Synthesis, characterization, biological evaluation and in silico screening of oxadiazinanones

Article abstract of DOI:10.1007/s00044-012-0313-6

A series of novel oxadiazinan-5-one namely 2-methyl-2-phenyl-1,3,4- oxadiazinan-5-one (6), 2-(3-hydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (7), 2-(4-hydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (8), 2-(2,4- dihydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (9) and 2-(2,5- dihydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (10) have been synthesized by the reaction of acetophenone and its derivatives with cyanoacetic acid hydrazide. The structural assignments of the products were done on the basis of IR, ¹H NMR, ¹³C NMR, MS, and analytical data. The in vitro antioxidant and antimicrobial activity of all the synthesized compounds (6-10) was tested by the DPPH and disk diffusion method, respectively. The synthesized compounds were screened against Gram-positive and Gram-negative bacterial and fungal strains. Compound (7) showed the highest inhibition comparable with the standard antibiotic drugs Ciprofloxacin and Amphotericin B. The antibacterial activity of the synthesized compounds was further investigated with the help of in silico docking study using Discovery studio 3.1, Molego Virtual Docker and LigandScout to predict the active sites.

Full text of DOI:10.1007/s00044-012-0313-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3085–3095
DOI 10.1007/s00044-012-0313-6
ORIGINAL RESEARCH
Synthesis, characterization, biological evaluation and in silico
screening of oxadiazinanones
•
Mehtab Parveen Akhtar Ali Mahboob Alam
•
•
•
Asad U. Khan Anis Ahmad
Received: 22 February 2012 / Accepted: 25 October 2012 / Published online: 10 November 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of novel oxadiazinan-5-one namely
2-methyl-2-phenyl-1,3,4-oxadiazinan-5-one (6), 2-(3-hydro-
xyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (7), 2-(4-hydro-
xyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (8), 2-(2,
4-dihydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-one (9)
and 2-(2,5-dihydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-
5-one (10) have been synthesized by the reaction of aceto-
phenone and its derivatives with cyanoacetic acid hydra-
zide. The structural assignments of the products were done
on the basis of IR, 1H NMR, 13C NMR, MS, and analytical
data. The in vitro antioxidant and antimicrobial activity of
all the synthesized compounds (6–10) was tested by the
DPPH and disk diffusion method, respectively. The syn-
thesized compounds were screened against Gram-positive
and Gram-negative bacterial and fungal strains. Compound
(7) showed the highest inhibition comparable with the
standard antibiotic drugs Ciprofloxacin and Amphotericin
B. The antibacterial activity of the synthesized compounds
was further investigated with the help of in silico docking
study using Discovery studio 3.1, Molego Virtual Docker
and LigandScout to predict the active sites.
Introduction
The development of protocols for the preparation of novel
heterocyclic derivatives have been a subject of great
interest because of increasing bacterial resistance adopted
by microorganism to traditional antimicrobial drugs
(Rajganarendar et al., 2010). A handful reports are avail-
able on the synthesis of substituted oxadiazinanones with
varied yields (Ranganarendar et al., 2005; Hassan et al.,
2002, 2004; Rodrigues et al., 2005). One of the remarkable
features of oxadiazinanones is the presence of twisted boat
conformation (Zukerman et al., 2009) and conformational
mobility of nitrogen in their ring structure (Rosling et al.,
1997; Sibi and Liu, 2001; Sibi et al., 2001; Hitchcock
et al., 2001). This flexibility makes these compounds an
ideal candidate for the design of biologically potent mol-
ecules which can act as lead compound in the development
of antioxidant and antimicrobial drugs.
It is well known that oxadiazinanes play a promising
role for scavenging the antimicrobial activity. Thus,
merging oxadiazinane skeleton in a variety of hetero-
cyclic or non-heterocyclic system found application in
drug development for treatment of bacterial, fungal,
convulsant, inflammatory, malarial, tuberculotic infec-
tion. Recently, oxadiazinanthione derivatives are
employed for treatment of crops protection from insect
pests and some Lepidopteron pest species (Tomlin,
2006). They were also found to act as agonists of nico-
tinic acetylcholine receptor affecting synapse in the
insect’s Central Nervous System (Tomizawa and Casida,
2005; Tan et al., 2007). In silico molecular docking
technique play a key role in the drug design and dis-
covery to predict the orientation of the docked mole-
cule(s) at the active site, so, the in silico studies of the
synthesized compounds were carried out to the predict
Keywords Oxadiazinanone ꢀ
Cyanoacetic acid hydrazide ꢀ Antimicrobial activity ꢀ
Antioxidant activity ꢀ Docking study
M. Parveen (&) ꢀ A. Ali ꢀ M. Alam
Department of Chemistry, Aligarh Muslim University,
Aligarh 202 002, India
e-mail: mehtab.organic2009@gmail.com
A. U. Khan ꢀ A. Ahmad
Interdisciplinary Biotechnology Unit, Aligarh Muslim
University, Aligarh 202002, India
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Med Chem Res (2013) 22:3085–3095
the active site of pathogenic ClpS (301F pdb) protein
and the results obtained are reported.
