Novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters: Synthesis and a new insight into their antioxidant and antimicrobial potential

Article abstract of DOI:10.1007/s00044-013-0793-z

A simple and efficient protocol for the synthesis of novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters 4(a-q) is described. Initially, p-anisaldehyde 1 was condensed (Mannich reaction) with acetone and ammonium acetate trihydrate afforded 2,6-bis(4-methoxyphenyl) piperidin-4-one 2. Then, methylation followed by oximationwith hydroxylamine hydrochloride (NH₂OH?HCl) furnished a key scaffold 4. Further, to explore the enhanced biological properties of the piperidin-4-one core i.e. the key scaffold 4 was conjugated with substituted benzoyl chlorides in the presence of anhydrous K₂CO₃ as base to obtain novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters 4(a-q) in excellent yields. The newly synthesized compounds were characterized by elemental analysis, IR, ¹H NMR, ¹³C NMR and mass spectroscopic techniques, and screened for their in vitro antioxidant and antimicrobial activities. Most of the compounds exerted positive efficacy towards the biological assays performed. Among the synthesized analogues, compounds 4l and 4m exhibited promising antioxidant activity and on the other hand compounds 4b and 4d manifested persuasive antibacterial activity, whereas compound 4b displayed stupendous antifungal activity against A. flavus strain. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0793-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1887–1898
DOI 10.1007/s00044-013-0793-z
ORIGINAL RESEARCH
Novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime
esters: synthesis and a new insight into their antioxidant
and antimicrobial potential
•
Salakatte Thammaiah Harini Honnaiah Vijay Kumar
•
•
Sannenahalli Krishnegowda Peethambar
•
Javarappa Rangaswamy Nagaraja Naik
Received: 13 January 2013 / Accepted: 12 September 2013 / Published online: 24 September 2013
ꢀ Springer Science+Business Media New York 2013
Abstract A simple and efficient protocol for the synthesis
of novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one
oxime esters 4(a–q) is described. Initially, p-anisaldehyde 1
was condensed (Mannich reaction) with acetone and ammo-
nium acetate trihydrate afforded 2,6-bis(4-methoxy-
phenyl)piperidin-4-one 2. Then, methylation followed by
oximation with hydroxylamine hydrochloride (NH2OH•HCl)
furnished a key scaffold 4. Further, to explore the enhanced
biological properties of the piperidin-4-one core i.e. the key
scaffold 4 was conjugated with substituted benzoyl chlorides
in the presence of anhydrous K2CO3 as base to obtain novel
2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime
esters 4(a–q) in excellent yields. The newly synthesized
compounds were characterized by elemental analysis, IR, 1H
NMR, 13C NMR and mass spectroscopic techniques, and
screened for their in vitro antioxidant and antimicrobial
activities. Most of the compounds exerted positive efficacy
towards the biological assays performed. Among the synthe-
sized analogues, compounds 4l and 4m exhibited promising
antioxidant activity and on the other hand compounds 4b and
4d manifested persuasive antibacterial activity, whereas
compound 4b displayed stupendous antifungal activity
against A. flavus strain.
Keywords 2,6-Bis(4-methoxyphenyl)-1-
methylpiperidin-4-one oxime esters ꢀ
Substituted benzoyl chlorides ꢀ Antioxidant ꢀ
Antimicrobial studies
Introduction
Free radicals are naturally present in living systems;
however, high amounts of free radicals can oxidise bio-
molecules, leading to tissue damage, cell death or degen-
erative processes, including aspects of ageing, cancer,
cardiovascular diseases, arteriosclerosis, neural disorders,
skin irritations and inflammation. Also, free radicals and
lipid peroxides play an important role in oxidative stress
(Gulc¸in, 2006). The harmful action of the free radicals can,
however, be blocked by antioxidant substances, which
scavenge the free radicals and detoxify the organism
(Kumaran and Karunakaran, 2006). This had attracted a
great deal of research interest in therapeutic antioxidant-
based drugs formulations. Besides to this, the emerging
resistance of microorganisms requires the careful use of
existing antimicrobial drugs. However, there is a need for
the design of novel antimicrobial agents, particularly for
the treatment of the infections of hospitalized patients and
protection of immune-suppressed or HIV-infected patients.
A potential approach is the design of innovative drugs, with
different mechanisms of action, in effort to avoid cross
S. T. Harini ꢀ J. Rangaswamy ꢀ N. Naik (&)
Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysore 570006, Karnataka, India
e-mail: drnaik_chem@yahoo.co.in; drnaik@chemistry.uni-
mysore.ac.in; drnaikchem@gmail.com
S. T. Harini
e-mail: st.harini988@gmail.com
J. Rangaswamy
e-mail: rangaswamyteertha@gmail.com
H. V. Kumar (&)
Department of Organic Chemistry, Indian Institute of Science,
Bangalore 560012, Karnataka, India
e-mail: vijaycftri@gmail.com
S. K. Peethambar
Department of Biochemistry, Kuvempu University,
Jnanasahydri, Shankargatta, Shimoga 575025, Karnataka, India
e-mail: peethambar.sk@gmail.com
123

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Med Chem Res (2014) 23:1887–1898
resistance (Pfeltz and Wilkinson, 2004). Heterocyclic
compounds provide scaffolds on which pharmacophores
can arrange to yield potent and selective drugs (Khan et al.,
2005). Heterocyclic compounds specifically, piperidinone-
based chemical entities with aryl substituents at C-2 and C-
6 of the piperidinone ring have been documented as potent
antioxidant and antimicrobial agents (Balasubramanian
et al., 2005). Recently, oxime derivatives have attracted
considerable attention in medicinal research due to their
antiphytoviral, antitumor and antifungal bioactivities
(Fig. 1) (Ravindra et al., 2009).
Lipophilic nature
MeO
O
O
N
Me N
R
MeO
Motivated by the aforementioned findings and in continu-
ation of our research interest on functionalization of new tri-
cyclic and heterocyclic compounds (Vijay Kumar and Naik,
2010; Vijay Kumar et al., 2011; Nagaraja Naik et al., 2011;
Rangaswamy et al., 2012) herein, we described the synthesis
of novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one
oxime esters 4(a–q) and evaluated their potential towards
antioxidant and antimicrobial activity. The proposed general
chemical motif of 2,6-bis(4-methoxyphenyl)-1-methylpi-
peridin-4-one oxime esters is depicted in Fig. 2.
Electronic effects
R = H, F, Br, Cl, NO2, OH, OCH3, CH3
Fig. 2 General projected chemical motif of 2,6-bis(4-methoxy-
phenyl)-1-methylpiperidin-4-one oxime esters
were performed by the subsequent reaction pathways illus-
trated in Scheme 1. The formation of new compounds was
confirmed by spectral and elemental analysis studies. The IR
spectrum displayed the sharp bands that appeared at 1,608
and 3,617 cm-1 due to C=O and NH functions, respectively;
1
on the other hand, H NMR spectrum revealed the signals
Results and discussion
resonated at d 1.91 (s, 1H, NH), 4.10 (m, 2H, CH), 2.98 (m,
4H, CH2) and 6.94–7.10 (m, 8H, Ar–H), which confirms the
formation of compound 2. IR spectrum of newly synthesized
compounds 4(a–q) exhibited bands at 1691–1728 cm-1 due
to the ester (–N–OCOR) group. 1H NMR spectrum of com-
pounds 4(a–q) exhibited signal at d 1.68–1.77 (s, 3H, N–
CH3) which confirms N-methylation and also the absence of
N–OH proton at d 2.10 (s, 1H, N–OH) confirms the synthesis
of piperidinone oxime esters.
