Synthesis of polyfunctionalized piperidone oxime ethers and their cytotoxicity on HeLa cells

Article abstract of DOI:10.1016/j.bmcl.2011.09.063

A series of twenty 2,6-diarylpiperidin-4-one O-methyloximes were synthesized with fluoro/chloro/bromo/methyl/methoxy/ethoxy/isopropyl substituents on various positions of the phenyl at C-2 and C-6 in association with/without methyl substituent on the seco

Full text of DOI:10.1016/j.bmcl.2011.09.063

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6678–6686
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of polyfunctionalized piperidone oxime ethers and their cytotoxicity
on HeLa cells
Paramasivam Parthiban a,b, Ramjee Pallela c, Se-Kwon Kim c, Dong Ho Park a, Yeon Tae Jeong b,
⇑
a Department of Biomedicinal Chemistry, Inje University, Gimhae 621 749, Gyeongnam, South Korea
b Division of Image Science and Engineering, Pukyong National University, Busan 608 737, South Korea
c Marine Bioprocess Research Center, Pukyong National University, Nam-Gu, Busan 608 737, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 May 2011
Revised 29 July 2011
Accepted 16 September 2011
Available online 21 September 2011
A series of twenty 2,6-diarylpiperidin-4-one O-methyloximes were synthesized with fluoro/chloro/
bromo/methyl/methoxy/ethoxy/isopropyl substituents on various positions of the phenyl at C-2 and
C-6 in association with/without methyl substituent on the secondary amino group and methyl/ethyl/iso-
propyl substituents on the active methylene centers. Regardless of their substitution all compounds pre-
dominantly exist in the chair conformation except 3m, which adopts a twist-boat conformation. All the
synthesized compounds were evaluated for their in vitro antiproliferative activity against human cervical
carcinoma (HeLa) cell line. The cytotoxicity of the test compounds was determined by measuring the
number of live cells after 24 h of treatment by MTT assay method. This preliminary SAR suggests some
lead molecules 3c–f, 3j–k, 4d–g, and 4i with a scope of further structural optimization of the piperidone
pharmacophore toward the development of anticancer drug synthesis.
Keywords:
Polyfunctionalized piperidin-4-ones
Stereochemistry
Epimerization
HeLa cells
Ó 2011 Elsevier Ltd. All rights reserved.
Cytotoxicity
MTT assay
Nitrogen containing heterocycles always signified a subject of
great interest due to their ubiquity in nature and massive presence
as part of the skeletal backbone of many therapeutic agents.1 Of
these heterocycles, piperidone pharmacophore is very momentous
by its broad-spectrum of biological actions.2 Introduction of vari-
ous substituents on the piperidone heterocycle and reduction of
the carbonyl as oxime functionality improved the biological effi-
cacy.3 As a part of our ongoing research4 toward the development
of new piperidone based compounds with structure diversity, for
this study, 2,6-diphenylperidin-4-one O-methyloxime 3a, 3-alkyl
analogs 3b–j, 3,5-dimethyl analogs 3k–o and some N-methyl ana-
logs 4d–i were synthesized as depicted in Scheme 1.
All compounds were characterized by their analytical and spec-
tral data, and thus their stereochemistries are established.5 Com-
pound 3a has no substitution on the heterocycle other than the
phenyl rings at C-2 and C-6, which paved the secondary amino
group. The vicinal couplings clearly suggest that 3a adopts a chair
conformation with an equatorial orientation of the phenyl rings on
both sides of the secondary amino group. Introduction of a methyl
group at C-3 of 3a along with/without halo/alkyl/alkoxy substitu-
ents on the phenyl at C-2 and C-6 (3b–h) did not alter the stereo-
chemistry. Likewise, the introduction of methyl group at ring
nitrogen of 3d–h also did not alter the stereochemistry of 4d–h.
