Synthesis, spectral studies and in vitro antibacterial evaluation of triaza and dioxa aza spiro derivatives

Article abstract of DOI:10.14233/ajchem.2013.12976

Six compounds 7,9-diphenyl-1,4-dioxa-8-azaspiro[4.5]decane 1-6 have been synthesized by Mannich reaction and cyclo condensation. The structures and stereochemistry established by IR, NMR studies. The purities were checked by elemental analysis. The synthesized compounds 1-6 adopt chair conformation with equatorial orientation of the aryl groups. All the compounds were screened for their antibacterial activity against Proteus mirabilis, Klebsiella oxytoca, Staphylococcus aureus and Salmonella paratyphi. The compound 5 exhibited excellent in vitro antibacterial activity in all species.

Full text of DOI:10.14233/ajchem.2013.12976

Asian Journal of Chemistry; Vol. 28, No. 8 (2016), 1777-1782
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19821
Anticoagulant Activity and Molecular Docking of 7,9-bis(4-Chlorophenyl)-
6-methyl-1,4-dioxa-8-azaspiro[4.5]decane through Target Protein Thrombin
1,*
2
3
3
N. MANIVANNAN , S. SUGUNAKALA , B. ELANCHEZHIAN and G. SELVANATHAN
1Department of Chemistry, V.R.S. College of Engineering and Technology, Arasur, Villupuram-607 107, India
2Department of Bio-informatics, A.V.C. College (Autonomous), Mannampandal, Mayiladuthurai-609 305, India
3Department of Chemistry, A.V.C. College (Autonomous), Mannampandal, Mayiladuthurai-609 305, India
*Corresponding author: E-mail: nmrmani@gmail.com
Received: 5 January 2016;
Accepted: 16 March 2016;
Published online: 30 April 2016;
AJC-17890
The present study reported the synthesis and anticoagulant activity of spiro compounds, the potential compound was subjected to study
their binding mode and interaction using bioinformatics tools. Among the five compounds tested, 7,9-bis(4-chlorophenyl)-6-methyl-
1,4,8-triaza spiro[4.5]decane has prolonged activated partial thromboplastin time and prothrombin time as 22.7s and 18.5s at 25 µg mL-1,
respectively. Hence, the mandatory way study was carried out for this compound by using bioinformatics method. The interaction results
shows that this compound binds in the active site of the target protein thrombin which is similar to that of existing inhibitor and it may also
considered as an anticoagulant drug in future.
Keywords: Anticoagulant, Prothrombin, Spiro and Thrombin inhibitor.
anticoagulant from the class of the direct thrombin inhibitors.
It has been studied for various clinical indications and in some
cases it offers an alternative to warfarin as the preferred orally
administered anticoagulant (blood thinner) [8,9].
Warfarin (4) used for preventing thrombosis as well as
embolism and inhibition of metastasis [10-13]. It is widely
being used to prevent strokes. Warfarin, as an anticoagulant,
suppresses the formation of calcium dependent clotting factors
II, VII, IX and X. It diminishes the regeneration of vitamin K
and vitamin K hydroquinone in the tissues that inhibits the
vitamin K-dependent carboxylation activity of the glutamyl
carboxylase enzyme. Inhibition of carboxylation stops the
clotting factors to be active [14-16].
Betrixaban (5) is an anticoagulant drug which acts as a
direct factor Xa inhibitor [17]. It is potent, orally active and
highly selective for factor Xa. Betrixaban has undergone human
clinical trials for prevention of embolism after knee surgery
[18] and prevention of stroke following atrial fibrillation [19].
Heparin (6) is a highly sulfated glycosaminoglycan,
widely used as an injectable anticoagulant and has the highest
negative charge density of any known biological molecule [20].
It can also be used to form an inner anticoagulant surface on
various experimental and medical devices such as test tubes
and renal dialysis machines. Although it is used principally in
medicine for anticoagulation, its true physiological role in the
body remains unclear, because blood anticoagulation is
INTRODUCTION
The asymmetric point of the molecules due to the chiral
spiro carbon is one of the significant criterions for their biolo-
gical activities. Spiro compounds have potential biological
properties [1]. A lot of synthetic methodologies have been
developed for constructing these spiro cycles; most of them
prefer cyclo addition or condensation reactions [2-4].
The extrinsic and intrinsic coagulation schemes congregate
at the activation of factor X to Xa.Activated factor X (fXa) play
an important role in the conversion of prothrombin to thrombin,
which creates blood clots by converting factor XI to XIa, VIII
to VIIIa, V to Va, fibrinogen to fibrin and XIII to XIIIa. Thus,
thrombin inhibition is a key mechanism in the coagulation
cascade and is also an attractive target enzyme for the therapy
of atrial fibrillation to avoid thromboembolism [5].
Edoxaban (1) is an anticoagulant drug which acts as a
direct factor Xa inhibitor. It was developed by Daiichi Sankyo
and approved in July 2011 in Japan for prevention of venous
thromboembolisms (VTE) following lower-limb orthopedic
surgery.
Rivaroxaban (2) is an oral anticoagulant invented and
manufactured by Bayer [6,7], and marketed as Xarelto. It is
the first orally active direct factor Xa inhibitor. Rivaroxaban
is well absorbed from the gut and maximum inhibition of factor
Xa occurs 4 h after a dose. Dabigatran (3) (Pradaxa) is an oral

