Synthesis, spectral characterization, crystal structure and molecular docking study of 2,7-diaryl-1,4-diazepan-5-ones

Article abstract of DOI:10.1016/j.molstruc.2016.03.021

In this study, a series of variously substituted r-2,c-7-diaryl-1,4-diazepan-5-ones 9-16 have been synthesized using Schmidt rearrangement and are characterized by IR, mass and 1D & 2D NMR spectral data. The proton NMR coupling constant and estimated dihedral angles reveal that the compounds 9-16 prefer a chair conformation with equatorial orientation of alkyl and aryl groups. Single crystal X-ray structure has been solved for compounds 9 and 11 which also indicates the preference for distorted chair conformation with equatorial orientation of substituents. The compounds 9-16 have been docked with the structure of Methicillin-resistant Staphylococcus aureus (MRSA) and the results demonstrate that compound 10 is having better docking score and glide energy than others and it is comparable to co-crystal ligand. Furthermore, all the compounds have been evaluated for their antibacterial and antioxidant activities. All the compounds show moderate antibacterial activity and only 11 exhibits better activity against S. aures and Escherichia coli. The compounds 11, 13 and 14 exhibit half of the antioxidant power when compared to the BHT and the remaining compounds show moderate activity.

Full text of DOI:10.1016/j.molstruc.2016.03.021

Accepted Manuscript
Synthesis, spectral characterization, crystal structure and molecular docking study of
2,7-diaryl-1,4-diazepan-5-ones
S. Sethuvasan, P. Sugumar, V. Maheshwaran, M.N. Ponnuswamy, S. Ponnuswamy
PII:
S0022-2860(16)30218-6
10.1016/j.molstruc.2016.03.021
MOLSTR 22332
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 26 January 2016
Revised Date: 5 March 2016
Accepted Date: 7 March 2016
Please cite this article as: S. Sethuvasan, P. Sugumar, V. Maheshwaran, M.N. Ponnuswamy, S.
Ponnuswamy, Synthesis, spectral characterization, crystal structure and molecular docking study of 2,7-
diaryl-1,4-diazepan-5-ones, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.03.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, spectral characterization, crystal structure and
molecular docking study of 2,7-diaryl-1,4-diazepan-5-ones
S. Sethuvasana, P. Sugumarb, V. Maheshwaranb, M. N. Ponnuswamyb and S. Ponnuswamya*
aP.G. & Research Department of Chemistry, Government Arts College (Autonomous),
Coimbatore - 641 018, India.
bCentre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy
Campus, Chennai - 600 025, India.
Abstract
In this study, a series of variously substituted r-2,c-7-diaryl-1,4-diazepan-5-ones 9-16
have been synthesized using Schmidt rearrangement and are characterized by IR, mass and
1D & 2D NMR spectral data. The proton NMR coupling constant and estimated dihedral
angles reveal that the compounds 9-16 prefer a chair conformation with equatorial orientation
of alkyl and aryl groups. Single crystal X-ray structure has been solved for compounds 9 and
11 which also indicates the preference for distorted chair conformation with equatorial
orientation of substituents. The compounds 9-16 have been docked with the structure of
Methicillin-resistant Staphylococcus aureus (MRSA) and the results demonstrate that
compound 10 is having better docking score and glide energy than others and it is
comparable to co-crystal ligand. Furthermore, all the compounds have been evaluated for
their antibacterial and antioxidant activities. All the compounds show moderate antibacterial
activity and only 11 exhibits better activity against S. aures and E. coli. The compounds 11,
13 and 14 exhibit half of the antioxidant power when compared to the BHT and the
remaining compounds show moderate activity.
Keywords: 2,7-diaryl-1,4-diazepan-5-ones, Chair conformation, Single crystal XRD,
Molecular docking, Antibacterial, Antioxidant.
*Corresponding author: Post Graduate and Research Department of Chemistry,
Government Arts College (Autonomous), Coimbatore-641 018, Tamil Nadu, India. Mobile
number: +919244645744, E-mail: kspons2001@gmail
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1. Introduction
ε-Caprolactam plays an important role for the preparation of modified nylons [1] and
nanogels [2]. The 1,4-diazepanone ring nucleus acts as anticonvulsant agent and is present in
the liposidomycin nucleoside antibiotics that inhibits bacterial peptidoglycan synthesis [3].
Furthermore, recent studies on 1,4-diazepan-5-ones reveal their antimicrobial [4],
antitrypanosomal and antiplasmodial activities [5]. However, a little information is only
available on the synthesis, stereochemistry and biological evaluation of 2,7-diaryl-1,4-
diazepan-5-ones. Hence, it is of interest to report the synthesis and characterisation of 2,7-
diaryl-1,4-diazepan-5-ones by the ring expansion of 2,6-diarylpiperidin-4-ones using Schmidt
rearrangement. Only a few 2,7-diaryl-1,4-diazepan-5-ones have been already reported using
Beckmann rearrangement of piperidin-4-ones oximes [4, 6, 7] as well as Schmidt
rearrangement of piperidin-4-ones [8-16].
The present work involves the synthesis of a new series of 2,7-diaryldiazepan-5-ones
9-16 from their corresponding piperidin-4-ones 1-8 using Schmidt rearrangement with a
modified procedure (Scheme 1). Earlier the Schmidt rearrangement of piperidin-4-ones have
been carried out in two steps i.e. converting piperidin-4-ones into hydrochlorides and
subjecting them to Schmidt rearrangement after isolation [8, 11-14]. The conversion in one
step with simple reaction conditions is reported now.
The structural characterization and analysis of solution state conformation are made
using IR, mass, 1D and 2D NMR spectral studies. Furthermore, the X-ray crystal structures
of 9 and 11 have been solved in order to confirm their preferred conformation in solid state.
Baliah et. al. [6] reported only the synthesis of diazepan-5-one 12 from its oxime derivative of
piperidin-4-one by Beckmann rearrangement. However, the spectral data and stereochemistry
of 12 have not been reported. Depending on the migratory aptitude of the C-3 and C-5
carbons in piperidin-4-ones 1-8, two kinds of isomers are possible for the products in the
Schmidt rearrangements. The spectral data of earlier studies [8, 11] confirmed the migration
of C-3 to form diazepan-5-ones 9-16 as expected product. All the synthesised compounds
have been tested for their antibacterial and antioxidant activities. In addition, molecular
docking studies have been carried out for compounds 9-16. The docking studies show that
the compounds inhibit at the active site of the target protein and can be utilized as potential
drug molecules.
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2. Experimental
2.1. Materials and methods
All the reported melting points which are uncorrected have been taken in open
capillaries using melting point apparatus with a calibrated thermometer. FT-IR spectra have
been recorded on a Bruker alpha spectrophotometer using KBr pellets. 1H and 13C NMR
spectra have been recorded in CDCl3 and DMSO-d6 on a Bruker NMR spectrometer (400 &
1
13
500 MHz for H and 100 & 125 MHz for C), using TMS as internal standard. Chemical
shifts (δ) are expressed in ppm where abbreviations s, bs, d, dd, and m stand for the resonance