compounds (6–10) showed two sets of absorption bands in
the range of 3,248–3,250 and 3,382–3,385 cm-1 due to two
N–H group stretchings and 1,680–1,695 cm-1 ascribed to
C=O stretching frequencies, respectively. In addition, the
absorption band at 3,500–3,549 cm-1 along with aromatic
overtones was ascribed to the aromatic phenols. The
shifting of band for carbonyl group in the IR spectra of all
the synthesized compounds and emerging of weak band at
1,465–1,471 cm-1 (N–N) ascertains the formation of ox-
adiazinan-5-one. Moreover, the absence of absorption band
at 2,200–2,250 cm-1 ascribed to C:N group revealed the
presence of cyclic compounds because of C:N soft
leaving group during SN2 cyclization process in which a
pair of electrons on the alkoxide ion is attracted to the
carbon bonded to the cyanide group, which then leaves to
generate the product. Furthermore, a strong band at
1,110–1,140 cm-1 explicated the formation of ether link-
age during product formation. Although the formation of
(6–10) may be interpreted through the nucleophilic attack
either of hydroxyl group or hydroxyl ion of acetophenone
formed during the attack of rear nitrogen of cyanoacetic
acid hydrazide on cyano group (Scheme 1).
Results
Chemistry
With the aim of obtaining new candidates that may be of
value in designing new, potent, selective and less toxic
antioxidant and antimicrobial agent, we herein report the
synthesis of oxadiazinan-5-ones (6–10) starting from ace-
tophenones with versatile and convenient intermediate
cyanoacetic acid hydrazide. The structural assignments of
1
the products were done on the basis of IR, H NMR, 13C
NMR, MS, and analytical data. The synthesized com-
pounds were also explored for their plausible biological
activities notably their antioxidant, antibacterial, and anti-
fungal activities with respect to standard drugs. Cyano-
acetic acid hydrazide reagent can act as an ambident
nucleophile i.e., both an N- and C-nucleophile. Reaction of
this reagent with various reactants leads to a number of
polyfunctional heterocyclic compounds of pharmacological
importance because of the availability of five active sites
(Ried and Schleimer, 1958). Oxadiazinanones Scheme 1
were prepared from the reaction of respective acetophe-
nones with cyanoacetic acid hydrazide in acetic acid as a
solvent. The reaction does not demand rigorously dried
solvents, reagents or inert atmosphere. The structures of the
synthesized compounds were elucidated on the basis their
spectral and micro analytical data. IR spectrum of syn-
thesized compounds (6–10) gave useful information for
resolving of structures of oxadiazinan-5-ones. All the
1
The H NMR Spectra of the compounds (6–10) exhib-
ited a sharp down field singlet at d 3.20-3.68 attributed to
40-NH proton while the other singlets at 9.14–9.95 ascribed
to 30-NH proton, respectively. The methylene protons in
between ether linkage and ketonic group were displayed as
two distorted doublets in the range of d 3.60–3.87 and
3.87–4.45 ppm due to two protons at CH2 (Ha and Hb). The
phenolic OH in case of (7–10) was found to resonate as
independent singlets at d 10.82, 10.58 and 10.24, 10.58 and
10.25, 10.68 ppm respectively. Aromatic protons appeared
as a multiplet in each case at d 7.32–7.71 ppm for five
Scheme 1 Tentative
mechanism for the formation of
titled compounds 6–10
R'
4
R'
4
3
R'
3
2
1
2
1
-HCN
5
5
6
6
2'
R
O
NH
NH
OH
NH
NH
R
3'
4'
R
1'
O
NH2
NH
6'
5'
O
NC
NC
O
O
Cyanoacetic acid hydrazide
R
H
H
R'
H
3-OH
R
R'
H
H
H
H
6
7
8
9
1
2
3
4
3-OH
4-OH
H
4-OH
4-OH
5-OH
2-OH
2-OH
4-OH
5-OH
2-OH
2-OH
5
10
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Med Chem Res (2013) 22:3085–3095
3087
Table 1 Antibacterial activity
of oxadiazinan-5-one
compounds 6–10
Compounds
Diameter of zone of inhibition (mm)
Gram-positive bacteria Gram-negative bacteria
S. Pyogenes
MRSA*
P. aeruginosa
K. pneumoniae
E. coli
6
19.1 0.2
19.4 0.2
19.3 0.2
18.1 0.1
18.6 0.2
22.0 0.4
–
18.2 0.4
19.1 0.4
18.3 0.4
17.1 0.2
17.6 0.5
21.0 0.4
–
24.2 0.4
26.2 0.4
24.4 0.4
24.4 0.2
24.1 0.2
31.5 0.2
–
14.1 0.4
15.1 0.2
14.6 0.2
14.1 0.2
14.8 0.4
19.5 0.2
–
17.4 0.2
25.4 0.2
18.4 0.4
17.2 0.5
17.2 0.1
27.5 0.4
–
7
Positive control (standard):
Ciprofloxacin and negative
control (DMSO) measured by
the halo zone test (unit, mm)
8
9
10
Standard
DMSO
* Methicillin resistant
Staphylococcus aureus (MRSA
?ve)
Table 2 MIC and MBC results of oxadiazinan-5-one compounds 6–10 positive control ciprofloxacin
Compounds
Gram-positive bacteria
Gram-negative bacteria
S. Pyogenes
MRSA*
P. aeruginosa
K. pneumoniae
E. coli
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
6
25
50
25
25
50
25
25
100
25
25
100
25
25
50
25
7
12.5
25
12.5
25
12.5
25
12.5
25
12.5
50
8
50
50
100
[100
100
25
50
100
100
100
25
9
50
100
100
12.5
50
100
100
12.5
50
50
100
100
25
50
10
50
50
50
50
50
Standard
6.2
6.2
12.5
6.2
6.2
MIC (lg/ml) minimum inhibitory concentration, i.e., the lowest concentration of the compound to inhibit the growth of bacteria completely,
MBC (lg/ml) minimum bacterial concentration, i.e., the lowest concentration of the compound for killing the bacteria completely
protons (6), d 6.76–7.24 ppm for four protons (7), d
6.72–7.59 ppm for four protons (8), d 6.72–7.59 ppm for
three protons (9) and d 6.73–6.80 and 7.25 ppm for three
(10). 13C NMR spectra provided a firm support for the
formation of compounds and their signals were in good
agreement with proposed structures of synthesized com-
pounds. All the compounds exhibited signals at d
165.5–173.3 due to (C=O). Signals at d 72.8–73.8 due to
CH2 of the oxadiazinan-5-one ring were also observed.
