Design, synthesis, characterisation, conformation and biological investigation of N-acyl r-2,c-6-bis (4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-ones

Article abstract of DOI:10.1016/j.molstruc.2017.05.036

In a wide research programme towards the study of piperidin-4-ones with efficient pharmacological effect, a new series of N-acyl r-2,c-6-bis(4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-ones 2–5 are synthesized and characterized by IR spectra, ¹

Full text of DOI:10.1016/j.molstruc.2017.05.036

Accepted Manuscript
Design, synthesis, characterisation, conformation and biological investigation of N-
acyl r-2,c-6-bis (4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-ones
V. Mohanraja, S. Ponnuswamy
PII:
S0022-2860(17)30619-1
10.1016/j.molstruc.2017.05.036
MOLSTR 23777
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
07 March 2017
05 May 2017
10 May 2017
Please cite this article as: V. Mohanraja, S. Ponnuswamy, Design, synthesis, characterisation,
conformation and biological investigation of N-acyl r-2,c-6-bis (4-methoxyphenyl)-c-3,t-3-
dimethylpiperidin-4-ones, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.
2017.05.036
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Graphical Abstract

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Highlights
 Five N-acyl piperidone-4-ones have been synthesized and characterized using NMR
spectra.
 Compounds 2-5 prefer to exist in a distorted boat conformation B1
 Compounds 1-5 exhibit superior anti-bacterial activity against pseudomonas species
 Only moderate antioxidant activities have been observed for these compounds 1-5
 Docking studies confirm the binding of these compounds with target protein CHK1.