Chemistry
In the present work, four-step synthetic strategies were
adapted for the synthesis of 2,6-bis(4-methoxyphenyl)-1-
methylpiperidin-4-one oxime esters 4(a–q). p-anisaldehyde
condensed (Mannich reaction) with acetone and ammonium
acetate afforded 2,6-bis(4 methoxyphenyl)piperidin-4-one 2
in 70 % yield. Later, compound 2 was treated with iodo-
methane and anhydrous potassium carbonate (anh. K2CO3)
in the presence of acetone to accomplish N-methylated
product 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one
3. In the next step, oximation of compound 3 with NH2
OH.HCl in the presence of sodium acetate trihydrate in
absolute alcohol furnished key scaffold 2,6-bis(4-methoxy-
phenyl)-1-methylpiperidin-4-one oxime 4. Further, a key
scaffold 4 was conjugated with substituted benzoyl chlorides
in the presence of t-BuOK as base afforded 2,6-bis(4-
methoxyphenyl)-1-methylpiperidin-4-one oxime esters 4(a–
q) in good yields. The synthesis of the target compounds
Biological studies
In vitro antioxidant assays
The antioxidant potential of novel 2,6-bis(4-methoxy-
phenyl)-1-methylpiperidin-4-one oxime esters 4(a–q) was
evaluated by means of different in vitro tests like 2,20-
diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
(RSA), 2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
(ABTS•1) radical scavenging assay, ferric ion-reducing
antioxidant power (FRAP) assay and cupric ion-reducing
antioxidant capacity (CUPRAC) assay. Butylated hydroxy
anisole (BHA) was used as an internal standard antioxidant.
Cl
Cl
Cl O
N
DPPH radical scavenging activity
Cl
N
DPPH radical scavenging assay is a standard assay for
antioxidant studies and offers a rapid technique for screening
the radical scavenging activity of any compounds or extracts.
N
Fig. 1 Structure of pharmacologically active oxime-derived drug
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Med Chem Res (2014) 23:1887–1898
1889
O
O
H3C
CH3
O
O
NH4OAc,3H2O, EtOH
rt, 20 min
N
H
+
H
H
MeO
OMe
(2)
MeO
OMe
(1)
(1)
MeI, K2CO3,
acetone,
reflux, 4 h
HO
N
N
O
NH2OH HCl, EtOH
N
NaOAc.3H2O, reflux, 2 h
Me
Me
MeO
OMe
MeO
OMe
(4)
(3)
O
Cl
R1
R1
H
H
H
H
H
H
H
COOMeH
OH
H
H
H
R2
H
H
H
H
Cl
H
NO2
R3
H
F
Br
Cl
H
NO2
H
H
H
H
OH
H
OH
OMe
OMe
OMe OMe
Me
R4
H
H
H
H
H
H
NO2
H
H
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R5
R4
t-BuOK
DMF, rt, 8 h
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
R2
R3
R2
R5
R1
O
R3
H
OH
H
OH
OH
H
H
OMe
H
O
N
N
R4
H
OH
OH
H
H
H
OMe
H
H
Me
MeO
OMe
H
H
4(a-q)
Scheme 1 Reaction protocol for the synthesis of 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters 4(a–q)
RSA of 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one
oxime esters 4(a–q) was tested using an ethanolic solution of
the ‘stable’ free radical, DPPH. Thus, antioxidant molecules
can quench DPPH free radicals (i.e. by providing hydrogen
atoms or by electron donation, conceivably via a free-radical
attack on the DPPH molecule) and convert them to a col-
ourless/bleached product (i.e. absorbance at 517 nm).
Hence, the more rapidly the absorbance decreases, more
potent the antioxidant activity of the compound. 50 %
inhibition concentrations (IC50) for newly synthesized
compounds were calculated and depicted in Table 1. Ini-
tially, 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one
oxime 4 exhibited certain degree of antioxidant activity.
Further, to enhance the RSA, piperidin-4-one oxime 4 was
coupled with different substituted benzoyl chlorides, and
thus showed significant enhancement of activity. This indi-
cates that electronic effects of substituents on phenyl ring
attached to piperidin-4-one oxime play a vital role for
enhanced antioxidant activity. Among the synthesized
compounds 4l and 4m containing two and three –OH groups,
respectively, on phenyl moiety displayed most potent DPPH
radical scavenging capacity of about 13–15 folds compared
to key scaffold 4 and indeed greater activity than the standard
as well. Thus, free-radical scavenging capacity is primarily
attributed to high reactivity of OH group (Heim et al., 2002).
Whereas, compounds 4(i–k) possessing one hydroxyl group
on the phenyl ring at different positions showed 7–10 folds
increased activity. On the other hand, compounds 4(n–p)
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Med Chem Res (2014) 23:1887–1898
Table 1 Concentration required for 50 % scavenging (IC50) of
DPPH and ABTS•? radical scavenging activities of the compounds
4(a–q) and a standard antioxidant BHA
Table 2 Antioxidant capacity of compounds 4(a–q) and a standard
BHA in FRAP and CUPRAC assays at the concentration of 10 lM
Tested
compounds
Fe3?-Fe2?
reducing abilitya
Cu2?-Cu?
reducing abilitya
a
Tested compounds
Scavenging activity (IC50
)
DPPH•
ABTS•?
4
0.1274
0.1321
0.2364
0.2651
0.2584
0.2431
0.2160
0.1954
0.1795
0.3296
0.3213
0.3325
0.3524
0.3735
0.3101
0.2962
0.2856
0.1543
0.3497
0.1636
0.1109
0.1634
0.2154
0.1924
0.1792
0.1568
0.1455
0.1312
0.2719
0.2654
0.2934
0.3628
0.3615
0.2455
0.2364
0.2317
0.1265
0.3247
4a
4b
4c
4d
4e
4f
4
165 0.1
152 0.3
86 0.5
92 0.2
98 0.1
186 0.1
120 0.9
125 0.4
132 0.7
19 0.7
21 0.1
15 0.9
12 0.8
11 0.2
32 0.1
56 0.2
71 0.4
143 0.5
13 0.6
380 0.8
375 0.9
152 0.6
121 0.3
135 0.2
136 0.1
280 0.7
312 0.4
350 0.3
29 0.8
34 0.9
25 0.1
19 0.7
18 0.6
68 0.1
80 0.5
93 0.3
368 0.5
20 0.1
4a
4b
4c
4d
4e
4f
4g
4h
4i
4g
4h
4i
4j
4k
4l
4j
4k
4l
4m
4n
4o
4p
4q
BHA
a
4m
4n
4o
4p
4q
BHA
a
The values are expressed as absorbance. High absorbance indicates
high reducing power
The values are expressed as lM concentration. Lower IC50 values
indicate higher radical scavenging activity
FRAP assay
possessing electron-donating group –OCH3 on phenyl ring
exhibited 3–5 folds increased DPPH activity. Whereas,
compounds 4(b–g) holding electron-withdrawing groups (F,
Br, Cl and NO2) registered the least activity compared to
other analogues.
