N-Benzyl-2,7-diphenyl-1,4-diazepan-5-one analogues: Synthesis, spectral characterization, stereochemistry, crystal structure and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2016.05.065

Three novel N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12 have been synthesized via two routes starting from 2,7-diphenyl-1,4-diazepan-5-ones 4-6 and N-benzyl-2,6-diphenylpiperidin-4-ones 7-9. The structural characterization and conformational analysis

Full text of DOI:10.1016/j.molstruc.2016.05.065

Accepted Manuscript
N-benzyl-2,7-diphenyl-1,4-diazepan-5-one analogues: Synthesis, spectral
characterization, stereochemistry, crystal structure and molecular docking studies
S. Sethuvasan, P. Sugumar, M.N. Ponnuswamy, S. Ponnuswamy
PII:
S0022-2860(16)30510-5
10.1016/j.molstruc.2016.05.065
MOLSTR 22579
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 15 April 2016
Revised Date: 15 May 2016
Accepted Date: 17 May 2016
Please cite this article as: S. Sethuvasan, P. Sugumar, M.N. Ponnuswamy, S. Ponnuswamy,
N-benzyl-2,7-diphenyl-1,4-diazepan-5-one analogues: Synthesis, spectral characterization,
stereochemistry, crystal structure and molecular docking studies, Journal of Molecular Structure (2016),
doi: 10.1016/j.molstruc.2016.05.065.
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N-Benzyl-2,7-diphenyl-1,4-diazepan-5-one analogues: Synthesis, Spectral
characterization, Stereochemistry, Crystal Structure and Molecular Docking Studies
S. Sethuvasana, P. Sugumarb, 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
Three novel N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12 have been synthesized
via two routes starting from 2,7-diphenyl-1,4-diazepan-5-ones 4-6 and N-benzyl-2,6-
diphenylpiperidin-4-ones 7-9. The structural characterization and conformational analysis of
1
13
these synthesized compounds have been carried out using IR, mass and H, C, DEPT-135
and 2D (COSY and HSQC) NMR spectral techniques. The N-benzyldiazepan-5-one 10 is
found to prefer chair conformation with equatorial orientation of alkyl and phenyl groups
while N-benzyldiazepan-5-ones 11 & 12 prefer to adopt twist-boat conformation with phenyl
rings at C-2 & C-7 occupying equatorial and pseudo-axial orientations, respectively. The
single crystal X-ray structure of compound 12 has been determined which also supports the
twist-boat conformation. In silico molecular docking study has also been performed and the
results show that the compounds 10-12 might exhibit inhibitory activity against HIV-1
protease. All the compounds are screened for their antibacterial activity against three
bacterial strains (Staphylococcus aureus, Escherichia coli and Bacillus cereus) and only
compound 11 shows moderate activity.
Keywords: N-benzyldiazepan-5-ones, Chair, twist-boat, Single crystal XRD, Molecular
docking
*Corresponding author: Post Graduate and Research Department of Chemistry,
Government Arts College (Autonomous), Coimbatore-641 018, Tamil Nadu, India. E-mail:
kspons2001@gmail
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1. Introduction
Synthesis of new molecules by the fusion of two or more biologically active
fragment is an important process in the fields of medicinal and pharmaceutical chemistry. A
wide range of diazepines has been identified as potential drugs which are used for many
therapeutic applications [1, 2]. Especially, 1,4-diazepines play a vital role in the drug
industry as antimicrobial [3, 4], anti-HIV [5], psychotropic [6] and anticancer agents [7].
Furthermore, N-benzyl derivatives of piperidines have been found to be active against human
cervical carcinoma (HeLa) cells [8]. This prompted us to synthesise a new system of
N-benzyl-1,4-diazepan-5-ones as target molecules and to study their stereochemistry and
biological activity. Very little information is available on the synthesis and stereochemistry
of 1,4-diazepan-5-ones [9]. Their nitroso [10-15], ethoxycarbonyl [16] and formyl [15]
derivatives prefer to exist in the flattened boat, alternate chair and twist-chair conformations
in the solution state. The delocalization of nitrogen lone pair into the П cloud results in
restricted rotation about N-NO and N-CO functions which is responsible for the drastic
change in the conformation of the ring and orientation of the substituents in these
compounds.
The present work involves the synthesis of three new N-benzyl-1,4-diazepan-5-ones
10-12 by adopting two methods: (i) Schmidt rearrangement of piperidin-4-ones 4-6 followed
by benzylation, (ii) benzylation of piperidin-4-ones 7-9 followed by Schmidt rearrangement.
The characterization of compounds 10-12 are made with the help of IR, mass, 1H, 1H (D2O),
13C, DEPT-135 NMR spectra and the NMR signals are unambiguously assigned using 2D
NMR (1H-1H COSY &1H-13C HSQC) spectra. The conformational analysis of compounds
10-12 has been carried out using NMR spectral data. In addition, the crystal structure has
also been solved for compound 12 to predict the preferred conformation in the solid state
using single crystal X-ray diffraction study. Furthermore, antibacterial activity and molecular
docking studies with the target protein are also carried out for compounds 10-12.
2. Experimental section
All chemicals were purchased from commercial suppliers and used without further
purification. The melting points were taken on an electrically heated block with a calibrated
thermometer and are uncorrected. The FT-IR spectra were recorded on Bruker alpha
1
spectrometer using KBr pellets. H and 13C NMR spectra were recorded on Bruker 400 MHz
and 100 MHz NMR spectrometer, respectively, using CDCl3 as solvent and tetramethylsilane
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(TMS) as an internal reference. All chemical shifts are reported in δ units and described as
being either singlet (s), broad singlet (bs), doublet (d), doublet of doublets (dd), triplet (t),
broad (br) and multiplet (m). Thin-layer chromatography (TLC) was performed and the
progress of the reaction was monitored on a pre-coated silica gel Merck plates. Mass spectra
were acquired on an EI spectrometer at 70 eV. Elemental analyses were performed on a
Carlo Erba 1108 CHN analyser.
All the piperidin-4-ones 1-3 [17], diazepan-5-ones 4-6 [9] and N-benzylpiperidin-4-
ones 7-9 [18, 19] have been synthesized by following the reported procedures. The analytical
data of N-benzyl-1,4-diazepan-5-ones 10-12 are presented in Table 1.
2.1.
2.1.1 Method A
A mixture of diazepan-5-ones 4-6 (0.01 mol), benzyl bromide (0.015 mol, 1.78 ml)
General procedure for the synthesis of N-benzyl-1,4-diazepan-5-ones 10-12
and anhydrous potassium carbonate (0.02 mol, 2.76 g) in DMF (30 ml) is stirred at room
temperature for 36-48 h. To this, an excess of ice-cold water is added and extracted using
dichloromethane (2×10 ml). The organic layer is washed with brine solution and dried over
anhydrous Na2SO4. The organic layer is then concentrated in a rotary evaporator to obtain
the crude product. The crude solid thus separated is purified by recrystallisation from
ethanol.
2.1.2. Method B
The N-benzylpiperidin-4-ones 7-9 (5 mmol) are added to a suspension of conc. H2SO4
(20 ml) and dichloromethane (40 ml) at 0ºC. Then NaN3 (sodium azide) is continuously
added over a period of 3 h and the resulting mixture is stirred at RT for 1 h. The resulting
solution is poured into crushed ice and alkalized with ammonia solution (20-30 ml to pH 11-
12). The organic layer is separated from the water fraction, extracted with DCM
(dichloromethane), dried using anhydrous Na2SO4 and concentrated in a rotary evaporator to
obtain the crude product. The crude product thus obtained is recrystallised using ethanol.
Here, among the two methods (Methods A & B), the overall percentage of yield in
method B (57-77%) is higher than that in the method A (53-61%, Table 1).
2.2. X-ray crystallography