1778 Manivannan et al.
Asian J. Chem.
achieved mostly by heparansulfate proteoglycans derived from
endothelial cells [21]. Few important anticoagulant drug
structures are shown in Fig. 1.
(10 µL) in each concentration (25, 50, 100, 150 and 200 µg/
mL), respectively and incubated for 1 min at 37 °C, followed
by activated partial thromboplastin time reagent (100 µL) was
added to the mixture and incubated for 5 min at 37 °C. There-
after, the clotting was induced by adding 0.02 M calcium
chloride (100 µL) and clotting time was recorded (Method
followed by Pacific hemostasis kit).
Prothrombin time (PT): In prothrombin time, the citrated
normal human plasma (90 µL) was mixed with 10 µL of spiro
compounds 7-11 in each concentration (25, 50, 100, 150 and
200 µg/mL) and incubated for 10 min, respectively. Then,
prothrombin time reagent (200 µL) pre-incubated for 10 min
at 37 °C was added and clotting time was recorded (Method
followed by Pacific hemostasis kit).
EXPERIMENTAL
All the chemicals and reagents used in the present study
were of laboratory grade and purchased from Sigma Aldrich.
Methods: The melting points were determined in open
capillaries and are uncorrected. IR spectra were recorded on
AVATAR 330 FT-IR Thermo Nicolet spectrometer in KBr
pellets. Elemental analysis was performed on an Elemental
Vario EL III (C, H, N and O & Cl) analyzer. The purity of the
compounds was checked by thin layer chromatography (TLC)
1
using silica gel plates. H NMR spectra were recorded on a
Preparation of compounds: The target spiro compounds
7-11 are prepared by Natarajan et al. [22].
BrukerAMX-300 NMR spectrometer operating at 300.03 MHz
for 1H with the following spectral parameters; acquisition time
= around 3.0 s, number of scans = 100 and spectral width =
10,330 Hz. Proton decoupled 13C NMR spectra were recorded
on a Bruker AMX-300 NMR spectrometer operating at 75.07
MHz for 13C with the following spectral parameters; acquisition
time around 0.5 s; number of scans = 1000; spectral width =
30,000 Hz.All NMR measurements were made in 5 mm NMR
tubes using solutions made by dissolving about 10 mg of the
material in 0.5 mL of DMSO-d6.
Retrieval of 3D structure: The 3D structure of the protein
was downloaded from Research Collaborator for Structural
Bioinformatics (RCSB), Protein Databank (PDB, http://
www.pdb.org). The PDB ID of the selected protein was found
to be 3DA9. The Water molecules and ligands attached to the
protein were removed by using Swiss PDB viewer.
Model quality assessment: Quality of the models was
assessed by using Ramachandran plot. It can be used in two
different ways. One is to show in theory which values, or
conformations, of the ψ and ϕ angles are possible for an amino-
acid residue in a protein. A second is to show the empirical
distribution of data points observed in a single structure in
usage for structure validation or else in a database of many
structures. Either case is usually shown against outlines for the
theoretically favoured regions. Here, the structural assessment
was done by using SAVS server (http://nihserver.mbi.ucla.edu/
SAVES) and it provides the values of Phi and Psi angle for the
amino acids present in the target protein.
Anticoagulant activity
Preparation of plasma: Blood was collected from indivi-
dual healthy donor through vein puncture without bleeding or
thrombosis and then it was mixed with 3.8 % tri sodium citrate
at 9:1 ratio. Further it was centrifuged for 20 min and the
plasma was stored at -40 °C until use.
Activated partial thromboplastin time (APTT): For
activated partial thromboplastin time assay, citrated normal
human plasma (90 µL) was mixed with spiro compounds 7-11
O
O
N
O
O
O
H
N
O
OH
R
O
O
N
N
H
N
O
S
NH
S
O
O
NH
N
H
O
Cl
N
O
N
O
Warfarin (4)
O
S
Rivaroxaban (2)
Cl
Edoxaban (1)
O
N
N
N
HO
O
O
S
NH
OH
O
H
N
O
O
N
O
O
O
OH
O
NH
O
S
Cl
O
O
HO
HO
H2N
N
O
O
O
S
O
O
O
Betrixaban (5)
HO
Dabigatran (3)
Heparin (6)
N
NH
Fig. 1. Few important anticoagulant drugs structure