multiplicities singlet, broad singlet, doublet, doublet of doublet and multiplet, respectively,
and coupling constants are given in Hz. Electron impact mass spectra have been recorded
using JEOL GCMS spectrometer. Elemental analyses have been carried out in Carlo Erba
1108 CHN analyzer and are within 0.4% of the calculated values.
All the parent r-2,c-6-diarylpiperidin-4-ones 1-8 have been prepared according to
the reported procedures [17].
2.2. General procedure for the synthesis of r-2,c-7-diaryl[1,4]diazepan-5-ones 9-16.
r-2,c-6-Diarylpiperidin-4-ones 1-8 (0.03 mol) in conc. HCl (2 ml) and
dichloromethane (60 ml) are stirred well in a beaker for a few minutes. To this stirred
solution, conc. H2SO4 (20 ml) is added in dropwise at 0-5 ºC for 1 h. Temperature of the
solution is allowed to rise at 25 ºC. While stirring, NaN3 (0.09 mol) is added over a period of
1h and stirring is continued for another 2-3 h. The solution is poured into crushed ice and
stirred well. Ammonium hydroxide is added slowly with stirring to reach pH=11. The
resulting mass is washed with water and about 20 ml of dichloromethane is added. The
organic layer is separated, dried over anhydrous Na2SO4 and concentrated for crystallization.
The crystals thus obtained are recrystallized from dichloromethane-pet ether (60-80 ºC) in the
ratio of 5:1.
2.3. Single crystal X-Ray studies
X-ray diffraction intensity data have been collected for the 2,7-diaryl-1,4-diazepan-
5-ones 9 and 11 on Bruker axs Kappa ApexII [18] single crystal X-ray diffractometer
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equipped with graphite mono-chromated MoKα (λ=0.7103 Å) radiation and CCD detector.
Crystals have been cut to suitable size and mounted on a glass fibre using cyanoacrylate
adhesive. The unit cell parameters have been determined from 36 frames measured (0.5° phi-
scan) from three different crystallographic zones and using the method of difference vectors.
An average four-fold redundancy per reflection has been used to collect the data at an
optimum resolution of 0.75 Å. The intensity data collection, frames integrations (Lorentz and
polarization) and decay correction have been done using SAINT-NT (version 7.06) [18]
software. Empirical absorption correction (multi-scan) has been performed using SADABS
[18] program. Crystal structures have been solved by direct methods using SHELXS-97 [19].
The phase sets with the best combined figure of merits reveal the positions of all non-
hydrogen atoms in both the structures. The structures have been then refined by full-matrix
least-squares procedures using SHELXL-97 [19].
2.4. Molecular docking studies
Molecular docking studies have been performed to examine the binding mode and
pattern of ligands with MRSA, for which the structural coordinates have been retrieved from
Protein Data Bank (PDB ID: 4D7I) [20]. The target protein is optimized after removal of the
water molecules from the crystal structure and partial atomic charges assigned according to
the force field. Minimization of target is performed until the average root mean square (rms)
deviation of the non-hydrogen atoms reached 0.3 Å (OPLS-2005 force field) to remove the
steric hindrance. Ligands are also minimized by OPLS-2005 force field. The results with the
docking score are compared with the co-crystal ligand. The docking studies are carried out
using Glide module of MAESTRO, Schrodinger suite [21]. The figures are generated using
Pymol [22].
2.5. Antibacterial evaluation
2.5.1 Test Organisms
Four human pathogenic bacterial strains viz., Staphylococcus aureus (ATCC 25923),
Streptococcus pyogenes (ATCC 19615), Micrococcus luteus (ATCC 9341) and Escherichia
coli (ATCC 25922) have been maintained on nutrient agar slants at refrigerated condition
until used in the present study.
2.5.2. Agar well diffusion method
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Agar well diffusion method [23] has been followed to determine the antibacterial
activity. Nutrient agar (NA) plates have been swabbed (sterile cotton swabs) with 8 hour old -
broth culture of respective bacteria. The well, measuring 6 mm in diameter has been made in
each of these plates using sterile cork borer. Each petri plate has been prepared by pouring
20 ml of appropriate agar media. The solutions of synthesized compounds 9-16 have been
prepared at a concentration of 1 mg/ml using DMSO. About 100 µl solutions of each
compound have been added into the wells of seeded plates. DMSO has been used as a
control for all the experiments. The plates have been incubated at 37°C for 18-24 h and
antibacterial activity has been determined by measuring the diameter of the inhibition zone
and the activity index has also been calculated. Triplicates have been maintained and the
experiment has been repeated thrice. For each replicates, the readings have been taken in
three different fixed directions and the average values have been reported.
2.5.3. MIC using MTT assay method
The MTT assay has been carried out as described by Mosman [24]. The 96 wells
plates have been prepared under aseptic conditions. Each culture plates have the volume of
100ꢀl which contain Mueller Hinton Broth (MHB) and compounds 9-16 are dissolved with
5% (v/v) DMSO. Using micropipette, serially descending concentrations (400, 200, 100, 50,
25, 12.5 and 6.25 ꢀg/ml) of compounds 9-16 have been prepared. A 10ꢀl of bacterial
suspension has been added to each well to achieve a density of 1.0×107 CFU/ml (McFarland
No. 0.5). The commercial antibiotic ampicillin has been used as positive control. The plates
have been placed in an incubator at 37 °C for 24 h. Then, to all these wells, 10 ꢀl of MTT
solution (5mg/ml of MTT in PBS) has been added. After 4 h, the medium has been discarded
and added 150 ꢀl of DMSO. The plates have been kept for 10 min in order to make the
complete dissolution. Optical absorbance has been measured at 490 nm.
2.6. Antioxidant evaluation
2.6.1. DPPH radical scavenging assay
The evaluation of antioxidant activity of newly synthesized compounds has been
done by DPPH radical scavenging activity (RSA) [25]. Solutions of all the synthesized
compounds 9-16 have been prepared in different concentrations (50, 100, 200, 300, 400 and
500 µg/ml) using methanol. The 1 ml of test compounds solutions (9-16) has been added
with 0.1 mM methanol solution of DPPH and shaken vigorously. The solution tubes have
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been incubated at room temperature in dark room for 30 min. The decrease in absorbance
has been measured at 517 nm using UV-Visible spectrophotometer and remaining DPPH has
been calculated. Butylated Hydroxytoluene (BHT) has been used as standard and the
experiment has been carried out in triplicate. From the results of this bioassay, RSA % has
been calculated using the following formula:
% Scavenging = [(Ac-As)/Ac × 100]
where Ac is the absorbance of the control and As is the absorbance of the
compound.
3. Results and discussion
3.1. Analysis of IR and Mass spectra
The IR spectra of the diazepan-5-ones 9-16 display the bands at 3281-3322 cm-1 for
amine NH and 3195-3216 cm-1 for amide NH. The strong band at 1651-1673 cm-1 is
indicative of the amide CO stretching which in turn confirms the formation of the products.
Mass spectral studies provide information regarding structural and molecular weight of a
