Proficient synthesis of biologically active pregnane derivatives and its glycoside - Experimental and theoretical approach

Article abstract of DOI:10.1016/j.molstruc.2013.08.048

Synthesis of a number of pregnane derivatives including the glycoside has been described in detail. These compounds were synthesized by reaction of 3β-acetoxy-5, 16-pregnadiene-20-one, derived from diosgenin and then treating it with different nucleophilic reagents. The structures of these newly synthesized compounds were established on the basis of their physical, chemical and spectral data. The molecular geometry of compounds were calculated in ground state by density functional theory method (DFT/B3LYP) using 6-31G (d,p) basis set. ¹H NMR chemical shifts were also studied using gauge-including atomic orbital (GIAO) approach, which were found in good agreement with the experimental values. The study of electronic properties such as UV-Vis spectral analysis, HOMO and LUMO energy calculations were performed with time dependent DFT (TD-DFT). Global and local reactivity descriptors were calculated to study the reactive sites within the molecules. These compounds were also evaluated for their anti-dyslipidemic (Triton model) and in vitro anti-oxidant activities. Out of these, compound 9 showed potent anti-dyslipidemic and anti-oxidant activity.

Full text of DOI:10.1016/j.molstruc.2013.08.048

Journal of Molecular Structure 1052 (2013) 112–124
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Proficient synthesis of biologically active pregnane derivatives and its
glycoside – Experimental and theoretical approach
a
a
a
b
b
Arun Sethi a, , Akriti Bhatia , Atul Maurya , Anil Panday , Gitika Bhatia , Atul Shrivastava ,
⇑
Ranvijay Pratap Singh a, Rohit Prakash a
a Department of Chemistry, University of Lucknow, Lucknow 226007, India
b Division of Biochemistry, Central Drug Research Institute, Lucknow, India
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis of a novel pregnane glycoside is described. All the reaction products were synthesized in a single step reaction.
Structures of synthesized compounds were established on the basis of their physical, chemical and spectral analysis (UV–Vis, 1H NMR, MS and IR).
Experimental 1H NMR chemical shifts and IR frequencies were compared with the theoretical values.
Chemical reactivity of the reactants studied with the aid of reactivity descriptors.
The newly synthesized compounds were screened for anti-dyslipidemic and anti-oxidant activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 April 2013
Received in revised form 24 August 2013
Accepted 24 August 2013
Available online 30 August 2013
Synthesis of a number of pregnane derivatives including the glycoside has been described in detail. These
compounds were synthesized by reaction of 3b-acetoxy-5, 16-pregnadiene-20-one, derived from dios-
genin and then treating it with different nucleophilic reagents. The structures of these newly synthesized
compounds were established on the basis of their physical, chemical and spectral data. The molecular
geometry of compounds were calculated in ground state by density functional theory method (DFT/
B3LYP) using 6-31G (d,p) basis set. 1H NMR chemical shifts were also studied using gauge-including
atomic orbital (GIAO) approach, which were found in good agreement with the experimental values.
The study of electronic properties such as UV–Vis spectral analysis, HOMO and LUMO energy calculations
were performed with time dependent DFT (TD-DFT). Global and local reactivity descriptors were calcu-
lated to study the reactive sites within the molecules. These compounds were also evaluated for their
anti-dyslipidemic (Triton model) and in vitro anti-oxidant activities. Out of these, compound 9 showed
potent anti-dyslipidemic and anti-oxidant activity.
Keywords:
Pregnanes
1H NMR
TD-DFT
Reactivity descriptors
Lipid lowering
Anti-oxidant activity
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
sis of a number of C-20 amino, nitro, hydroxyl, oxime and amid-
inohydrazone derivatives of pregnanes eliciting digitalis like
Pregnanes are one of the most versatile steroidal derivatives,
which have been dynamically studied. No other group of steroidal
derivates has exhibited such a vast range of biologically activity as
exhibited by pregnanes [1]. In fact synthesis of pregnane deriva-
tives has attracted the attention of number of research groups
belonging to different branches of science and technology [2].
Synthesis of a number of allopregnanolone and pregnanolone
analogues exhibiting significant GABA receptor binding property
has added a new dimension to the synthetic organic chemistry
activity have also been reported [5]. D-ring substituted 1,2,3-triaz-
olyl 20-keto pregnane derivatives exhibiting significant anti-can-
cer activity and were found to be active against DU-145 and PC-3
cell lines [6]. A series of steroid based trioxanes have also been syn-
thesized and evaluated for their anti-malarial activity [7]. Out of
these, pregnane based trioxanes showed better activity profile than
trioxanes derived from cholesterol and tigogenin. Besides this, a
number of 20-oximo pregnane derivatives have been evaluated
as inhibitor of human testicular 17
a-hydroxylase/C17,20-lyase
[3]. A series of novel 2b-piperazino-(20R)-5a-pregnane-3a, 20-diol
(P45017 ), thereby preventing the development and progression
a
N-derivatives were evaluated as anti-leukemic agents [4]. Synthe-
of benign prostatic hypertrophy and prostatic cancer [8–10]. More
recently a number of chalcone pregnenolones were found to pos-
sess significant antimicrobial activity [11]. Besides this the requi-
site side chain of loteprednol etabonate, a corticosteroid used for
⇑
Corresponding author. Tel.: +91 9415396239.
E-mail address: alkaarunsethi@rediffmail.com (A. Sethi).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.08.048

A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
113
anti-inflammatory and allergy related ophthalmic disorders has
been introduced in the 16 DPA moiety [12]. In continuation of
our synthesis and isolation of some novel pregnane derivatives
[13,14] and taking into account the biological importance of these
derivatives, we herein report the synthesis, characterization, lipid
lowering and anti-oxidant activity of newly synthesized pregnane
derivatives 4, 5, 7, 8, 9, 10 supported by theoretical studies. A de-
tailed study regarding the structural and spectroscopic properties
of these newly synthesized pregnanes helped in better under-
standing the chemical reactivity of these compounds. Based on
the optimized geometries, the HOMO–LUMO orbitals of 4, 5, 7, 8,
9, and 10 were generated using TD-DFT approach. Though few pa-
pers related to the Density Functional Theory of steroids [15] are
available, but not much literature is devoted to theoretical study
of pregnanes for evaluating the global and local reactivity descrip-
tors [16]. This prompted us to undertake a detailed chemical reac-
tivity study for better understanding the chemical behavior and
predicting the stability and activity of newly synthesized
pregnanes.
2.2.3. 3b-Acetoxy-5, 16-pregnadiene-20-one oxime (3)
Compound 1 was converted into compound 3 by reported
method [20] and was identified by its m.p. 226 °C, 1H NMR and
ESI-MS.
2.2.4. 20-(O-2-bromo ethyl)-oximino-3b-hydroxy-pregn-5, 16-diene
(4)
Compound 3 (500 mg, 1.40 mmol), NaH (96 mg, 4.01 mmol) in
dry tetrahydrofuran (THF) (25 mL) was stirred at 0 °C for 30 min.
To the reaction mixture 1,2-dibromoethane (0.5 mL) was added
and the reaction mixture was further stirred at room temperature
for 4 h (the reaction was monitored by TLC during this period).