Further evidences of structures (6–10) were given by (?)-
ESI mass spectroscopy. The mass spectra of (6), (7), (8),
(9), and (10) showed the molecular ion peak at m/z 192
(M)?, m/z 209 (M ? H)?, m/z 231 (M ? Na)?, m/z 258
(M ? H ? Na)?, and m/z 236 (M ? 2H)?, respectively.
Table 3 Antifungal activity of oxadiazinan-5-one compounds 6–10
positive control (Amphotericin B) and negative control (DMSO)
measured in terms of zone (unit, mm)
Compounds CA
AF
TM
PM
6
21.4 0.4 17.2 0.4 14.5 0.2 14.1 0.4
25.5 0.2 22.4 0.4 17.2 0.2 14.5 0.4
22.4 0.4 19.4 0.4 16.3 0.2 15.1 0.4
20.2 0.2 16.2 0.5 14.1 0.4 11.1 0.2
21.4 0.3 16.8 0.8 14.8 0.2 11.7 0.4
30.5 0.2 26.5 0.2 23.5 0.3 21.5 0.5
7
8
9
10
Standard
DMSO
–
–
–
–
Diameter of zone of inhibition (mm)
CA Candida albicans, AF Aspergillus fumigatus, TM Trichophyton
entagrophytes, PM Penicillium marneffei
Antimicrobial study
zone of inhibition 25.4 0.2 against Escherichia coli
(ATCC-8739) (bacterial strain) while as the MIC of com-
pound 7 is 12.5 lg/mL with zone of inhibition 22.4 0.4
against Aspergillus fumigates (fungal strain). The zones of
inhibition (mm) of every compound against Gram-positive
and Gram-negative bacteria are given in Table 1. The
zones of inhibition (mm) of every compound against fungal
The evaluation of preliminary antimicrobial testing of
compounds (6–10) is presented in Tables 1, 2, 3, and 4.
The in vitro antimicrobial activity demonstrated that the
compound (7) was most active among the five compounds
in terms of antibacterial activity as well as antifungal
activity. The MIC of compound (7) is 12.5 lg/mL with
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Med Chem Res (2013) 22:3085–3095
antibacterial studies where compound (7) (Figs. 2, 3) was
found to be the most active compound against E. coli strain
compared with the standard drug (Figs. 4, 5) shows almost
similar behavior in term of docking studies as well as
Ramachandran plot. The activity of the compound (7) can
also be explained, on structural basis (Fig. 6), which
involves removal of hydrogen atom by hydroxyl group on
the aromatic ring at the meta position as compared to ortho/
para position. The increase in activity may be regarded due
to low surface area which facilitates the guest relation with
Table 4 MIC and MFC of oxadiazinan-5-one compounds 6–10
CA
AF
TM
PM
Compounds MIC MFC MIC MFC MIC MFC MIC MFC
6
25
25
25
25
50
25
25
50
25
25
100
50
7
12.5
25
12.5
50
12.5
25
12.5
50
8
100
100
100
25
100
100
100
25
50
100
[100
100
25
9
50
50
50
100
100
25
50
10
50
50
50
50
Standard
6.25
12.5
6.25
12.5
CA Candida albicans, AF Aspergillus fumigatus, TM Trichophyton
mentagrophytes, PM Penicillium marneffei, MIC (lg/ml) minimum
inhibitory concentration, i.e., the lowest concentration of the com-
pound to inhibit the growth of fungus completely, MFC (lg/ml)
minimum fungicidal concentration, i.e., the lowest concentration of
the compounds for killing the fungus completely
strains are given in Table 3. MIC results of both bacterial
and fungal strains are shown in Tables 2 and 4. It may be
concluded that compound (7) bearing OH group at meta
position of the benzene ring is most potent followed by
compounds (9), (8), (6), and (10) bearing OH groups at
different position, respectively. The high potency of (7)
may be attributed to the presence of H-bond acceptor with
proper orientation.
Fig. 2 Binding mode of compound (ligand) 7 in the binding site of
P1 crystal form of E. coli ClpS (301Fpbd), it has 5 hydrogen bonds, it
shows hydrogen bond one between A:CYS101:HG–LIGAND:N and
LIGAND:H–A:CYS73:SG, and another hydrogen bonds between
A:LEU32:H–LIGAND:O, A:VAL88:H–LIGAND:O and LIGAND:
H–A:LYS84:O
The antibacterial data of compounds (6–10) was further
investigated on structural basis, molecular modeling and
docking study of tested compounds into P1 crystal form of
pathogenic protein ClpS (301F pdb) using MVD and Dis-
covery studio software was performed to predict the
affinity, orientation and surrounding surface (Fig. 1) of the
synthesized compounds at the active site. The different
bonds i.e., hydrogen bonds, van der Wall forces and
hydrophobic behavior formed with amino acids were in
good agreement with the predicted binding affinities
obtained by molecular docking studies as verified by
Fig. 3 Ramachandran plot of ClpS. The figure shows the pairs of
(psi, phi) angles for all active binding amino acid residues with
compound 7
Fig. 1 Showing surface consist of different intra and interactions
around the ligand (7)
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ring further decrease activity due to the lack of proper
orientation which ceases available lone pair to make
hydrogen bond with receptor (amino acids). Moreover
compound (10) (Fig. 6b) makes intramolecular hydrogen
bond between the ether linkage oxygen atom and phenolic
group at the position of 6 which checked the formation of
hydrogen bond with amino acids of pathogenic protein.