compound. The important IR stretching bands and molecular ion (M+) peaks are depicted in
Table 1.
3.2. Analysis of NMR spectra
The assignments of 1H and 13C NMR signals for compounds 9-16 are made with the
help of 1D and 2D NMR spectra. Based on the 1H NMR (D2O exchange) spectra, the amine
and amide NH protons are assigned. The DEPT-135 spectra are used to assist the assignment
of 13C NMR spectra. The characteristic 1H and 13C NMR signals of 9-16 are identified using
1D NMR spectra (1H, 13C and DEPT-135) and unambiguously assigned using 2D (COSY and
1
HSQC) NMR spectra (Tables 2 & 3). While recording the H NMR spectra of diazepan-5-
ones 14-16 in DMSO-d6, the amide NH protons appear as a doublet with the coupling
constant value of 2-4.5 Hz which is due to the coupling between amide NH and proton at C-
3. This observation suggests the migration of C-3 carbon during Schmidt rearrangement of
piperidin-4-ones 1-8 to diazepan-5-ones 9-16. The deshielding of C-3 carbon than C-6 in the
13C NMR spectra of 9-16 also supports the above prediction.
3.2.1. NMR spectral analysis of compound 9
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For the compound 9, the two singlets at 1.92 and 6.13 ppm are assigned to amine
and amide protons, respectively. Higher deshielding of amide proton is due to the presence
of electron withdrawing C=O group and its resonance with nitrogen. Since no coupling
partner is available at C-3 for C-2 proton, the H-2 benzylic proton appears as a singlet at 4.73
ppm. The chemical shift value of H-2 benzylic proton is comparable to that of 3-alkyl
analogues which is indicative of the axial orientation for the proton and equatorial orientation
for the aryl group at C-2.
3
The signal at 4.62 ppm appearing as doublet with J value of 11.0 Hz (3JH6a, H7a) is
assigned to the axial proton at C-7 (H-7a) which in turn confirms the equatorial position of
the aryl group at C-7. Further, the signal at 4.62 ppm is correlated with a doublet of doublet
at 3.10 ppm and a doublet at 2.69 ppm in its COSY spectrum (Fig. 1). This confirms that the
peaks at 3.10 and 2.69 ppm are due to the axial and equatorial protons at C-6, respectively.
The axial and equatorial protons can be assigned using their coupling constants. The signal at
3.10 shows coupling constants values of 11.5 and 15.0 Hz and the other one at 2.69 has the 2J
value of 15.5 Hz. The H-7a proton itself shows a doublet (3J=11.0 Hz) instead of a doublet
of doublet and the higher coupling constant value indicates that it is due to the diaxial
2
coupling. Hence, the doublet of doublet at 3.10 ppm (3JH6a, H7a=11.5 Hz and JH6a, H6e=15.0
Hz) and a doublet at 2.69 ppm (2JH6a, H6e =15.5 Hz) are assigned to the axial and equatorial
protons, H-6a & H-6e, respectively. The H-6e appears as a doublet instead of a doublet of
doublet which indicates that it has zero coupling with H-7a, since the magnitude of the
vicinal coupling approaches zero when the dihedral angle is 90°. The aromatic protons
appear as multiplet between 7.18-7.69 ppm. The two methyl groups at C-3 appearing at 1.65
and 1.09 ppm are assigned to 3-CH3 and 3-‘CH3, respectively. The assignments have been
made for the other compounds 10-16 in a similar manner and the complete assignments of 1H
and 13C NMR chemical shifts data are presented in Tables 2 and 3.
Correlation of protons with their coupling partners and one bond 1H-13C correlations
1
are made unambiguously using 1H-1H COSY and H-13C HSQC, respectively (Fig. 1 and 2).
1H-13C HMBC NMR of 9 gives correlations between carbons and protons over longer ranges
of about 2-4 bonds and the direct one-bond correlations are suppressed. By this the
correlations of carbonyl and ipso carbons with ring protons can be understood. Two, three
and four bond correlations are clearly depicted in Fig. 3. The complete correlation of 1H-1H
COSY, 1H-13C HSQC and 1H-13C HMBC for 9 are presented in Table 4.
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3.3. Conformational analysis
The preferred conformation of the diazepan-5-ones 9-16 has been arrived by
considering the vicinal coupling constants between the H-7a and H-6a & H-6e protons as
well as between H-2a and H-3a protons. The vicinal coupling constant data (3JH6a, H7a and
3JH6e, H7a) are employed to estimate the dihedral angles between the vicinal protons (Фcis=Ф
and Фtrans=ФH6a,H7a) by DAERM [26] and are used for the conformational analysis.
H6e,H7a
The extracted coupling constants and the estimated dihedral angles of 9-16 using DAERM
are presented in Table 5. The conformations of several substituted piperidin-4-ones have
been extensively studied in solution as well as in solid states which denote that the piperidine
ring exists in a chair conformation with equatorial orientation of aryl groups [17, 27-29]. The
extracted vicinal coupling constants and estimated dihedral angles for the reported piperidin-
4-ones 6 and 7 are JH2a, H3a= 10.3-10.5 Hz, JH5a, H6a= 11.5 – 12.0 Hz, JH5e, H6a= 2.3-3.0 Hz and
Фcis= 57-60 o, Фtrans=177-180 o [27].
The seven membered ring, in general, is more flexible than the six membered ring.
Hence, the diazepan-5-one ring may not adopt rigid chair conformation like piperidin-4-ones.
Here, the coupling constants and dihedral angles for diazepan-5-ones 9-16 are, JH2a, H3a= 7.6-
o
8.0 Hz, JH6a, H7a= 10.8 - 11.6 Hz, JH6e, H7a= 0 Hz and Фcis= 81 o & Фtrans= 159 . The cis
coupling constant of 0 Hz observed between H-7a and H-6e in the diazepan-5-ones 9-16
suggests a dihedral angle close to 90o. The dihedral angle of 81o estimated using DAERM
supports the above observation. The higher vicinal coupling constant values suggest that the
aryl groups at C-2 and C-7 and the C-3 /C-6 alkyl groups of diazepan-5-ones 9-16 prefer to
adopt the equatorial orientation. The observed values of coupling constants suggest that the
diazepan-5-ones 9-16 adopts a chair conformation. Among the two methyl groups at C-3 for
compounds 9-13, one is axially oriented and the other one is equatorially oriented. The
analysis of proton NMR data also reveals that H-6a is more deshielded than H-6e. Similarly
the more deshielded methyl group (3-CH3) is assigned as axial and the other methyl group (3-
‘CH3) as equatorial.
3.4. Single crystal X-Ray studies
The ORTEP plots [30] of 9 and 11 are shown in Fig. 4. In 11, there are two
crystallographically independent molecules in the asymmetric unit with similar structural
environment. The diazepine rings of 9 and 11 adopt distorted chair conformation (Fig. 5).
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The puckering parameters [31] and asymmetry parameters [32] are: q2 = 0.277(2)Å, q3 =
0.676(2)Å, φ2 = 199.5(5)° & ꢁCs(C-5) = 0.125.5(2)° for 9 and q2 = 0.309(2)Å[0.288(2)Å],
q3 = 0.652(2)Å[0.665(2)Å], φ2 = 191.4(4)°[ 5.2(4)°] & ꢁCs(C-5) = 113.4(2)°[ 0.120.8(2)°]
for the molecule A[B] of 11.
The planar phenyl rings substituted at C-2 and C-7 positions of the diazepine ring
are in equatorial orientation in both 9 and 11 as evidenced from the torsion angles [(N4-C3-
C2-C14/C5-C6-C7-C8) 168.67(2)°/159.2(2)° for 9; -169.09(2)° & 165.82(2)° /161.46(2)° &
-161.23(2)° for the two molecules in 11]. The best plane of diazepine ring orients at angles
of 60.59(11)° and 70.59(11)° with respect to the phenyl rings of 9 and 62.74(11)° &