Vol. 28, No. 8 (2016) AnticoagulantActivity and Molecular Docking of 7,9-bis(4-Chlorophenyl)-6-methyl-1,4-dioxa-8-azaspiro[4.5]decane 1779
O
Selection of potential drug candidates: Based on the
experimental results obtained from the analysis of activated
O
partial thromboplastin time and prothrombin time, the compound
CH3
Ethyl methyl ketone
7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-azaspiro[4.5]decane
was chosen, because this compound took prolonged time
requirement for clotting among the studied five compounds.
Docking process: Protein-ligand docking is used to check
the structure, position and orientation of a protein when it
interacts with small molecules like ligands. The software ‘Hex’
is used to calculate the binding energy requirements of ligand
with its receptors. In HEX, full rotation search mode is used
and shape only correlation type for docking. The docking
parameters employed are as follows:
Search mode: Full rotation; Correlation type: Shape only;
Radial filter: None; Post processing: None; Grid dimension:
0.6; Solution: 500, samples: 642; Receptor range: 180, samples:
642; Ligand range: 180, samples: 128; Twist range: 360;
Distance range: 40; Scan step: 1.0; Sub step: 2.
Ethanol
∆
CHO
Aldehyde
X
X
CHO
+
N
H
Aldehyde
X
X
NH4OAc
Piperidin-4-one (a-e
)
Ammonium acetate
Scheme-I: General reaction scheme for synthesis of piperidine-4-one (a-e)
compounds
O
CH3
Benzene/12 h/
∆
Y
Y
Anhydrous K2CO3
1,4-nucleophile
N
H
X
X
Piperidin-4-one
X
H
Cl
Cl
Y
O
NH
O
Y
Y
7
8
9
CH3
Protein ligand interaction analysis: By using ligand tool,
the interactions of the PDB inhibitor, β-phenyl-D-phenyl-
alanyl-N-propyl-L-prolinamide and the synthesized compound
7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-azaspiro-
[4.5]decane with the target protein thrombin were analyzed.
10 CH3 NH
11
N
H
CH3
O
X
X
Spiro 7-11
Scheme-II: General reaction scheme for synthesis of spiro (7-11) compounds
RESULTS AND DISCUSSION
3
2
The synthesis of piperidine-4-one derivatives were shown
in Scheme-I and the synthesis of spiro derivatives were shown
in Scheme-II (Fig. 2). Many synthetic approaches [23] have
been reported for the preparation of cyclopropane such as
intramolecular cyclization, addition of carbenes to olefins and
Michael initiated ring closure [24-30]. Spiro triazoles and their
derivatives can be conveniently synthesized from aldehyde/
ketone thiosemicarbazones and also from substituted thio-
semicarbazides by cyclization using suitable reagents. Earlier
FeCl3·6H2O [31], H2O2 [32] and m-CPBA [33] were used to effect
cyclization of steroidal and non-steroidal ketone thiosemicar-
bazones into corresponding 1,2,4-triazolidin-3-thiones.
Here we report a simple and effective procedure for
condensation reaction without catalyst and anticoagulant
activities of the following compounds (7-11). 6-Methyl-7,9-
diphenyl-1,4-dioxa-8-aza spiro[4.5]decane (7), 7,9-bis(4-
chlorophenyl)-6-methyl-1,4,8-triaza spiro[4.5]decane (8), 7,9-
bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-aza spiro[4.5]decane
(9), 7,9-bis(4-methoxyphenyl)-6-methyl-1,4,8-triazaspiro[4.5]
decane (10) and 7,9-bis(4-methoxyphenyl)-6-methyl-1,4-
dioxa-8-aza spiro[4.5]decane (11). The molecular structure
of the spiro compounds are given in Fig. 3.
Activated partial thromboplastin time and prothrombin
time analysis: Compounds were evaluated for their anti-
coagulant activity according to their PDBID:3DA9 values and
for anti-coagulant activity in vitro by the CT2 values of their
prothrombin times (PT). The CT2 value was defined as the
concentration required double the clotting time. In addition,
oral anticoagulant activity was evaluated by the ability of
compounds to prolong prothrombin time following oral
administration in human being.
X
X
X
O
NH
O
NH
O
Y
Ar
Phenyl
p-Cl-phenyl
p-Cl-phenyl
p-methoxy-phenyl
Compd.
1
4
CH3
CH3
CH3
CH3
7
8
9
10
11
Y
5
8
10
6
CH3 p-methoxy-phenyl
9
7
Ar
N
Ar
H
Fig. 2. General templates for synthesized compounds (7-11)
TABLE-1
ACTIVATED PARTIAL THROMBOPLASTIN TIME DATA OF
SPIRO COMPOUNDS WITH DIFFERENT CONCENTRATIONS
Compounds
Conc.
(µg/mL)
7
8
9
10
11
25
50
100
150
200
20.2s
31.4s
43.7s
55.8s
99.3s
18.3s
24.0s
41.0s
53.1s
89.9s
22.7s
31.8s
49.3
61.1s
110.6s
20.4s
36.9s
46.4s
68.7s
91.1s
20.6s
34.6s
46.8s
60.9s
109.2s
Retrieval of target protein: The target protein thrombin
with its inhibitor (PDBID:3DA9) was retrieved from the Protein
Databank. The structural analysis using Rasmol software shows
that the structure consists of 10 helices, 26 strands and 30
turns. The target protein structure shows in Fig. 6.
TABLE-2
PROTHROMBIN TIME DATA OF SPIRO
COMPOUNDS WITH DIFFERENT CONCENTRATION
Compounds
Conc.
(µg/mL)
7
8
9
10
11
25
50
100
150
200
18.6s
29.3s
36.7s
48.0s
63.0s
15.4s
21.6s
31.8s
42.2s
81.0s
18.5s
26.4s
38.3s
54.2s
97.2s
14.4s
29.6s
40.7s
56.0s
90.1s
17.9s
29.1s
40.0s
53.1s
98.3s
The activated partial thromboplastin time and prothrombin
time of synthesized spiro compounds are presented in Tables
1 and 2.