Docking studies
In the present investigation, an attempt was made to know
the ligand (synthesized compounds)-receptor interaction of
oxadiazinanones against pathogenic protein (ClpS; 301F
pdb) by performing docking studies using discovery studio
3.1 (Accelrys Software Inc, 2011), Molego Virtual Docker
(Thomsen and Christensen, 2006) and LigandScout
(Wolber et al., 2006). These are one of the most accurate
and commonly used tools to predict the binding orientation,
energy calculation, and surrounding interactions between
compound and protein. All the compounds were designed
and structure was analyzed using Chem Draw Ultra 3D
software and then these structures were energetically
minimized using MOPAC, and coordinates of compounds
were checked using PRODRG. The P1 crystal structure of
ClpS (301F pdb) was taken from PDB (www.rcsb.org/pdb).
Docking studies were carried out to distinguish the nature
and the extent of interaction of title product oxadiazina-
nones with P1 crystal of pathogenic strain. The docking
studies of a typical compound (7) are presented in Table 5.
It shows the interaction between protein and compound (7)
in the form of MolDock scores, hydrogen bond interac-
tions. The obtained data indicates that the MolDock score
is minimum indicating more than 75 % host–guest relation
between compound-receptor. Therefore, the compound (7)
shows maximum inhibitor action. Moreover, the obtained
docking result of (7) mentioned in Table 1 along with bond
distance showed three hydrogen bond interactions between
compound and receptor. The maximum inhibitor action and
drug-likeness of (7) can be predicted using Lipinski Rule of
Five (Table 7) which explains the molecule in terms of
drug and it must contain minimum number hydrogen
donors group (\5) and lipophilicity (Log P [ 5). Com-
pound (7) fully satisfies this criteria of drug, showing
almost similar behavior in term of docking studies, Ra-
machandran plot as well as structure performance (Figs. 2,
3) as compare with the reference drug (Figs. 4, 5).
Fig. 4 Binding mode of standard drug Ciprofloxacin (ligand) in the
binding site of P1 crystal form of E. coli ClpS (301Fpbd), it has 8
hydrogen bonds, it shows hydrogen bonds between A:LEU32:
H–LIGAND:F, A:LYS84:HZ1–A:HOH169:O, A:LYS84:HZ3–A:HO
H121:O, A:LYS84:HZ3–A:HOH169:O, A:LEU100:H–A:HOH1:O,
A:HOH1:O–A:VAL33:O, A:HOH120:O–A:VAL33:O and A:HOH
120:O–A:ASP35:OD2
Fig. 5 Ramachandran plot of ClpS. The figure shows the pairs of
(psi, phi) angles for all active binding amino acid residues with
standard antibiotics Ciprofloxacin
Antioxidant study
receptor (host) in case of ortho/para positions. There are
either large surface area or steric resistance (Fig. 6b, d)
ceasing bonding with receptor. Moreover introduction of
two hydroxyl groups on ortho-para positions on aromatic
The synthesized compounds (6–10) were subjected to free
radical scavenging activity by DPPH methods (Nami et al.,
2012; Shirwaington et al., 2004). This model of scavenging
activity by DPPH radical is extensively applied to evaluate
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Fig. 6 Ligand interaction a showing hydrogen bond acceptor (HBA),
hydrogen bond donor (HBD), aromatic (AR), and hydrophobic
behavior (H), b showing intramolecular hydrogen bond between OH
of Ar and ether linkage including HBA, HBD, AR, and H,
c ciprofloxacin showing HBA, HBD, AR, and H and d showing
steric resistance between methyl and 2-OH of Aromatic
Table 5 Amino acid residues and atoms of ligand 7 involved in hydrogen bonding, bond length, MolDock, ReRank, and HBond calculation
˚
Distance (A)
Residues
Atom ID
Pdb atom name
Ligand atom
MolDock score
rerank score
HBond
Cys
Cys
Leu
Ala
101 (A)
73 (A)
32 (A)
71 (A)
S (donor)
S (donor)
O (acceptor)
O (both)
3.22
3.52
2.01
3.59
-72.418
-57.34
-0.8004
N (donor)
O (acceptor)
O (acceptor)
O (both)
the antioxidant activity in shorter time that as compared to
other methods. The odd electron in the DPPH free radical
gives a strong absorption band at k = 517 nm, which is
purple in color. This property makes it suitable for spec-
trometric studies. The DPPH assay has often been used to
estimate the antiradical activity of antioxidant. The free
radical scavenging capabilities of the compounds were
measured in term of hydrogen donating of free radical
scavenging ability after adding methanolic solution of
(DPPH) to the sample solution of different concentrations.
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The synthesized compounds react with DPPH and convert
it into 1,1-diphenyl-2-picrylhydrazine. The extent of dec-
olourization is an indicative of potentiality of antioxidant
behavior of a particular compound. Ascorbic acid was used
as the reference compound. All the tests were performed in
triplicate. The compounds (6–10) were tested for antioxi-
dant property by DPPH method. The compound (7)
showed the highest IC50 value in antioxidant activity fol-
lowed by the compounds (9), (8), (6), and (10) respectively
and result were reported in Table 6.