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Design, synthesis, characterisation, conformation and biological investigation of
N-acyl r-2,c-6-bis (4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-ones
V. Mohanraja,b and S. Ponnuswamy a*
aP.G. & Research Department of Chemistry, Government Arts College (Autonomous),
Coimbatore - 641 018, India.
b P.G. & Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya
College of Arts and Science, (Autonomous) Coimbatore-641020, India.
Corresponding author : S.PONNUSWAMY,
Email: kspons2001@gmail.com
Mobile: +91- 9244645744
Abstract
In a wide research programme towards the study of piperidin-4-ones with efficient
pharmacological effect, a new series of N-acyl r-2,c-6-bis(4-methoxyphenyl)-c-3,t-3-
1
dimethylpiperidin-4-ones 2-5 are synthesised and characterized by IR spectra, H, 13C,
DEPT - 135 and 2D (COSY and HSQC) NMR and mass spectra. The parent compound 1
prefers to exist in a chair conformation whereas the extracted coupling constant, chemical
shifts and estimated dihedral angles show that the N-acyl piperdine-4-ones 2-5 prefer to exist
in a distorted boat conformation B1 (with C2 and C5 in prow and stern positions) with
coplanar orientation of N-C=O moiety. The existence of a fast N-CO rotational equilibrium
between the boat conformations B (I) and B (II) has also been observed. Anti bacterial
activity of the above test compounds 1-5 is determined against pseudomonas sp. and
salmonella sp. The antioxidant activities are determined by the ABTS, DPPH and superoxide
assays. Furthermore, molecular docking studies have been carried out for the compounds 1-5
with target protein CHK1.
Keywords: N-.Acylpiperidin-4-ones; NMR spectra; Distorted boat conformation;
Antibacterial activity, Antioxidant activity, Molecular docking study.
1. Introduction
The development of new synthetic methods for use in organic synthesis is one of the
areas of organic chemistry that has experienced a major renaissance during the recent past.
There has been much interest in the synthesis of heterocyclic systems due to their varied
physiological and biological activities [1]. 2, 6-Disubstituted piperidines are the main
constituents of a number of alkaloids which possess broad spectrum of biological activity
[2-10]. The 2,6-diarylpiperidin-4-ones and their derivatives have in recent years attracted
considerable attention due to their possible bio-significance [11-12]. The synthesis of
compounds with attachment of electron withdrawing groups like NO, COR, etc., at nitrogen
of a 2,6-disubstituted piperidine ring has been reported by our group, as well by others, to
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bring major changes in the conformation of the ring and the orientation of the substituents
[8,10-15]. The conformational preferences of piperidine ring are largely decided by the
extent of resonance and magnitude of the resonance energy caused by the delocalization of
nitrogen lone pair into the N-NO and N-C=O functions (Fig.1). The relative magnitudes of
A1,3-strain, torsional strain and 1,3-diaxial interaction are also the deciding factors [16]. The
cis-2, 6-diphenylpiperidin-4-ones with various substituents at 3 and 5 positions of the ring
have been reported much earlier and they prefer to occupy a chair conformation with
equatorial orientation of all the substituents (Fig.2). Though there appears in the literature
more studies on the conformations of N-acyl-cis-2, 6-diphenylpiperidin-4-ones, to the best
of our knowledge, no investigation has been performed on N-acyl-r-2, c-6-bis(4-
methoxyphenyl)-c-3, t-3-dimethylpiperidin-4-ones.
In the present work, r-2,c-6-bis(4-methoxyphenyl)-c-3, t-3-dimethylpiperidin-4-one
(1) and its N-phenylcarbamoyl, N-chloroacetyl, N-dichloroacetyl and N-morpholinoacetyl
substituted compounds 2-5, have been synthesized and their stereochemistry, studied using
IR spectra, 1H , 13C and 2D (COSY and HSQC) NMR spectra and dihedral angle estimation
by ratio method (DAERM) [17]. DEPT spectra are also recorded for all the compounds to
assist the assignment of 13C NMR spectra. The mass spectra for the all compounds have
been recorded to confirm the structure. The anti-bacterial activity studies on 1-5 have also
been carried out. The X-ray crystal structure and the NMR spectral data show that the parent
piperidin-4-one 1 prefers to adopt a chair conformation with equatorial orientation of anisyl
groups [13, 18]. However, perusal of NMR data of the N-acyl compounds 2-5 reveals that
all of them prefer to exist in a distorted boat conformation. All the compounds 1-5 are
docked with CHK1 protease protein using Schrodinger 9.5 software.
2. Experimental
2.1 Materials, methods and instruments
All the reported melting points were taken in open capillaries and are uncorrected.
The IR spectra were recorded using a SHIMADZU FT-IR 884008 spectrometer using KBr
pellets. The 1H and 13C NMR spectra were recorded in CDCl3 solution at room temperature
with a Bruker-AMX500 MHz NMR spectrometer, and the chemical shifts were referenced
to TMS. A 0.05 M solution of the sample prepared in CDCl3 was used for obtaining the 2D
NMR spectra and the tubes used for recording the NMR spectra were of 5-mm diameter.
Electron impact mass spectra were recorded using a JEOL GS mate spectrometer. Unless
otherwise stated, all the reagents and solvents were of high grade and purchased from
Sigma-Alrich chemicals, Bangalore, India and Merck chemicals, Worli, Mumbai. All the