The single crystal X-ray diffraction data were collected on a Bruker axs Kappa
APEXII CCD diffractometer employing graphite-monochromatized Mo Kαradiation (λ =
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0.71073 Å). The data were processed using the SAINT software program [20]. The structure
was solved by the direct methods using SHELXS-97 and refined on F2 by a full-matrix leasts-
quares technique using the SHELXL-97 package [21, 22]. The molecular graphics plots were
drawn using PLATON [23] and ORTEP-3 [24]. All H atoms were fixed geometrically and
allowed to ride on their parent C atoms, with C-H distance fixed in the range 0.93-0.97 Å.
The H atom displacement parameters were restricted to be 1.2Ueq for the parent atom.
Crystal data and refinement details are given in Table 2. Selected bond distances, bond
angles and torsion angles of the diazepan-5-one 12 are listed in Tables 3a & 3b. The
molecular structure with atom-numbering and displacement ellipsoids are drawn at 30%
probability level is shown in Fig. 1a. The overlapping of both the molecules present in the
asymmetric unit is shown in Fig. 1b [24]. The RMSD value between the overlapped
molecules is 0.272Å.
2.3. Molecular modeling study
All docking calculations were performed using the Induced Fit Docking module of
the Schrödinger suite. It performs flexible protein-ligand docking and searches for
favourable interactions between small ligand molecule and a typically larger receptor
molecule, usually protein. Docking process is divided into three steps. Step 1 is the Initial
Glide Docking, wherein protein preparation constrained refinement is carried out with a
maximum of 20 poses. Step 2 involves Prime Induced Fit, wherein the side chains are
optimized and refinement of residues take place if the ligand poses are within 5.0Ǻ. Step 3
consists of the Glide redocking stage using Standard Precision (SP) mode. The best-docked
structure/pose was chosen using the following three criteria: Glide score (G-score) function,
Glide Energy and the number of residual matches (hydrogen bonds) with the original drug
complex. All computational works were performed with various modules of Schrödinger
Suite 2011 [25] using Red Hat Enterprise Linux 5.0 interface running on Pentium D
workstation.
2.4. In vitro antibacterial evaluation
The minimum inhibitory concentration (MIC) of the N-benzyldiazepan-5-ones 10-12
against the three human pathogens [Staphylococcus aureus (ATCC 25923), Escherichia coli
(ATCC 25922) and Bacillus cereus (ATCC 10876)] was analyzed by resazurin reduction
assay described by Sarker et al [26].
2.4.1. Method to prepare resazurin dye solution
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The resazurin dye solution was made by dissolving a 270 mg tablet in 40 ml of sterile
distilled water. The vortex mixer was used to ensure that the resazurin solution was well
dissolved and form a homogeneous solution.
2.4.2. Preparation of the activity plates
The 96 wells plates were prepared under aseptic conditions. A volume of 200 ꢀl of
compounds (1mg/ml) in 5% (v/v) dimethyl sulfoxide was pipetted out into the first row of the
96 wells plate. To all other remaining wells, 100 ꢀl of nutrient broth was added to the
bacterial cells. The serial dilutions were performed using micropipette with sterile pipette
tips such that each well had 100 ꢀl of the test material in serially descending concentrations.
To all these wells 10 ꢀl of resazurin dye solution was added. A 10 ꢀl of bacterial suspension
(5 × 106 cells/ml) was added to each well to achieve a concentration of 5 × 105 cells/ml. The
commercial antibiotic, streptomycin was used as positive control in the assay plate. The
plates were placed in an incubator at 37°C for 18-24 h. The color change was then observed
visually. The color changes from blue to pink or colorless were recorded as a reduction of
dye by the viable bacteria. The lowest concentration at which no color change occurred was
taken as the MIC value.
3. Results and discussion
3.1. Chemistry
The benzylation on 1,4-diazepan-5-ones is performed by adopting two routes which
are given in Scheme 1. Both the routes yielded only mono benzylated product. The N-
benzyl-1,4-diazepan-5-ones 10-12 are synthesized from 1,4-diazepan-5-ones 4-6 and from
N-benzylpiperidin-4-ones 7-9, respectively.
The N-benzyldiazepan-5-ones 10-12 are
characterized using spectral and analytical data. The absence of amine NH (3292-3003 cm-1)
and the presence of amide NH (3184-3203 cm-1) and amide CO bands (1654-1671 cm-1) in
their FT-IR spectra confirm the benzylation at amine nitrogen. This is further confirmed
1
from the H (D2O) NMR spectra which show the presence of amide NH proton at 5.72-6.04
ppm. In addition, new signals appear for N-benzyl methylenic protons at 3.43-3.73 ppm.
These CH2 protons of 10 & 11 are diastereotopic in nature. Furthermore, the molecular ion
(M+) peaks and fragmentation patterns for the compounds 10-12 in their mass spectra support
the formation of the products. The complete analytical data for compounds 10-12 are
presented in Table 1.
3.2. NMR spectral assignment
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The structure of N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12 is confirmed from
1
the IR, H and 13C NMR spectral data. DEPT-135 spectra are also recorded for all the
13
1
compounds which support the assignment of C NMR signals. The signals of the H NMR
spectra of
N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12 are assigned based on their positions,
1
multiplicities and intensity. In addition, 2D (1H-1H COSY, H-13C HSQC) NMR spectra are
used for the unambiguous assignment of 1H & 13C NMR signals and are presented in Tables 4
& 5, respectively, along with those of parent diazepan-5-ones 4-6 [10].
3.3. Analysis of 1H and 13C NMR signals of compound 10
The compound 10 has AMX and AB spin systems for the heterocyclic ring protons.
The H-6a, H-6e and H-7a protons belong to the AMX spin system. The proton signal
appearing at 4.24 ppm as a doublet correlates with the doublet at 3.00 ppm and a doublet of
doublet at 3.17 ppm in the COSY spectrum. In fact, all the signals are expected to show
doublet of doublet. However, two signals show only doublets which reveal that one of the
couplings is zero due to the expected dihedral angle of 90o between these vicinal protons
[27]. The signal at 4.24 ppm may be assigned to H-7 proton since it correlates with other two
3
proton signals. It shows only a doublet with the J value of 7.6 Hz. However, the expected
diaxial coupling constant would be 9-11 Hz and the decrease in diaxial coupling constant
may be due to ring distortion. Further, the H-7 proton occupies the axial position and it
makes an approximate angle of 90o with the equatorial proton at C-6 (H-6e) which results in
zero coupling (3JH7a
, H6e=0 Hz). The equatorial proton at C-6 (H-6e) appears as doublet at
3.00 ppm and it has only a geminal coupling with H-6a (2JH6a, H6e = 16.4 Hz). The doublet of
doublet at 3.17 ppm is assigned to axial proton at C-6 (H-6a). The extracted coupling
constant of 10-12 along with their estimated dihedral angles using DAERM (Dihedral Angle
Estimation by Ratio Method) [28] are presented in Table 4 along with their parent diazepan-
5-ones 4-6.
The H-3 proton appears as a multiplet at 3.59 ppm due to its coupling with CH3 and H-2
protons. The H-2 & H-3 protons belong to AB spin system. Thus, the doublet appearing at
3.78 ppm is assigned to H-2 benzylic proton. It has a J value of 8.4 Hz and it confirms that
both the protons at C-2 and C-3 are diaxially oriented (H-2a & H-3a, respectively). Hence, the
phenyl group at C-2 and the methyl group at C-3 occupy equatorial orientations. The doublet
at 0.59 is assigned to CH3 protons at C-3. Two doublets are observed for the methylenic