Materials and methods
Melting points were determined on a Kofler apparatus
and are uncorrected. The IR spectra were recorded on
KBr pellets with Shimadzu IR-408 Perkin-Elmer 1800
1
(FTIR) and its values are given in cm-1. The H NMR
and 13C NMR spectra were run in DMSO-d6 on a Bruker
Avance-II 400 MHz instrument with TMS as internal
standard: J values are in Hertz. Chemical shifts are
reported in ppm (d) relative to the solvent peak. Mass
spectra were recorded on a JEOL D-300 mass spec-
trometer. Elemental analyzes (C, H, N) were conducted
using Carlo Erba analyzer model 1108. Thin layer
chromatography (TLC) glass plates (20 9 5) were
coated with silica gel (E-Merck G254, 0.5 mm thick-
ness) and exposed to iodine vapors to check the purity
as well as the progress of reaction. Acetophenone,
m-hydroxy acetophenone, p-hydroxy acetophenone, and
2,4-dihydroxy acetophenone were purchased from
Sigma–Aldrich Chemicals Pvt. Ltd. Other chemicals
used were of analytical grade without further purifica-
tion. Cyanoacetic acid hydrazide reagent was synthe-
sized by usual method.
Experimental
General method for the preparation of oxadiazinan-5-
one derivatives (6–10)
To a solution of acetophenone or its derivatives (1–5),
(1 mmol) in acetic acid (10 mL) was added cyano acetic
hydrazide in equimolar ratio in same solvent. The reaction
mixture was stirred for 4 h. The progress as well as purity
of reaction was monitored by TLC. On completion of
reaction the precipitate was formed. The precipitate was
suspended in water (30 mL), neutralized with saturated
aqueous NaHCO3 and filtered using suction. It was washed
with water and ether was added. The ethereal layer was
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2-(2,4-dihydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-
washed with water and dried over anhydrous sodium sul-
fate. Crude product was obtained by removing the solvent
and was crystallized from chloroform–methanol to furnish
oxadiazinanone-5-one (6–10).
5-one (9)
It was crystallized with CHCl3–EtOH as yellowish colored
solid; Yield: 65 %, m.p. 185–87 °C; Anal. Calc. for
C10H12N2O4; C, 53.55; H, 5.40; N, 12.50; O, 28.55; found:
2-methyl-2-phenyl-1,3,4-oxadiazinan-5-one (6)
C, 53.51; H, 5.43; N, 12.51; O, 28.52; IR m
cm-1: 3466
KBr
Max
(OH), 3250 (N–H), 3385 (N–H), 1695 (C=O), 1470 (N–N),
1140 (–O–); 1H NMR (400 MHz, DMSO-d6, d, ppm): 1.65
(3H, s, CH3), 3.33 (1H, s, NH, D2O exchangeable), 3.75,
4.11 (Ha, Hb, dis. d, CH2), 6.72–6.78 (2H, m, H2, H6, Ar–H),
7.51–7.59 (1H, d, Ar–H), 9.28 (1H, s, NH, D2O exchange-
able), 10.24 (1H, s, 4-OH, D2O exchangeable), 10.58 (1H, s,
2-OH, D2O exchangeable) 13C NMR (100 MHz, DMSO-d6,
d, ppm): 25.2 (CH3), 73.3 (C60), 91.3 (C20), 108.1 (C3),
113.4 (C5), 121.0 (C1), 129.3 (C6), 153.1 (C2), 157.5 (C4)
and 173.1 (C=O); MS (ES?) m/z: 235(M ? H)?.
It was crystallized with CHCl3–EtOH as white colored
solid; Yield: 65 %, m.p. 162–65 °C; Anal. Calc. for
C10H12N2O2: C, 62.49; H, 6.29; N, 14.57; O, 16.65. Found:
KBr
Max
C, 62.49; H, 6.30; N, 14.48; O, 16.73; IR m
cm-1: 3244
(N–H), 3382 (N–H), 1680 (conjugated C=O), 1465 (N–N),
1110 (–O–); 1H NMR (400 MHz, DMSO-d6, d, ppm): 1.80
(3H, s, CH3), 3.28 (1H, s, NH, D2O exchangeable), 3.74,
4.0 (Ha, Hb dis. d, CH2), 7.32–7.71 (5H, m, Ar–H), 9.95
(1H, s, NH, D2O exchangeable); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 25.1 (CH3), 72.8 (C60), 94.1 (C20),
126.3 (C2 & 6), 128.4 (C3 and 5), and 129 (C4), 149.6 (C1)
and 165.5 (C=O); MS (ES?) m/z: 192 (M)?.