After the reaction was complete, THF was evaporated under re-
duced pressure and the solid residue obtained was extracted with
chloroform, washed with water, dried over anhydrous sodium sul-
phate and concentrated in vacuum. Column chromatography of the
resultant residue afforded (325 mg, 65% yield) compound 4 as syr-
upy solid. [a]
– 30° (CHCl3), 1H NMR (300 MHz,CDCl3) d (ppm)
D
5.77 (m, 1H, H-16), 5.38 (m, 1H, H-6), 4.60 (m, 2H,@NOCH2), 3.94
(m, 2H, C@NAOCH2CH2), 3.58 (m, 1H, H-3), 2.03 (s, 3H, CH3-21),
1.03 (s, 3H, 19-CH3), 0.84 (s, 3H, 18-CH3); 13C NMR (75 MHz,
CDCl3), d171.44 (C-20), 159.94 (C-17), 152.22 (C-16), 142.80 (C-
5), 123.41 (C-6), 73.86 (@NOACH2), 71.97 (C-3), 57.63 (C-14),
53.46 (C-9), 45.67 (C-13), 42.71 (C-4), 39.36 (C-12), 37.63 (C-1),
36.22 (C-10), 35.72 (@NOACH2CH2Br), 34.28 (C-15), 32.63 (C-8),
31.28 (C-7), 29.75 (C-2), 22.93 (C-11), 19.62 (C-19), 17.88 (C-21),
14.77 (C-18); MS m/z = 435 [M+], 437 [M++2], 388 [435-2CH3AOH],
295 [435-CH2CH2BrAH2OACH3], 251[435-C4H7BrNOA2CH3], 118
[435-C9H14OAC4H7BrNOACH3].
2. Experimental
2.1. Materials and methods
All solvents used were of laboratory grade and were purified
and dried according to standard procedures prior to their use. Thin
layer chromatography (TLC) on Silica Gel ‘G’ (Qualigen, India)
coated plates were used for monitoring the progress of reaction
and purity of the compounds. Column chromatography was per-
formed using silica gel (60–120 mesh) (Acme, India) as stationary
phase. 1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on either Advance FT NMR (400 MHz) (13C, 100 MHz) or
2.2.5. 3b-Hydroxy-16a-phenyl-pregn-5-en-20-one (5)
400 mg of compound 2 was dissolved in 35 mL of dry THF and
then 15 mL of phenyl magnesium bromide (in THF) was added to
it. The reaction mixture was stirred at 10 °C for 5 h (The reaction
monitored by TLC during this period). After the reaction was com-
plete, THF was removed under reduced pressure and the solid res-
idue obtained was extracted with chloroform, washed with water,
dried over anhydrous sodium sulphate and concentrated in vac-
uum. Column chromatography of the resultant residue afforded
Advance DRX (300 MHz)
(
13C, 75 MHz) or Advance DPX FT
(200 MHz) (13C, 50 MHz) (Bruker, Switzerland) using TMS as an
internal reference. Fast Atom Bombardment (FAB-MS) was re-
corded on JEOL SX 102/DA 6000 (Jeol, Japan), using m-nitro benzyl
alcohol as matrix (The matrix peak appeared at m/z 136, 137, 154,
289 and 307) whereas Electronspray ionization (ESI-MS) was re-
corded on MICRO-MASS QUATTRO II triple quadrupole (Microcass
Altricem, United Kingdom) mass spectrometer. Optical rotations
were recorded on SEPA-300 digital polarimeter (Horiba, Japan).
IR spectra were recorded on Perkin Elmer FTIR spectrometer with
the range from 4000 to 400 cmꢁ1. The spectra were analyzed using
Spectrum™ Software suite. The spectra were measured with
4 cmꢁ1 resolution and 1 scan co-addition. The ultraviolet absorp-
tion (UV) spectra was examined in the range 200–600 nm using
(275 mg, 75% yield) white solid. m.p. 70 °C, [a]
– 23° (CHCl3), 1H
D
NMR (300 MHz,CDCl3) d (ppm) 7.32–7.11 (m, 5H, Ar H’s), 5.37(m,
1H, H-6), 3.85 (m, 1H, H-16), 3.57 (1H, m, H-3), 2.71 (d, 1H, H-
17, J = 9.2 Hz), 2.03 (s, 3H, CH3-21), 1.03 (s, 3H, CH3-19), 0.77 (s,
3H, CH3-18); 13C NMR (50 MHz, CDCl3), d208.66 (C-20), 147.21
(C-5), 141.21 (C-10), 128.9 (C-30 and C-50), 127.5 (C-20 and C-60),
126.2 (C-40), 121.67 (C-6), 74.45 (C-3), 72.09 (C-17), 57.82 (C-14),
50.44 (C-9), 46.11 (C-13), 42.63 (C-4), 39.39 (C-10), 37.68 (C-12),
36.94 (C-1), 34.76 (C-16) 32.56 (C-15), 32.39 (C-8), 32.18 (C-7),
31.99 (C-2), 30.10 (C-21), 21.42 (C-11), 19.81 (C-19),14.42 (C-18);
MS m/z = 392 [M+], 331 [392-COCH3-H2O], 332 [392-COCH3-OH]
301 [392-COCH3-H2O-2CH3), 285 [392-2CH3AC6H5], 270 [285-
CH3]. Anal. Calc. for C27H36O2: C, 82.62; H, 9.24. Found: C, 82.28;
H, 9.36.
a
ELICO BL-200 UV–Vis spectrophotometer equipped with a
10 mm quartz cell in chloroform. Melting points were determined
in open capillary tubes and were uncorrected. Triton WR-1339 was
purchased from sigma chemical company, St. Louis, MO, USA. TG
test kits and Total cholesterol test kits were purchased from Merck.
2.2. Synthesis of pregnane derivatives (1–10)
2.2.6. 3b-Hydroxy-16a, 17a-epoxypregn-5-en-20-one (6)
Compound 1 was converted into compound 6 by reported
method [21,22]. It was identified by its m.p. 214 °C (lit m.p
216 °C), 1H NMR and ESI-MS.
The compounds 1–10 were synthesized as given in (Scheme 1).
2.2.1. 3b-Acetoxy-5, 16-pregnadiene-20-one (1)
Diosgenin was converted into compound 1 by reported method
2.2.7. 3b, 17
en-20-one (7)
a-Dihydorxy-16a-[2(2-hydroxy ethoxy) ethoxy] pregn-5-
[17] and identified [18] by its m.p. 168 °C, 1H NMR and ESI-MS.
1000 mg of compound 6 was dissolved in 10 mL of freshly
distilled diethylene glycol and 1.4 mL of boron trifluoride ether-
ate was added to it. The reaction mixture was stirred at 30 °C for
30 h (reaction was monitored by TLC during this period). Ice-cold
solution of sodium bicarbonate was added and the aqueous
2.2.2. 3b-Hydroxy-5, 16-pregnadiene-20-one (2)
Deacetylation of compound 1 by zemplen method [19] yielded
compound 2, identified by its m.p. 214 °C (lit m.p. 216 °C) [12]1H
NMR and ESI-MS.