Both the methyl groups at N-1 and C-3 preferred the equatorial
disposition. Similarly to methyl, the introduction of ethyl 3i and
isopropyl 3j groups at C-3 of 3a also did not affect the stereochem-
istry significantly. They also adopt the analogous stereochemistry
as 3b. However, the decrease in the 3J2a,3a of 3j indicates that there
may be a least possible population of boat conformation for 3j and
its N-methyl analog 4i.
On the other hand, the incorporation of methyl group on both
sides of the active methylene centers C-3 and C-5 partially/com-
pletely modify the stereochemistry of the synthesized compounds
3k–o. Although the 3,5-dimethyl substituted piperidones 1k–o
exist in the chair conformation with an equatorial orientation of
all substituents, the oxime derivatives 3k, 3l, 3n and 3o under-
went epimerization to retain the chair conformation. One of the
methyl groups, which is syn to the oxime moiety was epimerized
to adopt an axial orientation at C-5 to stabilize the chair confor-
mation. In fact, in order to relieve from the severe allylic 1,3-
interaction between the methyl at C-5 and N–O, the methyl at
C-5 was epimerized. But a prominent change was noticed on
compound 3m, which underwent a conformational transforma-
tion instead of the epimerization observed in analogous com-
pounds and thus adopts a twist-boat conformation.
All the synthesized compounds 3a–4i were evaluated for their
in vitro antiproliferative activity against human cervical carcinoma
(HeLa) cell line. The cytotoxicity of the test compounds was deter-
mined by measuring the number of live cells after 24 h of treatment
by MTT assay.6 The IC50 values of all compounds are summarized
along with their stereochemical structures in Table 1 for better
⇑
Corresponding author.
E-mail address: ytjeong@pknu.ac.kr (Y.T. Jeong).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.063

P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
6679
O
R3
R2
O
O
R3
R2
R3
R2
a
b
O
O
R1
R
R1
R
R1
R
R1
R
R1
R
R1
R
N
CH3
N
H
H
H
1a-1o
c
2d-2i
c
CH3COONH4
CH3
CH3
O
O
N
N
R3
R2
R3
R2
R1
R
R1
R
R1
R
R1
R
N
CH3
N
H
3a-3o
4d-4i
Entry
R
R1
R2
R3
Entry
R
H
R1
R2
R3
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
CH
2CH3
H
a
b
c
d
e
f
i
j
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH(CH3)2
CH3
H
Br
H
CH3
CH3
CH3
CH3
CH3
k
l
Cl
CH3
H
H
H
H
CH3
F
CH(CH3)2
OCH3
CH3
m
n
o
H
CH3
CH3
OCH3
H
H
OCH2CH3
CH3
g
h
Scheme 1. Reagents and conditions: (a) Ethanol/warm; (b) methyl iodide/anhydrous K2CO3/dry acetone/reflux; (c) methoxylamine hydrochloride/sodium acetate trihydrate/
ethanol/reflux.
structure–activity comprehension. Besides, the standard drugs
Camptothecin and Etoposide were also analyzed under identical
conditions and their IC50 values are also reproduced in the Table 1.
The HeLa cell line was obtained from American Type Culture
Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Med-
ium (DMEM) was purchased from BioWhittakerÒ, whereas fetal
bovine serum (FBS) and other cell culture materials were pur-
chased from Gibco BRL Life Technologies, USA. Paraformaldehyde
and Bisbenzimide Hoechst 33342 stain were procured from Sig-
ma–Aldrich Corp., St. Louis, MO, USA, and MTT [3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrasolium bromide] was purchased
from Biosesang Inc., Korea.
microplate reader (Tecan Austria GmbH). Effects of the test com-
pounds on cell viability were calculated using cells treated with
DMSO as control. The data were subjected to linear regression
analysis and the regression lines were plotted for the best
straight-line fit. The IC50 (inhibition of cell viability to 50%) concen-
trations were calculated using the respective regression equation
as shown in Table 1.