The reducing capacity of compounds can be measured by
the direct reduction of potassium ferricyanide (III) to
potassium ferrocyanide (II) which leads to the formation of
the intense Perl’s Prussian blue complex, which has a
strong absorbance at 700 nm. Generally, increase in the
absorbance of the reaction mixture indicates increase in the
reducing capacity due to increase in the formation of the
complex. The absorbance values of all the synthesized
compounds are depicted in Table 2. Compounds 4(b–g)
having electron-withdrawing groups (F, Cl, Br and NO2)
on the phenyl ring at different positions displayed less
absorbance values, while the compounds 4(i–k) possessing
–OH group at ortho, meta and para position enhance the
absorbance value reflecting higher but slightly lower
activity than the standard. The introduction of benzoyl
chlorides containing more number of hydroxy groups to
compound 4 which led to compounds 4l and 4m resulted in
higher ferric ion-reducing power than the standard.
ABTS•1 radical scavenging assay
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime
esters 4(a–q) were subjected to ABTS•1 radical scaveng-
ing activity. Here the technique is based on the direct
production of the blue/green ABTS•? chromophore
through the reaction between ABTS and potassium per-
sulphate. The reduction of ABTS•? concentration is
directly related to the antioxidant capacity of the com-
pounds being tested. In this assay, all the synthesized
compounds displayed certain degree of ABTS•1 radical
scavenging capacity. Compounds 4l and 4m bearing more
hydroxyl groups on phenyl skeleton exhibited dominant
activity among all the synthesized compounds and as well
as the standard (Table 2). On the other hand, substitution of
electron-withdrawing group like (F, Br, Cl and NO2) in
compounds 4(b–g) does not showed appreciable enhanced
antioxidant activity.
CUPRAC assay
CUPRAC assay is based on the reduction of cupric (II) ion
to cuprous (I) ion by antioxidants. The observed antioxi-
dant potential for the ethanolic solutions of compounds can
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Med Chem Res (2014) 23:1887–1898
1891
be related to the presence of various functional groups,
such as hydroxyl and methoxy groups. The reducing power
of all the tested compounds increased with increase in their
concentration. Compounds 4l and 4m having the highest
polyphenolic content revealed the highest absorption than
the standard BHA and act as superior cupric ion reducer.
Compounds 4(i–k) having single hydroxyl group and 4(n–
p) bearing one or more methoxy group also exhibited the
marked cupric ion-reducing ability but slightly less than
compounds 4l and 4m. Rest of the compounds, 4a without
any substitution, 4(b–h) possessing electron-withdrawing
groups and 4q bearing methyl group, exhibit moderate
antioxidant capacity (Table 2).
position showed excellent antibacterial activity against P.
aeruginosa and E. coli at concentrations of 1,000 and
500 lg/mL compared to the standard drug streptomycin.
The more lipophilic nature of piperidinone core along with
the presence of electron-withdrawing groups like chloro/
nitro in para-position of the phenyl rings may contribute
for this enhanced antibacterial activity. Halogens like flu-
oro and chloro are very useful to modulate the electronic
effects on phenyl ring and has strong inductive electron-
attracting effects; moreover, these atoms may also influ-
ence the steric characteristics and the hydrophilic–hydro-
phobic balance of the molecules (Rossello et al., 2002).
Compounds 4c and 4e having bromo and chloro group on
benzoyl ester moiety exhibited appreciable activity. The
replacement of fluoro group by nitro showed moderate
activity, whereas introduction of electron-donating hydro-
xyl and methoxy groups in compounds 4(i–p) leads to
marked decrease in activity.
Antimicrobial studies
Antibacterial studies
The potentiality of the synthesized compounds as antimi-
crobials was appraised for their antibacterial studies against
different strains of human pathogens namely Escherichia
coli ATCC 25922 (Gram -Ve), Staphylococcus aureus
ATCC 25923 (Gram ?Ve) and Pseudomonas aeruginosa
ATCC 27853 (Gram -Ve). The results obtained as zone of
inhibition (mm) are presented in Table 3 and revealed that
compounds 4b and 4d possessing electronegative fluoro
(F) and chloro (Cl) group on benzoyl ester moiety at para-
Antifungal studies
Newly synthesized compounds 4(a–q) were also screened
for their antifungal activity against three pathogenic fungal
species namely Aspergillus flavus MTCC 3306, Candida
albicans MTCC 3017 and Chrysosporium keratinophilum
MTCC 2827. The compounds were dissolved in DMSO
and antifungal activity was determined by well plate
Table 3 Inhibitory zone (diameter) mm of the synthesized compounds 4(a–q) against tested bacterial strains by well plate method
Compound name
Escherichia coli
Staphylococcus aureus
Pseudomonas aeroginosa
Concentration (lg/mL)
1000
500
1000
500
1000
500
4
1
2
0.01
0.02
0
1
1
3
0.02
0.02
0
2
9
6
8
7
3
3
2
4
4
3
4
3
1
2
3
1
1
2
0.01
0.01
0
1
4a
0.01
0.01
0.02
0.01
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.01
0.01
4b
17 0.03
10 0.01
16 0.02
12 0.01
12 0.02
0.01
10 0.01
13 0.01
0.02
12 0.01
19 0.02
0.01
15 0.02
0.01
12 0.02
4c
6
8
9
7
4d
16 0.01
11 0.02
4e
7
3
4
2
3
5
3
4
4
1
3
3
1
0.02
0.01
0.01
0.02
0.01
0.02
0.02
0.01
0.01
0.02
0.02
0.01
0.01
9
7
6
5
4
6
5
4
5
3
4
5
3
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.02
8
5
6
4
3
3
2
4
3
3
6
3
2
0.01
0.02
0.01
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.02
0.01
0.01
4f
7
9
4
5
6
4
3
6
2
5
4
3
0.02
0.01
0.01
0.01
0.01
0.02
0.01
0.02
0.01
0.02
0.02
0.01
8
9
5
4
5
3
5
4
4
6
5
2
0.01
0.01
0.02
0.02
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.02
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
Streptomycin
16 0.01
10 0.01
15 0.02
10 0.01
16 0.01
13 0.02
123

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Med Chem Res (2014) 23:1887–1898
Table 4 Inhibitory zone (diameter) mm of the synthesized compounds 4(a–q) against tested fungal strains by well plate method
Compound name
Aspergillus flavus
Chrysosporium keratinophilum
Candida albicans
Concentration (lg/mL)
1000
500
1000
500
1000
500
4
1
1
0.01
0.02
0
1
1
2
9
6
8
7
4
5
3
5
1
2
0
3
3
4
5
1
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.02
1
1
7
4
7
5
3
5
2
3
0
1
1
2
3
3
2
2
0.01
0.01
0.02
0.01
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.01
1
3
0.03
0.01
1
2
0.01
0.01
4a
0.01
4b
12 0.03
10 0.02
16 0.02
0.01
10 0.02
0.01
10 0.02
4c
5
8
7
3
4
2
3
1
0
1
3
5
4
2
2
0.01
0.02
0.01
0.02
0.01
0.01
0.01
0.01
0.02
0.01
0.02
0.01
0.02
0.02
0.01
4
5
6
2
3
1
2
0
1
0
1
4
3
3
1
0.01
0.01
0.02
0.01
0.01
0.02
0.01
0.02
0.02
0.01
0.01
0.02
0.02
0.01
0.01
9
8
4d
11 0.01
10 0.02
4e
7
7
3
5
2
1
2
3
6
5
4
3
1
0.01
0.02
0.01
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.02
0.01
0.01
4f
6
8
4
6
3
2
3
8
7
6
3
2
0.01
0.01
0.02
0.02
0.01
0.02
0.01
0.02
0.01
0.01
0.02
0.02
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
Fluconazole
13 0.01
11 0.02
17 0.02
15 0.01
22 0.02
20 0.02
method at concentration of 1,000 and 500 lg/mL. The
results are depicted in Table 4. Among the tested com-
pounds, compound 4b possessing electronegative fluoro
group on benzoyl ester moiety has emerged as active
antifungal agent against A. flavus compared with the
standard fluconazole. Whereas, other compounds showed
weak to moderate antifungal activity against all the tested
fungal strains compared to the standard.