114
A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
O
O
HO
Diosgenin
O
21
20
17
18
O
12
R1ON
11
9
19
13
14
16
1
4
a
2
10
15
8
b
7
RO
3
5
6
HO
HO
R=Ac
1.
2.
5.
g
RO
O
O
3. R=H, R1=H
c
3'
2'
1'
4
. R=H, R1= CH2-CH2-Br
17
O
4'
5'
16
6'
HO
6.
d,e,f
O
OH
17
R'
16
RO
3'
4'
1'
2'
7. R = H, R' = OCH2CH2OCH2CH2OH
1'
3'
2'
4'
8. R = Ac, R' = OCH2CH2OCH2CH2OAc
9. R = H, R' = CH2CH2CH2CH3
O
O
OAc
OH
OAc
AcO
OH
HO
Ac2O/Py
HO
OAc
O
O
OAc
OAc
6'
OH
O
h
O
1'
4'
AcO
HO
(I)
5'
AcO3'
10.
AcO 2'
(II)
(a) NH2OH.HCl/CH3COONa/MeOH (b) BrCH2CH2Br/NaH/THF (c) H2O2/NaOH
(d) HOCH2CH2OCH2CH2OH/BF3.Et2O (e) Acetic anhydride/Py (f) C4H9MgBr
(g) C H MgBr/BF .Et O
(h) SnCl4/DCM
6
5
3
2
Scheme 1.
phase was extracted with dichloromethane. The organic layer
was washed with water, dried over anhydrous sodium sulphate
and concentrated in vacuum. The crude product was purified
by column chromatography on silica gel (hexane/ethyl acetate
80:20) to afford (852 mg, 85% yield) of compound 7. m.p.
served], 406 [436-2CH3], 363 [436-COCH3A2CH3] 346 [363-OH],
347 [436-COCH3-CH3-CH2@OH], 331 [436-OCH2CH2OCH2CH2OH],
288 [331-COCH3]. Anal. Calc. for C25H40O6: C, 68.7; H, 9.23.
Found: C, 68.22; H, 9.33.
124 °C, [
a]
– 23° (CHCl3), 1H NMR (CDCl3,300 MHz) d (ppm)
D
5.35 (1H, m, H-6); 4.57 (1H, m, H-16), 3.71–3.65 (6H, m, OCH2-
ACH2AOACH2ACH2AOH), 3.58–3.54 (3H, m, OCH2ACH2AOACH2-
ACH2AOH, H-3), 2.08 (3H, s, CH3-21), 1.19 (3H, s, CH3-19), 0.93
(3H,s, CH3-18); 13CNMR (75 MHz,CDCl3) d (ppm) 212.6 (C-20),
141.5(C-5), 121.2(C-6), 96.0 (C-17), 81.4 (C-16), 77.5 (OACH2-
ACH2AOACH2ACH2AOH), 72.3 (C-3), 71.7 (OACH2ACH2-
2.2.8. 3b-acetoxy-17
pregn-5-en-20-one (8)
Conventional acetylation of compound 7 with acetic anhydride
and pyridine yielded compound 8 as syrupy compound. [
a-hydroxy 16a [2(2-acetoxy ethoxy)-ethoxy]
a]
–
D
30.0°(CHCl3), 1H NMR (CDCl3, 300 MHz) d (ppm) 5.44 (1H, m,
H-6); 5.18 (1H, t, H-16), 4.65–4.59 (3H, m,OACH2ACH2AOACH2
ACH2AOAc and H-3), 4.23–4.14 (4H, m, OACH2ACH2AOACH2
ACH2AOAc), 3.70–3.64 (2H, m, OACH2ACH2AOACH2ACH2A),
2.10 (3H, s, CH3-21), 2.04 (3H, s, OAc), 1.97,(3H, s, OAc), 1.01 (3H,
s, CH3-19), 0.86 (3H, s, CH3-18); MS m/z = 520 [M+], 445 [520-CH3-
COOHACH3], 385 [520-2CH3COOHACH3].
AOACH2ACH2AOH),
70.1
(OACH2ACH2AOACH2ACH2AOH),
65.2 (OACH2ACH2AOACH2ACH2AOH), 61.4 (C-14), 48.7 (C-9),
42.2 (C-13), 41.9 (C-4), 40.0 (C-12), 36.8 (C-1), 33.1 (C-10),
32.2 (C-15), 31.2 (C-8), 30.5 (C-7), 29.6 (C-2), 28.2 (C-21), 23.6
(C-11), 22.7 (C-19), 19.7 (C-18); MS m/z = 436 [M+, not ob-

A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
115
2.2.9. 3b, 17
a
-Dihydorxy-16
a
-(butyl)-pregn-5-en-20-one (9)
control group (group I) by single intraperitoneal injection (1 mL/
kg ) of freshly prepared solution of triton WR-1339 (400 mg/kg) di-
luted with normal saline solution (400 mg/mL). Animals of all
groups were given 0.2% (w/v) aqueous gum acacia suspension or-
ally. The animals of group III, IV, V, VI, VII and VIII were fed orally
(10 mL/kg) with compounds 4, 5, 7, 8, 9 and 10, macerated with 2%
aqueous gum acacia at a dose of 250 mg/kg simultaneously with
triton. However group IX, instead of the pregnane derivatives
was fed with reference drug-Gemfibrozil macerated with 2% aque-
ous gum acacia at a dose of 250 mg/kg simultaneously with triton.
200 mg of compound 6 was dissolved in 20 mL of dry THF and
then 5 mL of butyl magnesium bromide (in THF) was added to it.
The reaction mixture was refluxed on water bath for 6 h (reaction
monitored by TLC during this period). After the reaction was com-
plete THF was removed under reduced pressure and then 100 mL
water added to the residue. The reaction mixture was extracted
with chloroform thrice. The combined organic extract was dried
over anhydrous sodium sulphate and evaporated to dryness. Col-
umn chromatography of the residue afforded (150 mg, 75% yield)
of compound 9. m.p. 110 °C, [a]
– 98° (CHCl3), 1H NMR (CDCl3,
D
300 MHz) d (ppm) 5.35 (1H, m, H-6), 3.53 (1H, m, H-3), 2.25(3H,
s, CH3-21), 2.94 (1H, m, H-16), 1.85–1.60 (6H, m, ACH2ACH2ACH2
A), 1.05 (3H, s, CH3-19), 0.95 (3H, s, CH3-18), 0.88 (3H, t, ACH2
ACH3, J = 6 Hz); MS m/z 388 [M+], 355 [388-H2OACH3], 345 [388-
CH2CH2CH3], 321 [388-CH2CH2CH3AH2OACH3AH], 311[388-CH2
CH3AH2OA2CH3]. Anal. Calc. for C25H40O3: C, 77.27; H, 10.38.
Found: C, 76.98; H, 10.44.
2.3.3. Biochemical analysis
After 18 h of fasting, the rats were injected with heparin solu-
tion through tail vain at the dose of 1 mL/kg. The animals were
anaesthetized with sodium pentothal solution (50 mg/kg, i.p) pre-
pared in normal saline after 15 min of treatment. The blood was
withdrawn from retro-orbital sinus with the help of glass capillary
coated with EDTA (3 mg/mL blood). The plasma was separated
from the blood which was then centrifuged at 4 °C for 10 min.