60.14(9)° (molecule A) and 73.76(9)° & 65.29(1)° (molecule B) of 11, respectively.
In 9 & 11, one of the methyl groups (C-20) substituted at C-3 occupies equatorial
orientation whereas the other (C-21) occupies axial orientation (N1-C2-C3-C20/ N1-C2-C3-
C21 -175.68(18)°/-53.8(2)° for 9; -175.13(16)° & 174.52(18)°/-53.7(2)° & 52.1(2)° for 11.
The chlorine atom and methyl group in both the molecules of 9 and 11 lie in the plane of
phenyl rings which is evidenced from the deviation from the attached rings (0.014(1)Å & -
0.060(1)Å of Cl-1 & Cl-2 for 9; -0.009(4)Å[0.033(3)Å] & 0.096(4)Å[0.038(3)Å]C-22 C-23
For 11(A[B]). In 9 and 11, the molecules are stabilized by N-H...O, C-H...Cl and C-H...N
types of hydrogen bondings. In both the compounds the molecules are linked through N-
H...O hydrogen bond into cyclic centrosymmetric R2 dimer [33] as shown in Fig. 6.
2
3.5. Docking Analysis
Methicillin-resistant Staphylococcus aureus (MRSA) is gram-positive human
bacterial pathogen which is highly connected with infections in hospitals. It is acquiring
resistance to the β-lactam antibiotics and vancomycin [34], which necessitates the search to
find alternative drugs. The structural information retrieved from the “Protein Data Bank”
(PDB ID: 4D7I) has been used as the template for this study. The diazepanones 9-16 are
shown to be effective inhibitor against MRSA. X-ray crystal structures have been utilized to
predict the expected binding mode and electronic properties enabled optimization to
diazepanones as a potent second-generation lead. The results obtained from this study would
be useful in both understanding the inhibitory mode of diazepan-5-ones 9-16 and accurately
predicting the activities of newly designed inhibitors on the basis of docking scores. These
models also provide some beneficial clues in structural modification for designing new drug.
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A view of the X-ray crystal structure of the MRSA active site shows the key hydrogen
contacts
between
co-crystal
[6-(4-(((3-fluorophenethyl)amino)methyl)phenyl)-4-
methylpyridin-2-amine] and enzyme and this is illustrated in Fig. 7. The surface diagrams for
eight diazepanone molecules docked at the active site of MRSA are depicted in Fig. 8. The
molecules predominantly interact with Glu243 target residue, in the docked pose of MRSA.
Scaffolds containing methoxy substituted phenyl rings in the structure are placed well into
the pocket lined by hydrophobic residues; the polar heads bearing carbonyl and hydroxyl
groups interact through H-bonds with the polar side chains of the amino acids at the active
site. Molecules 13 and 14 show more number of H-bond interactions at the active site
residues. Among them, following interactions Glu243, Tyr239, His128, Asp220, Arg132,
Gln129, Trp329 and Asn248 have also been noticed in the co-crystal ligand of MRSA, apart
from this, interaction with other residues like Trp59, Leu162, Val163, Lys200, Ile235,
Glu240 and Asp300 has been found in remaining compounds. Overall, molecule 10 [2,7-
bis(4-chlorophenyl)-3,3-dimethyl-1,4-diazepan-5-one] is having better docking score and
glide energy is comparable to co-crystal ligand of MRSA. The docking score, glide energy
and hydrogen bonding interactions are presented along with the co-crystal ligand (Fig. 9. &
Table 6) docked at the active site of target for all molecules. The results indicate that the
identified compounds have good and efficient inhibitory activities against the chosen target.
3.6. Antibacterial activity assay
All the synthesized compounds 9-16 have been screened for antibacterial activity
against four bacterial strains using agarose well diffusion method and by minimum inhibitory
concentration (MIC) method [23, 24]. The solvent, DMSO used for the preparation of
compounds does not show inhibition against the tested organisms. The results reveal that
most of the synthesized compounds exhibit antibacterial activity against S. aureus, S.
pyogenes, and E. coli whereas M. luteus strain shows no activity. The results are presented in
Tables 7 & 8. Compounds 10, 11, 15 and 16 dictate a certain degree of antibacterial activity
except M. luteus. Moreover, compounds 11 and 15 show better activity among the all
compounds.
The minimal inhibitory concentration has been screened by MTT assay method [24].
Here, DMSO has been used as a negative control and ampicillin as a positive control.
Among all the compounds 9-16, only the compound 11 shows comparable results with the
standard drug ampicillin against S. aureus and E. coli strains. The other compounds show
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significant to weak bactericidal inhibitory activity against S. aureus, S. pyogenes, E. coli and
M. luteus. All the compounds 9-16 exhibit better activity against S. aureus when compared
with other strains.
3.7. Antioxidant activity
The DPPH radical scavenging method [25] is the most common tool to inspect the
antioxidant capacity of the specific compounds. The synthesized compounds 9-16 interact
with the free radicals in the DPPH and inhibit the oxidation exhibiting a certain degree of
radical scavenging activity. The antioxidant results are expressed in terms of IC50 in µM as
presented in Table 9. From the results, the compounds 11 (24.68 ± 0.48), 13 (30.29 ± 0.29)
and 14 (26.90 ± 0.39) show significant antioxidant activities equal to half of the antioxidant
power of standard BHT. All the remaining compounds exhibit moderate antioxidant activity.
In general, presence of electron donating groups in the aromatic ring of compounds 11 (4-
CH3) and 13 (4-OCH3) show better antioxidant activity than the electron withdrawing groups
in the aromatic rings of compounds 9 (2-Cl) and 10 (4-Cl). The compound having Cl at the
ortho position of the aromatic ring (9) shows higher activity than the Cl at the para position
(10). The increase in bulkiness of groups at the C-3 carbon of the diazepanone ring results in
the decrease of antioxidant activity (14>15). The substituted aromatic rings show batter
activity than the unsubstituted phenyl rings (9-11, 13 and 14> 12). Among the diazepanones
9-16, the antioxidant activity order can be presented as 11> 14> 13> 9> 10> 12> 16> 15.
4. Conclusion
In summary, diazepan-5-ones 9-16 have been synthesized using Schmidt
rearrangement in good yields by adopting simple one step procedure. All the compounds are
characterized using IR, mass, 1D and 2D NMR spectral studies. The stereochemistry of
1
13
compounds 9-16 has been studied with the help of H and C NMR data. The diazepan-5-
ones 9-16 prefer to adopt chair conformation with equatorial orientation of alkyl groups at C-
3/C-6 and aryl groups at C-2 and C-7. Single crystal X-ray diffraction studies have been
carried out for the compounds 9 and 11 which show the preference for distorted chair
conformation with equatorial orientation of substituents. The in silico docking study has
been carried out for all the compounds which reveals the better docking score of the
compound 10 and the comparable glide energy to co-crystal ligand of MRSA. The in vitro
evaluation of antibacterial activities reveal that the compound 11 has better activity against S.
aureus and E. coli strains and the remaining compounds exhibit moderate activity when
11