scavenging capacity than the standard because of having the
highest phenolic content. The presence of hydroxyl group at
para-position of phenyl rings is perhaps a greater determi-
nant for antioxidant activity. Compounds 4(i–k), 4(n–p)
having electron-donating groups like –OH and –OCH3
exhibit good antioxidant activity but slightly less than the
standard. The compounds 4(b–e) having electron-with-
drawing groups like fluoro, bromo and chloro demonstrated
moderate activity. The rest of the compounds 4(f–h) and 4q
exhibited slightly lessto moderate activity. On theotherhand
antimicrobial study was also undertaken to appraise their
antimicrobial potential against different strains of human
pathogens. Compounds 4b and 4d showed excellent anti-
bacterial activity against tested bacterial strains as compared
to the standard drug. Among the screened samples com-
pound 4b emerged as active antifungal agent against A. fla-
vus. The compounds holding electron-donating hydroxyl
and methoxy groups 4(i–p) showed marked decrease in
antimicrobial activity. A careful observation of the results
implies that the lipophilic nature of piperidinone skeleton
and the electronic effects of the halogen substituent on the
benzoyl ester moiety were significant towards increased
antimicrobial activity. Our results prompt that, further
studies on these compounds may be operating as a positive
reinforce of the tendency for the construction of novel
chemical entities with better pharmacological profiles than
the standard drugs.
Conclusion
In wide search programme towards novel and efficient
pharmacophore agents, we have reported a convenient four-
step reaction protocol for the synthesis of novel series of
2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime
esters 4(a–q) through the pathway involving Mannich
reaction of p-anisaldehyde with acetone and ammonium
acetate followed by N-methylation and oximation. Further,
in order to improve the pharmacological activity of piperi-
din-4-one oxime core, esterification of 2,6-bis(4-methoxy-
phenyl)-1-methylpiperidin-4-one oxime 4 with substituted
benzoyl chlorides was successfully done by employing a t-
BuOK-assisted reaction. The newly synthesized compounds
4(a–q) were screened for their antioxidant and antimicrobial
activity using assorted in vitro models. It is noteworthy that
compounds 4l and 4m possessed dominant radical
123

Med Chem Res (2014) 23:1887–1898
Experimental
1893
solvent along with excess methyl iodide was stripped off
from the reaction mixture under reduced pressure. Addition
of distilled water (20 ml) followed by aqueous ammonia
(5 ml) yielded a crude solid. The crude solid thus obtained
was filtered and washed with distilled water. Crystalliza-
tion from ethanol yielded the compound 3 (83 %).
Materials and methods
All the reagents used were purchased from commercial
suppliers without further purification. Melting points were
determined by using an open capillary method and are
uncorrected. Thin-layer chromatography (TLC) was per-
formed with aluminium sheets–Silica gel 60 F254 pur-
chased from Merck. The compounds were purified by using
column chromatography with silica gel (60–120 mesh)
using hexane:ethylacetate (8:2) as eluent. IR: Nicolet 5700
Yield 83 % as brown solid, M.p 138–140 ꢁC. IR (KBr)
1
m
(cm-1): 3092-2918 (Ar–CH), 1608 (C=O). H NMR
max
(300 MHz, DMSO-d6) d ppm: 6.79–6.90 (m, 6H, Ar–H),
3.83 (s, 6H, OCH3), 3.50 (m, 2H, CH), 2.96-2.70 (m, 4H,
CH2), 1.73 (s, 3H, N–CH3). 13C NMR (100 MHz, DMSO-
d6) d ppm: 206.8, 158.0, 135.6, 127.8, 127.0, 114.08,
113.5, 113.3, 63.8, 63.5, 55.8, 50.3, 55.0, 41.05. Mass (m/
z): (M?) 325.10. Anal.calcd. for C20H24NO3, C, 73.82; H,
7.12; N, 4.30; O, 14.75 found: C, 73.80; H, 7.10; N, 4.28;
O, 14.73 %.
1
FT–IR spectrophotometer; H NMR and 13C NMR spectra
were acquired at 300 and 100 MHz, respectively, by using
DMSO as a solvent for all the compounds. Mass spectra
were obtained by Waters–Q–TOF ultima spectrometer.
Micro analytical data were obtained by Elemental–Vario
EL–III.
Synthesis of 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-
4-one oxime (4)
Synthesis of 2,6-bis(4-methoxyphenyl)piperidin-4-one (2)
To the ethanolic solution of 2,6-bis(4-methoxyphenyl)-1-
methylpiperidin-4-one (2 mmol) in a two-neck round-bot-
tomed flask fitted with a reflux condenser, sodium acetate
trihydrate (3 mmol) was added. The mixture was heated to
boil for 15 min. Then, hydroxylamine hydrochloride
(2.3 mmol) was added to the above mixture and was heated
to reflux for further 2 h. After cooling, when the mixture
was slowly poured into ice-cold water (50 ml) with con-
stant stirring, the crude product was precipitated. Crystal-
lization from absolute ethanol yielded the pure compound 4
(89 %).
The experimental procedure adopted for the condensations
was as follows: a mixture of acetone (2 mmol), p-anisal-
dehyde (4 mmol) and ammonium acetate (2 mmol) in
10 ml of ethanol was heated until the acetate dissolved
(Noller and Baliah, 1948) and a yellow colour developed.
After cooling, the reaction mixture was taken in ether
(20 ml) then addition of conc. HCl (1 ml) to the clear fil-
trate afforded the hydrochloride of the 2,6-bis(4-
methoxyphenyl) piperidin-4-one. The free base was
obtained by treating a suspension of the hydrochloride with
aqueous ammonia, followed by dilution with water. The
crude solid thus obtained was filtered. Crystallization from
ethanol yielded the compound 2 (70 %).
Yield 89 % as brown solid, M.p 243–244 ꢁC. IR (KBr)
max
m
(cm-1): 3102-2956 (Ar–CH), 3411 (N–OH). 1H
NMR (300 MHz, DMSO-d6) d ppm: 6.94–7.10 (m, 8H,
Ar–H), 1.75-1.48 (m, 4H, CH2), 1.72 (s, 3H, CH3), 3.30 (m,
2H, CH), 3.80 (s, 6H, OCH3), 2.12 (s, 1H, N–OH). 13C
NMR (100 MHz, DMSO-d6) d ppm: 159.5, 159.4, 156.5,
131.2, 131.1, 128.8, 128.2, 126.8, 126.0, 114.3, 114.2,
113.8, 113.5, 69.8, 69.5, 55.8, 55.1, 42.8, 37.5, 31.2. Mass
(m/z): (M?) 340.28. Anal.calcd. for C20H24N2O3, C, 70.56;
H, 7.11; N, 8.23; O, 14.10 found: C, 70.53; H, 7.09; N,
8.20; O, 14.08 %.