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solvents were distilled prior to use. The parent piperdin-4-one 1 was prepared by following
the literature procedure [19].
2.2 Synthesis
2.2.1 Synthesis of r-2,c-6-bis(4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-one (1)
3-Methylbutanone (10.7 ml, 100 mmol), p-methoxybenzaldehyde (24.2 ml, 200
mmol), and ammonium acetate (7.7 g, 100 mmol) were dissolved in 80 ml of distilled
ethanol and heated over boiling water bath, with constant shaking until an yellow colour
developed and changed into orange. The solution was left undisturbed for 14 hrs. The solid
thrown out was filtered and purified by recrystallisation from ethanol.
2.2.2 N-Acyl piperidones 2-5: General procedure
The compounds 2-4 were prepared by the reaction of phenylisocyanate
(1.6 ml, 15 mmol), chloroacetyl chloride (1.2 ml, 15 mmol) and dichloroacetyl chloride (1.5
ml, 15 mmol), respectively, on the r-2,c-6-bis(4-methoxyphenyl)-c-3,t-3-dimethylpiperidin.-
4-one (1) (2 g, 5.9 mmol) in benzene medium using triethylamine (2 ml, 15 mmol) as a
catalyst ( Scheme 1). The N-morpholinoacetyl piperidin-4-one 5 was prepared by the
reaction of N-chloroacetylpiperidin-4-one 3 (2 g, 5 mmol) with morpholine (1 ml, 12 mmol)
in benzene medium using triethylamine (2 ml, 15 mmol) as a catalyst. The reaction mixture
for synthesis of 2-5 was stirred at room temperature for 6 h. The organic layer was
separated, dried over anhydrous Na2SO4 and concentrated. The resulting product was
o
purified by recrystallisation from benzene and pet-ether (60 - 80 C) in a 2:1 ratio.
Analytical data of the compounds 1-5 are reported in Table 1.
2.3 Anti-bacterial activity
2.3.1 Preparation of 24 hours old culture of test organism
Two human pathogenic bacterial strains viz., pseudomononas species and salmonella
species have been used as test organism. 50 ml of Nutrient broth was prepared, sterilized
and inoculated with the test organism. It was incubated at 37 oC for 24 hours. To determine
antibacterial activity of test compounds, standard bacterial cultures of organisms 1.5 x
108cfu/ml were used. This was achieved by diluting the overnight test cultures with sterile
nutrient broth until an O.D. value is obtained that is equivalent to the O.D. values of 0.5
Mcfarland standards. Further, this culture broth was diluted (i.e) 1:100 dilution to bring a
count of 1.5 x 106 cfu/ml of the culture broth. Quantitatively the activities of the test
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compounds 1-5 can be explained from the reduction in the OD values when compared to
those of the DMSO control for each dilution [20].
2.4 Antioxidant activity of the compounds 1-5
2.4.1 DPPH• method
The antioxidant activity of the compounds 1-5 is determined in terms of hydrogen
donating or radical scavenging ability using the 1, 1-diphenyl-2-picrylhydrazyl stable
radical DPPH, according to the method of Blois [21]. Various concentrations of samples
were prepared and the volume was adjusted to 100 µl with methanol. 5 ml of 0.1 mM
methanolic solution of DPPH was added and shaken vigorously. The tubes were allowed to
stand for 20 minutes at 27 ºC. The absorbance of the sample was measured at 517 nm.
Radical scavenging activity of the samples was expressed as IC50. Concentration of the
sample necessary to decrease initial concentration of DPPH• by 50% (IC50) under the
experimental condition was determined. Lower value of IC50 indicates a higher antioxidant
activity. Rutin and BHT were used as standard antioxidants in DPPH assay. The capacity to
scavenge the DPPH radical was calculated using the following equation:
DPPH Scavenging effect (%) = (A0 – A1/A0) × 100.
Where,
A0 =the absorbance of the standard;
A1 =the absorbance of the sample.
2.4.2 Antioxidant activity by ABTS•+ assay
The total antioxidant activity of the samples was measured by ABTS radical cation
decolourisation assay according to the method of Re et al. [22]. ABTS•+ was produced by reacting
7 mM ABTS aqueous solution with 2.4 mM potassium persulfate in the dark for 12-16 h at room
temperature. Prior to assay, this solution was diluted in ethanol (about 1:89, v/v) and equilibrated
at 30 ºC to give an absorbance of 0.700±0.02 at 734 nm. The stock solution of the sample extracts
were diluted such that after introduction of 10 µl aliquots into the assay, they produced between
20% and 80% inhibition of the blank absorbance. After the addition of 1 ml of diluted ABTS
solution to 10 µl of sample or Trolox standards (final concentration 0-15 µM) in ethanol,
absorbance was measured at 30 ºC exactly 30 minutes after the initial mixing. Appropriate solvent
blanks were also run in each assay. Triplicate determinations were made at each dilution of the
standard, and the percentage inhibition was calculated for the blank absorbance at 734 nm and
then was plotted as a function of Trolox concentration. The unit of Trolox equivalent antioxidant
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capacity (TEAC) is defined as the concentration of Trolox having equivalent antioxidant activity
expressed as µM/g sample extract on dry matter.
•−
2.4.3 Superoxide radical (O2 ) scavenging activity
The assay was based on the capacity of the extracts to inhibit formazan formation by
scavenging the O2•− generated in riboflavin-light-nitrobluetertrazolium (NBT) system [23]. Each 3
ml reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 20 µg riboflavin, 12