A careful analysis of Table 1 provides the structure–activity cor-
relations as indicated by their IC50 values. Compound 3a is a simple
piperidone molecule and it has no substitution on the phenyl as
well as active methylene centers and ring nitrogen, which shows
activity at an IC50 of 121 lM. The introduction of a methyl group
Cells were cultured in T-75 tissue culture flasks (Nunc, Den-
mark) at 37 °C in a 5% CO2 humidified incubator using appropriate
media supplemented with DMEM containing 10% heat-inactivated
on one of the active methylene centers of the piperidone moiety
3a affords 3b, which fairly improves the efficacy as noticed IC50
of 113
an incredible improvement in its efficacy (IC50 57
the replacement of methyl by isopropyl 3j exhibits activity at an
IC50 of 49 M. Further the incorporation of a methyl group at the
ring nitrogen of 3i improved the IC50 of 4i from 57 to 41 M.
l
M. However, the replacement of methyl by ethyl 3i shows
FBS, 100 units/mL Penicillin and 100
lg/mL Streptomycin. Cells
lM). Similarly,
were seeded in a 96 well microtiter plate containing 100 lL med-
ium at a final density of 2 ꢀ 104 cells/well at identical conditions.
l
After overnight incubation, the cells were treated with different
l
concentrations of test compounds (6.25–100
l
g/mL) or DMSO
A remarkable improvement is observed by the introduction of a
bromo substituent on para-position of the phenyl at C-2 and C-6
(3c), which discloses an excellent inhibition of the growth of the
(carrier solvent) in a final volume of 200 L. After 24 h, 10
l
lL of
MTT (5 mg/mL) was added to each well and the plate was incu-
bated at 37 °C in the dark for 4 h. Then the media along with
MTT was removed and the formazan crystals were solubilized by
HeLa cells at an IC50 of 25.02
three-fold higher than the standard drug Camptothecin
(8.93 M); however, it is very closer and even comparable to the
Etoposide standard (23.33 M). The replacement of para-bromo
lM in 24 h. This IC50 is nearly
adding DMSO (100
l
L/well). Finally, the reduction of MTT was
l
quantified by reading the absorbance at 570 nm by GENiosÒ
l

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P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
Table 1
aCytotoxic effect of compounds 3a–4i on HeLa cells
Compound
Structure
Yield
bLinear regression equation [log]: Y = A + Bx
R value
cIC50 in
lM
OCH3
H
N
H
H
3a
96
Y = ꢁ59.47669 + (70.42453)x
0.97
120.68 15.03
H
N
H
H
H
OCH3
H
N
H
H
6
4
5
3b
95
Y = ꢁ51.73964 + (66.89936)x
0.97
112.72 2.27
2
N 1
3
CH3
H
H
H
OCH3
H
N
H
H
Br
3c
CH3
93
Y = 4.00027 + (43.70457)x
0.99
25.02 2.07
N
H
H
H
Br
OCH3
H
N
H
H
Cl
CH3
3d
93
Y = ꢁ2.07215 + (41.57979)x
0.99
48.97 6.6
N
H
H
H
Cl

P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
6681
Table 1 (continued)
Compound
Structure
Yield
bLinear regression equation [log]: Y = A + Bx
R value
cIC50 in
lM
OCH3
H
N
H
H
F
3e
94
Y = ꢁ20.54996 + (58.6915)x
0.99
49.18 2.14
CH3
N
H
H
H
F
OCH3
F
H
N
H
H
3f
89
Y = ꢁ9.32307 + (51.13401)x
0.99
38.42 7.55
CH3
N
H
H
H
F
OCH3
H
N
H
H
H3C
3g
93
Y = ꢁ34.93378 + (59.77543)x
0.98
81.64 4.05
CH3
N
H
H
H
H3C
OCH3
H
N
H
H
H3CO
CH3
N
3h
95
Y = ꢁ57.94737 + (62.12321)x
0.97
154.34 8.06
H
H
H
H3CO
(continued on next page)

6682
P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
Table 1 (continued)
Compound
Structure