Yield 70 % as brown solid, M.p 148–150 ꢁC. IR (KBr)
m
max (cm-1): 3030-2956 (Ar–CH), 1608 (C=O), 3617 (N–
1
H). H NMR (300 MHz, DMSO-d6) d ppm: 6.94–7.10 (m,
8H, Ar–H), 4.10 (m, 2H, CH), 3.81 (s, 6H, OCH3), 2.98 (m,
4H, CH2), 1.91 (s, 1H, NH). 13C NMR (100 MHz, DMSO-
d6) d ppm: 206.8, 158.0, 135.4, 127.3, 127.0, 126.9, 114.0,
114.0, 113.9, 113.9, 64.0, 55.6, 50.3, 50.1. Mass (m/z):
(M?) 311.05. Anal.calcd. for C19H21NO3, C, 73.29; H,
6.80; N, 4.50; O, 15.41 found: C, 73.25; H, 6.84; N, 4.51,
O, 15.41 %.
General procedure for the synthesis of 2,6-bis(4-methoxy-
phenyl)-1-methylpiperidin-4-one oxime esters 4(a–q) To
a stirring solution of 2,6-bis(4-methoxyphenyl)-1-meth-
ylpiperidin-4-one oxime (1 mmol) in N,N-dimethylform-
amide (DMF) at room temperature, potassium tert-
butoxide (0.1 mmol) was added, after stirring for 25 min
substituted benzoyl chlorides (0.9 mmol) was added in
drop-wise fashion to the reaction mixture and the stirring
was continued for further 8 h at room temperature. Pro-
gress of the reaction was monitored by TLC using hexane:
Synthesis of 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-
4-one (3)
A mixture of 2,6-bis(4-methoxyphenyl)piperidin-4-one
(2 mmol), potassium carbonate (K2CO3) (2 mmol) and
iodomethane (2.5 mmol) in acetone was refluxed for 4 h.
Progress of the reaction was monitored by TLC using
hexane:ethylacetate (8:2) as solvent. After cooling, the
123

1894
Med Chem Res (2014) 23:1887–1898
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-chlo-
ethyl acetate (8:2) mixture as mobile phase. Then, the
reaction mixture was quenched with brine solution (5 ml).
The organics was separated and the aqueous layer was
extracted with diethyl ether (2 9 10 ml). The combined
organics were dried over anhydrous sodium sulphate and
concentrated under reduced pressure. Purification of the
piperidinone oxime esters was accomplished by column
chromatography over silica gel, eluting with a solvent
system of hexane:ethyl acetate (80:20). The fractions
containing products 4(a–q) were collected and concen-
trated under reduced pressure.
robenzoyl oxime (4d) Yield 81.25 % as yellow solid, M.p
(cm-1): 3069-2856 (Ar–CH),
227–229 ꢁC. IR (KBr) m
max
1728 (NOCOR). 1H NMR (300 MHz, DMSO-d6) d ppm: 6.
68–7.85 (m, 12H, Ar–H), 3.28 (m, 2H, CH), 3.81 (s, 6H,
OCH3), 1.78-1.53 (m, 4H, CH2), 1.70 (s, 3H, N–CH3). 13
C
NMR (100 MHz, DMSO-d6) d ppm: 164.5 159.4, 158.8,
158.5, 147.4, 147.3, 146.8, 132.3, 131.7, 131.5, 121.7, 121.
4, 115.1, 114.9, 112.4, 112.2, 110.5, 110.3, 108.8, 69.6, 69.
8, 56.6, 56.7, 37.6, 31.6, 40.8, 30.2. Mass (m/z): (M?) 478.
07. Anal.calcd. for C27H27ClN2O4, C, 67.71; H, 5.68; Cl, 7.
40; N, 5.85; O, 13.36 found: C, 67.51; H, 5.18; Cl, 7.48; N,
5.25; O, 13.16 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-ben-
zoyl oxime (4a) Yield 87.41 % as brown solid, M.p
(cm-1): 3128-2978 (Ar–CH),
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-3-chlo-
229–231 ꢁC. IR (KBr) m
max
1692 (NOCOR). 1H NMR (300 MHz, DMSO-d6) d ppm: d
6.79–8.21 (m, 13H, Ar–H), 3.80 (s, 6H, OCH3), 3.31 (m,
2H, CH), 1.75-1.50 (m, 4H, CH2), 1.72 (s, 3H, N-CH3),.
13C NMR (100 MHz, DMSO- d6) d ppm: 165.6, 159.8,
158.5, 158.1, 133.5, 132.2, 132.8, 132.6, 131.5, 131.4, 128.
9, 127.8, 127.3, 127.6, 125.5, 125.4, 114.8, 114.9, 113.6,
113.5, 68.8, 68.9, 55.8, 55.1, 42.1, 36.5. Mass (m/z): (M?)
444.28. Anal.calcd. for C27H28N2O4, C, 72.95; H, 6.35; N,
6.30; O, 14.40 found: C, 72.55; H, 6.34; N, 6.32; O, 14.
41 %.
robenzoyl oxime (4e) Yield 73.50 % as brown solid, M.p
(cm-1): 3030-2956 (Ar–CH),
252–254 ꢁC. IR (KBr) m
max
1705 (NOCOR). 1H NMR (300 MHz, DMSO-d6) d ppm: 6.
68–7.85 (m, 12H, Ar–H), 3.80 (s, 6H, OCH3), 1.73-1.48
(m, 4H, CH2), 1.75 (s, 3H, N–CH3), 3.31 (m, 2H, CH),. 13
C
NMR (100 MHz, DMSO- d6) d ppm: 164.5 159.4, 158.8,
158.5, 147.4, 147.3, 146.8, 132.3, 131.7, 131.5, 121.7, 121.
4, 115.1, 114.9, 112.4, 112.2, 110.5, 110.3, 108.8, 69.6, 69.
8, 56.6, 56.7, 37.6, 31.6, 40.8, 30.2. Mass (m/z): (M?) 478.
19. Anal.calcd. for C27H27ClN2O4, C, 67.71; H, 5.68; Cl, 7.
40; N, 5.85; O, 13.36 found: C, 67.54; H, 5.15; Cl, 7.44; N,
5.21; O, 13.16 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-flu-
orobenzoyl oxime (4b) Yield 76.0 % as brown solid, M.p
(cm-1): 3128-2917 (Ar–CH),
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-ni-
173–175 ꢁC. IR (KBr) m
max
1695 (NOCOR). 1H NMR (300 MHz, DMSO-d6) d ppm: 6.
68–8.19 (m, 12H, Ar–H), 3.84 (s, 6H, OCH3), 3.32 (m, 2H,
CH), 1.73-1.47 (m, 4H, CH2), 1.68 (s, 3H, N–CH3). 13C
NMR (100 MHz, DMSO-d6) d ppm: 168.6, 165.7, 159.5,
159.51, 156.5, 131.93, 131.25, 131.26, 128.82, 128.83,
127.5, 126.05, 126.04, 115.6, 115.3 114.5, 114.6, 113.66,
113.65, 69.8, 69.9, 55.5, 55.51, 43.8, 37.4, 32.85. Mass (m/
z): (M?) 462.19. Anal.calcd. for C27H27FN2O4, C, 70.11;
H, 5.88; F, 4.11; N, 6.06; O, 13.84 found: C, 70.09; H, 5.