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compared with standard drug ampicillin. The compounds 11, 13 and 14 exhibit the activity
equal to half of the antioxidant power of standard drug and the remaining compounds show
moderate activity, as seen from the antioxidant assay.
Acknowledgements
SS thanks UGC, New Delhi for the award of Rajiv Gandhi National Fellowship
(RGNFS). PS thanks the UGC, New Delhi for the financial support in the form of a Research
Fellowship in Science for Meritorious Students. We wish to thank (SAIF) IIT, Chennai and
(SIF) Vellore for NMR spectra.
Additional information
Crystallographic data (CIF and FCF) for the structures of compounds 9 and 11
reported in this paper have been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication numbers, CCDC 1442870 and CCDC 1442857, respectively.
Copies of the data can be obtained free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1 EZ, UK. (fax: +44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk).
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All captions
Scheme 1
Synthesis of r-2,c-7-diaryl[1,4]diazepan-5-ones 9-16.
Figure captions
Figure 1
1H-1H COSY NMR spectrum of 9.
Figure 2
1H-13C HSQC NMR spectrum of 9.
Figure 3
1H-13C HMBC NMR spectrum of 9.
Figure 4
The ORTEP plot of the molecules 9 and 11.
Figure 5
Chair conformation of compounds 9 and 11.
Figure 6
Crystal packing of compounds 9 and 11.
Figure 7
Interaction of protein with the co-crystal
Figure 8
Surface diagram showing the binding of 9-16 within the hydrophobic pocket of MRSA.
Figure 9
Interactions of 9-16 docked complex with MRSA.
15