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protons at 3.72 and 3.43 ppm. Thus, the appearance of separate signals for the CH2 protons
proves its diastereotopic nature. The aromatic protons appear between 7.14 and 7.58 ppm.
1
13
The assignment of NMR signals of compounds 11 & 12 using H, C NMR, and 2D
1
13
NMR spectra has also been made in a similar way. The complete assignment of H and C
NMR signals is presented in Tables 4 and 5.
3.4. Conformational analysis of N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12
The conformational analysis of 1,4-diazepan-5-ones 4-6 has been carried out in
solution and solid states. In both the states, the diazepan-5-ones 4-6 prefer to adopt a chair
conformation with the equatorial orientation of bulky groups Fig. 2 [10, 14]. Further,
N-benzylpiperidin-4-ones 7-9 are also reported to exist in a chair conformation, similar to
piperidin-4-ones 1-3, with the equatorial orientation of alkyl and aryl groups in solution and
solid states [18, 19].
The preferred conformation of the N-benzyl-1,4-diazepan-5-ones 10-12 has been
arrived by considering the vicinal coupling constants between the H-7 and H-6a & H-6e
protons and between H-2 and H-3 protons. The vicinal coupling constant data (3JH6a, H7 and
3JH6e, H7) are employed to estimate the dihedral angles between the vicinal protons
(Фcis=ФH6e,H7a and Фtrans=ФH6a,H7a) by DAERM [28] and are used for the conformational
analysis (Table 6). The appearance of Bohlmann bands (2803-2865 cm-1) in the IR spectra of
10-12 indicates that atleast two axial hydrogens on carbons adjacent to the nitrogen atom are
interacting with the nitrogen lone pair in the diazepanone ring [29, 30].
The seven-membered 1,4-diazepan-5-one rings are, in general, more flexible than
the six-membered piperidin-4-one rings. Hence, the destabilizing strain factors (viz., 1,3-
diaxial interaction, gauche interaction, 1,4-diaxial hydrogen-alkyl interaction and bond
eclipsing interaction) may force the ring to change the chair (CE) conformation of the parent
precursors 4-6 (Fig. 2). However, the attachment of benzyl group at the ring nitrogen in the
piperidin-4-ones 1-3 has not altered the chair conformation of its precursor [18, 19]. Hence,
it is of interest to find out the preferred conformation of the flexible rings due to the
attachment of benzyl substituent at amine nitrogen. The possible conformations for N-
benzyl-1,4-diazepan-5-ones 10-12 are shown in Fig. 3.
3.4.1. N-Benzyl-3-methyl-2,7-diphenyl-1,4-diazepan-5-one (10)
In the chair conformation (CE), alkyl and aryl substituents occupy equatorial
orientation. The expected vicinal coupling constants between H-2 & H-3, H-6a & H-7, and
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H-6e & H-7 would be around 8-9 Hz, 10-11 Hz, and 0 Hz, respectively. The cis coupling
(JH7, H6e) constant of 0 Hz observed in the parent diazepanones 4-6 suggests a dihedral angle
of ca. 90º and the trans dihedral angle ΦH7a, H6a would be 159º [10]. Here, the observed
coupling constant and estimated dihedral angles for compound 10 are, JH2, H3= 8.4 Hz,
JH6a, H7= 7.6 Hz, JH6e, H7= 0 Hz and ΦH7a, H6a= 161º, ΦH7a,H6e= 79º. Thus, the coupling
constants and dihedral angles of compound 10 are similar to its parent precursor 4
(JH2, H3= 7.8 Hz, JH6a, H7= 10.6 Hz, JH6e,H7= 0 Hz and ΦH7a, H6a= 159º, ΦH7a, H6e= 81º, Table 6).
Hence, similar to parent diazepan-5-one 4, compound 10 also prefers to adopt a chair
conformation (CE) with equatorial orientation of phenyl and methyl groups (Fig. 4). Further,
the possibility of the chair (CA & TC) and boat (BE, BA & TB) conformations is
eliminated.
3.4.2. N-Benzyl-2,7-diphenyl-1,4-diazepan-5-ones (11 & 12)
As discussed earlier, the vicinal coupling constant and dihedral angles for the chair
(CE) conformation is JH2, H3=8-9 Hz, JH6a, H7=10-11 Hz, JH6e, H7=0 Hz and ΦH7, H6a =159º,
ΦH7,
=90º. The extracted coupling constant and estimated dihedral angles for the
H6a
compounds 11 and 12 are JH2, H3=8.1-8.8 Hz, JH6a, H7=8.8-10.6 Hz, JH6e, H7= 2.4 Hz and
ΦH7, H6a =175º, ΦH7, H6e =55º. Thus, the increase in the cis coupling constant and dihedral
angle values of 11 & 12 when compared with that of the parent 5 & 6, respectively, do not
support the CE conformation for the compounds 11 & 12. Hence the diazepan-5-ones 11 &
12 prefer a different conformation when compared to diazepan-5-one 10.
In CA conformation, all the alkyl and aryl groups occupy axial orientations as well
as the ring protons occupy equatorial orientations. The cis and trans coupling constants for
benzylic protons would be expected in the range of 2-4 Hz. Further, the cis and trans
dihedral angles are expected around 60º. In addition, 1,3-diaxial interaction is inevitable in
CA which destabilizes the chair conformation and tends to move the ring towards twist (TC
& TB) or boat (BA & BE) conformations. Hence, the possibility of CA conformation is
ruled out for the compounds 11 & 12.
The expected vicinal coupling constants in the twist-chair (TC) conformation are
JH2, H3= 9-11 Hz, JH7, H6a= 4-5 Hz, JH7, H6e= 2-3 Hz or JH2, H3= 2-3 Hz, JH7, H6a= 9-11 Hz,
JH7, H6e= 4-5 Hz. Here, the expected vicinal coupling constants coincide with the extracted
vicinal coupling constants for compound 11 (JH2, H3= 8.8 Hz, JH7, H6a= 8.8 Hz & JH7, H6e= 2.4
Hz). However, the twist-chair conformation does not favor a partial double-bond character at
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the amide C-N bond and thus amide carbon behaves like a ketone carbon. The amide carbon
signals of 11 & 12 are observed at 174-177 (δ, ppm) in their 13C NMR spectra which
eliminates the possibility of TC conformation.
The protons at C-2 and C-7 are axial and the protons at C-3 and C-6 are equatorial in
the boat conformation BA. The expected coupling constants would be 4-5 Hz for cis (Ja,e)
coupling and 9-10 Hz for trans (Ja,a) coupling. In addition, 1,3-diaxial interaction is also
possible between the two phenyl groups at C-2 and C-7 which occupy axial orientation.
However, in the absence of allylic strain [31], the phenyl groups need not be forced to occupy
axial orientations. Thus, the diaxial coupling (8.8-10.0) observed between H-2 and H-3 for
the compounds 11 and 12 eliminates the possibility for BA conformation.
The phenyl groups at C-2 & C-7 and alkyl groups at C-3 and C-6 are occupying
equatorial and axial orientations, respectively, in BE conformation. Here, only cis coupling
would be possible between H-2 & H-3. For the compounds 11 & 12, the observed diaxial
coupling constant values between H-2 and H-3 (JH2, H3= 8.8-10.0 Hz) does not support the BE
conformation. Furthermore, the alkyl substituent at C-3 and C-6 would feel the 1,4-diaxial
hydrogen-alkyl interaction in this conformation. Thus, all these observations do not support
the boat conformations with equatorial phenyl groups. Hence, the conformation BE is not
considered further.
In the twist-boat (TB) conformation, the alkyl and aryl groups at C-2 & C-3 and C-6
& C-7 are equatorial, pseduo-equatorial & pseduo-axial, respectively. The extracted vicinal
coupling constants for the compounds 11 and 12 (Table 6) are comparable with the expected
values of TB conformation. The predicted TB conformation is further supported by the
X-ray crystal structure of 12. Hence, it is concluded that N-benzyl-2,7-diphenyl-1,4-
diazepan-5-ones 11 and 12 prefer to adopt twist-boat conformation (TB) with the phenyl
group at C-7 occupying pseudo-axial orientation and the phenyl group at C-2 occupying
equatorial orientation (Fig. 5).
The increase in the cis (Ja,e) coupling constant from 0 to 2.4 Hz and decrease in trans
(Ja,a) coupling constant from 10.7 to 8.8 Hz as well as a decrease in the cis dihedral angle
(81→55º) and increase in trans dihedral angle (159→175º) between C-6 and C-7 are
observed when compared with that of parent compounds 5 & 6. This may be due to an