The plasma was diluted with normal saline in the ratio 1:3 (v/v)
and further used for TC (Total cholesterol), TG (Triglyceride), PL
(Phospholipids) and LCAT (Lecithin-cholesterol-acyl-transferase)
assessment using the standard reported protocol [23].
2.2.10. 3b-(2-3, 4, 6-tetra-O-acetyl-b-D-galactopyranosyl)oxy-pregn-
5, 16-diene-20-one (10)
Conventional acetylation of
anhydride (12.5 mL) and fused sodium acetate yielded 1,2,3,4,6
penta-O-acetyl-b- -galactopyranose (II), m.p 136 °C [lit m.p
142 °C]. Stannic chloride (1.0 mL) was added to a solution of
1,2,3,4,6 penta-O-acetyl-b- -galactopyranose (II) (500 mg) and
D-galactose (2.5 g) with acetic
D
2.3.4. Generation of superoxide anions and hydroxyl radicals
Superoxide anions (Oꢁ2) and hydroxyl radicals (ÅOH) were gen-
erated enzymatically by known protocol [1] in the absence or pres-
ence of compounds 4, 5, 7, 8, 9, and 10 at concentrations of 100
D
compound 2 (420 mg) in dichloromethane (20 mL). The mixture
was stirred at 20 °C for 4 h under nitrogen condition. The mixture
was diluted with dichloromethane and washed with water for
decomposition of stannic chloride. The organic layer was washed
with 1 M NaOH and water, dried over anhydrous sodium sulphate
and then concentrated in vacuum. Column chromatography of the
resultant residue afforded (128 mg, 30% yield) compound 10 as
and 200 (lg/mL). The amount of formazone formed in case of
superoxide anions and malondialdehyde (MDA) content in case
of hydroxyl radicals generated in both experimental and reference
samples were estimated spectrophotometrically at 560 nm [24,25].
syrupy residue. [a]
– 42.14° (CHCl3), 1H NMR (CDCl3, 200 MHz)
D
2.4. Computational study
d (ppm) 6.70 (m, 1H, H-16), 5.44 (1H, m, H-6), 5.07 (dd, 1H, H-20,
J = 8.1 and 3.3 Hz), 5.22 (d, 1H, H-10, J = 4 Hz), 5.36 (dd, 1H, H-30,
J = 8 and 3.1 Hz), 4.33 (m, 1H, H-40), 4.28–4.17 (m, 2H, H-60a and
H-60b), 4.08 (d, 1H, H-50, J = 7 Hz), 3.90 (m, 1H, H-3), 2.13 (s, 3H,
CH3-21), 2.06 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.98 (s, 6H, OAc),
1.04 (3H, s, CH3-19), 0.83 (3H,s, CH3-18), MS m/z = [M+] 644 (not
The molecular geometry optimization for compounds 1–10
were carried out with Gaussian 03 W [26] program package using
B3LYP functional [27] with the standard 6-31G (d,p) basis set. The
1H NMR isotropic shielding of compounds 4, 5, 7, 8, 9, 10 were cal-
culated with the help of GIAO method [28,29] using B3LYP/6-31G
(d,p) method. UV–Vis spectra, electronic transitions and electronic
properties such as HOMO–LUMO were further computed with the
help of time-dependant DFT (TD-DFT) method. Presentation graph-
ics including visualization of the molecular structures were done
with the help of CHEMCRAFT software [30] and Gauss View [31].
observed)
509[644-2CH3COOHACH3],
480[644-2CH3-
COOHACOCH3AH], 393[644-C12H16OACH3COOHACH3], 331 (su-
gar + 1-OH], 211 [331-2CH3COOH], 138 [aglycon + 1-C12H16O],
120 [120-H2O], 105 [120-CH3].
2.3. Lipid lowering and anti-oxidant activity
2.3.1. Experimental animals
3. Result and discussion
Charles Foster strain rats (adult, male) weighing 200–225 g
were housed in polyacrylic cages with not more than six animals
per cage and maintained under standard laboratory conditions
[temperature (25–26 °C), humidity (60–80%) and 12/12 light/dark
cycle]. The animals were allowed free access to standard dry pellet
diet and water ad libitum. The animals were acclimatized for
10 days before starting the experiment. All the experimental stud-
ies were approved by Central drug research institute, ref No. 120/
10/Biochem/IAEC) constituted under CPCSEA and GLP annual wel-
fare board, Government of India.
3.1. Synthesis
Diosgenin a naturally occurring sapogenin was converted to 3b-
acetoxy pregn-5, 16-diene-20-one compound 1 by earlier reported
method [17]. Reaction of compound 1 with hydroxylamine hydro-
chloride in presence of sodium acetate yielded compound 3 [20]. A
novel steroidal oximino ether derivative 4 was synthesized by
treating compound 3 with 1,2-dibromoethane in dry THF in pres-
ence of base NaH [32]. In the 1H NMR spectrum of compound 4,
two multiplets at d 4.60 and d 3.94 are due to methylene groups
bonded to oxygen (@NAOCH2) and halogen (C@NAOCH2CH2Br).
In the 13C NMR spectrum the presence of carbon signals at d
73.86 (@NOACH2) and d 35.72 (@NOACH2CH2Br) further con-
firmed the introduction of ethyl group into the oxime 4. The pres-
ence of bands at 1659.0 cmꢁ1 for C@N stretching together with a
band at 668.31 cmꢁ1 for CABr stretching in the IR spectrum further
confirmed the proposed structure. The ESI-MS of compound 4
2.3.2. Induction of hyperlipidemia
Adult male rats were divided into nine groups of six rats in each
group, namely control (group I), triton treated (group II), triton + 4
(group III), triton + 5 (group IV), triton + 7 (group V) triton + 8
(group VI), triton + 9 (group VII), triton + 10 (group VIII) and tri-
ton + Gemfibrozil (100 mg/kg) (group IX). After fasting for over-
night, hyperlipidemia was induced in rats of all groups except

116
A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
Table 1
Effect of compounds 4, 5, 7, 8, 9, 10 on plasma total cholesterol, phospholipids, triglycerides, lecithin cholesterol acyl-transferase levels in triton induced hyperlipidemic rats.