Yield
bLinear regression equation [log]: Y = A + Bx
R value
cIC50 in
lM
OCH3
H
N
H
H
3i
95
Y = ꢁ45.72953 + (73.21416)x
0.97
57.27 3.85
C2H5
N
H
H
H
OCH3
H
N
H
H
3j
93
Y = ꢁ17.80104 + (56.36107)x
0.96
49.54 1.62
N
CH(CH3)2
H
H
H
OCH3
F
H
N
H
H
6
4
5
3k
85
Y = 0.09372 + (44.63047)x
0.99
37.86 5.05
2
1
3
CH3
N
CH3
H
H
F
OCH3
H
N
H
H
H3C
3l
78
Y = ꢁ30.78913 + (63.75446)x
0.98
54.88 3.46
CH3
N
CH3
H
H
H3C

P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
6683
Table 1 (continued)
Compound
Structure
Yield
bLinear regression equation [log]: Y = A + Bx
R value
cIC50 in
lM
H
Ar
H
OCH3
N
H3C
3
6
2
4
3m
55
Y = ꢁ13.53982 + (48.80922)x
0.98
50.96 1.61
N
1
5
CH3
H
H
Ar
H
Ar = para-CH(CH3)2Ph
OCH3
H
H
N
H
H3CO
3n
76
Y = ꢁ35.01759 + (55.23339)x
0.96
93.91 3.38
CH3
N
CH3
H
H
H3CO
OCH3
H
N
H
H
C2H5O
3o
71
Y = ꢁ35.39224 + (53.82258)x
0.97
98.02 9.74
CH3
N
CH3
H
H
C2H5O
OCH3
H
N
H
H
Cl
4d
90
Y = ꢁ21.30888 + (62.78321)x
0.97
36.27 1.94
CH3
N
H
H3C
H
Cl
(continued on next page)

6684
P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
Table 1 (continued)
Compound
Structure
Yield
bLinear regression equation [log]: Y = A + Bx
R value
cIC50 in
lM
OCH3
H
N
H
H
F
CH3
N
4e
91
Y = ꢁ25.19937 + (65.48558)x
0.97
40.73 2.63
H
H3C
H
F
OCH3
H
N
H
H
H3C
CH3
N
4g
91
Y = ꢁ19.85852 + (58.03169)x
0.99
47.68 2.94
H
H3C
H
H3C
OCH3
H
N
H
H
H3CO
CH3
N
4h
92
Y = ꢁ19.31948 + (47.05231)x
0.96
80.62 5.33
H
H3C
H
H3CO
H
OCH3
N
H
H
4i
93
Y = ꢁ0.933 + (45.94471)x
0.99
41.69 7.05
C2H5
N
H
H3C
H
Camptothecin
Etoposide
Y = 29.02966 + (42.56504)x
Y = 2.27708 + (41.80905)x
0.99
0.98
8.93 0.54
23.33 0.92
a
Exponentially growing cells were treated with different concentrations of test compounds for 24 h and cell growth inhibition was analyzed through MTT assay.
Mean percent decrease in cell number of five independent experiments was used to calculate the linear regression equation. Linear regression: Y = A + Bx (A = Y-intercept;
b
B = slope of the line; x = x-scale).
c
IC50 is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control cultures in the absence of an inhibitor. The values
represent the mean SD of five individual observations.

P. Parthiban et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6678–6686
6685
Figure 1. Light and fluorescent micrographs of normal and treated HeLa cells. Phase contrast microscope images of (A) normal cells (B) the cells treated with IC50
concentration (25 M) of the most active lead compound 3c. Early signs of apoptosis was characterized by the blebbing of cell membrane (inset a) followed by cell membrane
destruction (inset b). Fluorescence microscope images of (C) normal cells (D) the cells treated with 25 M of 3c, stained with Hoechst 33342. Extremely condensed chromatin
l
l
(Pyknotic nuclei) is marginated into a horseshoe-shaped structure (inset a & b). The cells were detected by fluorescence light microscopy at 360 nm/470 nm excitation/
emission.
by chloro (3d) or fluoro (3e) decrease its efficiency nearly to half
but the N-methylation of 3d and 3e register an enhanced efficacy
without any cytomorphological abnormalities (Fig. 1A). The cells
treated with IC50 concentration (25 M) of 3c for 24 h showed evi-
l
(IC50 of 4d and 4e are 36 and 41
non-methylated counterparts. Analogously, a two-fold improve-
ment is observed for 4g–h (IC50 48 and 81
tion on 3g–h.