86; F, 4.14; N, 6.03; O, 13.82 %.
trobenzoyl oxime (4f) Yield 80.88 % as yellow solid, M.p
231–232 ꢁC. IR (KBr) m
(cm-1): 3028-2956 (Ar–CH),
max
1715 (NOCOR). 1H NMR (300 MHz, DMSO-d6) d ppm: 6.
68–7.85 (m, 10H, Ar–H), 3.85 (s, 6H, OCH3), 3.30 (m, 2H,
CH), 1.82-1.50 (m, 4H, CH2), 1.70 (s, 3H, N–CH3). 13C
NMR (100 MHz, DMSO-d6) d ppm: 166.3, 161.3, 158.5,
147.9, 147.7, 145.8, 134.3, 133.4, 132.6, 131.9, 130.8, 130.
1, 128.9,128.5, 127.4, 121.2, 120.5, 116.3, 115.8, 111.
4,110.5, 67.8, 56.9, 55.8, 40.8, 37.9, 30.9. Mass (m/z):
(M?) 489.13. Anal.calcd. for C27H27N4O6 C, 66.25; H, 5.
56; N, 8.58; O, 19.61 found: C, 66.28; H, 5.54; N, 8.60; O,
19.60 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-bro-
mobenzoyl oxime (4c) Yield 75.93 % as off-white solid,
(cm-1): 3058-2914 (Ar–
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-2,4-dini-
M.p 215–217 ꢁC. IR (KBr) m
max
1
trobenzoyl oxime (4g) Yield 86.54 % as yellow solid, M.
(cm-1): 3152-3010 (Ar–
CH), 1701 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: d 6.68–8.19 (m, 12H, Ar–H), 1.75-1.50 (m, 4H,
CH2), 1.72 (s, 3H, N–CH3), 3.30 (m, 2H, CH), 3.83 (s, 6H,
OCH3). 13C NMR (100 MHz, DMSO-d6) d ppm: 165.5,
159.6, 159.4, 156.5, 139.2, 131.8, 131.6, 131.5, 131.1, 128.
4, 128.3, 128.1, 126.5, 126.0, 114.3, 114.2, 113.8, 113.5,
69.8, 69.9, 55.2, 55.0, 42.8, 37.5, 31.8. Mass (m/z): (M?)
522.09. Anal.calcd. for C27H27BrN2O4, C, 61.96; H, 5.20;
Br, 15.27; N, 5.35; O, 12.23 found C, 61.93; H, 5.18; Br,
15.24; N, 5.32; O, 12.25 %.
p 238–240 ꢁC. IR (KBr) m
max
1
CH), 1711 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.68–8.94 (m, 9H, Ar–H), 3.79 (s, 6H, OCH3), 3.25
(m, 2H, CH), 1.78-1.54 (m, 4H, CH2), 1.76 (s, 3H, N–
CH3). 13C NMR (100 MHz, DMSO-d6) d ppm: 165.3, 161.
3, 154.7, 147.9, 147.7, 145.8, 134.3, 133.4, 132.6, 131.9,
130.8, 130.1, 128.9, 128.5, 127.4, 121.2, 120.5, 116.3, 115.
8, 111.4,110.5, 68.6, 67.8, 56.9, 55.8, 38.9, 31.9. Mass (m/
z): (M?) 534.08. Anal.calcd. for C27H26N4O8, C, 60.67; H,
123

Med Chem Res (2014) 23:1887–1898
1895
4.90; N, 10.48; O, 23.95 found: C, 60.65; H, 4.91; N, 10.
40; O, 23.94 %.
77 (s, 3H, N–CH3). 13C NMR (100 MHz, DMSO-d6) d
ppm: 168.5,166.2, 162.2, 148.3, 147.3, 147.2, 146.5, 134.0,
133.8, 132.6, 130.9, 130.5, 130.2, 127.0, 121.3, 120.2, 116.
3, 115.8, 112.4, 110.5, 68.2, 67.8, 56.0, 55.8, 40.2, 38.1, 30.
5. Mass (m/z): (M?) 506.19. Anal.calcd. for C27H28N2O5,
C, 70.42; H, 6.13; N, 6.08; O, 17.37 found: C, 70.44; H, 6.
11; N, 6.01; O, 17.14 %.
2-((2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-
ylideneaminooxy)carbonyl)phenyl acetate (4h) Yield 84.
20 % as white solid, M.p 221–223 ꢁC. IR (KBr) m
max
(cm-1): 3069-2919 (Ar–CH), 1721 (NOCOR). 1H NMR
(300 MHz, DMSO-d6) d ppm: 6.68–8.18 (m, 10H, Ar–H),
3.83 (s, 6H, OCH3), 3.30 (m, 2H, CH), 1.83-1.54 (m, 4H,
CH2), 1.73 (s, 3H, N–CH3), 2.28 (s, 3H, COCH3). 13C
NMR (100 MHz, DMSO-d6) d ppm: 170.5, 165.3, 161.3,
159.7, 147.9, 147.7, 145.8, 134.3, 133.4, 132.6, 131.8, 130.
5, 130.0, 128.7,128.4, 127.6, 121.0, 120.4, 116.7, 114.8,
111.4,110.5, 68.6, 65.8, 56.1, 55.8, 40.9, 31.5, 19.5. Mass
(m/z): (M?) 502.31. Anal.calcd. for C29H30N2O6, C, 69.31;
H, 6.02; N, 5.57; O, 19.10 found: C, 69.30; H, 6.04; N, 5.
51; O, 19.11 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-3,5-
dihydroxybenzoyl oxime (4l) Yield 75.39 % as brown
solid, M.p 261–263 ꢁC. IR (KBr) m max (cm-1): 3120-2917
1
(Ar–CH), 1716 (NOCOR). H NMR (300 MHz, DMSO-
d6) d ppm: 6.68–8.10 (m, 11H, Ar–H), 5.34 (s, 2H, OH), 3.
80 (s, 6H, OCH3), 3.28 (m, 2H, CH), 1.82-1.57 (m, 4H,
CH2), 1.72 (s, 3H, N–CH3). 13C NMR (100 MHz, DMSO-
d6) d ppm: 165.4, 159.3, 147.8, 147.5, 145.0, 134.3, 143.4,
142.6, 141.9, 130.8, 130.1, 130.9, 125.5, 121.4, 121.2, 115.
5, 115.8, 112.2, 111.4, 110.5, 68.6, 67.9, 55.9, 55.8, 40.8,
37.9, 30.9. Mass (m/z): (M?) 476.28. Anal.calcd. for
C27H28N2O6, C, 68.05; H, 5.92; N, 5.88; O, 20.15 found: C,
68.04; H, 5.91; N, 5.85; O, 20.16 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-2-hy-
droxybenzoyl oxime (4i) Yield 79.35 % as brown solid,
(cm-1): 3081-2872 (Ar–
M.p 215–217 ꢁC. IR (KBr) m
max
1
CH), 1708 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.68–8.94 (m, 12H, Ar–H), 5.35 (s, 1H, OH), 3.80 (s,
6H, OCH3), 3.30 (m, 2H, CH), 1.72-1.49 (m, 4H, CH2), 1.
68 (s, 3H, N–CH3). 13C NMR (100 MHz, DMSO-d6) d
ppm: 168.2, 165.3, 161.3, 159.7, 147.9, 147.7, 145.8, 134.