2-(2,5-dihydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-
5-one (10)
2-(3-hydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-
It was crystallized with CHCl3–EtOH as yellowish colored
one (7)
solid; Yield: 65 %, m.p. 197–200 °C; Anal. Calc. for
C10H12N2O4; C, 53.55; H, 5.40; N, 12.50; O, 28.55; found:
The compound (7) was crystallized with CHCl3–MeOH as
yellowish white colored solid; Yield: 70 %, m.p.172–75 °C;
Anal. Calc. for C10H12N2O3; C, 57.67; H, 5.81; N, 13.46; O,
C, 53.52; H, 5.43; N, 12.49; 0, 28.54; IR m
cm-1: 3465
KBr
Max
(OH), 3248 (N–H), 3386 (N–H), 1693 (C=O), 1471 (N–N),
1140 (–O–); 1H NMR (400 MHz, DMSO-d6, d, ppm): 1.67
(3H, s, CH3), 3.68 (1H, s, NH, D2O exchangeable), 3.76,
4.13 (Ha, Hb, dis. d, CH2), 6.73–6.80 (2H, m, H3, H4, Ar–H),
7.25 (1H, d, H6 Ar–H), 9.96 (1H, s, NH, D2O exchangeable),
10.25 (1H, s, 5-OH, D2O exchangeable), 10.68 (1H, s,
2-OH, D2O exchangeable) 13C NMR (100 MHz, DMSO-d6,
d, ppm): 25.3 (CH3), 73.8 (C60), 91.5 (C20), 108.1 (C3),
113.8 (C4), 121.9 (C1), 129.9 (C6), 153.5 (C2), 157.9 (C5)
and 173.3 (C=O); MS (ES?) m/z: 257 (M ? Na)?.
KBr
23.06; found: C, 57.69; H, 5.81; N, 13.45; O, 23.09; IR m
Max
cm-1: IR m cm-1: 3435 (OH), 3248 (N–H), 3383 (N–H), 1685
(C=O), 1468 (N–N), 1120 (–O–); 1H NMR (400 MHz,
DMSO-d6, d, ppm): 1.81 (3H, s, CH3), 3.67 (1H, s, NH, D2O
exchangeable), 3.87, 3.94 (Ha, Hb, dis. d, CH2), 6.76–7.24 (4H,
m, H2, H4, H5, H6, Ar–H), 9.14(1H, s, NH, D2O exchangeable),
10.82 (1H, s, 3-OH, D2O exchangeable); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 25.2 (CH3), 73.2 (C60), 93.0 (C20), 113.2
(C2), 113.4 (C4), 115.3 (C6), 117.7 (C5), 149.9 (C1), 159.2
(C3) and 172.7 (C=O); MS (ES?) m/z: 209 (M ? H)?.
Antimicrobial activity
2-(4-hydroxyphenyl)-2-methyl-1,3,4-oxadiazinan-5-
The in vitro antimicrobial activities of acetophenone ox-
adiazinanones (6–10) were evaluated using the bacterial
cultures of Staphylococcus Pyogenes (clinically isolated),
Methicillin resistant Staphylococcus aureus (MRSA ?Ve),
Pseudomonas aeruginosa (ATCC-9029), Klebsiella pneu-
monia (clinically isolated), and Escherichia coli (ATCC-
25922) and fungal cultures of Candida albicans (ATCC-
90028), Candida albicans, Aspergillus fumigates, and
Aspergillus niger by the disk diffusion method
(Cruickshank et al., 1975; Collins, 1976; Khan, 1997;
Varma, 1998) and subsequently the Minimum Inhibitory
Concentration (MIC) of all the compounds were deter-
mined. Ciprofloxacin was used as standard drug in case of
bacteria and Amphotericin B in case of fungi, whereas the
disk poured in DMSO (dimethyl sulphoxide) was used as a
one (8)
It was crystallized with CHCl3–MeOH as white colored solid;
Yield: 70 %, m.p. 193–95 °C; Anal. Calc. for C10H12N2O3;
C, 57.67; H, 5.81; N, 13.46; O, 23.06; found: C, 57.70; H,
5.84; N, 13.43; O, 23.03; IR mKMBarx cm-1: 3440 (OH), 3249 (N–
1
H), 3384 (N–H), 1690 (C=O), 1470 (N–N), 1134 (–O–); H
NMR (400 MHz, DMSO-d6, d, ppm): 1.73 (3H, s, CH3), 3.33
(1H, s, NH, D2O exchangeable), 3.60, 3.87 (Ha, Hb, dis. d,
CH2), 6.72-6.78 (2H, m, H3, H5, Ar–H), 7.49–7.59 (2H, m,
H2, H6, Ar–H), 9.29 (1H, s, NH, D2O exchangeable), 10.58
(1H, s, 4-OH, D2O exchangeable); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 25.0 (CH3), 73.4 (C60), 93.5 (C20), 113.4
(C3), 115.5 (C5), 128.4 (C2), 128.6 (C6), 139.1 (C1), 158.5
(C4) and 173.0 (C=O); MS (ES?) m/z: 231 (M ? Na)?.
123

Med Chem Res (2013) 22:3085–3095
3093
negative control. The MIC was assisted by the macro
dilution test using standard inoculums of 105 c.f.u./mL.
The susceptibility was assessed on the basis of diameter of
zone of inhibition against Gram-positive and Gram-nega-
tive strains of bacteria. Inhibition zones were measured and
compared with the controls. The bacterial zones of inhi-
bition values are given in Table 3.
taken from each tube and spread on agar plates. The
number of c.f.u. were counted after 48 h of incubation at
35 °C. MFC was defined as the lowest drug concentration
at which 99.9 % of the inoculums were killed. The mini-
mum inhibitory concentration and minimum fungicidal
concentration are given in Table 4.
Minimum inhibitory concentrations (MICs) were deter-
mined by broth dilution technique. The nutrient broth,
which contained logarithmic serially two fold diluted
amount of test compound and controls were inoculated
with approximately 5 9 105 c.f.u./mL of actively dividing
bacteria cells. The cultures were incubated for 24 h at
37 °C, and the growth was monitored visually and spec-
trophotometrically. The lowest concentration (highest
dilution) required to arrest the growth of bacteria was
regarded as minimum inhibitory concentration (MIC). To
obtain the minimum bacterial concentration (MBC),
0.1 mL volume was taken from each tube and spread on
agar plates. The number of c.f.u. was counted after 18–24 h
of incubation at 35 °C. MBC was defined as the lowest
drug concentration at which 99.9 % of the inoculums were
killed. The minimum inhibitory concentration and mini-
mum bactericidal concentration are summarized in
Table 4.
