Could X-ray analysis explain for the differing antimicrobial and antioxidant activity of two 2-arylamino-3-nitro-coumarins?

Article abstract of DOI:10.1007/s10870-010-9918-0

X-Ray analyses of 4-(naphthalen-1-ylamino)- 3-nitro-chromen-2-one and 3-nitro-4-phenylamino-chromen- 2-one showed that thementioned compounds crystallize in the space groups P1- (triclinic crystal system; unit cell parameters: a = 8.087(2) ?, b = 9.241(3)

Full text of DOI:10.1007/s10870-010-9918-0

J Chem Crystallogr (2011) 41:545–551
DOI 10.1007/s10870-010-9918-0
ORIGINAL PAPER
Could X-Ray Analysis Explain for the Differing Antimicrobial
and Antioxidant Activity of Two 2-Arylamino-3-Nitro-Coumarins?
•
Niko S. Radulovic Goran A. Bogdanovic
•
´
Polina D. Blagojevic Vidosav S. Dekic
´
•
•
´
´
´
Rastko D. Vukicevic
´
Received: 2 March 2010 / Accepted: 28 November 2010 / Published online: 9 December 2010
Ó Springer Science+Business Media, LLC 2010
Abstract X-Ray analyses of 4-(naphthalen-1-ylamino)-
3-nitro-chromen-2-one and 3-nitro-4-phenylamino-chromen-
2-oneshowedthat the mentionedcompoundscrystallize in the
space groups P1- (triclinic crystal system; unit cell parame-
derivatives might explain the observed significant difference
of the antimicrobial and antioxidant activities and spectral
properties of two 4-arylamino-3-nitro-coumarin derivatives.
˚
ters: a = 8.087(2) A, b = 9.241(3) A, c = 10.911(3) A,
˚
˚
Keywords 4-Arylamino-3-nitro-coumarins Á Synthesis Á
X-ray analysis Á Conformation Á Antimicrobial activity Á
Antioxidant capacity
a = 93.77(3)°, b = 102.51(3)°, c = 106.44(2)°, V =
3
756.4(4) A and Z = 2) and P212121 (orthorhombic crystal
˚
˚
system; unit cell parameters: a = 4.9274(9) A, b =
˚
˚
14.725(3) A, c = 17.866(4) A, a = b = c = 90°, V =
3
1296.3(5)A andZ = 4), respectively. The analysesofcrystal
˚
Introduction
structures and gas phase conformations, inferred from single
X-ray crystallographic and molecular modeling experiments,
respectively, showed that the changes in p delocalization
of the farmacoactive formal 3-amino-2-nitro-acrylic acid
In the continuation of our investigation [1] of biologi-
cally active coumarin derivatives we synthesized two
2-arylamino-3-nitro-coumarins: 4-(naphthalen-1-ylamino)-
3-nitro-chromen-2-one (1) and 3-nitro-4-phenylamino-
chromen-2-one (2), Fig. 1. Previous research has shown
that 4-amino-3-nitrocoumarins, tested for pharmacological
activity in mice and rabbits, had neurotropic activity, gen-
erally inhibiting spontaneous locomotor activity, decreasing
hyperactivity induced by phenamine, and prolonging thio-
pental sleep [2]. The same study [2] also pointed out to the
most active of the derivatives containing a secondary amino
group, compound 2, that inhibited phenamine hyperactivity
2-5-fold and prolonged thiopental sleep 2-fold. Compounds
possessing a 4-amino-3-nitrocoumarin moiety were also
interesting due to the previously noted steric inhibition of
resonance of the C-3 NO2 group by the C-4 amino group in
4-morpholinyl-3-nitro-chromen-2-one, evident from their
IR and UV spectra [3]. Both compounds (1 and 2) have
previously been synthesized [3, 4], however their crystal
structures and antimicrobial and antioxidant activities have
not been published thus far. Bearing this in mind, the goal of
this work was set to establish the true conformations of
the two compounds in question and to possibly mutually
correlate the observed structural and biological activity data.