The positional isomer of 3e, that is, compound 3f shows an
improvement in its inhibition from the IC50 of 49 to 38 M and a
further improvement noticed by the incorporation of another
methyl group on another active methylene center. Though it is less
for 3k, a remarkable progress observed for compounds 3l and 3n
(55 and 94 lM, respectively) than 3g (82 lM) and 3h (154 lM) by
the insertion of methyl on both the active methylene centers C-3
l
M, respectively) than their
dent morphological changes with blebbing of cellular membrane,
chromatin condensation and fragmentation followed by the forma-
tion of apoptotic bodies (Fig. 1B, inset a). Most of the treated cells
exhibited the symptoms of apoptosis but the damage was severe in
some cells, with the cell membrane rupture and the subsequent re-
lease of cytoplasm as observed in Figure 1B (inset b). The above re-
sults of light microscopy were consistent with that of fluorescence
microscopy using Hoechst 33342 stain for control and treated cells
(Figs. 1C and D, respectively). Bright condensed chromatin (pykno-
sis) was observed in the 3c treated cells, which represents the early
signs of apoptosis. This leads to a deformed nucleus/cytoplasm
consistency and the margination of chromatin into a horseshoe
shaped structure (Fig. 1D, inset ‘a’ and ‘b’). Consequent to this pro-
cess, fragmentation of the nucleus (karyorrhexis) was also ob-
served in the treated cells.
lM) by the N-methyla-
l
and C-5; in fact, nearly a two-fold improvement in their efficiency.
The replacement of para-methyl by para-isopropyl in 3l (55
provides only a marginal improvement (IC50 of 3m is 51 M).
lM)
l
The altered morphology of exposed cells (1 ꢀ 105/well) at dif-
ferent concentrations was studied after 24 h using phase contrast
microscope (DMI6000B, Leica Microsystems, Wetzlar, Germany).
Subsequently, the cells were Hoechst stained to observe the nucle-
ar/chromosomal condensation occurred by the treatment of the
test compound. For staining the cells, 96 well microtiter plates
were used to culture the cells (1 ꢀ 104 cells/well) in three repli-
cates to treat with the ideal lead compound 3c. Then the cells were
incubated at 37 °C for overnight and the media was removed to
wash the cells twice with phosphate buffered saline (PBS) and
fixed with 4% paraformaldehyde in PBS for one day at ꢁ4 °C. Fur-
The structure–activity correlations indicate the improvement in
IC50 by the substitution of methyl on both the active methylene
centers and secondary amino group besides the halo substitution
on the phenyl groups. As a result, the preliminary investigation
suggests some lead molecules with good cytotoxicity of <50
of IC50 after 24 h against the HeLa cells. Particularly, 3c exhibits
an IC50 of 25.02 M and is comparable to the standard drug Etopo-
side (23.33 M); thus deserves further structural optimization of
lM
l
l
this pharmacophore to attain betterment in the anticancer drug
synthesis.
ther, the cells were stained with 1 lg/mL of the fluorescent DNA-
binding dye, Bisbenzimide Hoechst 33342 stain and incubated
for 20 min at room temperature to reveal nuclear condensation/
aggregation due to the effect of the test compound. The Hoechst-
stained cells were visualized and photographed under fluorescence
microscope (CTR 6000; Leica, Wetzlar, Germany) and reproduced
in Figure 1.