unsymmetrical twisting of the ring which results in pseudo-axial orientation of phenyl group
at C-7 and pseudo-equatorial orientation of the methyl group at C-6.
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1
3.4.3. Shielding and deshielding effect of α, β-protons and carbons in H and 13C NMR
spectra
In the 1H NMR spectra, α-protons (H-2 & H-7) and β-protons (H-3, H-6a & H-6e) of
10-12 are less deshielded (∆δ=+ 0.04-0.56 ppm) when compared with their parent
compounds 4-6. The introduction of benzyl group at amine nitrogen is not influencing the
α- and β-protons when compared to that of hetero conjugate groups (-NO, -CHO, -COOEt,
etc.) [9-12]. However, significant shielding and deshielding for α & β-carbons of 10-12 are
noted when compared to their parent compounds 4-6 in 13C NMR spectra. Greater shielding
is felt by the β-carbons (C-3 & C-6) when they are having bulky substituents at C-3 and C-6.
For the compounds 10 & 11, α-carbon C-7 is shielded (∆δ= - 2.23-2.38 ppm) and another
α-carbon C-2 is deshielded (∆δ= + 1.20-2.54 ppm). The reverse is true for the compound 12
[(∆δ= -0.50 (C-2), +1.21 (C-7)]. The shielding of α-carbons can be explained by the
eclipsing interaction between one of the α-carbons (C-2 or C-7) and methylenic carbon in the
benzyl group at N-1. The shielding and deshielding of chemical shifts of the α- and β-protons
and carbons are presented in Tables 7 & 8.
3.5. X-ray crystal structure of compound 12
The N-benzyl-1,4-diazepan-5-one 12 is crystallized with two crystallographically
independent molecules in the asymmetric unit. The study on asymmetry parameters, torsion
angles and least-squares planes reveals that the diazepanone ring in both the molecules adopts
twist-boat conformation (Fig. 6) with puckering [32] and asymmetry parameters [33] are: q2
= 1.113(2)Å, q3 = 0.054(2)Å, φ2 = 342.02(3)° & ꢁCs(C-3A) = 15.6(2)° for molecule A and
q2 = 1.115(2)Å, q3 = 0.073(2)Å, φ2 = 340.02(1)° & ꢁCs(C-3B)= 12.4(2)° for molecule B,
respectively. The torsion angles shown in the figure (Fig. 6) are supporting the above said
twist-boat conformation for both molecules (A & B).
The planar phenyl rings substituted at C-2 and C-7 positions of the diazepanone ring
in both the molecules occupy equatorial orientation. The corresponding torsion angles are
[C14-C2-C3-N4] 178.7(2)° & -173.1(2)° and [C5-C6-C7-C8] 145.9(2)° & 147.2(2)° for
molecules A & B. The best plane of diazepanone ring orients at angles of 72.5(1)° &
80.5(1)° (Molecule A) and 56.6(1)° & 87.8(1)° (Molecule B) with respect to the two phenyl
rings, respectively. The benzyl group substituted at the N-1 position of the diazepanone ring
is oriented at an angle of 49.60(16)° (Molecule A) & 42.45(13)° (Molecule B), respectively.
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In compound 12, the methyl groups (C-20 & C-21) substituted at C-3 & C-6 occupy
equatorial & axial orientations and the corresponding torsion angles are [N1-C2-C3-C20=] -
173.5(2)° & -168.5(2)°; [C21-C6-C7-N1=] -136.1(2)° & 136.6(2)°, for molecules A & B of
compound 12, respectively. The bond lengths and bond angles are comparable with each
other. The sum of the bond angles around the hetero nitrogen atoms N-1 [352.4° & 352.7°]
& N-4 [359.4° & 359.7°] for molecules A & B shows that the atoms N-1 & N-4 are in sp2
hybridized state.
3.5.1. Packing features
The packing of the molecules viewed down along the c-axis is shown in Fig. 7. The
molecules are stabilized by N-H...O type of intermolecular interactions in addition to van der
Waals forces (Table 9). The molecules at (x, y, z) and the symmetry related molecules at
(1/2-x,-1/2+y,1/2-z) & (1/2-x,1/2+y,1/2-z) are linked through intermolecular N4A-
H4A...O1B & N4B-H4B...O1A hydrogen bonds into cyclic centrosymmetric R2 (8) dimers
2
[34].
3.6. Molecular docking studies for the compounds 10-12
In this study, glide docking has been used to study the binding orientations to predict
the binding affinity of the compounds 10-12. The results obtained would be useful to
understand the inhibitory mode of the molecule based on docking scores. This will also
provide some beneficial clues in a structural modification to design new inhibitors for the
treatment of HIV. The structural information and the data for the target were collected from
“Protein Data Bank” (PDB ID: 1AJX) [35]. A view of the crystal structure of the compounds
10-12 in the HIV-1 protease active site along with hydrogen contacts is shown in Fig 8.
The disease acquired immune deficiency syndrome (AIDS) is caused by the
retrovirus (HIV) human immunodeficiency virus [36]. The HIV-1 protease is essential for
maturation of the virus into infectious viral particles and this enzyme is considered, as a
target for drugs against AIDS [37]. HIV PR is an aspartyl protease that is functional as a
dimer of two identical subunits with 99 amino acid residues [38]. Guidance in the design of
inhibitors has come from the crystal structures of HIV PR complexed with different inhibitors
[39]. There is a significant difference in the conformation of the protease in these inhibitor
complex structures compared to the native structure [40]. The analysis of these inhibitors in
complexation with the protease is presented here.
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The changes in the active site area between different inhibitor HIV PR complexes
are generally small (within 0.3-0.8 Å rms deviation) [41]. The amino acids Ile50/47 are
symmetrically positioned and in close contacts with the inhibitors. The aspartic acids are in
contact with the inhibitor, due to the asymmetric positioning of inhibitor in the active site.
The conformational change of the diazepanone ring and the shift in the side chain of
compounds induce the interactions with the HIV PR.
For comparison, we performed the docking studies for similar N-benzyl derivatives
of diazepanones, namely N-benzyl-3-methyl-2,7-diphenyl-1,4-diazepan-5-one (10), N-
benzyl-3-ethyl-2,7-diphenyl-1,4-diazepan-5-one (11), N-benzyl-3,6-dimethyl-2,7-diphenyl-
1,4-diazepan-5-one (12) and including Co-Crystal ((4r,5s,6s,7r)-1,3-Dibenzyl-4,7-
bis(phenoxymethyl)-5,6-dihydroxy-1,3 diazepan-2-one).
In compound 12, the nitrogen and keto O atoms interact with ILE-50 at distances of
3.13 & 3.4Å. Similarly in compound 10, the nitrogen atom in the diazepanone ring interacts
with ILE-47 at 3.23Å. In compound 11, the oxygen and nitrogen atoms in the diazepanone
ring are in contact with the aspartic acids (ASP-29 and ASP-30) at distances of 2.77Å and
3.20Å.) This is due to the asymmetric positioning of inhibitor in the active site. All the N-
benzyl-1,4-diazepan-5-ones 10-12 show appreciable interactions, good docking scores and
glide energy. The Co-Crystal ligand also docked well at the active site and makes contact
with ILE-50 and ASP-25.
The view of the N-benzyl-1,4-diazepan-5-ones 10-12 docked at the active site of the
protein is shown in Fig. 8. The relevant docking scores and energy details are given in Table
10. Since the docking scores of diazepan-5-ones 10-12 are similar, it is difficult to judge the
suitable inhibitor. However the parameters are valuable and useful to design inhibitors
comprising seven-membered ring as a core structure.
3.7. Antibacterial activity
The MIC of the compounds 10-12 is determined visually in the resazurin dye
reduction assay. The change of color of resazurin solution from blue to pink indicates that
the microbial cells are viable. The enzyme oxidoreductase present inside the microbial cells
converts the resazurin to resorufin which is pink in color. When the color of the dye remains
blue, it indicates that there is no activity of viable cells. The compounds 10-12, when added,
kill the bacterial cells during incubation. This is known from the blue or purple color of dye
in the respective cells. The pink color formation in the cells even after treating with test
12