Antioxidant studies
The compounds (6–10) were tested for their antioxidant
property using 1,1-diphenylpicrylhydrazyl (DPPH) method.
In this procedure, drug stock solution (1 mg/mL) was
diluted to final concentration of 2, 4, 6, 8, 10 and 12 in
methanol. Methanolic DPPH solution (1 mL, 0.3 mmol)
was added to 3.0 mL of drug solution of different concen-
trations. The tube was kept at an ambient temperature for
30 min and the absorbance was measured at 517 nm. The
scavenging activity was calculated by following formula: %
inhibition = [(AControl - ASample)/AControl] 9 100. Where
AControl is the absorbance of the L-ascorbic acid (Standard)
and ASample is the absorbance of different compounds. The
methanolic DPPH solution (1 mL, 0.3 mM) was used as
control. The inhibitory concentration (IC50) value represents
the concentration required to exhibit 50 % antioxidant
activity (Fig. 7) The IC50 values were calculated by the
linear regression of plots where the abscissa represents the
concentration of the compounds (lg/mL). Explicitly, IC50 is
the average percentage of antioxidant activity. Results in the
form of percent inhibition are tabulated in the Table 6.
Antifungal studies
Antifungal activity was also done by disk diffusion method
using Candida albicans, Aspergillus, fumigatus, Penicil-
lium marneffei and Trichophyton mentagrophytes (recul-
tured) in DMSO by agar diffusion method. Sabourands
agar media was prepared by dissolving peptone (1 g),
D-glucose (4 g), and agar (2 g) in distilled water (100 mL)
and adjusting pH to 5.7. Normal saline was used to make a
suspension of spore of fungal strain for lawning. A loopful
of particular fungal strain was transferred to 3 mL saline to
get a suspension of corresponding species. Twenty milli-
liters of agar media was poured into each petri dish. Excess
of suspension was decanted and the plates were dried by
placing in an incubator at 37 °C for 1 h. Using an agar
punch, wells were made and labeled. A control was also
prepared in triplicate and maintained at 37 °C for
3–4 days. The fungal activity of each compound was
compared with Amphotericin B used as standard drug.
Inhibition zones were measured and compared with the
control. The fungal zones of inhibition values are given in
Table 5. The nutrient broth, which contained logarithmic
serially two fold diluted amount of test compound and
controls was inoculated with approximately 1.6 9 104–
6 9 104 c.f.u./mL. The cultures were incubated for 48 h at
35 °C and the growth was monitored. To obtain the min-
imum fungicidal concentration (MFC), 0.1 mL volume was
In silico studies
The retrieved protein ClpS (301F pdb) was improved using
import and preparation option of MVD software, and
90
Ascorbic Acid
6
7
80
8
9
70
10
60
50
40
30
20
10
0
2
4
6
8
10
Conc.µ/ml
Fig. 7 Antioxidant activity of compounds 6–10
123

3094
Med Chem Res (2013) 22:3085–3095
Table 7 In silico ADMET properties of synthesized compounds
Compound
Mutagenic
Tumerogenic
Irritation
Reproductive effect
cLog P
Solubility
Drug-likeness
Drug score
6
No
No
No
No
No
No
No
No
No
No
No
No
0.19
-0.10
-0.10
-0.40
-0.40
-1.28
-0.98
-0.98
-0.68
-0.68
4.18
2.34
1.58
2.28
2.56
0.96
0.93
0.57
0.93
0.94
7
No
No
8
Medium
No
Medium
No
9
10
No
No
Cruickshank R, Duguid JP, Marmion BP, Swain RHA (eds.) (1975)
Medicinal microbiology 12th edn., vol. 2 Churchill Livingstone,
London, pp 196–202
Hassan EM, Yardley PA, Theodore SS, Wayne PA (2002) 3-Thioxo-
[1,2,4]-oxadiazinan-5-one derivatives.US0061883Al. A61K
031/5395; C07D 273/04
Hassan EM, Theodore SS, Magid A-G, John BA, Sie-Yearl C,
Geraldin MR, Mar-lee M, John BL, Steven AJ, Elaine QM
(2004) Design, synthesis, and biological evaluation of thio-
containing compounds with serum HDL-cholesterol-elevating
properties. J Med Chem 47:681–695. doi:10.1021/jm030219z
Hitchcock SR, Nora GP, Casper DM, Squire MD, Maroules CD,
Ferrence GM, Szczepura LF, Standard JM (2001) X-Ray
crystallographic and 13C nuclear magnetic resonance studies of
3,4,5,6-tetrahydro-2H-1,3,4-oxadiazin-2-ones derived from
ephedrine and pseudoephedrine. Tetrahedron 57:9789–9798.