The cytomorphological abnormalities occurred by the effect of
the lead compound 3c were observed under a phase contrast
microscope. The control showed normal healthy and intact nuclei
References and notes
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yield 93%; 1H NMR: d = 3.83 (dd, J5a,6a = 11.72 Hz; J5e,6a = 2.92 Hz, 1H, H-6a),
3.50–3.46 (2H, d of H-2a and dd of H-5e are overlapped), 2.40 (m, 1H, H-3a),
1.91 (dd, J5e,5a = 13.56 Hz; J5a,6a = 11.72 Hz,1H, H-5a), 1.82 (br s, 1H, NH), 0.86 (d,
J = 6.60 Hz, 3H, CH3 at C-3), 3.86 (s, 3H, CH3 of oxime ether), 7.38 (dd, J = 8.10,
1.80 Hz, 4H, H-200 and H-600), 7.31 (d, J = 9.88 Hz, 2H, H-20000), 7.29 (d, J = 8.44 Hz,
2H H-60000) ppm; 13C NMR: d = 68.50 (C-2), 60.08 (C-6), 43.14 (C-3), 34.42 (C-5),
158.51 (C@N), 11.79 (CH3 at C-3), 61.34 (CH3 of oxime ether), 141.09 (C-2’),
142.20 (C-6’), 133.15 (C-20000), 133.42 (C-60000), 129.26, 128.66, 128.59, 128.05
(other aryl carbons) ppm; IR: 1637 (C@N stretching) cmꢁ1; HRMS: m/z: 363.10
[M+H]+; Anal. Cacld for C19H20Cl2N2O: C, 62.82; H, 5.55; N, 7.71. Found C, 62.79;
H, 5.52; N, 7.70; 3m: 3,5-Dimethyl-2,6-bis(4-isopropylphenyl)piperidin-4-one
O-methyloxime. Colorless semi-solid, yield 55%; 1H NMR: d = 3.75 (d,
J2,3 = 5.84 Hz, 1H, H-2), 3.64 (d, J5,6 = 9.16 Hz, 1H, H-6), 3.20 (quintet, 1H, H-5),
2.79 (quintet, 1H, H-3), 1.28 (d, J = 7.5 Hz, 3H, CH3 at C-3), 1.15 (d, J = 6.96 Hz,
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Struct. 2011, 990, 44.
5. Analytical and spectral data of some representative compounds: 3a: r-2,c-6-
Diphenylpiperidin-4-one O-methyloxime. Off-white semi-solid, yield 96%; 1H
NMR: d = 3.94 (dd, J2a,3a = 11.72 Hz; J2a,3e = 2.56 Hz, 1H, H-2a), 3.86 (dd, H-6a,
overlapped with the methyl protons of oxime ether moiety), 3.44 (td,
J5e,6a = 13.92 Hz, 1H, H-5e), 2.58 (td, J3e,2a = 13.56 Hz, 1H, H-3e), 2.35 (t,
J3a,3e = 12.84 Hz, J3a,2a = 12.08 Hz 1H, H-3a), 1.99 (t, J5a,5e = 12.84 Hz,
J5a,6a = 12.44 Hz 1H, H-5a), 1.92 (br s, 1H, merged with H-5a), 3.86 (s, CH3 of
oxime ether), 7.48 (dd, 4H, H-200 and H-600, J = 6.76, 4.80 Hz), 7.35 (t, 4H, H-2000
and H-6000, J = 7.32, 7.68 Hz), 7.29 (d, 2H, H-20000 and H-60000, J = 7.32 Hz) ppm; 13C
NMR: d = 62.16 (C-2), 60.81 (C-6), 40.53 (C-3), 34.02 (C-5), 157.25 (C@N), 61.37
(CH3 of oxime ether), 143.65 (C-20), 143.58 (C-60), 128.72, 128.69, 127.80,
i
3H, CH3 at C-5), 3.83 (s, 3H, CH3 of oxime ether), 2.95–2.83 (m, 2H, CH of Pr),
1.23 (d, J = 6.96 Hz, 12H, CH3 of iPr), 7.20 (dd, 4H, H-2’’’ and H-6’’’), 7.36 (dd, 4H,