Experimental schedule
Total cholesterola
Phospholipida
Triglyceridea
LCATb
Control
Triton treated
85.55 6.70
244.44 20.17***
(+2.95 F)
86.60 5.77
232.80 16.93***
(+2.62 F)
85.80 7.00
258.56 20.14***
(+3.01 F)
65.20 3.88
40.14 2.00***
(ꢁ38%)
Triton + 4
229.99 18.66NS
(ꢁ6%)
217.27 18.88NS
(ꢁ7%)
226.66 18.96*
(ꢁ12%)
40.30 3.12**
(+20%)
Triton + 5
208.33 16.66*
(ꢁ15%)
189.54 14.82*
(ꢁ18%)
215.71 18.76*
(ꢁ17%)
51.84 3.80**
(+23%)
Triton + 7
232.49 19.79NS
(ꢁ5%)
209.83 17.80*
(ꢁ10%)
233.09 19.84*
(ꢁ10%)
52.44 4.00**
(+23%)
Triton + 8
223.05 20.00NS
(ꢁ9%)
210.56 19.39*
(ꢁ10%)
235.85 21.22NS
(ꢁ9%)
43.92 2.77NS
(+9%)
Triton + 9
189.83 13.21**
(ꢁ22%)
170.25 14.44***
(ꢁ26%)
200.95 16.72**
(ꢁ22%)
58.80 3.80***
(+32%)
Triton + 10
Triton + Gemfibrozil
217.49 18.33*
(ꢁ11%)
212.39 15.99NS
(ꢁ9%)
233.66 16.98*
(ꢁ10%)
48.00 2.77*
(+16%)
158.99 18.33***
(ꢁ35%)
160.56 13.20***
(ꢁ31%)
177.13 14.00***
(ꢁ32%)
61.66 3.92***
(+34%)
Unit: amg/dl, bg/dl. Each value is mean SD of six rats. *Pn0.05, **Pn0.01, ***Pn0.001, NS = non-significant. Triton treated group was compared with control. Triton plus drug and
Gemfibrozil treated groups compared with triton only.
formation of this fragment proved the presence of oximo ethyl
Table 2
derivative at C-20.
Effect of compounds 4, 5, 7, 8, 9, 10 on free radical generation (superoxide anions and
Synthesis of C-16 substituted pregnane derivative 5 was carried
out by treating compound 2 with phenyl magnesium bromide in
dry THF. In the 1H NMR of compound 5, the absence of vinylic pro-
ton signal of C-16, together with presence of one proton multiplet
at d 3.85 due to C-16 methine proton and a five proton multiplet
from d 7.32 to d 7.11 due to aromatic protons suggested the intro-
duction of aromatic ring at C-16. Besides this, the presence of one
proton doublet at d 2.71 presumably due to C-17 proton confirmed
the introduction of this group at C-16. The magnitude of the cou-
hydroxyl radicals) in rat liver microsomes in vitro.
Compounds
Dose (lg/
Superoxide anionsa
Hydroxyl radicalsb
mL)
Control
4
–
100
177.29 14.40
EXP 160.43 13.00NS
(ꢁ9%)
104.68 7.89
EXP 96.31 8.00NS
(ꢁ8%)
200
EXP 154.85 10.62*
(ꢁ13%)
EXP 88.30 6.11*
(ꢁ15%)
5
7
100
200
EXP 151.59 12.77*
(ꢁ14%)
EXP 94.48 7.97*
(ꢁ10%)
pling of C-17 proton doublet (J = 9.2 Hz) due to J16bH-17 H confirms
a
a
. In the 13C NMR
EXP 145.24 10.70*
(ꢁ18%)
EXP 90.78 6.88*
(ꢁ13%)
the orientation of the side chain at C-16 to be
spectrum the presence of carbon signals at d 141.2 (C-10), d 128.9
(C-30 and C-50), d127.5 (C-20 and C-60), d 126.2 (C-40), d 72.0 (C-
17), together with the aromatic CAH stretching at 3027.1 cmꢁ1
and C@C-C aromatic ring stretching at 1601.8 and 1493.9 cmꢁ1
in the IR spectrum, and the fragments at m/z 331, m/z 301 and
m/z 285 in the ESI-MS spectra further confirmed the proposed
structure.
100
200
EXP 163.30 15.12NS
(ꢁ8%)
EXP 97.31 7.77NS
(ꢁ7%)
EXP 151.33 12.77*
(ꢁ15%)
EXP 90.11 6.84*
(ꢁ14%)
8
100
200
EXP 168.79 14.12NS
(ꢁ5%)
EXP 97.51 8.12NS
(ꢁ7%)
EXP 154.79 14.00*
(ꢁ12%)
EXP 92.44 6.82*
(ꢁ13%)
Epoxides are versatile organic tools as both building blocks and
synthetic intermediates. Reaction of compound 6 with diethylene
glycol in presence of BF3.Et2O led to the formation of compound
7. The 1H NMR spectrum of compound 7 showed the presence of
a broad multiplet of six protons from d 3.71 to d 3.65 due to C-40,
C-30 and C-20 methylene protons, a three proton multiplet from d
3.58 to d 3.54 due to C-10 methylene and C-3 methine proton along
with a one proton multiplet at d 4.57 due to methine proton at C-
16 suggested the introduction of side chain at C-16. In the 13C NMR
spectrum of compound 7 the presence of carbon signals at d 96.0
(C-17), d 81.4 (C-16), d 77.5 (C-30), d 71.7 (C-20), d 70.1(C-10) and
d 66.0 (C-40), together with the bands at 1150.5, 1053.5 and
982.0 cmꢁ1 for CAOAC stretching, CAO stretching vibration for
primary alcoholic group and rocking vibration for methyl respec-
tively further proved the proposed structure. The ESI-MS of com-
pound 7 did not record the M+ or the protonated molecular ion
peak, but the fragment at m/z 346 and m/z 331 definitely proved
the presence of tertiary hydroxyl group at C-17 and a 2(2-hydro-
xy-ethoxy) ethoxy group at C-16.
9
100
200
EXP 150.34 12.12*
(ꢁ15%)
EXP 89.60 6.84*
(ꢁ14%)
EXP 125.33 10.00***
(ꢁ29%)
EXP7 6.22 5.30***
(ꢁ27%)
10
100
200
EXP 161.55 13.66NS
(ꢁ9%)
EXP 96.00 5.79NS
(ꢁ8%)
EXP 153.33 11.20*
(ꢁ14%)
EXP 88.40 4.70*
(ꢁ15%)
Standard
drug
–
57.32 3.84***
42.81 1.88***
(ꢁ68% over control)
(ꢁ59% over control)
(Allopurinol) (20
mL)
l
g/
(Mannitol) (100
mL)
lg/
Units: anmol uric acid formed/min; bnmol formazone formed/min. Each value is the
mean SD. *Pn0.05, **Pn0.01, ***Pn0.001, NS = non significant as compared to the
systems without drug treatment.
recorded the molecular ion peak for Br91at m/z 437. The fragment
at m/z 295 obtained due to loss of side chain at C-17 followed by
loss of methyl radical confirmed the introduction of ethyl bromide
group into the oxime. The RDA fragmentation due to D5 bond
yielded fragments, which after the loss of side chain at C-17
followed by loss of methyl radical gave fragment at m/z 118. The
Acetylation of compound 7 with acetic anhydride and pyridine
gave the expected diacetyl derivative 8. In the 1H NMR spectrum of
compound 8 the downfield shifting of C-3 methine proton and C-40
methylene protons of the side chain at C-16, which appeared as
multiplet from d 4.65 to d4.59 along with the appearance of two

A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
117
Table 3
Percentage activity change (anti-hyperlipidemic and anti-oxidant) with changes in structural features of compound 1, 4, 5, 7, 8, 9, 10.