mM EDTA, 0.1 mg NBT and 1 ml of sample solution (50-250 µg/ml). Reaction was initiated by
illuminating the reaction mixture with different concentrations of sample extracts for 90 seconds.
Immediately after illumination, the absorbance was measured at 590 nm. Identical tubes with
reaction mixture kept in dark served as blanks. The percentage inhibition of superoxide anion
generation was calculated using the following formula: % of inhibition = [(A0 – A1)/A0] × 100
where, A0 is the absorbance of the control, and A1 the absorbance of the extract/standard. The
results were compared with the commercially available antioxidants such as rutin and BHT.
2.5 Molecular Docking Studies
Checkpoint kinase 1, commonly referred to as CHK1 is a serin/threonine-specific
protein kinase, which in humans is encoded by the CHK1 gene CHK1 is required for the
initiation of DNA damage checkpoints and has recently been shown to play a role in the
normal cell cycle. In addition to mediating cell cycle checkpoints, CHK1 also contributes to
DNA repair processes, gene transcription, embryo development, cellular responses to HIV
infection and somatic cell viability. The DNA damage response is a network of signalling
pathways that leads to activation of checkpoints, DNA repair and apoptosis to inhibit
damaged cells from progressing through the cell cycle.
CHK1 protein was modeled computationally and its active sites were predicted using
site map(Schrodinger 9.5) followed by docking studies. The preparation of the modeled
CHK1 was achieved by a protein preparation wizard, piperidone and it was prepared using
the ligprop program and docking procedure was performed using (XP) extra precision glide
(Schrodinger 9.5) which produces the least number of inaccurate poses and calculate the
accurate binding energy of the 3D structure of a known protein with a ligand. Docking
studies were carried out using piperidone as an inhibitor against the model of CHK1 [24].
3. Results and discussion
In the IR spectra of the compounds 2-5, the amide >C=O stretching bands are
observed at 1639 – 57 cm-1 in addition to the ring carbonyl stretching band at 1705-20 cm-1.
In the IR spectrum of the compound 2, NH stretching band around 3265 cm-1 is absent and
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the new NH (3301 cm-1) and carbonyl stretching (1651 cm-1) bands are observed due to the
CONHPh moiety. The absence of N-H stretching band around 3265 cm-1 and the presence
of additional carbonyl stretching band at approximately 1639-57 cm-1 in the IR spectra of
the compounds 3-5 indicate the formation of 3-5. In the mass spectra, the presence of
molecular ion peaks at m/z 339, 458, 415, 449.85.and 424 for compounds 1-5, respectively,
and their fragmentation patterns confirm the structures of 1-5. (Table 1)
1
13
The H and C NMR spectral data of N-acylpiperidin-4-ones 2-5 are presented in
1
Table 2. Analysis of H and 13C NMR spectral data of the N-acylpiperidin-4-ones 2-5
shows the isochronous nature of proton and carbon signals at room temperature (Figs. 3-4).
1
The H NMR spectral signals of N-acylpiperidin-4-ones 2-5 are assigned based on their
positions, multiplicities and intensities as well as in comparison with the parent amine 1. In
addition, COSY spectra are recorded for N-phenylcarbamoyl and N-chloroacetylpiperidin-4-
ones, 2 & 3, to confirm the assignments. Further, the unambiguous assignment of 13C NMR
signals for all the compounds 2-5 has been made using their DEPT spectra (Fig. 5). The 1H-
13C HETCOR spectra are also recorded (Figs. 6-7) to validate the assignment of 1H and 13C
1
NMR signals. Analysis of H & 13C NMR spectral data of N-acylpiperidin-4-ones 2-5
indicates the preference for a non-chair conformation.
3.1 Orientation of N-C=O group
The N-C=O function may adopt either a coplanar or a perpendicular orientation with
respect to the C2-N-C6 plane of the piperidine ring system. The N-acylpiperidin-4-ones 2-5
show the isochronous proton and carbon signals in their NMR spectra. If the delocalisation
of the lone pair of electrons on the nitrogen of the N-acylpiperidin-4-ones 2-5 is sufficient to
create a substantial double bond character along the N-CO bond, the adjacent carbons and
protons may give rise to anisochronous signals as in the case of N-nitroso-r-2, c-6-
diphenylpiperidines [25]. The absence of anisochronous signals in the RT NMR spectra of
these N-acylpiperidin-4-ones 2-5 reveals that the N-CO group might adopt a coplanar
orientation with the C2-N-C6 plane with very low N-CO rotational barrier (G#). The
broadening of benzylic proton signals (H2 & H6) has been observed in the case of N-
acylpiperidin-4-ones 3-5. This broadening of -proton signals may be due to the presence of
rotational equilibrium about N-CO bond in these systems, which could be possible only in
the coplanar orientation of the N-C=O moiety with respect to C2-N-C6 plane of the
piperidine ring. Further, the absence of anisochronous signals in the N-acylpiperidin-4-ones
2-5 may be due to the low rotational barrier for the N-CO rotation compared to that of N-
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NO. The -proton signals in the case of N-phenylcarbamoyl piperidin-4-one 2 are not broad
and it may be due to the lower rotational barrier for N-CONHPh moiety compared with
other N-acyl functions [26-27] and to the extended conjugation through Ph-NH linkage of
the phenylcarbamoyl group. The lower energy barrier of N-CONHPh function is due the