H-2’’ and H-6’’) ppm; 13C NMR: d = 67.88 (C-2), 66.56 (C-6), 42.59 (C-3), 40.00
(C-5), 164.39 (C@N), 21.16 (CH3 at C-3), 15.90 (CH3 at C-5), 61.30 (CH3 of oxime
i
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ether), 33.89 (CH of Pr), 24.12 (CH3 of Pr), 141.13 (C-2’), 142.41 (C-6’), 148.34,
148.29 (C-2’’’’ and C-6’’’’), 127.93, 127.54, 127.24, 126.92 (other aryl carbons)
ppm; IR: 1645 (C@N stretching) cmꢁ1; HRMS: m/z: 392.28 [M]+. 3o: t-3,c-5-
Dimethyl-r-2,c-6-bis(4-ethoxyphenyl)piperidin-4-one O-methyloxime. Color-
less semi-solid, yield 71%; 1H NMR: d = 3.42 (d, 1H, H-2a J2a,3a = 10.24 Hz);
4.02 (m, 5H, d of H-6a overlapped with the ethoxy methylene protons); 3.59
(sextet, 1H, H-5e); 2.58 (sextet, 1H, H-3a); 0.92 [d, J = 6.96 Hz, 3H, CH3 at C-5
(ax)], 0.84 [d, J = 6.60 Hz, 3H, CH3 at C-3 (eq)]; 3.88 (s, 3H, CH3 protons of oxime
ether); 4.02 (m, –O–CH2–CH3 on the phenyl), 1.41 (m, –O–CH2–CH3 on the
phenyl); 7.37 (d, 2H, J = 8.44 Hz), 7.32 (d, 2H, J = 8.80 Hz) (phenyl protons of H-
2’’ and H-6’’); 6.88 (d, 2H, J = 8.40 Hz), 6.86 (d, 2H, J = 8.80 Hz) (phenyl protons of
H-2’’’ and H-6’’’) ppm; 13C NMR: d = 68.97 (C-2), 62.39 (C-6), 38.87 (C-3), 34.65
(C-5), 164.30 (C@N), 11.90 [CH3 at C-3 (eq)], 11.29 [CH3 at C-5 (ax)], 61.25 (CH3
of oxime ether), 63.39 (-O-CH2-CH3 on the phenyl), 14.88 (–O-CH2-CH3 on the
phenyl), 133.94 (C-2’), 135.26 (C-6’), 157.95 (C-2’’’’), 158.51 (C-6’’’’), 128.87,
127.88, 114.38 (other aryl carbons) ppm; IR: 1640 (C@N stretching) cmꢁ1; MS
(ES): m/z: 396.24 [M]+; Anal. Cacld for C24H32N2O3: C, 72.70; H, 8.13; N, 7.06.
Found C, 72.72; H, 8.16; N, 7.07; 4d: t-1,3-Dimethyl-r-2,c-6-bis(4-
chlorophenyl)piperidin-4-one O-methyloxime. Viscous liquid, yield 90%; 1H
NMR: d = 2.85 (d, J2a,3a = 10.64 Hz, 1H, H-2a), 3.17 (dd, J5a,6a = 12.08 Hz;
J5e,6a = 3.32 Hz, 1H, H-6a), 3.39 (dd, J5e,5a = 13.92 Hz; J5e,6a = 3.32 Hz,1H, H-5e),
2.50 (m, 1H, H-3a), 2.02 (dd, J5e,5a = 13.92 Hz; J5a,6a = 12.08 Hz, 1H, H-5a), 1.68 (s,
1H, N–CH3), 0.79 (d, J = 6.60 Hz, 3H, CH3 at C-3), 3.84 (s, 3H, CH3 of oxime ether),
7.38–7.29 (m, 8H, aryl protons), ppm; 13C NMR: d = 76.96 (C-2), 68.64 (C-6),
43.10 (C-3), 34.95 (C-5), 157.76 (C@N), 12.63 (CH3 at C-3), 41.58 (N–CH3), 61.37
(CH3 of oxime ether), 141.43 (C-2’), 142.77 (C-6’), 132.89 (C-2’’’’), 133.06 (C-6’’’’),
129.48, 128.87, 128.72, 128.51 (other aryl carbons) ppm; IR: 1636 (C@N
stretching) cmꢁ1; HRMS: m/z: 376.10 [M]+; Anal. Cacld for C20H22Cl2N2O: C,
63.67; H, 5.88; N, 7.42. Found C, 63.68; H, 5.90; N, 7.40.