Percentage activity change (Anti-hyperlipidemic and anti-oxidant) Structural features
TC
(%)
PL
(%)
TG
(%)
LCAT
(%)
Superoxide
anions
Hydroxyl
radicals
1
4
5
7
8
ꢁ4
ꢁ10
ꢁ7
ꢁ18
ꢁ10
ꢁ10
ꢁ4
13
20
23
23
9
ꢁ6%, ꢁ12%
ꢁ9%, ꢁ13%
ꢁ14%, ꢁ18%
ꢁ8%, ꢁ15%
ꢁ5%, ꢁ12%
ꢁ6%, ꢁ10%
ꢁ8%, ꢁ15%
ꢁ10%, ꢁ13%
ꢁ7%, ꢁ14%
ꢁ7%, ꢁ13%
C@O at C-20, Double bonds at C16-C17 and C5-C6
C@N at C-20, Double bonds at C16AC17 and C5AC6
C@O at C-20, Phenyl ring at C-16, Double bond at C5AC6
C@O at C-20, CAOAC linkage at C-16, Double bond at C5AC6
ꢁ6
ꢁ15
ꢁ5
12
ꢁ17
ꢁ10
ꢁ9
ꢁ9
CAOAC linkage at C-16 and acetate group at C-3 and C-16 side chain, C@O at C-20, Double
bond at C5AC6
9
ꢁ22
ꢁ26
ꢁ9
ꢁ22
ꢁ10
32
16
ꢁ15%, ꢁ29%,
ꢁ9%, ꢁ14%
ꢁ14%,ꢁ27%
ꢁ8%, ꢁ15%
Butyl side chain at C-16, C@O at C-20, Double bond at C5AC6
10 ꢁ11
C-3 glycoside
singlets of three proton each at d 2.04 and d 1.97, suggested the
diacetyl nature of compound 8.
(2.6-fold) and TG (3-fold) as given in Table 1. Treatment with
compounds 4, 5, 7, 8, 10 (dose 250 mg/kg p.o) caused mild de-
crease in plasma levels of TC, PL and TG by 6%, 7%, 12% in case
of 4, 15%, 18%, 17% in case of 5, 5%, 10%, 10% in case of 7, 9%,
10%, 9% in case of 8 and 11%, 9%, 10% in case of 10 when com-
pared to triton. In case of 9 the decrease in plasma levels of TC,
PL and TG was 22%, 26%, 22% respectively, which was more sig-
nificant in comparison to compounds 4, 5, 7, 8, 10. These data
were compared with standard drug Gemfibrozil (dose 100 mg/
kg) which showed decrease in plasma levels of TC, PL and TG
by 35%, 32% and 31% respectively. Triton causes drastic increase
in plasma lipid levels by enhancing activity of 3-hydroxy-3-
methyl-glutaryl CoA (HMG-CoA) reductase and also inhibits activ-
ity of lipases responsible for hydrolysis of plasma lipids [38,39].
Involvement of LCAT in the regulation of blood lipids is also sug-
gested and it plays a key role in lipoprotein metabolism. The test
compounds 4, 5, 7, 8, 9, 10 activated plasma LCAT in hyperlipi-
demic animals, as a result LCAT converts free cholesterol into cho-
lesteryl ester (a more hydrophobic form of cholesterol), which is
then sequestered into the core of a lipoprotein particle, eventually
synthesizing the less harmful HDL (High Density lipoprotein). The
levels of LCAT were increased by 20% in case of 4, 23% for 5, 23%
for 7, 9% for 8 and 16% for 10. Compound 9 showed a significant
increase of 32% in LCAT level as compared to triton treated
(Table 1).
Reaction of epoxide with n-butyl magnesium bromide in dry
THF resulted in the formation of compound 9. In the 1H NMR spec-
trum of compound 9, a three proton triplet at d 0.88 (J = 6 Hz) along
with a broad multiplet of six protons from d 1.85 to 1.60 due to
methylene groups of the side chain suggested the introduction of
the butyl group. The ESI-MS recorded the molecular ion at m/z
388, besides this the fragment at m/z 345 and m/z 321 due to loss
of side chain at C-16 further proved the proposed structure.
Conjugating the pregnane with appropriate carbohydrate moi-
ety not only increases their hydrophilic character but it also in-
creases it efficacy and reduced toxicity relative to the parent
pregnane [33]. In our effort to synthesize some novel pregnane
glycoside, condensation of glycosyl donor 1,2,3,4,6 penta-O-acet-
yl-D-galactose (II) with pregnane genin 2 in the presence of Lewis
acid catalyst stannic chloride yielded the product 10. The 1HNMR
of compound 10 not only confirmed that it is a pregnane glyco-
side but it also helped in ascertaining the configuration of the gly-
cosidic linkage. The anomeric proton was observed as a doublet at
d 5.22 (J = 8.1 Hz). The value of large coupling constant, due to
diaxial coupling confirmed the glycosidic linkage to be b. The
downfield shifting of one proton multiplet of the methine proton
at C-3 centered around d 3.5 (in the genin) and now appearing at
d 3.90 (in the glycoside) was in conformity with the proposed
structure that the sugar was glycosidically linked to C-3 hydroxyl
The scavenging potential of compounds 4, 5, 7, 8, 9, and 10 on
group. The presence of vibrational bands at 1180.0 cmꢁ1
,
in vitro generation of oxygen free radical at 100 and 200
lg/mL
1040.1 cmꢁ1 and 661.8 cmꢁ1 corresponding to CAOAC stretch,
symmetric CAO stretching and OACAO deformation for acetyl
groups further confirms the conjugation of the acetylated sugar
with the pregnane moiety. In the FAB-MS of 10, fragment at m/
z 509 obtained by successive loss of two acetic acid molecules
and methyl group, and at m/z 480 obtained by successive loss
of two acetic acid molecules and C-17 keto-methyl chain sug-
gested that an acetylated sugar is glycosidically linked to preg-
nane moiety in compound 10 .
against formation of Oꢁ2 and OH in enzymatic system was also
studied (Table 2). The generation of superoxide anion in an enzy-
matic system was found to be inhibited by 4 (9%), 5 (14%), 7
Å
(8%), 8 (5%), 9 (15%) and 10 (9%) at a dose of 100
lg/mL and (8%),
(10%), (7%), (7%), (14%) and (8%) at a dose of 200
lg/mL respec-
tively. The enzymatic generation of hydroxyl free radicals was also
inhibited by 4 (13%), 5 (18%), 7 (15%), 8 (12%), 9 (29%) and 10 (14%)
at a dose of 100
(15%) at a dose of 200
pound 9, the inhibition was found to be (29%) at a dose of 100
mL for superoxide ion and (27%) at a dose of 200 g/mL for hydro-
l
g/mL and (15%), (13%), (14%), (13%), (27%) and
g/mL respectively (Table 2). In case of com-
g/
l
l
l
3.2. Anti-hyperlipidemic and anti-oxidant activity
xyl free radical generation which again was quite significant. The
involvement of hydroxyl free radicals (OH.) has been found to be
a major causative factor for peroxidative damage to lipoproteins,
which is responsible for inhibition and progression of atherosclero-
sis in hyperlipidemic subjects [40]. Results of in vitro study suggest
that compound 9 showed noteworthy decrease in plasma levels of
TC, TG, PL and LCAT thus showing a marked improve in the lipid
profile of triton induced hyperlipidemic rats and also possessed
anti-oxidant property as it potentially inhibited the in vitro gener-
ation of both O2ꢁ and OH. free radicals in an enzymatic system at
Hypercholesterolemia is an essential risk factor for cardiovascu-
lar diseases [23]. When plasma cholesterol exceeds the optimum
required level, it results in the development of atherosclerosis
and stroke [34]. The treatment of hyperlipidemia reduces cardio-
vascular events [35]. Furthermore hypercholesterolemia increases
oxidative stress by production of endothelial superoxide anions.