3, 133.4, 131.5, 130.0, 128.5, 128.4, 127.6, 121.2, 120.6,
116.7, 114.6, 111.4,110.5, 68.6, 65.8, 56.1, 55.0, 40.9, 38.
7, 31.5. Mass (m/z): (M?) 460.16. Anal.calcd. for
C27H28N2O5, C, 70.42; H, 6.13; N, 6.08; O, 17.37 found: C,
70.42; H, 6.13; N, 6.08; O, 17.37 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-3,4,5-tri-
hydroxybenzoyl oxime (4m) Yield 87.17 % as brown solid,
(cm-1): 3101-2972 (Ar–
M.p 245–247 ꢁC. IR (KBr) m
max
1
CH), 1691 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.94–7.10 (m, 10H, Ar–H), 5.32 (s, 3H, OH), 3.84 (s,
9H, OCH3), 3.30 (m, 2H, CH), 1.71-1.52 (m, 4H, CH2), 1.
68 (s, 3H, N–CH3). 13C NMR (100 MHz, DMSO-d6) d
ppm: 165.4, 159.3, 147.8, 147.5, 145.0, 134.3, 143.4, 142.
6, 141.9, 130.8, 130.1, 130.9, 125.5, 121.4, 121.2, 115.5,
115.8, 112.2, 111.4, 110.5, 68.6, 67.9, 55.9, 55.8, 40.8, 37.
9, 30.9. Mass (m/z): (M?) 492.24. Anal.calcd. for
C27H28N2O7 C, 65.84; H, 5.73; N, 5.69; O, 22.74 found: C,
65.81; H, 5.75; N, 5.67; O, 22.72 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-3-hy-
droxybenzoyl oxime (4j) Yield 69.56 % as brown solid,
(cm-1): 3162-2958 (Ar–
M.p 193–195 ꢁC. IR (KBr) m
max
1
CH), 1700 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.68–8.94 (m, 12H, Ar–H), 5.30 (s, 1H, OH), 3.81 (s,
6H, OCH3), 3.31 (m, 2H, CH), 1.80-1.50 (m, 4H, CH2), 1.
73 (s, 3H, N–CH3). 13C NMR (100 MHz, DMSO-d6) d
ppm: 165.4, 159.3, 147.8, 147.5, 145.0, 134.3, 143.4, 142.
6, 141.9, 130.8, 130.1, 130.9,125.5, 121.4, 121.2, 115.5,
115.8, 112.2, 111.4,110.5, 68.6, 67.9, 55.9, 55.8, 40.8, 37.
9, 30.9. Mass (m/z): (M?) 506.16. Anal.calcd. for
C27H28N2O5, C, 70.42; H, 6.13; N, 6.08; O, 17.37 found: C,
70.41; H, 6.10; N, 6.04; O, 17.17 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-
methoxybenzoyl oxime (4n) Yield 81.34 % as white solid,
(cm-1): 3178-2911 (Ar–
M.p 212–214 ꢁC. IR (KBr) m
max
1
CH), 1689 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.94–7.10 (m, 12H, Ar–H), 3.81 (s, 9H, OCH3), 3.30
(m, 2H, CH), 1.73-1.50 (m, 4H, CH2), 1.70 (s, 3H, N–
CH3). 13C NMR (100 MHz, DMSO-d6) d ppm: 168.1, 164.
7, 160.8, 158.1, 146.0, 147.1, 147.0, 146.9, 133.7, 131.8,
131.7, 122.0, 118.8, 115.7, 114.2, 112.9, 111.7, 106.8, 105.
0, 101.8, 69.8, 69.4, 56.1, 56.0, 55.2, 55.4, 40.5, 37.4. Mass
(m/z): (M?) 474.20. Anal.calcd. for C28H30N2O5, C, 65.84;
H, 5.73; N, 5.69; O, 22.74 found: C, 65.81; H, 5.75; N, 5.
67; O, 22.72 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-hy-
droxybenzoyl oxime (4k) Yield 76.24 % as brown solid,
(cm-1): 3079-2952 (Ar–
M.p 233–235 ꢁC. IR (KBr) m
max
1
CH), 1713 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.68–8.94 (m, 12H, Ar–H), 5.35 (s, 1H, OH), 3.85 (s,
6H, OCH3), 3.33 (m, 2H, CH), 1.79-1.53 (m, 4H, CH2), 1.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-2,4-
dimethoxybenzoyl oxime (4o) Yield 74.50 % as brown
123

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Med Chem Res (2014) 23:1887–1898
solid, M.p 219–221 ꢁC. IR (KBr) m max (cm-1): 3205-2950
(Ar–CH), 1711 (NOCOR). H NMR (300 MHz, DMSO-
added and shaken vigorously. The tubes were then incu-
bated in the dark room at RT for 20 min. A DPPH blank
was prepared without compound and ethanol was used for
the baseline correction. Changes (decrease) in the absor-
bance at 517 nm were measured using a UV–visible
spectrophotometer and the remaining DPPH was calcu-
lated. The percent decrease in the absorbance was recorded
for each concentration and percent quenching of DPPH was
calculated on the basis of the observed decreased in
absorbance of the radical. The radical scavenging activity
was expressed as the inhibition percentage and was cal-
culated using the formula:
1
d6) d ppm: 6.68–8.12 (m, 10H, Ar–H), 3.80 (s, 12H,
OCH3), 3.30 (m, 2H, CH), 1.72-1.53 (m, 4H, CH2), 1.69 (s,
3H, N–CH3). 13C NMR (100 MHz, DMSO-d6) d ppm: 168.
5, 164.1, 160.5, 159.1, 146.9, 147.3, 147.4, 147.1, 133.6,
131.5, 131.2, 121.0, 119.8, 115.6, 115.2, 112.5, 111.8, 105.
8, 105.0, 101.8, 69.5, 69.4, 56.3, 56.0, 55.7, 55.4, 40.5, 37.
4, 31.2. Mass (m/z): (M?) 504.33. Anal.calcd. for
C29H32N2O6, C, 69.03; H, 6.39; N, 5.55; O, 19.03 found: C,
69.05; H, 6.34; N, 5.53; O, 19.01 %.
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-3,4,5-
trimethoxybenzoyl oxime (4p) Yield 76.36 % as brown
solid, M.p 247–249 ꢁC. IR (KBr) m max (cm-1): 3058-2977
Radical scavenging activityð%Þ ¼ ½ðAo ꢁ A1Þ=Ao ꢂ 100ꢃ
where Ao is the absorbance of the control (blank, without
compound) and A1 is the absorbance of the compound.
1
(Ar–CH), 1702 (NOCOR). H NMR (300 MHz, DMSO-
d6) d ppm: 6.68–8.13 (m, 10H, Ar–H), 1.77-1.49 (m, 4H,
CH2), 1.69 (s, 3H, N–CH3), 3.30 (m, 2H, CH), 3.78 (s,
15H, OCH3). 13C NMR (100 MHz, DMSO-d6) d ppm: 168.
5, 164.1, 160.5, 159.1, 146.9, 147.3, 147.4, 147.1, 133.6,
131.5, 131.2, 121.0, 119.8, 115.6, 115.2, 112.5, 111.8, 105.
8, 105.0, 101.8, 69.5, 69.4, 56.3, 56.1, 56.0, 55.7, 55.4, 40.