doi:10.1016/S0040-4020(01)00967-X
Khan ZK (1997) In vitro and vivo screening techniques for bioactivity
screening and evaluation, Proc. Int. Workshop UNIDO-CDRI,
210–211
Nami SAA, Alam M, Husain A, Parveen M (2012) Synthesis,
characterization, thermal and antioxidant studies of potassium
dihydrobisphenothiazinyl borate and its transition metal com-
plexes. Spectrochimica Acta Part A Mol Biomol Spectrosc
96:729–735. doi:10.1016/j.saa.2012.07.033
Table 8 Lipinski rule of five for compounds 6–10
Molecular
weight
(compound)
Hydrogen
bond (donor) bond
(acceptor)
Hydrogen
Log P Molar
refractivity
192.00 (6)
208.00 (7)
208.00 (8)
224.00 (9)
224.00 (10)
2
3
3
4
4
3
0.514 51.025
0.219 52.689
0.219 52.688
-0.075 54.353
-0.075 54.353
4
4
5
5
missing bond order, hybridization state, angle, and flexi-
bility for achieving reliable potential binding site in
receptor. The energy minimized ligands (synthesized
compounds) drawn with ChemDrwa Ultra (2D & 3D) were
predicted ADMET Table 7, drug-likeness and ligand
structural properties (Fig. 6, c for ciprofloxacin) achieved
through organic chemistry portal Lipinski’s Rule of Five
Table 8 and LigandScout, respectively. Discovery studio,
MVD, and Ligand scout were used to perform molecular
docking and energy profile of ligand-receptor interaction,
independently.
Rajganarendar E, Rami RAS, Shaik FP (2010) Synthesis of 1,3,5-
triazinane-2-thiones and 1,3,5-oxadiazinane-4-thiones linked
with isoxazoles. Ind J Chem 49(B):119–222
Ranganarendar E, Afzal M, Ramu K (2005) Microwave-assisted
synthesis of some new isoxazolyl-[1, 3, 5]-triazinan-2-ones and
isoxazolyl-[1, 3, 5]-oxadiazinan-4-ones in dry media. Indian J
Chem 44(B):376–380
Conclusions
Ried W, Schleimer B (1958) Cyan-acethydrazid zur Darstellung von
Stickstoff-Heterocyclen, II. 1-Acyl-3.5-dimethyl-pyrazole als
Acylierungsmittel. Angew Chem 70:164. doi:10.1002/ange.
19580700607
Rodrigues A, Olivato PR, Rittner R (2005) Synthesis of (5R)-4-
methyl-5-phenyl-1,3,4-oxadiazinan-2-one and Some N-Acyl
derivatives from (R)-phenylglycine. Synthesis 2:578–582. doi:
10.1055/s-2005-872101
Rosling A, Fulop F, Sillanpaa R, Mattinen J (1997) 2-[Arylamino
(imino)]perhydropyrido-[1,2-d] [1,3,4]oxadiazine and 2-[Aryl-
amino(imino)]-perhydropyrrolo[1,2-d]-[1,3,4] oxadiazine: het-
erocyclic ring systems involving a bridge-head nitrogen. J
Heterocycles 45:242–927. doi:10.3987/COM-97-7760
Shirwaington A, Rajendran K, Kumar CD (2004) In vitro antioxidant
studies of Annona squamosa Linn. leaves. Indian J Exp Biol
42:803–807
The present work reports the in vitro antimicrobial and
antioxidant activities of synthesized oxadiazinan-5-one
derivatives of acetophenones. It is inferred from biological
and in silico docking studies that all compounds exhibit
substantial antibacterial and antifungal activity against
different strains of bacterium and fungus, respectively.
Moreover, oxadiazinanones moiety also showed good
antioxidant activity.
References
Sibi MP, Liu M (2001) Enantioselective conjugate addition of
hydroxylamines to pyrazolidinone acrylamides. Org Lett 3:4181–
4184. doi:10.1021/ol016807t
Sibi MP, Venketraman L, Liu M, Jasperse CP (2001) A new approach
to enantiocontrol and enantioselectivity amplification: chiral
Accelrys Software Inc (2011) Discovery studio modeling environ-
ment Release 3.1 San Diego: Accelrys Software Inc
Collins AH (1976) Microbiological methods, 2nd edn. Butterworth,
London
123

Med Chem Res (2013) 22:3085–3095
3095
relay in Diels–Alder reactions. J Am Chem Soc 123:8444–8445.
doi:10.1021/ja016396b
Tomlin CDS (2006) Ed. The pesticide manual. 14th edn. British Crop
Protection Council, Alton; Hampshire, UK. 1350
Tan J, James JG, Robert MH (2007) Agonist actions of neonicotinoids
on nicotinic acetylcholine receptors expressed by cockroach
neurons. Neurotoxicology 28:829–842. doi:10.1016/j.neuro.
2007.04.002
Thomsen R, Christensen MH (2006) MolDock: A new technique for
high-accuracy molecular docking. J Med Chem 11:3315–3321.
doi:10.1021/jm051197e
Tomizawa M, Casida JE (2005) Neonicotinoid insecticide toxicology:
mechanisms of Selective Action. Annu Rev Pharmacol Toxicol
45:247–268. doi:10.1146/annurev.pharmtox.45.120403.095930
Varma RS (1998), Antifungal Agents: Past, present and future
prospects; ed., National Academy of Chemistry & Biology:
Lucknow, India
Wolber G, Dornhofer AA, Langer T (2006) Efficient overlay of small
organic molecules using 3D pharmacophores. J Comput Aided
Mol Des 20:773–788. doi:10.1007/s10822-006-9078-7
Zukerman JS, Lucas SM, Alessandro R, Elisangela V, Paulo OR (2009)
4-Methyl-3-(2-phenoxy-acet-yl)-5-phenyl-1,3,4-oxadiazinan-
2-one. Acta Cryst 65(E):1468. doi:10.1107/S1600536809020285
123