´
´
N. S. Radulovic (&) Á P. D. Blagojevic
Department of Chemistry, Faculty of Science and Mathematics,
ˇ
University of Nis, Visegradska 33, 18000 Nis, Serbia
e-mail: vangelis0703@yahoo.com
ˇ
ˇ
´
G. A. Bogdanovic
VINCA Institute of Nuclear Science, Laboratory of Theoretical
ˇ
Physics and Condensed Matter Physics, 522, 11001 Belgrade,
Serbia
´
V. S. Dekic
Department of Chemistry, Faculty of Science and Mathematics,
ˇ ˇ
University of Pristina, Pristina, Serbia
´
R. D. Vukicevic
Department of Chemistry, Faculty of Science and Mathematics,
´
´
University of Kragujevac, R. Domanovica 12, 34000
Kragujevac, Serbia
123

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J Chem Crystallogr (2011) 41:545–551
4-(Naphthalen-1-ylamino)-3-nitro-chromen-2-one
(1, C19H12N2O4)
R
NH
O
NO2
O
Yield: 74%. M. p., IR and UV previously published [3]. 1H
NMR (CDCl3): d = 6.76 (m, 1H, C11-H), 7.03 (dd,
J = 8.4; 1.3 Hz, 1H), 7.29–7.34 (m, 2H), 7.44–7.54
(m, 2H), 7.62–7.71 (m, 2H), 7.94–8.15 (m, 3H), 11.73 (brs,
1H, N–H) ppm. 13C NMR (CDCl3): d = 112.4, 117.3,
118.2, 121.8, 123.5, 123.7, 125.6, 127.7, 127.8, 128.2,
128.5, 128.9, 129.2, 134.5, 134.8, 135.0, 151.6, 153.1,
153.2 ppm. HRMS(EI): M? (C19H12N2O4) 332.0789,
requires 332.0797 (D = -0.8 mmu). C19H12N2O4 : calcd.
C, 68.67; H, 3.64; N, 8.43; O, 19.26; found C, 68.69; H,
3.65; N, 8.40.
(1) R = 1-naphthyl
(2) R = phenyl
Fig. 1 The structures of the synthesized compounds 1 and 2
Experimental
General Methods
Microanalysis of carbon, hydrogen and nitrogen were
carried out with a Carlo Erba 1106 microanalyser. MS (EI,
70 eV), including HRMS(EI), spectra were recorded on a
Finnigan-MAT 8230 BE mass spectrometer. The IR
measurements were carried out with a Perkin-Elmer 457
grating FT instrument in KBr tablets. UV spectra (in
chloroform) were measured on a Perkin-Elmer Lambda
15 UV–VIS spectrophotometer. The NMR spectra were
recorded on a Varian Gemini 200 (1H at 200 MHz, 13C
at 50 MHz) spectrometer, using CDCl3 as the solvent.
Chemical shifts are expressed in d (ppm) using TMS
(Me4Si) as an internal standard. For TLC, silica gel plates
(Kiesel 60 F254, Merck) were used.
3-Nitro-4-phenylamino-chromen-2-one (2, C15H10N2O4)
Yield: 85%, M. p., IR and UV previously published [4]. 1H
NMR (CDCl3): d = 6.95 (m, 2H, C11-H and C15-H),
7.14–7.61 (m, 7H), 11.38 (brs, 1H, N–H) ppm. 13C NMR
(CDCl3): d = 112.3, 117.5, 118.2, 123.5, 124.9 (two C),
128.3, 130.3 (two C), 135.1, 138.6, 151.6, 153.1, 154.1
ppm. HRMS(EI): M? (C15H10N2O4) 282.0635, requires
282.0641 (D = -0.6 mmu). C15H10N2O4 : calcd. C, 63.83;
H, 3.57; N, 9.92; O, 22.67; found C, 63.81; H, 3.58; N,
9.90.
Starting Material
Single Crystal X-Ray Experiments
According to a previously published procedure [3],
4-hydroxy-coumarin (Merck, Hohenbrunn, Germany) was
nitrated in acetic acid by 72% HNO3 to give 4-hydroxy-
3-nitro-coumarin, which was treated with POCl3 in N,
N-dimethylformamide to give 4-chloro-3-nitro-coumarin.
All other chemicals were commercially available (ani-
line and 1-naphthylamine were purchased from Sigma–
Aldrich) and used as received, except that the solvents
were purified by distillation.