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compounds 10-12 or commercial drug (Streptomycin) indicates the presence of viable cells.
Thus, the least dilution in which the color remains blue is taken as the MIC value of the
respective compound.
The compounds 10-12, tested in this assay show activity against the bacterial
pathogens. The MIC values of the test material are tabulated in Table 11. Both Gram-
positive and Gram-negative bacteria are susceptible to the test compounds. The results
indicate that only compound 11 exhibits moderate activity and the other compounds 10 and
12 show no activity even at 100 ꢀg/ml.
4. Conclusion
In the present study, three new N-benzyl-1,4-diazepan-5-ones 10-12 have been
synthesized by following two methods and the structures are characterized using IR, mass,
NMR spectral techniques. The stereochemistry has been studied for the synthesized
1
compounds using H and 13C NMR data. The compound 10 prefers to adopt CE
conformation whereas the compounds 11 & 12 are shown to prefer TB conformation with
equatorial and pseudo-axial orientations phenyl rings. The X-ray crystal structure of 12
shows that it is in twist-boat conformation in solid state. In silico molecular docking studies
are carried out for all the synthesized compounds and the results reveal that the compounds
10-12 are having similar binding interactions, docking score and glide energy when
compared with Co-Crystal ligand. The in vitro antibacterial screening indicates that the
compounds exhibit no activity even at 100 ꢀg/ml and only compound 11 shows moderate
activity.
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 structure of compounds 12 reported in
this paper have been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication numbers, CCDC1453761, respectively. Copies of the data can be
13