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Table captions
Table 1
Analytical data of diazepan-5-ones 9-16
Table 2
1H NMR chemical shift values of diazepan-5-ones 9-16 [δ (ppm)]
Table 3
13C NMR chemical shift values of diazepan-5-ones 9-16 [δ (ppm)]
Table 4
1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC correlations of 9
Table 5
Coupling constants (Hz) and dihedral angles (°) of compounds 9-16
Table 6
Hydrogen bond interactions of 9-16 and co-crystallized ligand with amino acids at the active
site of MRSA
Table 7
Zone of inhibition in mm at 100 µg/ml concentration of 9-16 with selected bacterial strains
Table 8
Minimum inhibitory concentration (MIC) of 9-16 against selected bacterial strains
Table 9
Screening results of DPPH radical scavenging activity of 9-16.
16

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Table 1
Elemental analysis
Found (Calcd) %
IR stretching frequencies (cm-1)
compound Molecular weight m.p. (ºC) Yield (%)
C
H
N
NH (amine) NH (amide) C=O (amide)
62.47
5.66
8.08
363.49
363.58
322.73
294.00
354.18
340.12
354.10
354.23
284-286
202-204
144-146
72
69
75
81
71
69
70
68
3288
3282
3281
3285
3285
3322
3307
3294
3204
3198
3203
3195
3216
3202
3214
3206
1654
1652
1651
1668
1653
1666
1673
1666
9
(62.82) (5.55) (7.71)
63.08 5.64 7.39
(62.82) (5.55) (7.71)
77.94 8.27 9.01
(78.22) (8.13) (8.69)
10
11
12
13
14
15
16
208-210
-
-
-
[Lit.6 210]
71.50
7.28
8.26
172-174
180-182
138-140
133-135
(71.16) (7.39) (7.90)
70.79 7.26 8.51
(70.56) (7.11) (8.23)
71.42 7.24 8.21
(71.16) (7.39) (7.90)
70.83 7.47 7.64
(71.16) (7.39) (7.90)