Data were collected on an Enraf–Nonius CAD-4 diffrac-
˚
tometer [5] using Mo Ka radiation (k = 0.71073 A) and
x/2h scans in 1.93 to 26.97° h range for compound 1 and
1.79 to 25.97° h range for compound 2. Cell constants and
an orientation matrix for data collection, obtained from 25
centered reflections in the h range 13.13 to 15.91° for 1 and
11.45–14.44° for 2 corresponded to triclinic and ortho-
rhombic cells respectively. Unit cell dimensions and other
crystallographic data for both compounds are given in
Table 1. The data were corrected for Lorentz and polari-
zation effects [6].
General Procedure for the Preparation of 2-Arylamino-
3-Nitro-Coumarins
The structures were solved by direct methods [7] and
difference Fourier methods and refined on F2 by full-matrix
least-square method [8]. In both crystal structures all H
atoms were easily determined from DF maps but at the
final stage of refinement they were placed at calculated
positions (for the purpose of decreasing the number of
refined parameters) using a riding model. An exception was
done for H atoms bonded to N atoms which were refined
isotropically without any constrain. All non-hydrogen
atoms were refined with anisotropic thermal parameters.
Compound 2 crystallizes in the noncentrosymmetric space
group P212121. However, since the compound itself is not
The solution of 4-chloro-3-nitro-coumarin (11 g, 49 mmol)
and the appropriate arylamine (49 mmol) in ethanol
(30 mL) in the presence of catalytic amounts of triethyl-
amine (1 mL) was refluxed for 2–6 h. After cooling, the
precipitated solid was filtered off, washed with ethanol
and recrystallized from ethyl acetate. The purity of
the synthesized compounds was checked by TLC. Suit-
able crystals for X-ray analyses were obtained by slow
evaporation of the chloroform solutions of the two
compounds.
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J Chem Crystallogr (2011) 41:545–551
547
Table 1 Crystallographic data
for compounds 1 and 2
1
2
Empirical formula
C19H12N2O4
332.31
C15H10N2O4
282.25
Formula weight
Crystal size (mm3)
Color; habit
0.42 9 0.36 9 0.36
Orange; prismatic
293(2)
0.36 9 0.10 9 0.10
Yellow; stick
293(2)
Temperature (K)
˚
Wavelength (A)
0.71073
0.71073
Crystal system
Space group
Triclinic
Orthorhombic
P212121
P1-
Unit cell dimensions
˚
a (A)
8.087(2)
9.241(3)
10.911(3)
93.77(3)
102.51(3)
106.44(2)
756.4(4)
2
4.9274(9)
14.725(3)
17.866(4)
90
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
90
90
3
˚
V (A )
1296.3(5)
4
Z
Dcalc (Mg m-3
)
1.459
1.446
Absorption coefficient (mm-1
h range for data collection (°)
Index ranges
)
0.104
0.107
1.93 to 26.97
1.79 to 25.97
0 B h B 10, -11 B k B 11,
-13 B l B 13
0 B h B 6, 0 B k B 18,
-20 B l B 20
Reflections collected
Independent reflections
Data/parameters
3532
2894
3294 (Rint = 0.0211)
3294/230
2505 (Rint = 0.0479)
2505/194
Goodness-of-fit on F2
1.025
0.964
Final R indices [I [ 2r(I)]
Largest diff. peak and hole (e.A
R1 = 0.0475, wR2 = 0.1266
0.229 and -0.192
R1 = 0.0462, wR2 = 0.0847
0.181 and -0.182
-3
)
˚
chiral, the determination of the absolute configuration was
without any significance for our study.
(ATCC 6633), Sarcina lutea (ATCC 9341), Staphylococcus
aureus (ATCC 6538), Gram-negative bacteria: Escherichia
coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031),
Pseudomonas aeruginosa (ATCC 9027), Salmonella enter-
ica (ATCC 13076), and fungal organisms Aspergillus niger
(ATCC 16404), Candida albicans (ATCC 10231), and
Sacharomyces cerevisiae (ATCC 9763).
The software used for the preparation of the materials
for publication: PLATON [9, 10], WinGX [11], PARST
[12] and ORTEPIII [13].
Crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic Data
Centre, CCDC Nos. 641435 and 641436 for compounds 1
and 2, respectively. Copies of this information may be
obtained free of charge from The Director, CCDC, 12
Union Road, Cambridge, CB2 1EZ, UK (fax: ?44-1223-
336033; e-mail: deposit@ccdc.cam.ac.uk or www: http://
www.ccdc.cam.ac.uk).