1780 Manivannan et al.
Asian J. Chem.
Compound
7
Compound
8
Compound 9
Compound 10
Fig. 3. Molecular structure of the spiro compounds
Compound 11
150
100
50
25 50 100 150 200
0
7
8
9
10
11
Compounds
Fig. 4. Activated partial thromboplastin time data of spiro compounds with
different concentrations
120
25 50 100 150 200
100
80
60
40
20
0
7
8
9
10
11
Compounds
Fig. 6. Target protein
Fig. 5. Prothrombin time data of spiro compounds with different concen-
tration
Tertiary structure analysis: The PDB structure was
validated using Ramachandiran plot from SAVS Server. The
results showed that the modeled structure contains 85.2 % of
amino acids are present in most favoured regions which show
that the retrieved structure has good quality. Ramachandran
plot for 3DA9 protein was given in Fig. 7.
Structure of ligand: The structure of ligand done by using
open babel software the. cdx molecular file for this chemical
compound was converted in to pdb file. The ligand structure
are shown in Fig. 8.
Energy minimization: The process of energy minimization
is very important for docking and this was done with the help of
SWISS PDB Viewer software and the values for bonds, angles,
torsion, improper, non-bonded, electro statistics, constraints and
the total are given in Table-3. The computations were done in
vacuo with the GROMOS 96, 43B1 parameters set without
reaction field.The results shows that the total energy for thrombin is
-12365.702 kJ/mol and this energy were minimized to -16334.184
kJ/mol. The energy minimization values are shown in Table-3.