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obtained free of charge, on application to CCDC, 12 Union Road, CambridgeCB2 1 EZ, UK.
(fax: +44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk).
14

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All captions
Scheme 1
Synthesis of N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12
Figure captions
Figure 1
(a) ORTEP plot of the compound 12 with displacement ellipsoids drawn at 30% probability
level
(b) The overlapping of molecules (A & B) for the compound 12 present in the asymmetric
unit
Figure 2
chair conformation (CE) of 2,7-diphenyl-1,4-diazepan-5-ones 4-6
Figure 3
Possible conformations for compounds 10-12
Figure 4
Preferred conformation for compounds 10
Figure 5
Preferred conformation for compounds 11 and 12
Figure 6
Torsion angles for the compound 12 showing the seven-membered diazepanone ring adopts
twist-boat conformation for both molecules
Figure 7
Packing of the molecules for the compound 12 viewed down on c-axis and the formation of
dimer through N-H…O types of hydrogen bonds is shown in dotted lines
Figure 8
A view of the N-benzyl-1,4-diazepan-5-ones 10-12 in the protein active site showing the key
hydrogen contacts
18

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Table captions
Table 1
Analytical data of N-benzyl-2,7-diphenyl-1,4-diazepan-5-ones 10-12
Table 2
Crystal data of N-benzyl-3,6-dimethyl-2,7-diphenyl-1,4-diazepan-5-one (12)
Table 3a
Selected bond lengths (Å) and bond angles (°) for compound 12
Table 3b
Selected torsion angles (°) for compound 12
Table 4
1H NMR chemical shift values of N-benzyl-1,4-diazepan-5-ones 10-12 [δ (ppm)]
Table 5
13C NMR chemical shift values of N-benzyl-1,4-diazepan-5-ones 10-12 [δ (ppm)]
Table 6
Coupling constants (Hz) and dihedral angles (°) of compounds 10-12
Table 7
1H NMR chemical shift of the α- and β-protons (∆δ, ppm) for the compounds 10-12
Table 8
13C NMR chemical shift of the α- and β-carbons (∆δ, ppm) for the compounds 10-12
Table 9
Hydrogen-bond geometry (Å, °) for compound 12
Table 10
Hydrogen bond interactions of N-benzyl-1,4-diazepan-5-ones 10-12 with amino acids at the
active site of HIV protein
Table 11
Minimum inhibitory concentration (MIC) of compounds 10-12 against selected bacterial
strains
19