Antimicrobial Assay
A disk diffusion method, according to the NCCLS [14],
was employed for the determination of the antimicrobial
activity of the compounds 1 and 2. The following nutritive
media were used: Antibiotic Medium 1 (Difco Laborato-
ries, Detroit Michigan USA) for growing Gram-positive
and Gram-negative bacteria and Tripton soy agar
(TSA—Torlak, Belgrade) for A. niger, C. albicans, and
S. cerevisiae. Nutritive media were prepared according to
the instructions of the manufacturer. All agar plates were
Test Microorganisms
The in vitro antimicrobial activities of the compounds 1
and 2 were tested against a panel of laboratory control
strains belonging to the American Type Culture Collection
Maryland, USA—Gram-positive bacteria: Bacillus subtilis
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J Chem Crystallogr (2011) 41:545–551
prepared in 90 mm Petri dishes with 22 mL of agar, giving
a final depth of 4 mm. 0.1 mL of a suspension of the tested
microorganisms (108 cells per mL) were spread on the
solid media plates. Sterile filter paper disks (‘‘Antibiotica
Test Blattchen’’, Schleicher and Schuell, Dassel, Germany,
6 mm in diameter) were impregnated with 50 lL of the
solutions of compounds 1 and 2 in methanol, 1 mg/mL,
giving 50 lg per disk (all solutions were filter-sterilized
using a 0.45 lm membrane filter), and placed on inocu-
lated plates. These plates, after standing at 4 °C for 2 h,
were incubated at 37 °C for 24 h for bacteria and at 30 °C
for 48 h for the fungi. Standard disks of Ampicillin and
Nystatin (origin—Institute of Immunology and Virology
‘‘Torlak’’, 30 lg of the active component, diameter 6 mm)
were used individually as positive controls, while the disks
imbued with 50 lL of pure methanol were used as a neg-
ative control. The diameters of the inhibition zones were
measured in millimeters using a ‘‘Fisher-Lilly Antibiotic
Zone Reader’’ (Fisher Scientific Co. USA). Each test was
performed in quintuplicate and repeated three times; the
results were then analyzed for statistical significance and
mean values are presented.
Results and Discussions
Compounds 1 and 2 (Fig. 1) have been prepared by
reactions of 4-chloro-3-nitrocoumarin with aniline and
1-naphthylamine as nucleophiles. The elucidation of their
structures was achieved by spectral methods (1H and 13C
NMR, IR, UV and HRMS, chemical analysis) and cor-
roborated by X-ray crystallographic analysis.
Both molecules (Figs. 2, 3) contain the same structural
part shown in Fig. 1. We have found no other published
crystal structures containing this molecular fragment.
However, there are three crystal structures [16–18] that
possess the same structural part without the N2–H group.
Generally both molecules are comprised from two aro-
matic parts mutually connected through the N2 atoms.
These parts are approximately planar and mutually form
different angles between their mean planes [70.5(1) and
55.5(1)° for 1 and 2 respectively]. In both compounds N2
and C10 form a single bond (Table 2). The N2–C3 bond is
significantly shorter than N2–C10 with bond length typical
for N–C aromatic bond that means that N2 participates in
electron p delocalization from the O1–C1 ten-membered
ring. The same conclusion one can find in the fact that
N2–H group is coplanar with O1–C1 ring in both crystal
structures.
Determination of the Total Antioxidant Capacity
The total antioxidant capacity of the synthesized compounds
1 and 2 was evaluated by the method of Prieto et al. [15].
The antioxidant capacity of their methanol solutions
(100 lg/mL) was measured spectrophotometrically using a
phosphomolybdenum method, based on the reduction of
Mo(VI) to Mo(V) by the sample analyte and the subsequent
formation of green phosphate/Mo(V) compounds with a
maximum absorption at k = 695 nm. A 0.1 mL aliquot of
sample solution (100 lg/mL) was combined in an Eppen-
dorf tube with 1 mL of the reagent solution (0.6 M sulfuric
acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). The tubes were capped and incubated in a
boiling water bath at 95 °C for 90 min. After the samples
had cooled to room temperature, the absorbance of the
aqueous solution of each was measured at 695 nm, against a
blank solution, using a Perkin-Elmer Lambda 15 UV–VIS
spectrophotometer. A typical blank solution contained 1 mL
of reagent solution and the appropriate volume of methanol
used for the dissolution of the samples and it was incubated
under the same conditions as the other samples. Stock
solutions of a-tocopherol acetate were prepared in methanol
just prior to use. Exact concentrations were determined
spectrophotometrically on the basis of the absorption coef-
ficients from the literature. The total antioxidant capacity
was expressed as equivalents of a-tocopherol acetate (lmol/
mL), as well as the ratio between the activity of a-tocopherol
acetate and that of the synthesized compounds at the same
mmol/mL concentration.