increase in bulkiness of the substituent. Increase in the bulkiness of the substituents
destabilises the planar ground state rather than the perpendicular transition state leading to a
lowering of rotational barrier.
Further support for the coplanar orientation of N-C=O function in the compounds 2-
1
13
5 can be derived from the H and C NMR spectral data of -proton and carbon signals.
The higher amount of deshielding of -protons by 1 - 2.7 ppm and shielding of -carbons
by 4 - 7.5 ppm in N-acyl compounds 2-5 compared with the parent amine 1 support the
coplanar orientation of N-C=O group. Hence, it may be concluded that the rotation about
N-CO bond may be fast at room temperature compared to the NMR time scale and it results
in a time averaged signal due to fast N-CO rotational equilibria. In addition, the reduction in
the intensity as well as the number of Bohlmann bands [28, 29] around 2850 cm-1 in the IR
spectra of the N-acylpiperidin-4-ones 2-5 compared to that of the parent amine 1 indicates
that the lone-pair of electrons on nitrogen is in conjugation with N-C=O function which in
turn could be possible only when the carbonyl groups in 2-5 are coplanar to the C2-N-C6
plane of the piperidine ring.
3.2 Conformation of the ring
The preferred conformation for the N-acyl compounds 2-5 has been arrived at by
considering the vicinal coupling constants between the H6 and H5a & H5e protons. The
coupling constant values of the N-acylpiperidin-4-ones 2-5 have been extracted from their
1H NMR spectra and are presented along with that of parent piperidin-4-one 1 in the Table
3. Except N-phenylcarbamoylpiperidin-4-one 2, all the other N-acylpiperidin-4-ones show
an unsymmetrical triplet for the H6 benzylic proton instead of doublet of a doublet.
However, for all these compounds 3-5, the H5a & H5e protons show distinct double doublets.
Hence the cis & trans vicinal coupling constants for the H6 benzylic protons of N-acyl
piperidin-4-ones 3-5 are extracted from its coupling partners H5a & H5e. In the case of N-
phenylcarbamoylpiperidin-4-one 2, the H6 benzylic proton shows a doublet of a doublet.
The H2 benzylic protons in these compounds 2-5 show a singlet since these do not have a
coupling partner at C3 (Figs. 3 & 4).
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The vicinal coupling constant data of compounds 2-5 are employed to estimate the
dihedral angles between the vicinal protons (cis = 6a,5e and trans = 6a,5a) by DAERM and
are presented in Table 4. Perusal of the data in the Tables 2–4 led to the following
observations: (i) there is a significant deshielding of benzylic protons at C2 and C6 positions.
(ii) The H2 benzylic protons are more deshielded than H6 benzylic protons .There is a
3
significant change in the vicinal coupling constants (3J6, 5e & J6, 5a) as well as dihedral angles
when compared to the parent piperidin-4-one 1, indicating a preference for non-chair
conformation. (iii) The C2 and C6 benzylic carbons are significantly shielded in the N-acyl
compounds 2-5 compared with that of parent piperidin-4-one 1. (iv) C2 benzylic carbons are
more shielded compared to C6 benzylic carbons.
The preferred conformation of the N-acyl piperidin-4-ones 2-5 can be derived by
using the above points. The parent piperidin-4-one 1 has been shown to prefer a chair
conformation with equatorial orientation of aryl groups. The possible conformations for the
N-acylpiperidin-4-ones 2-5 with coplanar orientation of N-C=O function are depicted in Fig
8. All these N-acylpiperidin-4-ones 2-5 show a similar trend in the spectral data when
compared with its parent amine 1. Hence the NMR spectral data of N-
phenylcarbamoylpiperidin-4-one 2 is considered as a reference for discussion. In the chair
conformation CE of N-phenylcarbamoylpiperidin-4-one 2, which is similar to the parent
amine 1, the dihedral angles expected between H6 benzylic proton and H5e & H5a protons are
around 60o and 180o, respectively, (cis = 6a, 5e ≈ 60o and trans = 6a,5a ≈ 180o). However,
the observed cis coupling constant (3J6a,5e) values of 6.0 Hz for the compound 2 (Table 4)
and the corresponding dihedral angles of 44o (Table 4) could not be explained using the CE
conformation. In addition, in the chair conformation CE, the benzylic protons at C2 & C6
occupy axial positions and are in the “out of plane” (shielding) region and the observed
deshielding of  = 2.72 & 1.05 ppm from that of the parent cannot be explained using the
model proposed by Paulsen and Todt for the anisotopic effect of the amides [30]. Hence,
the possibility of chair conformation CE is ruled out.
The >N–C=O moiety has been shown to adopt a coplanar orientation with reference
to C2 – N – C6 plane due to the delocalization of lone-pair of electrons on ring nitrogen into
the carbonyl -cloud. It results in the change of hybridization of the nitrogen from sp3 to
sp2. In the chair conformation CE, the p-anisyl groups at the equatorial orientation alpha to
the nitrogen exhibit a severe non-bonded interaction viz., A1,3 strain, with the coplanar N-
C=O function. In order to relieve this A1,3 strain, an alternate chair form CA can be
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considered. In this CA conformation, the aryl groups are forced to occupy the axial
orientation and this conformation would be destabilized by the 1, 3-diaxial interaction
between aryl groups as well as aryl groups and C3-axial methyl group. If both CE & CA