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Table 2
C-4’/C-4’’ C-4’/C-4’’
Aromatic
protons
Compound
H-2a
H-3a
H-6a
H-6e
H-7a
3-CH3
3-‘CH3
3-CH2CH3
6-CH3
NH
CONH
OCH3
CH3
7.69-7.18
(m)
4.73 (s)
3.93 (s)
3.90 (s)
3.87 (s)
3.89 (s)
3.64 (s)
3.71 (s)
3.61 (s)
-
3.10 (dd)
3.11 (dd)
3.14 (dd)
3.09 (dd)
3.13 (dd)
3.10 (dd)
3.10 (dd)
3.04 (dd)
2.69 (d)
2.58 (d)
2.62 (d)
2.56 (d)
2.60 (d)
2.61 (dd)
2.61 (dd)
-
4.62 (d)
4.08 (d)
4.06 (d)
4.03 (d)
4.05 (d)
4.07 (d)
4.08 (d)
b
1.65 (s)
1.57 (s)
1.58 (s)
1.53 (s)
1.57 (s)
0.81 (d)
-
1.09
0.96
0.94
0.88
0.94
-
-
-
-
-
-
-
-
-
-
1.92 (br.s)
1.90 (br.s)
1.89 (br.s)
1.88 (br.s)
1.86 (br.s)
1.96 (br.s)
1.97 (br.s)
2.00 (br.s)
6.13 (s)
6.30 (s)
6.11 (s)
6.21 (s)
5.95 (s)
5.91 (s)
5.71 (s)
5.75 (s)
9
7.38-7.26
(m)
-
-
-
-
-
-
10
11
12
13
14
15
16
7.31-7.06
(m)
-
-
2.30 (s)
7.36-
7.17(m)
-
-
-
-
-
-
-
3.78, 3.77
(s)
7.35-6.80
(m)
-
-
7.33-6.82
(m)
a
3.58 (m)
b
-
a
1.09 (m, CH2),
0.85 (m, CH3)
3.78, 3.77
(s)
7.33-6.82
(m)
-
-
7.30-6.81
(m)
0.81 (d)
-
-
0.72 (d)
b
(a) 3.80-3.75 (δ, ppm) H-3a merged with C-4’/C-4’’OCH3, (b) 3.80-3.74 (δ, ppm) H-3a and H-7a merged with C-4’/C-4’’OCH3

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Table 3
C-4’/C-4’’ C-4’/C-4’’
Aromatic ipso
Compound
C-2
C-3
C-5
C-6
C-7
3-CH3
21.93
3-‘CH3
3-CH2CH3
-
6-CH3
-
Aromatic carbons
OCH3
CH3
carbons
129.81, 129.71, 128.89,
128.79, 128.74, 127.29,
127.14, 127.04
141.29, 139.65,
133.99, 132.59
67.41
56.68
174.65
45.05
56.78
28.39
-
-
9
142.86, 140.29, 129.55, 128.97, 128.48,
133.87, 133.54 127.75
141.92, 139.34, 129.39, 128.88, 128.24,
137.60, 137.38 126.36
73.08
73.69
73.87
73.17
70.45
55.80
56.16
56.02
56.16
54.91
174.58
175.32
175.18
175.19
175.83
46.84
47.12
47.06
47.03
47.59
60.38
60.96
61.17
60.55
59.03
21.31
21.54
21.45
21.37
19.90
30.41
30.54
30.41
30.56
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
11
12
13
14
21.14,
21.10
128.72, 128.34, 128.21,
127.92, 127.75, 126.43
-
-
-
144.65, 142.12
55.29,
55.25
55.27,
55.26
55.41,
55.27,
55.25
159.18, 159.05, 129.27, 127.45, 114.02,
137.05, 134.42 113.49
159.23, 159.00, 128.65, 127.86, 127.48,
137.23, 134.59 113.96, 113.91, 113.70
129.32, 128.73, 127.47,
25.72 (CH2),
10.06 (CH3)
159.21, 159.00,
69.44
70.45
59.14
54.40
176.18
178.49
47.56
46.07
60.69
64.43
-
-
-
-
-
-
114.22, 113.95, 113.93,
137.29, 134.39
15
16
113.78
159.20, 158.99, 128.83, 128.65, 113.87,
135.67, 134.70 113.66
19.71
-
14.60
55.25