The common structural part (Fig. 1) in the two mole-
cules has quite different conformations- orientation of the
N2–H group is opposite in the two molecules. In 1 this
Fig. 2 The molecular geometry and atom labeling scheme of 1.
Displacement ellipsoids are drawn at the 40% probability level for
non-H atoms
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J Chem Crystallogr (2011) 41:545–551
549
Table 3 The geometry of hydrogen bonding and selected C–HÁÁÁO
˚
interactions (A, °) for compounds 1 and 2
Compound D–HÁÁÁA
D–H
DÁÁÁA
HÁÁÁA
D–H–A
1
N2–H1N2ÁÁÁO4i 0.92(2) 2.542(2) 1.75(2) 143(2)
C11–H11ÁÁÁO2ii 0.93
0.93
3.47
2.60
156
C7–H7ÁÁÁO4iii
3.46
2.58
157
2
N2–H1N2ÁÁÁO2iv 0.82(2) 2.907(4) 2.14(3) 156(3)
C5–H5ÁÁÁO3i
C5–H5ÁÁÁO2iv
0.93
3.20
3.37
3.42
2.55
2.47
2.56
128
163
153
0.93
C13–H13ÁÁÁO3v 0.93
Symmetry codes: (i) x,y,z; (ii) -x ? 1,-y ? 1,-z; (iii) x ? 1,
?y ? 1, ?z; (iv) -x ? 1, ?y ? 1/2,-z ? 1/2; (v) x - 1/2,-y ?
1/2,-z ? 1
Fig. 3 The molecular geometry and atom labeling scheme of 2.
Displacement ellipsoids are drawn at the 40% probability level for
non-H atoms
the average displacement of atoms in the O1-C1 ring from
˚
its mean plane is considerably higher in 1 (0.074 A) than in
˚
2 (0.017 A). Intramolecular N2–HÁÁÁO4 hydrogen bond in
the crystal structure of 1 influences also the bond lengths
and electron p delocalization in the C3–C2–C1–O1–C9
fragment (Table 2). Due to this hydrogen bonding the
˚
Table 2 Selected bond lengths (A) for compounds 1 and 2
1
2
˚
N1–O4 bond distance is about 0.04 A longer than N1–O3,
C2–C3
C2–N1
C2–C1
O4–N1
O1–C9
O1–C1
N2–C3
N2–C10
N2–H1N2
N1–O3
C4–C9
C4–C5
C4–C3
C1–O2
C9–C8
C8–C7
C5–C6
C7–C6
1.410(2)
1.428(2)
1.448(3)
1.248(2)
1.367(2)
1.379(2)
1.326(2)
1.434(2)
0.92(2)
1.386(5)
1.448(4)
1.422(5)
1.225(4)
1.386(4)
1.379(4)
1.335(4)
1.438(5)
0.82(3)
not being the case in 2.