126.86, 126.77 (other aryl carbons) ppm; IR: 1637 (C@N stretching) cmꢁ1
;
HRMS: m/z:281.15 [M+H]+; Anal. Cacld for C18H20N2O: C, 77.11; H, 7.19; N, 9.99.
Found: C, 77.10; H, 7.20; N, 10.00; 3b: t-3-Methyl-r-2,c-6-diphenylpiperidin-4-
one O-methyloxime. Faint yellow semi-solid, yield 95%; 1H NMR: d = 3.53 (m, d
of H-2a and dd of H-5e are overlapped), 3.88 (dd of H-6a overlapped with the
methyl protons of oxime ether), 2.53 (sextet, 1H, H-3a), 2.01 (t, H-5a), 1.87 (s,
1H, NH), 0.88 (d, J = 6.24 Hz, 3H, CH3 at C-3), 3.87 (s, CH3 of oxime ether), 7.45 (t,
4H, H-200 and H-600), 7.37–7.26 (m, 6H, H-2000, H-6000, H-20000 and H-60000) ppm; 13C
NMR: d = 69.36 (C-2), 60.89 (C-6), 42.92 (C-3), 34.16 (C-5), 159.39 (C@N), 61.67
(CH3 of oxime ether), 11.96 (CH3 at C-3), 142.24 (C-20), 143.31 (C-60), 128.55,
128.48, 128.12, 128.05, 127.70, 126.82 (other aryl carbons) ppm; IR: 1642 (C@N
stretching) cmꢁ1; HRMS: m/z: 295.17 [M+H]+; Anal. Cacld for C19H22N2O: C,
77.52; H, 7.53; N, 9.52. Found: C, 77.50; H, 7.52; N, 9.50; 3c: t-3-Methyl-r-2,c-6-
bis(4-bromophenyl)piperidin-4-one O-methyloxime. Brown semi-solid, yield
93%; 1H NMR: d = 3.82 (dd, J5a,6a = 11.72 Hz; J6a,5e = 2.92 Hz, 1H, H-6a), 3.50–3.46
(m, 2H, d of H-6a and dd of H-5e are overlapped), 2.40 (sextet, 1H, H-3a), 1.90
(dd, J5e,5a = 13.56 Hz; J5a,6a = 11.72 Hz, 1H, H-5a), 1.77 (br s, 1H, NH), 0.86 (d,
J = 6.60 Hz, 3H, CH3 at C-3), 3.86 (s, 3H, CH3 of oxime ether), 7.45 (dd, 4H,
J = 10.24, 8.44 Hz), 7.32 (dd, 4H, J = 8.76, 2.56 Hz) ppm; 13C NMR: d = 68.58 (C-2),
60.15 (C-6), 43.09 (C-3), 34.36 (C-5), 158.51 (C@N), 61.41 (CH3 of oxime ether),
11.82 (CH3 at C-3), 141.56 (C-2’), 142.67 (C-6’), 121.34 (C-20000), 121.63 (C-60000),
131.65, 131.60, 129.66, 128.44 (other aryl carbons) ppm; IR: 1637 (C@N
stretching) cmꢁ1; HRMS: m/z: 451.00 [M+H]+; Anal. Cacld for C19H20N2OBr2: C,
50.47; H, 4.46; N, 6.20. Found C, 50.50; H, 4.45; N, 6.21; 3d: t-3-Methyl-r-2,c-6-
bis(4-chlorophenyl)piperidin-4-one O-methyloxime. Pale yellow semi-solid,
6. Mosmann, T. J. Immunol. Methods 1983, 65, 55.