.5(3)
-179.6(2)
-39.8
-74.0
-178.2
33.3
72.7(3)
76.8
179.1(4)
178.7
1
8

-3
Table 3. The MIC values [mg dm ] corresponding to presented complex and reference compounds, tested
against Gram-negative and Gram-positive bacteria. Standard deviations are given when MIC was calculated as
a mean value of four tests.
Phx-1
MIC
Questiomycin A
[Ag(Phx-1)
2
NO
3
]
AgNO
MIC
3
SD
MIC
SD
20
MIC
23
SD
SD
1.3
E. coli (G-)
>375
>375
>375
>375
82
5.2
5.9
23
12
94
12
P. fluorescens (G-)
A. faecalis (G-)
S. aureus (G+)
>375
>375
59
23
164
47
41
20
81
12
B. licheniformis (G+) 234
R. erythropolis (G+) 375
81
234
35
47
5.9
-3
MIC of reference samples: nifuroxazide tested against S. aureus – 0.73 mg dm ; chloramphenicol tested
-3
against E. coli – 2.9 mg dm .
1
9

Table 4. Comparison of the selected experimental and the theoretical IR and Raman bands of Phx-1 ligand an its Ag(I)
complex.
Phx-1
2 3
[Ag(Phx-1) NO ]
IR
R
DFT
PED [%]*
IR
R
DFT
General assignment*
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
9
9
8
8
8
8
7
7
6
6
399
286
694
623
617sh
587
579sh
533
490
394
370
354
312
288
267
229
220sh
173
148
125
108
063
71
no
3619
3485
1704
1612
1612
1566
1558
1554
1473
1385
1366
1342
1307
1273
1257
1236
1217
1158
1134
1116
1098
1044
959
ν(N1-H3)-67 - ν(N1-H2)-25
ν(N1-H2)-52 + ν(N1-H3)-33
ν(C=O)-70
3452, 3375
3288, 3219
1710, 1694
1630sh
1617
1574
no
no
no
3594, 3557
3469, 3345
ν
ν
a
(NH
(NH
2
)
no
s
2
)
1693
1619
no
1711, 1696 1722, 1712
ν(C=O)
ν(C1=C2)-24 + ν(C2-N1)-7
ν(C1=C2)-16 + ν(C11=N10)-9
ν(C11=N10)-21 + ν(C9-C91)-13
no
1624
1613
1536
1562
1523
1475
1403
1367
1353
1314
1279
1263
1247
1222
1162
1150
1123
1099
1059
960
ν(C=C)R1 + ν(C-N)
ν(C=C)R1 + ν(C=N)
ν(C=N)
1619
1577
no
1591
no
δ(NH
2
)-57
2
δ(NH )
1534
1492
1400
1366
1353
1313
1289
1268
1228
1222sh
1174
1151
1126
1110
1065
972
ν(C11=N10)-31 + ν(C7-C8)-11
1529
1489
1383
1370
1362sh
1320
1284
1269
1231
1223
1173
1153
1129
1107
1053
972
1532
1491
1381
1365
1365
1313
1287
1266
1232
1223
1177
1154
1133
1109
1055
975
ν(C=N)
ν(C6-C7)-10 + ν(C9-C91)-9 + ν(C91-N10)-8
ν(C2-C3)-10 + ν(C1-C11)-7
ν(C-C)R3 + νC-
ν(C-C)R1
δ(CH
3
)-57
3
δ(CH )
ν(C1-C11)-14 + ν(C2-N1)-8 + ν(C11-C41)-7
ν(C6-C7)-18 + ν(C7-C8)-14 + ν(C51-C91)-14
ν(C4-C41)-9 + ν(C11-C41)-6
ν(C51-O13)-12 + ν(C7-C13)-9
ν(C51-O13)-8 + ν(C2-N1)-7
ν(C91-N10)-22
ν(C-C)R1,R2
ν(C-C)R3,R2
ν(C-C)R1,R2
ν(C-O)
ν(C-O)
νC-
ν(C-C)R1
ν(C-O)
δ(C-H)R3
ν(C-O)
ν(C3-C4)-15 + ν(C12-C41)-7
ν(C7-C13)-19 + ν(C51-O5)-14
δ(C8-H)-22 + δ(C9-H)-10
ν(C41-O5)-25
r
ρ (NH
2
)-14 + ν(C12-C41)-11
r 2
δ (NH )
ν(C41-O5)-11 + ν(C7-C8)-6
ν(C41-O5)-15 + ν(C7-C13)-6
ν(C12-C41)-18
ν(C-O)
ν(C-O)
ν(C-CH
14
915
905
920
922
906
86
no
882
882
884
882
3
)
56
856
856
π(C1-H)-92
860
865
857
π(C-H)R1,R3
43
no
833
ν(C91-N10)-6 + ν(C51-C91)-5
π(C8-H)-59 + π(C9-H)-20
δ(C41-O5-C51)-5 + δ(C11-N10-C91)-5
ν(C2-C3)-7 + ν(C7-C13)-7
ν(C11-C41)-7 + ν(C4-C41)-6
ν(C11-C41)-5 + δ(C2-C3-O1)-5
837
no
843
νC
π(C-H)R3
12
815
805
808
no
807
60
763
773
763
766
781
δ(C-O-C) + δ(C=N-C)
ν(C-C)R1
44
745
748
747
749
748
62
665
647
672
no
657
ν(C-C)R2
33
no
622
645
646
628
ν(C-C)R2
1
1
1
77, 118
43, 76
53sh
177, 123
no
169
νaAg
νsAg
νAg
162
151
150
NO
3
1
1
8
426, no
042
1425, no
1043
no
1436, 1330
1035
νa(NO3) - ν
νs(NO3) – ν
π(NO3) – ν
23
789
7
36, 715
731, 716
691, 674
δ(NO3) – ν
*
terms related to oxazine ring are boldfaced
2
0

Figure captions
Figure 1. Representative aminophenoxazinones with skeleton numbering system of Phx-1.
Figure 2. Molecular structure with thermal ellipsoids at 30% probability and labeling scheme for
[
Ag(Phx-1) NO ].
2 3
Figure 3. A packing diagram for [Ag(Phx-1) NO ], showing formation of a molecular chain
2
3
along the crystallographic b axis. Ligands from four neighboring molecules are shown. For the
sake of clarity, only N-H···O type hydrogen bonds are shown and hydrogen atoms not involved
in H-bonding are omitted. Symmetry codes: (i) 3/2-x, y-1/2, 3/2-z; (ii) x, y-1, z.
Figure 4. Infrared spectra of Phx-1 ligand and its Ag(I) complex in the "fingerprint" region.
Bands discussed and listed in table 4 are only labeled; * denotes the NO group vibration.
3
Figure 5. Comparison of MIC values for all tested Gram-positive and Gram-negative bacteria
exposed on [Ag(Phx-1) NO ] and AgNO solutions.
2
3
3
2
1

2
2

2
3

2
4

2
5

2
6

2
7