conformations have higher magnitude of destabilizing interactions then other possible
conformations may be assumed with boat ring (B1-B6). In all these boat forms B1–B6,
different destabilizing interactions like A1,3 strain (eg. B2, B3 & B6), 1, 3-diaxial interactions
(eg. B5), bond eclipsing interactions, gauche interactions, etc., would be involved. In such
instances, an optimum geometry would be reached by unsymmetrically twisting the ring
skeleton.
In the chair conformation CA, benzylic protons (H2 & H6) occupy equatorial
orientations and they are in the ‘deshielding region’ (inplane) of the amide plane. Hence the
observed deshielding can be reasonably explained using the Paulsen and Todt’s model [30].
However, the Jcis (J6a, 5e) and Jtrans (J6a, 5a) coupling constant values for the benzylic proton of
C6 would be expected in the range of 2-4Hz. Also the dihedral angles between H6a & H5e
(cis) and H6a & H5a (trans) protons are expected around 56-60o. But the observed coupling
constant values (compound 2.: Jcis = 6.0 Hz & Jtrans = 12.0 Hz) and the dihedral angles (cis
= 44o and trans = 164o) are different and cannot be explained using the conformation CA.
Hence the possibility of conformation CA is also ruled out. In the boat conformations B1-B6,
the observed deshielding of benzylic protons may be explained using the boat forms B1, B2
& B5 since in these conformations the benzylic protons are expected to be present in the
“deshielding region” of the amide function. The extracted coupling constants and the
estimated dihedral angles between H6 benzylic proton and H5 (axial & equatorial) protons
could be explained only by using boat form B1. In addition, the magnitude of coupling
constants (Jcis = 6.0 Hz & Jtrans = 12.0 Hz) and the dihedral angles (cis = 44o & trans 164o)
eliminate the possibility of an equilibrium between two boat conformations B1 & B2.
At least, two sets of signals are expected for -protons and -carbons if the
equilibrium between B1 and B2 is slow when compared to NMR time scale. However, in
these compounds 2-5, only isochronous proton and carbon signals have been observed
which indicate a fast equilibrium. In the N-C=O function, the conjugation of nitrogen lone
pair with >C=O creates a partial double bond character at N-C bond and leads to restricted
rotation around this bond which in turn is responsible for the fast rotational equilibrium
(since the double bond character is less in these compounds 2-5 when compared to N-NO
system). The observed coupling constants and the dihedral angles cannot be explained even
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if a fast equilibrium is assumed between B1 & B2. The Jtrans values are different for the N-
acyl compounds 2-5 (for 2: 12.0 Hz; 3 & 5: 10.0 Hz & 4 : 10.5 Hz) when compared to the
other reported N-acyl systems (Jtrans= 6-8 Hz). The higher value of Jtrans (10-12 Hz) and trans
(158-164o) would not have been possible if the equilibrium is assumed between B1 & B2.
Hence the possibility of an equilibrium between B1 and B2 is eliminated and for all these N-
acyl compounds 2-5 the rotational equilibrium with B1 conformation is assumed (Fig. 9).
In this B1 conformation, the C6 benzylic proton occupies axial position. It makes a
trans angle around 160o [H6a–C6–C5–H5a] and a cis angle around 45o [H6a–C6– C5–H5e] with
C5 axial and equatorial protons, respectively. Hence, the observed coupling constant values
for all the N-acyl compounds 2-5 (Jtrans = 10-12 Hz & Jcis = 6.0 – 6.5 Hz, Table 4) .and the
estimated dihedral angles using DAERM (trans = 158-164o & cis = 38-44o) can be
explained using the boat conformation B1. In all these N-acyl compounds 2-5, the magnitude
of desheilding of H2 benzylic protons are more compared to H6 benzylic protons [for eg. 2 :
 of H2 = +2.72 ; H6 = +1.05]. In the boat conformation B1, there exists gauche
interaction between C2-Ar group and the equatorial methyl group at C3 (Fig.10). In order to
avoid this interaction, a slight twist along N-C2-C3 may takes place which brings the H2
proton well into the deshielding region of the amide plane. Hence, the H2 protons are more
deshielded than H6 protons in all these N-acyl compounds, when compared to the parent
amine 1, (Table 5).
Support for the above speculation is obtained from the magnitude of shielding of
carbons observed in the 13C NMR spectrum (Table 6). For example, in compound 2, the C2
& C6 carbons are shielded by -7.59 and -3.76 ppm, respectively, compared with that of the
parent piperidin-4-one 1. It again confirms that the carbonyl group is coplanar to the C2-N-
C6 plane of the piperidine ring causing gamma-eclipsing interaction between C-O and N-C
bonds. Hence, the shielding of -carbons (C2 & C6) is ascribed to the gamma eclipsing
interaction between N1-C2 / N1-C6 bond with C-O bond. The magnitude of shielding of C2
benzylic carbon ( = -7.59 ppm) is more than the C6 benzylic carbon ( = -3.76 ppm).
Due to gauche interaction between C2-Ar & C3-Me groups (Fig.10), the ring undergoes a
slight twist along N-C2-C3 bonds. The slight twisting along N-C2-C3 bonds would reduce the
dihedral angle between N-C-O and C-N-C2 planes and the  -eclipsing interaction between
the C-O & N-C2 bonds may be more compared to that of C-O & N-C6 bonds. The shielding
of  - carbons is attributed to the -anti effect induced at the -carbon by the N-C bond.
Hence, based on the coupling constants, chemical shifts and estimated dihedral angles, it is
10