Vol. 28, No. 8 (2016) AnticoagulantActivity and Molecular Docking of 7,9-bis(4-Chlorophenyl)-6-methyl-1,4-dioxa-8-azaspiro[4.5]decane 1781
TABLE-3
ENERGY MINIMIZATION REPORT
Energy
minimization
Non-
bonded
Electro-
statics
Cons-
traints
Protein name
Bonds
Angles
Torsion
Improper
Total
Before
After
935.29
181.66
1537.42
1027.22
1599.07
1392.13
290.81
248.68
-8821.03
-10194.1
-7907.27
-8989.72
0.000
0.000
-12365.70
-16334.18
Thrombin in complex
with inhibitor
3da9_protein
180
135
90
45
0
-45
-90
-135
Fig. 9. Before docking of 7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-
azaspiro[4.5]decane (9) with thrombin
-180
-135
-90
-45
0
45
90
135
180
Phi (°)
Fig. 7. Ramachandran plot
Fig. 10. After docking of 7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-
azaspiro[4.5]decane (9) with thrombin
Fig. 8. 3D-Ligand structure of thrombin inhibitor
Ser(235), Val(255), Ser(256), Trp(257), Gly(258), Glu(259).
Similarly, the 7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-
azaspiro[4.5]decane also produces hydrophobic interactions
with the same active sites and also it produces π-π interactions
with the residues His(79), Tyr(83).The binding mode analysis
of β-phenyl-D-phenylalanyl-N-propyl-L-prolinamide and 7,9-
bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-aza spiro[4.5]decane
were shown in Figs. 11 and 12, respectively.
Docking: Docking is the process of binding of receptor
with its ligand. The thrombin protein was docked with the
compound 7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-aza
spiro[4.5]decane using HEX software. The requirement of
binding energy E of thrombin protein lies between -263.23 and
-102.38 kJ/mol for molecular docking. The molecule before
and after docking are given in Figs. 9 and 10, respectively.
Analysis of interactions: Interactions of the compound
7,9-bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-aza spiro[4.5]-
decane for their binding mode analysis of thrombin with its
inhibitor, we noticed that the inhibitor produces hydrophobic
interactions with the active sites residues of His(79), Tyr(83),
Trp(86), Glu(130), Asn(131), Leu(132), Ile(209), Cys(231),
Conclusion
In the present study cyclo condensation method was
reported for the synthesis of spiro compounds and are evaluated
for their anticoagulant activity. Among the five compounds,
compound 9 was identified as potent anticoagulant (25 µg

1782 Manivannan et al.
Asian J. Chem.
of Technology (IIT), Chennai, India for providing spectral data
and UGC, New Delhi for economical support.
CYS
231
GLU
130
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VAL
255
LEU
132
TRP
257
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prothrombin time assay. The binding mode analysis of 7,9-
bis(4-chlorophenyl)-6-methyl-1,4-dioxa-8-aza spiro-[4.5]-
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The authors are thankful to Mr. Saravanan for his valuable
suggestions and support. Thanks are also due to Indian Institute