Therefore both hyperlipidemia and oxidative stress plays an
important role in atherogenesis [36,37].
Administration of triton WR-1339 to experimental animals
caused significant increase in plasma levels of TC (2.9-fold), PL
100 lg/mL and 200 lg/mL concentration. With its anti-oxidant

118
A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
1
2
E = –1121.44226751 a.u.
E = –968.76655480 a.u.
E = –1024.05665548 a.u.
E = –3673.79742737 a.u.
3
5
4
6
E = –1201.05457144 a.u.
E = –1043.97342578 a.u.
7
8
E = –1428.10437124 a.u.
E = –1733.42167543 a.u.
9
10
E = –1202.47145245 a.u.
E = –2190.17881838 a.u.
Fig. 1. Optimized geometry of compounds 1–10 using B3LYP/6-31G(d, p) level of theory.
potential, compound 9 may thus prove to reduce oxidative stress in
hyperlipidemic animals thereby preventing occurrence of athero-
genic events.
The percentage decrease of lipid parameters (TC, PL, TG) for
compound 9 remains to be highest (22%, 26%, 22%) as compared
to other compounds indicating that introduction of butyl side

A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
119
Table 4
chain at C-16 effects the lipid lowering efficacy of the compound to
a greater extent. Similarly, introduction of phenyl ring at C-16 po-
sition in compound 5 also causes a significant decrease of all three
lipid parameters (15%, 18%, 17%), but this decrease is lower in com-
parison when butyl is introduced at C-16. However minor decrease
in TC, TG and PL levels is observed for compounds 4, 7, 8 and 10.
Similar observations are made regarding decrease in levels of both
superoxide and hydroxyl free radicals, where again significant de-
crease is observed for compound 9. The structural features respon-
sible for the changes in the anti-hyperlipidemic and anti-oxidant
activity taking 16-DPA as the initial moiety (Compound 1) are
summarized in Table 3.
Comparison between experimental and theoretical 1H NMR chemical shifts d (ppm)
from TMS for studied compounds 4, 5,7, 8, 9, 10.
Compounds Assignment
d (exp.)
d (Cacl.)
4
H-16
H-6
5.77
5.38
4.60
6.31
5.45
4.20
@NOCH2
3.94
3.58
AC@NAOCH2ACH2ABr
H-3
3.58
2.03
1.03
0.84
3.68
2.01
1.14
1.12
CH3-21
CH3-19
CH3-18
5
ArAH0s
7.32–
7.11
5.37
3.85
3.57
2.71
2.03
7.33–
7.11
5.46
3.90
3.72
2.53
1.83
H-6
H-16
H-3
3.3. Molecular structure
The optimized geometries for compounds 1–10 were obtained
at B3LYP/6-31G (d,p) level of theory as shown in Fig. 1. All the com-
pounds were in the ground state corresponding to C1 point group
symmetry. Conversion of C@O to oxime C@NAOH derivative is evi-
dent from the elongation in bond length from 1.22 Å (C@O) to
1.29 Å (C@N) in compound 3, because nitrogen is lesser electroneg-
ative in comparison to oxygen. Introduction of side chain (BrACH2-
ACH2ABr) at C-20 position in compound 3 also caused slight
increase in N-O bond length corresponding to 1.38 Å (C@NAOAH)
in compound 3 to 1.40 Å (C@NAOACH2ACH2ABr) in compound 4.
The introduction of phenyl group at C-16 position in compound 5 is
evident from the conversion of C16AC17 double bond in com-
pound 2 (1.34 Å) to C16AC17 single bond (1.56 Å) in compound
5. The CAC and CAO bond lengths in compound 6 is 1.49 Å and
1.45 Å which is very much close to the normal CAC and CAO bond
lengths generally observed in case of reported oxiranes
(CAC = 1.46 Å and CAO = 1.44 Å). However, opening of the oxirane
ring led to the introduction of diethylene glycol at C-16 and hydro-
xyl group at C-17 position in compound 7. This compound showed
an increase in C16AC17 bond length from 1.46 Å to 1.57 Å, and this
appears to be due to the opening of the three membered strained
epoxide ring, thereby releaving it from angle as well as torsional
strain. Similar observation was made in case of compound 8 and
compound 9 where the C16AC17 bond length were calculated as
1.57 Å and 1.56 Å respectively. In case of compound 10, glycosida-
tion at C-3 position also leads to slight increase in CAO bond length
now calculated at 1.44 Å, against the bond length observed for the
acceptor molecule 2 at 1.42 Å.
H-17
CH3-21
CH3-19
CH3-18
1.03
0.77
0.86
0.79
7
H-6
H-16
5.35
4.57
3.71–
3.65
3.58–
3.54
5.40
4.79
4.05–
3.40
3.66–
3.21
A(OACH2ACH2AOACH2ACH2AOH)
A(OACH2ACH2AOACH2ACH2AOH),
H-3
CH3-21
CH3-19
CH3-18
2.08
1.19
0.93
2.18
1.04
0.65
8
H-6
H-16
5.44
5.18
4.65–
4.59
5.40
4.79
4.05–
3.24
A(OACH2ACH2AOACH2ACH2AOAc),
H-3
4.23–
4.14
3.70–
3.64
2.10
2.04
1.97
1.01
0.86
A(OACH2ACH2AOACH2ACH2AOAc)
A(OACH2ACH2AOACH2ACH2A)
CH3-21
OAc
OAc
CH3-19
CH3-18
2.18
2.01
1.92
1.04
0.62
9
H-6
H-3
CH3-21
H-16
5.35
3.53
2.25
2.94
1.85–
1.60
1.05
0.95
0.88
5.40
3.69
2.27
3.35
1.68–
1.15
1.07
0.73
0.93
A(CH2ACH2ACH2)
CH3-19
CH3-18
A(CH2ACH3)
3.3.1. 1H NMR analysis
The Gauge-including atomic orbital (GIAO) 1H NMR chemical
shift calculations of the compounds 4, 5, 7, 8, 9, 10 were made
using B3LYP method in conjugation with 6-31G (d,p) basis set
[29]. The experimental and calculated values of 1H NMR chemical
shifts of the newly synthesized compounds 4, 5, 7, 8, 9, 10 are gi-
ven in Table 4. The correlation between the experimental and cal-
culated 1H NMR chemical shift values of these compounds are
plotted in graphs (2a–2f) in Fig. 2. All the six correlation graphs fol-
low the linear equation (y = mx + c), where y = theoretical 1H NMR
chemical shift and x = experimental 1H NMR chemical shift (d
ppm). These graphs shows good correlation between the experi-
mental and the calculated results with the coefficient of regression
(R2) being in the range of 0.97 ꢂ 0.99.