5, 37.4, 31.2. Mass (m/z): (M?) 534.14. Anal.calcd. for
C30H34N2O7, C, 67.40; H, 6.41; N, 5.24; O, 20.95 found: C,
67.36; H, 6.31; N, 5.20; O, 20.98 %.
2,20-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•1
)
radical scavenging assay The ABTS•1 cation was produced
by the reaction between 7 mmol ABTS in H2O and 2.
45 mmol potassium persulphate, stored in the dark at room
temperature for 12 h (Re et al., 1999). Before usage, the
ABTS•1 solution was diluted to get an absorbance of 0.
700 0.025 at 734 nm with phosphate buffer (0.1 M, pH 7.
4). Then, 1 ml of ABTS•1 solution was added to the com-
pounds 4(a–q) solution in ethanol at different concentrations
(10, 25, 50, 100, 200 and 500 lM/ml). After 30 min, the
percentage inhibition at 734 nm was calculated for each
concentration relative to a blank absorbance (ethanol). The
scavenging capability of ABTS•1 radical was calculated
using the following equation:
2,6-Bis(4-methoxyphenyl)-1-methylpiperidin-4-one O-4-meth-
ylbenzoyl oxime (4q) Yield 81.55 % as off-white solid, M.
(cm-1): 3030-2917 (Ar–
p 216–218 ꢁC. IR (KBr) m
max
1
CH), 1698 (NOCOR). H NMR (300 MHz, DMSO-d6) d
ppm: 6.68–7.60 (m, 10H, Ar–H), 7.48 (m, 1H, C6H5CH), 6.
31 (d,1H, COCH), 1.75 (s, 3H, N–CH3), 1.69–1.55 (m, 4H,
CH2), 3.30 (m, 2H, CH), 3.82 (s, 6H, OCH3), 2.13 (s, 3H,
C6H5CH3). 13C NMR (100 MHz, DMSO-d6) d ppm 171.8,
159.5, 147.9, 147.6, 147.5, 146.8, 146.1, 136.1, 131.6, 130.
8, 128.6, 128.5, 127.9, 127.6, 126.9, 121.3, 121.5, 115.5,
114.5, 112.0, 111.5, 69.8, 68.9, 56.5, 56.4, 41.6, 31.5, 21.3.
Mass (m/z): (M?) 458.26. Anal.calcd. for C28H30N2O4, C,
73.34; H, 6.59; N, 6.11; O, 13.96 found: C, 73.31; H, 6.55;
N, 6.09; O, 13.94 %.
ABTSꢄþscavenging effectð%Þ ¼ ½ðAc ꢁ AsÞ=Acꢃ ꢂ 100
where, Ac is the absorbance of the initial concentration of the
ABTS•1 and As is the absorbance of the remaining con-
centration of ABTS•1 in the presence of the compounds.
Ferric ion-reducing antioxidant power (FRAP) assay
The reducing power of the synthesized compounds was
determined according to the method of Oyaizu (Oyaizu,
1986). The compounds having 10 lM were mixed with 2.
5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of
1 % potassium ferric cyanide and then incubated at 50 ꢁC
for 20 min. To this mixture 2.5 mL of 10 % trichloroacetic
acid was added and the mixture was centrifuged at
3000 rpm for 20 min. The upper layer (2.5 mL) was mixed
with 2.5 mL of deionised water and 0.5 mL of 0.1 % ferric
chloride and the absorbance was measured at 700 nm using
a spectrophotometer (Shimadzu 160A). Increases of
absorbance of the reaction mixture indicate higher reducing
power. Mean values from three independent samples were
calculated for each compounds.
Antioxidant evaluation
2,20-Diphenyl-1-picrylhydrazyl (DPPH) radical scaveng-
ing assay (RSA) The evaluation of antioxidant activity of
newly synthesized compounds was done by DPPH radical
scavenging assay (Blois, 1958). Internal standard BHA and
the synthesized compounds 4(a–q) of different concentra-
tions were prepared in distilled ethanol, 1 mL of each
compound solutions having different concentrations (10,
25, 50, 100, 200 and 500 lM) were taken in different test
tubes, 4 mL of 0.1 mM ethanol solution of DPPH was
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Med Chem Res (2014) 23:1887–1898
1897
Cupric ion-reducing antioxidant capacity (CUPRAC)
assay In order to determine the cupric ions (Cu2?)-
reducing ability of 2,6-bis(4-methoxyphenyl)-1-methylpi-
peridin-4-one oxime esters 4(a–q), the method of Huang
was used (Huang and Kuo, 2000). For the assay, 0.25 mL
CuCl2 solution (0.01 M), 0.25 mL ethanolic neocuproine
solution (7.5 9 10-3 M) and 0.25 mL CH3COONH4 buf-
fer solution (1 M) were added to a test tube, followed by
mixing of compounds at 10 lM. Then, total volume was
adjusted to 2 mL with distilled water and the solution was
mixed well. The tubes were stoppered and kept at room
temperature. Absorbance was measured at 450 nm against
a reagent blank 30 min later. Increased absorbance of the
reaction mixture indicates increased reduction capability.
poured into each Petri dish. Excess of suspension was
decanted and plates were dried by placing in incubator at
37 ꢁC for 1 h. Using sterile cork borer punched carefully,
wells were made on these seeded agar plates and different
concentrations of the test compounds in DMSO were added
into each labelled well. A control was also prepared for the
plates in the same way using solvent DMSO. The Petri
dishes were prepared in triplicate and maintained at 25 ꢁC
for 72 h. Antifungal activity was determined by measuring
the diameter of inhibition zone. Activity of each compound
was compared with fluconazole as the standard. Zones of
inhibition were determined for compounds 4(a–q).
Statistical analysis
Antimicrobial studies
Assays were carried out in triplicate for 3–5 separate
experiments. The amount of compound needed to inhibit
DPPH free radical and ABTS•1 cation by 50 % (IC50) was
graphically estimated using a linear regression algorithm.
The antimicrobial assays were carried out in triplicate and
standard deviation was calculated.
Antibacterial studies The antibacterial activities of newly
synthesized compounds 4(a–q) were determined by well
plate method in Mueller–Hinton Agar (Arthington-Skaggs
et al., 2000). The in vitro antibacterial activity was carried
out against 24-h-old cultures of bacterial strains. In this
work, E. coli ATCC 25922 (Gram -Ve), Staphylococcus
aureus ATCC 25923 (Gram ?Ve) and Pseudomonas
aeruginosa ATCC 27853 (Gram -Ve) were used to
investigate the activity. The test compounds were dissolved
in dimethyl sulphoxide (DMSO) at concentration of 1,000
and 500 lg/mL. Twenty millilitres of sterilized agar media
was poured into each pre-sterilized Petri dish. Excess of
suspension was decanted and plates were dried by placing
in an incubator at 37 ꢁC for an hour. About 60 ml of 24-h-
old culture suspension was poured and neatly swabbed with
the pre-sterilized cotton swabs. Six millimetre diameter
wells were then punched carefully using a sterile cork borer
and 30 ml of test solutions of different concentrations were
added into each labelled well. The plates were incubated
for 24 h at 37 ꢁC. The inhibition zone that appeared after
24 h around the well in each plate was measured as zone of
inhibition in mm. Experiments were triplicates and stan-
dard deviation was calculated.
Acknowledgments The authors are thankful to NMR Research Center,
Indian Institute of Science, Bangalore for providing spectral data.
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