Both molecules possess only one significant H donor
(N2–H group) but four oxygen atoms as possible H
acceptor. From that reason, the C–HÁÁÁO weak hydrogen
bonds (Table 3) play a significant role in the intermolecular
interactions. However, in the crystal packing of 1 the pÁÁÁp
interactions are predominant, where the p stacking exists in
two approximately orthogonal directions (Fig. 4). Perpen-
˚
dicular distance between aromatic rings is 3.28 A for
˚
O1–C1 rings and 3.45 A for C10–C19 rings. The shortest
˚
interplanar distance is 2.979(3) A between N1 and O4
1.2124(19)
1.383(3)
1.407(3)
1.463(2)
1.192(2)
1.391(3)
1.365(3)
1.368(3)
1.382(4)
1.228(3)
1.392(4)
1.386(5)
1.464(5)
1.208(4)
1.370(5)
1.376(5)
1.373(5)
1.377(5)
atoms what can be explained by their positive and negative
partial atomic charge. In the crystal packing of 2 (Fig. 5)
the intermolecular hydrogen bonding is more important
group forms a rather strong intramolecular N–HÁÁÁO
hydrogen bond (Table 3) while in 2 this group is directed
in the opposite way forming an intermolecular hydrogen
bond to O2 atom. Orientation of the NO2 group is also very
different in the two molecules. In 1 the NO2 group is
coplanar with O1–C1 ring (2.8° is the angle between their
mean planes) while in 2 it forms an angle to the O1–C1
ring of 47.1(3)°. As a consequence of these structural dif-
ferences (and possible O2ÁÁÁO3 repulsion), the O1–C1 ring
in 1 is less planar and O2 is displaced from its mean plane
˚
for 0.345(2) A. For example the C2–C1–O1–C9 torsion
Fig. 4 Perspective view of the crystal lattice fragment of
representing p stacking in two directions
1
angles are 12.3(2) and -1.5(5)° in 1 and 2, respectively;
123

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J Chem Crystallogr (2011) 41:545–551
and E. coli were the most susceptible bacterial strains of all
tested to compound 1, and A. niger and C. albicans among
the fungal organisms. Significant reductions of bacterial
growth were noted in the cases of medically important
pathogens S. lutea and B. subtilis. Compound 1 was com-
pletely inactive towards S. cerevisiae (however, this strain
was also the most resistant to Nystatin), although, at the
same time, being relatively unselective in its action
towards the tested bacteria. It seems that the Gram-positive
strains were equally resistant to compound 1 as the
Gram-negative bacteria, although it is recognized that the
presence of certain cell-wall lipopolysaccharides is to be
considered responsible for the greater resistance of Gram-
negative bacteria [19].
The antioxidant capacity assay results revealed a very
similar pattern of activity. The total antioxidant capacity of
compound 1 (2102 lmol/L), at the concentration of
100 lg/mL, expressed as equivalents of a-tocopherol ace-
tate (lmol/L), was ca. 263 times greater than that of
compound 2 (8 lmol/L). The ratio between the activity of
the synthesized compounds 1 and 2 and that of a-tocoph-
erol acetate, at the same mol/mL concentration, were 6.985
and 0.023, respectively, thus rendering compound 1 seven
times more active, and compound 2 forty-four times less
active that a-tocopherol acetate.
Fig. 5 Packing diagram of 2 viewed down the a axis. The dashed
lines represent intermolecular hydrogen bonds
than the pÁÁÁp interactions which exist only between O1–C1
rings but with a perpendicular distance between the aro-
˚
˚
matic rings of 3.76 A (0.5 A longer than in 1). The reason
for this structural behavior (the intermolecular interactions)
could be found in the fact that the N2–H group (only sig-
nificant H donor in both crystal structures) in 2 is not
involved in the intramolecular hydrogen bonding as in the
case of structure 1 (Fig. 2).
The conformations equivalent to the ones found for
compounds 1 and 2 in their crystal lattices (both com-
pounds in both types of conformations) were optimized to
correspond to the energetic minima by the use of MM?,
and AM1 force fields (at semiempirical level), with the
Polak-Ribiere (conjugate gradient) minimization method
with an energy convergence criterion of 0.04 kJ/mol,
incorporated in the HyperChem 7 software package. The
obtained results showed clearly that the gas-phase confor-
mation with intramolecular hydrogen bonding (analogous
to the one shown in Fig. 2) corresponds to the global
energetic minimum for both compounds 1 and 2, and that
these lie 20.7 and 18.8 kJ/mol, respectively, below the
conformations analogous to the one represented in Fig. 3.