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concluded that the N-acyl piperidin-4-ones 2-5 prefer the N-CO rotational equilibrium
between B1 conformations [B1 (I) & B1 (II)] with a slight twist along N-C2-C3 bonds and C2
& C5 in prow and stern positions with a coplanar N-C=O group (Fig. 9).
4. Antibacterial activity of compounds 1-5
The biological activity against the synthetic test compounds of substituted piperidin-
4-ones 1-5 is assessed using bacterial isolates that are obtained from processed clinical
samples. Anti-bacterial activity of the above test compounds is determined against the
pseudomonas sp. and salmonella sp. The minimum inhibitory concentration (MIC) for
compounds 1-5 has been assessed by two fold serial dilution methods. The results are
compared with standard drugs viz., chloramphenicol and gentamycin (Tables 7 & 8). The
results indicate that all the compounds 1-5 possess a broad spectrum of activity against the
tested microorganisms even at 6.25 g/ml. Compounds 1-5 exhibit superior activity against
pseudomonas species and significant activity against salmonella species when compared to
the above standard drugs. Compound 4 containing dichloroacetyl group shows superior
antibacterial activity against pseudomonas when compared to all other groups.
5. Antioxidant
The DPPH radicals scavenging method, ABTS assay methods and Superoxide
-
radicals (O2 ) scavenging activity are the most common tools to inspect the antioxidant
capacity of the specific compounds. The synthesized compounds 1-5 interact with the above
free radicals, inhibit the oxidation and there by exhibiting a certain degree of radical
scavenging activity. The results of the compounds 1-5 are compared with standard Rutin
and BHT and are presented in Table 9. The values of IC50, the effective concentration at
which 50% of the radicals are scavenged, are tested to evaluate the antioxidant activities.
Generally, a lower IC50 value demonstrates greater antioxidant activity. With ABTS
radicals, the compound 4 is found to possess a better antioxidant activity, compounds 1, 3 &
5 show a moderate activity and compound 2 shows a very poor activity when compared to
the standards. In the DPPH assay, the antioxidant activity of all the compounds 1-5 is found
to be moderate when compared to the standards. With superoxide assay, the compound 5
shows a better activity, compounds 2-4 show a moderate activity, and compound 1 shows
poor activity when compared to the standards. The ABTS, DPPH and superoxide
scavenging abilities are found to be in the order of 4>1>5>3>2, 4>2>3>5>1 and
5>3>2>4>1, respectively.
6. Molecular Docking Studies
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6.1 Binding mode of r-2,c-6-bis(4-methoxyphenyl)-c-3,t-3-dimethyl-piperidin-4-one (1)
into CHK1
Docking simulation of 1 into CHK1 results in the formation of single hydrogen bond
interaction with bond distance of 2.11Å and it is observed that side chain hydrogen atom of
positive charged residue of ASN 59 interacts with oxygen atom of 1. The Glide Score and
Glide Energy values for piperidin-4-one 1 are observed to be -5.710 kcal/mol and - 47.773
kcal/mol (Table-10). Furthermore, a number of hydrophobic interactions (LEU 82, LEU
137, PHE 149, ALA 36 VAL 23, TYR 86 & CYS 87) are found between piperidin-4-one 1
into CHK1 protein (Fig.11).
6.2 Binding mode of N-phenyl carbamoyl-r-2, c-6-bis(4-methoxyphenyl)-c-3, t-3-
dimethylpiperidin-4-one(2) into CHK1
Docking simulation of 2 within the active site of the checkpoint kinase1 has been
analyzed. The Glide Score and Glide Energy values for 2 are observed as -2.473 kcal/mol
and -31.04 kcal/mol, respectively (Table-10). Upon examining the docking features
between compound 2 and CHK1, two hydrogen bond interactions are found to be present.
First one is the side chain hydrogen atom of the positive charged residue of LYS 38 and it is
well interacted with oxygen atom of the compound 2 (with bond length 2.35Å). The second
one is the backbone hydrogen atom of the negative charged residue of GLU 17 which is
strongly interacted with oxygen atom of the compound 2 (with bond distance 2.18Å).
Furthermore, a number of hydrophobic interactions (LEU 82, LEU 84,PHE 149, ALA 36
VAL 23, LEU 137 & LEU 15) are found between compound 2 into CHK1 protein
(Figs.11&12).
6.3 Binding mode of N-chloroacetyl-r-2, c-6-bis(4-methoxyphenyl)-c-3, t-3-dimethyl-
piperidin-4-one (3) into CHK1
Docking simulation of compound 3 within the active site of the checkpoint kinase1
has been analyzed. The Glide Score and Glide Energy values for compound 3 are -4.42
kcal/mol and - 53.504 kcal/mol, respectively. On examination of docking features between
compound 3 and checkpoint kinase1, only one hydrogen bond interaction is found to be
present. The hydrogen atom of the CYS 87 is well interacted with oxygen atom of the
compound 3. Furthermore, a number of hydrophobic interactions (TYR 86, ALA 36, TYR
86, LEU 84, VAL 23, VAL 68, TYR 20 & LEU 15) are found between compound 3 into
CHK1 protein (Fig. 12).
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6.4 Binding mode of N-dichloroacetyl-r-2, c-6-bis(4-methoxyphenyl)-c-3, t-3-