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Table 1
Yield (%)
IR cm-1
Elemental analysis Found (Calcd)%
MS m/z
Compound
M. P. (ºC)
M. P. (ºC)
(M+)
Method A
Method B
amide NH amide CO
C
H
N
53
50
61
81.30 (81.05)
6.79 (7.07)
7.36 (7.56)
136-138 °C
114-116 °C
146-148 °C
136-138 °C
114-116 °C
146-148 °C
57
68
78
370.00
384.98
384.24
3184
3188
3203
1654
1656
1671
10
11
12
81.44 (81.21)
80.90 (81.21)
7.06 (7.34)
7.58 (7.34)
7.10 (7.29)
7.07 (7.29)

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Table 2
Parameters
Values
CCDC number
1453761
Empirical formula
Formula weight
C52 H56 N4 O2
769.01
Temperature
293(2) K
Wavelength
0.71073 Å
Crystal system, space group
Unit cell dimensions
Monoclinic, P21/n
a = 13.944(5) Å
b = 20.486(5) Å
c = 16.555(5) Å
β = 113.479(5) °
4338(2) Å3
Volume
4, 1.178 Mg/m3
0.072 mm-1
Z, Calculated density
Absorption coefficient
F(000)
1648
Crystal size
Theta range for data collection
Limiting indices
Reflections collected / unique
Completeness to θ = 28.38
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2σ(I)]
R indices (all data)
0.21 x 0.19 x 0.16 mm
1.62 to 28.38°
-18<=h<=17, -27<=k<=27, -21<=l<=22
42454 / 10748 [Rint = 0.0363]
98.8%
Full-matrix least-squares on F2
10748 / 2 / 527
1.015
R1 = 0.0608, wR2 = 0.1585
R1 = 0.1239, wR2 = 0.1941
0.467 and -0.206 e.Å-3
Largest diff. peak and hole

ACCEPTED MANUSCRIPT
Table 3a
Angles
Lengths
Atoms
Atoms
Molecule B
1.470(3)
1.528(3)
1.518(3)
1.462(3)
1.516(3)
1.231(2)
1.343(3)
1.509(3)
1.514(3)
1.550(3)
1.474(2)
1.528(3)
1.451(3)
Molecule A Molecule B
Molecule A
1.465(2)
C2-N1
C2-C3
N1-C2-C3
N1-C2-C14
C3-C2-C14
N4-C3-C20
N4-C3-C2
C20-C3-C2
O1-C5-N4
O1-C5-C6
N4-C5-C6
C5-C6-C21
C5-C6-C7
C21-C6-C7
N1-C7-C8
N1-C7-C6
111.20(16)
115.77(15)
113.88(16)
108.59(18)
109.31(16)
111.60(2)
110.48(16)
115.56(18)
115.84(17)
109.05(18)
108.16(16)
112.31(18)
121.30(2)
1.527(3)
C2-C14 1.533(3)
C3-N4 1.461(3)
C3-C20 1.514(3)
C5-O1
C5-N4
C5-C6
1.229(2)
1.342(3)
1.510(3)
122.00(2)
121.60(2)
122.60(19)
115.95(18)
112.86(18)
112.23(16)
109.65(18)
116.99(16)
113.14(17)
113.40(2)
C6-C21 1.525(3)
116.28(19)
111.54(19)
112.02(17)
110.03(19)
116.64(16)
111.91(16)
C6-C7
C7-N1
C7-C8
1.552(3)
1.476(3)
1.526(3)
C22-N1 1.462(3)
N1-C22-C23 112.78(18)
C22-N1-C2
C22-N1-C7
C2-N1-C7
C5-N4-C3
112.91(16)
117.13(16)
122.41(15)
123.46(18)
112.92(17)
116.04(18)
123.01(15)
123.67(18)

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Table 3b
Angles
Atoms
Molecule A Molecule B
N1-C2-C3-N4
C14-C2-C3-N4
N1-C2-C3-C20
C14-C2-C3-C20
O1-C5-C6-C21
N4-C5-C6-C21
O1-C5-C6-C7
N4-C5-C6-C7
C5-C6-C7-N1
C21-C6-C7-N1
N1-C7-C8-C13
N1-C7-C8-C9
N1-C2-C14-C15
N1-C2-C14-C19
N1-C22-C23-C24
N1-C22-C23-C28
C23-C22-N1-C2
C23-C22-N1-C7
C14-C2-N1-C22
C3-C2-N1-C22
C14-C2-N1-C7
C3-C2-N1-C7
C8-C7-N1-C22
C6-C7-N1-C22
C8-C7-N1-C2
C6-C7-N1-C2
O1-C5-N4-C3
C6-C5-N4-C3
C20-C3-N4-C5
C2-C3-N4-C5
-53.1(2)
-48.3(2)
173.11(17)
178.69(17)
-173.44(18) -168.49(18)
52.7(3)
-9.9(3)
58.5(3)
-10.5(3)
165.0(2)
113.3(2)
-71.2(2)
11.9(2)
166.38(18)
114.6(2)
-69.1(2)
9.8(2)
136.08(18)
-74.6(3)
106.7(3)
-75.2(3)
103.5(2)
39.2(4)
136.62(19)
-64.4(3)
117.5(2)
-80.8(3)
99.5(2)
49.1(3)
-145.3(2)
-132.2(2)
-147.2(2) -155.57(18)
62.9(3)
-49.6(3)
176.4(2)
97.8(2)
-36.1(3)
79.0(2)
53.9(3)
-58.5(2)
169.52(17)
90.3(2)
-41.7(2)
81.3(2)
-146.68(19) -144.61(18)
-67.5(3)
66.8(2)
-66.2(2)
67.9(2)
-163.60(19)
20.0(3)
-164.0(2)
20.5(3)
-170.44(19)
67.1(2)
-173.3(2)
64.7(3)