Thus, if only considering the data gathered in molecular
modeling experiments one could expect these compounds
not to vary significantly in their spectral properties and
biological activity associated with the coumarin moiety of
the molecules. This does not seem the case here, since the
conformation of compound 1 determined by X-ray analy-
sis, and highly possibly that present in solutions, diverged
quite strongly from the one optimized for the gas-phase. In
order to address this matter we performed additional cal-
culations using two molecules in the gas phase (since
structure 2 has a number of intermolecular hydrogen
bonds) to see how that might affect the total energy. The
solvent effects were modeled by adding a periodic box of
pre-equilibrated water molecules for an aqueous salvation
The results of the antimicrobial assays of compounds 1
and 2 are presented in Table 4. In a disk diffusion assay at
the dose of 50 lg/disk, the compounds showed markedly
different antimicrobial activities (Table 4): in the case of
compound 1 strong to moderate activity in reducing the
microbial growth compared to the standard antibiotics
(Ampicillin and Nystatin), and no activity observed what
so ever in the case of compound 2. Klebsiella pneumoniae
Table 4 The antimicrobial activity (growth inhibition zone, mm,
including disk diameter, 6 mm) of the synthesized compounds 1 and 2
(50 lg per disk)
Microorganism Compound Compound Ampicillin* Nystatine*
1
2
S. aureus
S. enterica
S. lutea
9
na
na
na
na
na
na
na
na
na
na
19
18
20
19
17
na
20
nt
nt
nt
nt
nt
nt
nt
nt
17
19
9
9
15
16
E. coli
P. aeruginosa 10
K. pneumoniae 16
B. subtilis
A. niger
15
16
16
na
C. albicans
S. cerevisiae
nt
nt
na not active, nt not tested
* 30 lg per disk of the active principle
123

J Chem Crystallogr (2011) 41:545–551
551
study. The results of these two further molecular modeling
experiments revealed that the two possible conformations
(with and without intramolecular hydrogen bonding) lay
much closer in energy and that the balance between the two
in crystalline state and solution might be reversed if addi-
tional factors are considered. However, these results still
suggest that the true reasons behind the changes in the IR
and UV spectra of bulky 4-aminosubstituted 3-nitrocou-
marins noted previously [3] are not solely steric in nature
(naphthyl group being more voluminous than the phenyl
group, hence expecting a greater deviation in the spectra of
compound 1).
coumarin core, and these, judging from the crystal struc-
tures of compounds 1 and 2, do not contribute significantly
to the overall hydride in the case of the tilted NO2 in
compound 2 (Fig. 3). Thus, leading to no activity in the
antimicrobial assay, and significantly reduced antioxidant
capacity. It also follows that since in compound 1 the
C1–O2 carbonyl is slightly out of the plane of the coumarin
core that the major stabilization in this molecule comes
from resonance forms of C-type, and that the carbonyl
delocalization deems less important than that of the nitro
group. This nitro group stabilization could be likewise
strengthened by the intramolecular hydrogen bond forming
(C-type resonance structures tend to augment the negative
charge on O4).
An explanation of the differing antioxidant and antimi-
crobial activities of the two compounds can be, however,
rationalized by taking into account the crystal structure
conformations of both molecules. One can first consider the
molecular moiety that could be credited for the observed
activities. The possible antimicrobial pharmacophor in
these molecules (that can be regarded as formal 3-amino-
2-nitro-acrylic acid derivatives), depicted in Fig. 6,
resembles the one found in many natural antimicrobial and
cytotoxic sesquiterpene a,b-unsaturated c-lactones [20],
acting as a Michael acceptor of biological nucleophiles,
e.g. thiol groups of proteins. The same pharmacophor could
be responsible for the observed antioxidant activity, at least
in part in the first free radical-forming step. Modes of
action, stabilization of the intermediary formed chemical
species (an anion or a radical-cation), have been graphi-
cally represented as resonance structures of types A–C
(Fig. 6). The influence of the loss of this delocalization
stabilization can be exemplified by a rather dramatic
decline in activity of a fungal metabolite, multiformin D
[21], due to the saturation of an a,b-double bond of a
carbonyl [21]. In the cases of compounds 1 and 2 formally
two acceptors of the negative charge or the unpaired
electron are available—the carbonyl C1–O2 and the nitro
group cross-conjugated to the amino group N2H-R. The
resonance forms of type C will not exert their full impact
on stabilization if the nitro group is not coplanar with the
Acknowledgments The authors acknowledge the Ministry of Sci-
ence and Technological Development of Serbia for financial support
(project number 172061).
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N
N
N
N
H
O
H
O
H
O
H
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N
O
N
O
N
O
N
O
O
O
O
O
N
H
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A
B
C
N
O
Nu
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Fig. 6 The possible antimicrobial and antioxidant pharmacophor of
the 4-arylamino-3-nitro-coumarin derivatives 1 and 2 (the structure
on the left) and the resonance structures of the intermediary species
explaining the likely mode of action of the pharmacophor
123