dimethylpiperidin-4-one(4) into CHK1
Docking simulation of compound 4 within the active site of the checkpoint kinase1
has been analyzed. The Glide Score and Glide Energy values for compound 4 are observed
to be -2.840 kcal/mol and -21.835 kcal/mol. Examination of docking features between
compound 4 and checkpoint kinase1 reveals only one hydrogen bond interaction between
compound 4 into CHK1. Furthermore, a number of hydrophobic interactions (LEU 151,
TYR 86, ALA 36,VAL 23 & LEU 15) are found between compound 4 into CHK1 protein
(Fig.12).
6.5 Binding mode of N-morpholinoacetyl-r-2, c-6-bis(4-methoxyphenyl)-c-3, t-3-
dimethylpiperidin-4-one(5) into CHK1
Docking simulation of compound 5 within the active site of the checkpoint kinase1
has been analyzed. The Glide Score and Glide Energy values for compound 5 are observed
to be -3.892 kcal/mol and -16.751 kcal/mol (Fig. 11). On examination of docking features
between compound 5 and checkpoint kinase1, only one hydrogen bond interaction is found
to be present. The side chain hydrogen atom of the positive charged residue of LYS 38 is
well interacted with oxygen atom of the compound 5 with bond distance of 2.46Å.
Furthermore, a number of hydrophobic interactions (LEU 137, LEU 15, CYS 87, ALA 36,
VAL 23, LEU 84, VAL 68, ILE 146, LEU 62, PHE 149 & TYR 20) are found between
compound 5 into CHK1 protein (Figs. 11 & 12). The Glide Score and Glide Energy for all
compounds 1-5 are given in Table. 9.
Conclusion
N-Phenylcarbamoyl, N-chloroacetyl, N-dichloroacetyl and N-morpholinoacetyl
derivatives 2-5 of r-2, c-6-bis(4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-one (1) have
been synthesized and characterized. The preferred conformation of these compounds 1-5
have been determined using IR spectra and 1H, 13C, DEPT & 2D (1H, 1H-COSY & 1H-13C-
HETCOR) NMR spectra. The mass spectra for all the compounds have been recorded to
confirm the structure. On the basis of the coupling constants, chemical shifts and estimated
dihedral angles it is found that the N-acyl piperidin-4-ones 2-5 prefer to exist in a distorted
boat conformation B1 ( with C2 and C5 in prow and stern positions) with coplanar orientation
of N-C=O moiety. The existence of a fast N-CO rotational equilibrium between the distorted
boat conformations B1 has also been observed. The antibacterial activity of substituted
piperidin-4-ones 1-5 is assessed against the bacterial isolates that were obtained from
processed clinical samples. Anti-bacterial activity of the above test compounds is
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determined against pseudomonas sp. and salmonella sp. The results clearly show that all the
five compounds demonstrate a significant anti-bacterial activity. The results of antioxidant
studies show that most of the compounds posses moderate activity when compared to the
standards Rutin and BHT. The compounds 1-5 have been docked with the structure of
CHK1 and the results demonstrate that all the compounds show good binding affinity.
Acknowledgement
We thank Dr. T. Viswanathan for antibacterial activity studies.
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Figure captions:
Fig.1: Delocalisation along N-X=Y function.
Fig. 2: Allylic strain or A1,3 strain.
1
Fig. 3: H NMR spectrum of compound 2
Fig. 4: 13C NMR spectrum of compound 2
Fig. 5: DEPT 135 spectrum of compound 2
1
Fig. 6: H-1H COSY spectrum of compound 2
Fig. 7: HSQC spectrum of compound 2
Fig. 8: Possible conformations of N-acylpiperidin-4-ones 2-5.
Fig. 9: Conformational equilibrium in N-acyl piperidin-4-ones 2-5
Fig. 10: Gauche interacting between C2-Ar and C3-Me group
Fig. 11: Docked structure of target protein CHK1 with compound 1 and 2
Fig. 12: Hydrophobic interactions found between compounds 2-5 and CHK1
.

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Fig. 1
Fig. 2

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Compound
Z
R
2
3
4
C6H5NCO
-NHC6H5
ClCOCH2Cl -CH2Cl
ClCOCHCl2 -CHCl2
CH2 N
O
5
Morpholine
Scheme-1

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1
Fig. 3: H NMR spectrum of compound 2

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Fig. 4: 13C NMR spectrum of compound 2

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Fig. 5: DEPT 135 spectrum of compound 2

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1
Fig. 6: H-1H COSY spectrum of compound 2

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Fig. 7: HSQC spectrum of compound 2

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Fig. 8: Possible conformations of N-acylpiperidin-4-ones 2-5. Ar = p-anisyl

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Fig. 9: Conformational equilibrium in N-acyl piperidin-4-ones 2-5
Fig. 10: Gauche interacting between C2-Ar and C3-Me groups

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Compound 1
Compound 2
Compound 5
Fig.11: Docked structure of target protein CHK1 with compounds 1, 2 and 5

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2
3
4
5
Fig.12: Hydrophobic interactions found between compounds 2-5 and CHK1.