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Table 4
Chemical shift
1H-1H COSY 1H-13C HSQC
1H-13C HMBC
(δ, ppm)
21.93 (3-CH3), 56.68 (C-3), 132.59 (C-2’), 139.65 (C-1’), 174.65
(CONH)
H-2a, 4.73 (s, 1H)
-
67.41 (C-2)
H-6a, 3.10 (dd, 1H)
H-6e, 2.69 (d, 1H)
H-7a, 4.62 (d, 1H)
3-CH3, 1.65 (s, 3H)
3-‘CH3, 1.09 (s, 3H)
H-6e, H-7a 45.05 (C-6)
56.78 (C-7), 174.65 (CONH)
H-6a
45.05 (C-6)
56.78 (C-7), 141.29 (C-1’’), 174.65 (CONH)
67.41 (C-2), 132.59 (C-2’’), 141.29 (C-1’’)
28.39 (3-‘CH3), 56.68 (C-3), 67.41 (C-2)
21.93 (3-CH3), 56.68 (C-3), 67.41 (C-2), 174.65 (CONH)
H-6a
56.78 (C-7)
-
-
21.93 (3-CH3)
28.39 (3-‘CH3)

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Table 5
Compound coupling constants (Hz)
Dihedral angles (°)
3JH2a, H3a 2JH6a, H6e 3JH6a, H7a 3JH6e, H7a
ФH6e,H7a
81
ФH6a,H7a
-
-
15.0
15.2
15.2
15.5
15.2
14.0
14.0
-
11.5
11.6
11.6
11.5
11.6
10.8
10.8
-
0
0
0
0
0
0
0
-
159
159
159
159
159
159
159
-
9
81
81
10
11
12
13
14
15
16
-
-
-
81
81
8.0
7.6
8.0
81
81
-

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Table 6
Hydrogen Bonding
Glide Energy
Compounds
Co-Crystal
Glide Score
(kcal/mol)
Interactions Distance (Å)
-7.52011
-49.9386
N-H…O(GLU243
N-H…O(TYR357)
-
2.9
3.8
(PDB ID: 4D7I)
-
-6.79254
-7.86139
-55.1623
-57.6659
9
10
O…H-O(TYR357)
O…H-N(ARG247)
O…H-N(ARG247)
N-H…N(ARG247)
2.87
2.93
3.4
-6.3959
-50.2444
-51.0804
-47.7864
11
12
13
2.8
-8.24083
-7.00516
N-H…O(HEM901) 3.2
O-H…O(HEM901) 3.4
O…H-O(GLU243)
O…H-N(GLN129)
O-H…O(HIS128)
N-H…O(ASP220)
2.8
2.9
2.8
3.3
N-H…O(HEM901) 3.0
O…H-N(ARG132)
O-H…O(TYR239)
O…H-N(TRP329
N-H…O(ASN248)
O…H-N(ASN248)
N-H…O(GLU243)
N…H-N(ARG132)
O…H-N(ARG132)
O…H-O(GLU243)
3.0
3.0
2.8
3.2
2.8
3.0
3.4
3.3
2.9
-7.53081
-48.6479
14
-7.48146
-8.16355
-49.2091
-49.199
15
16
O-H…O(HEM901) 2.7
O…H-O(GLU243)
O-H…O(ASP220)
O…H-N(HIS128)
2.5
2.8
3.1
N-H…O(HEM901) 3.2

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Table 7
Diameter of inhibition zone (mm)
Cpds
M. luteus
E.coli
-
S. aureus
S. pyogenes
-
-
-
-
-
-
-
-
-
12.29 ± 0.73
-
9
10.92 ± 0.45 10.11 ± 0.32
16.19 ± 0.32 11.12 ± 0.34
11.65 ± 0.27
14.16 ± 0.71
-
10
11
-
-
-
-
12
10.29 ± 0.33
-
13
10.29 ± 0.69 11.72 ± 0.22
14.48 ± 0.26 16.95 ± 0.35
11.11 ± 0.38 11.09 ± 0.29
24.12 ± 0.23 28.63 ± 0.31
14
15
10.19 ± 0.51
11.43 ± 0.95
22.43 ± 0.65
16
Ampicillin
Table 8
IC50 Concentration µM
Cpds
M. luteus
S. aureus
S. pyogenes
E. coli
60.72 ± 0.31
95.97 ± 0.13
97.68 ± 0.34
93.99 ± 0.18
80.14 ± 0.06
85.57 ± 0.21
101.91 ± 0.13
93.81 ± 0.32
89.91 ± 0.20
100.28 ± 0.12
24.15 ± 0.34
92.88 ± 0.34
9
64.91 ± 0.18
25.85 ± 0.07
87.00 ± 0.27
70.42 ± 0.33
64.46 ± 0.28
45.63 ± 0.33
61.73 ± 0.17
18.57 ± 0.19
93.69 ± 0.34
70.74 ± 0.17
82.05 ± 0.29
87.81 ± 0.17
81.21 ± 0.20
75.94 ± 0.23
81.21± 0.08
17.02 ± 0.15
88.02 ± 0.08
31.63 ± 0.21
83.77 ± 0.17
84.28 ± 0.20
64.45 ± 0.11
81.08 ± 0.35
74.46 ± 0.17
19.87 ± 0.26
10
11
12
13
14
15
16
Ampicillin

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Table 9
Compounds DPPH (IC50 ± SDa) µM
64.96 ± 0.50
77.80 ± 0.83
24.68 ± 0.48
87.73 ± 0.36
30.29 ± 0.29
26.90 ± 0.39
135.86 ± 0.91
124.83 ± 0.38
12.46 ± 0.12
9
10
11
12
13
14
15
16
BHT
a The results are average of triplicate analysis. Lower IC50 values indicate higher radical scavenging activity.

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