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Table 4
Aromatic
protons
Compound
H-2
H-3
H-6a
H-6e
3.00 (d)
2.65 (d)
2.94 (dd)
2.65 (d)
-
H-7
3-CH3
0.59 (d)
0.81 (d)
-
3-CH2CH3
6-CH3
N-CH2-Ph
NH
CONH
5.92 (s)
5.75 (bs)
5.72 (s)
5.77 (bs)
6.04 (d)
5.75 (bd)
3.72 (d)
3.43 (d)
3.78 (d)
3.70 (d)
3.85 (d)
3.77 (d)
3.69 (d)
3.65 (d)
3.59 (m)
3.82 (ddq)
a
3.17 (dd)
3.14(dd)
3.20 (dd)
3.15 (dd)
3.64 (q)
3.08 (dq)
4.24 (d)
4.13 (d)
4.31 (dd)
4.14 (d)
4.07 (d)
3.79 (d)
-
-
-
2.07 (bs)
-
7.14-7.58 (m)
7.23-7.43 (m)
7.14-7.52 (m)
7.20-7.44 (m)
7.08-7.34 (m)
7.20-7.40 (m)
10
4
-
-
-
0.95 (m, CH2)
0.71 (m, CH3)
3.73 (d)
a
-
11
5
0.85 (t, CH2)
1.12 (m, CH3)
3.65 (m)
4.25 (dq)
3.86 (ddq)
-
-
-
2.03 (s)
-
1.03 (d)
0.79 (d)
-
-
0.94 (d)
0.70 (d)
3.90 (dd)
-
12
6
-
2.06 (s)
(a) 3.53-3.48 (δ ppm) H-3 merged with HB of N-CH2-Ph

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Table 5
Compound
C-2
73.64 50.84 173.99 36.57 57.22
71.1 54.7 175.7 47.5 59.6
71.30 56.59 174.49 38.22 58.27
70.1 59.3 176.1 47.4 60.5
70.50 50.05 177.23 40.41 66.11
71.0 54.2 178.4 45.9 64.9
C-3
C-5
C-6
C-7
3-CH3
21.95
19.8
-
3-CH2CH3
6-CH3
N-CH2-Ph Aromatic ipso carbons
Aromatic carbons
129.49, 128.74, 128.65, 128.29, 127.91,
127.32, 126.97, 126.47
-
-
-
-
56.93
145.23, 142.70, 138.15
142.1, 144.7
10
4
-
126.4, 127.7, 128.0, 128.6
-
27.00 (CH2)
10.14 (CH3)
25.6 (CH2)
10.1 (CH3)
129.42, 129.34, 128.81, 128.61, 128.58,
128.27, 127.74, 127.24, 126.97, 126.57
56.92
145.13, 142.20, 138.21
142.0, 144.8
11
5
-
59.25
-
126.4, 127.6, 127.7, 127.8, 128.5,
129.19, 128.53, 128.48, 128.17, 128.14,
127.96, 127.16, 127.07, 126.98
126.7, 127.6, 127.7, 127.8, 128.0, 128.3,
128.5, 128.7
19.81
19.6
-
-
15.74
14.6
141.70, 140.14, 139.02
142.2, 143.2
12
6

ACCEPTED MANUSCRIPT
Table 6
coupling constants (Hz)
Dihedral angles (°)
Compound
3JH2, H3
8.4
2JH6a, H6e
16.4
14.1
16.0
14.1
-
3JH6a, H7
3JH6e, H7
ФH6e,H7
ФH6a,H7
161
159
175
159
-
7.6
0
0
79
81
55
81
-
10
4
7.8
10.6
8.8
8.8
2.4
0
11
5
7.8
10.7
9.6
10.0
8.1
-
12
6
-
10.6
-
-
-

ACCEPTED MANUSCRIPT
Table 7
α-protons
β-protons
Compound
H-2
H-7
H-3
-0.23
-
H-6a
+0.03
+0.05
+0.56
H-6e
+0.35
+0.28
-
+0.08
+0.08
+0.04
+0.11
+0.17
+0.28
10
11
12
+0.39
Table 8
α-carbons
β-carbons
Compound
C-2
C-7
-2.38
-2.23
+1.21
C-3
C-6
-10.93
-9.18
-5.49
+2.54
+1.20
-0.50
-3.86
-2.71
-4.15
10
11
12
Table 9
D-H H…A D…A
D-H…A
>DHA
N4A-H4A...O1Bi
N4B-H4B...O1Aii
0.86 2.15
0.86 2.09
2.946(3) 154
2.902(3) 157
Symmetry Equivalent Position
(i) 1/2-x,-1/2+y,1/2-z
(ii) 1/2-x,1/2+y,1/2-z

ACCEPTED MANUSCRIPT
Table 10
Compound Glide Score Glide Energy
Interactions
Distance(Å)
N-H…O(ILE50)
O…H-O(ILE50)
N…H-O(ASP30)
O…H-N(ASP30)
O…H-N(ASP29)
N-H…O(ILE47)
N-H…O(ILE50)
O-H…O(ASP25)
O-H…O(ASP25)
3.1
3.4
3.2
3.0
2.8
3.2
2.8
2.7
2.7
-8.71922
-50.8854
12
-9.1433
-7.31637
-7.70963
-52.492
-48.6696
-59.0774
11
10
Co-Crystal
Table 11
Minimum inhibitory concentration (MIC) ꢀg/ml
Compound
Bacterial pathogens
Staphylococcus aureus
Escherichia coli
Bacillus cereus
NA
NA
25
NA
10
11
12.50
6.25
NA
NA
NA
12
1.56
1.56
0.78
Streptomycin
NA-No inhibition even at 100 ꢀg/ml