10
H-16
H-6
6.70
5.44
5.36
5.22
5.07
4.33
4.28–
4.17
4.08
3.90
2.13
7.28
5.51
5.87
5.76
5.30
4.53
4.05–
3.92
3.85
3.46
2.00
H-30
H-10
H-20
H-40
H-60
H-50
H-3
CH3-21
CH3-19
CH3-18
1.04
0.83
1.13
1.09
3.4. IR analysis
The selected experimental and calculated vibrational wave-
numbers along with the corresponding vibrational assignments
for compound 4, 5, 7, 8, 9 and 10 are given in Table 5. Any dis-
crepancy observed between the experimental and the theoretical
values may be attributed to the fact that the experimental results
corresponds to solid phase, whereas the theoretical results belong
to gaseous phase. In order to obtain good correlation between the
two values, the calculated harmonic frequencies were scaled
down via scaling factor 0.9608 [41]. In the IR spectrum of
compound 4, the presence of
a broad band in the region

120
A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
Fig. 2. Linear regression graphs for compound 4 (2a), compound 5 (2b), compound 7 (2c), compound 8 (2d), compound 9 (2e) and compound 10 (2f) between the
experimental and theoretical 1H NMR chemical shifts at B3LYP/6-31G.
3600–3400 cmꢁ1 for hydrogen bonded OH group centered around
3405.2 cmꢁ1 was seen in the spectrum corresponding to the cal-
culated value at 3129.7 cmꢁ1. The characteristic C@N and NAO
stretching vibrations of oxime are observed at 1659.0 cmꢁ1 and
1045.3, 962.1 cmꢁ1, with the corresponding theoretical values at
1672.1 cmꢁ1 and 1039.2, 974.5 cmꢁ1 respectively [42]. The band
for CABr stretch is observed at 668.3 cmꢁ1 with the calculated va-
lue at 692.7 cmꢁ1. In the IR spectrum of compound 5, the car-
bonyl group stretch is observed at 1702.3 cmꢁ1 along with the
bands observed at 3027.1 cmꢁ1, 1601.8 cmꢁ1 and 1493.9 cmꢁ1
corresponding to CAH aromatic stretch and C@C aromatic stretch
1150.0 cmꢁ1 with the calculated value being at 1162.0 cmꢁ1
,
CAO stretching for primary OH at 1053.5 cmꢁ1 with the calcu-
lated value at 1047.0 cmꢁ1 and rocking vibration for methyls at
982.0 cmꢁ1 [43] the calculated value being 941.7 cmꢁ1. In addi-
tion, the C@O stretch is observed at 1698.5 cmꢁ1 along with
asymmetric and symmetric CAH stretching vibrations at 2926.6
and 2852.4 cmꢁ1 respectively. In the IR spectrum of compound
8, the corresponding vibrational frequencies for acetate groups
are observed at 1242.7 cmꢁ1
O@CAOAC stretch, OCO deformation and CCO deformation
respectively [44,45]. The calculated values are at 1256.8 cmꢁ1
653.7 cmꢁ1 and 574.9 cmꢁ1 respectively. The experimental FT-IR
spectrum of compound
shows vibrations at 3437.4 cmꢁ1
,
657.5 cmꢁ1
,
581.0 cmꢁ1 for
,
respectively. The IR spectrum of compound
7 showed the
characteristic stretching vibrations for CAOAC stretching at
9
,

A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
121
Table 5
Selected Experimental and Calculated IR frequencies, vibrational assignments for compound 4, 5, 7, 8, 9, 10.
Calculated IR
Experimental IR
Vibrational assignments
Compound 4
3129.7
2961.9
3405.2
2927.6
2856.1
1659.0
Hydrogen bonded OH stretch
Asymmetric CAH stretch
Symmetric CAH stretch
C@N stretch
1672.1
1429.2 and 1388.5
1039.2
1443.8 and 1379.8
1045.3
CH3, CH2 deformation
NAO stretch
974.5
962.1
692.7
668.3
CABr
Compound 5
3137.4
3080.8
3395.0
3027.1
2926.7
2852.6
Hydrogen bonded OH stretch
CAH aromatic stretch
Asymmetric CAH stretch
Symmetric CAH stretch
C@O stretch
2961.7
1795.2
1702.3
1637.8, 1538.7, 1437.4
1393.8
777.1
1601.8 and 1493.9
1379.5
761.9
C@CAC aromatic ring stretch deformation vibration of methyl of CH3CO
CH2 rocking
Compound 7
3669.7
2971.5
3406.7
2926.6
2852.4
1698.5
1379.5
1150.0
1053.5
982.0
Hydrogen bonded OH stretch
Asymmetric CAH stretch
Symmetric CAH stretch
C@O stretch
Deformation vibration of methyl of CH3CO
CAOAC stretch
1781.0
1396.3
1162.0
1047.0
941.7
CAO stretching vibration for primary OH
Rocking vibrations for methyls
Compound 8
3666.8
2988.2
3417.1
2917.9
2849.7
1733.3
1373.3
1242.7
657.5
Hydrogen bonded OH stretch
Asymmetric CAH stretch
Symmetric CAH stretch
C@O (ester)
Deformation of methyl of CH3CO
O@CAOAC stretch
1783.1
1380.4
1256.8
653.7
OCO deformation
574.9
581.0
CCO deformation
Compound 9
3134.5
2986.4
2961.5
1786.3
3437.4
2931.8
2852.8
1701.8
Hydrogen bonded OH stretch
Asymmetric CAH stretch
Symmetric CAH stretch
C@O stretch
1441.3
1451.9
CH3, CH2 deformation
1392.8
1223.2 and 1150.3
1376.8 and 1355.6
1223.9 and 1152.1
CH3deformation, CH2 wagging
CH3 rocking, CH2 twisting
Compound 10
2937.5
2882.6
1767.0
1397.4
1169.8
1051.8
2924.5
2851.9
1728.7
1377.9
1180.0
1040.1
661.8
Asymmetric CAH stretch
Symmetric CAH stretch
C@O (ester)
Deformation of methyl of CH3CO
CAOAC stretch
Symmetric CAO stretch
OACAO deformation (acetate)
667.8
2931.8 cmꢁ1 and 2852.8 cmꢁ1 corresponding to hydrogen bonded
2986.4 cmꢁ1 and 2961.5 cmꢁ1 respectively. The vibrational bands
observed at 1376.8 cmꢁ1 and 1355.6 cmꢁ1 corresponds to defor-
mation mode for methyls and wagging vibration for methylenes
respectively. Further vibrational bands at 1223.9 cmꢁ1 and
1152.1 cmꢁ1 corresponds to rocking vibration for methyls and
twisting vibration for methylenes respectively [43]. The IR spec-
OH along with both asymmetric and symmetric CAH stretching
vibrations. The calculated values corresponds to 3134.5 cmꢁ1
,
Table 6
Experimental wavelengths (nm) and energy (eV) for compounds 4, 5, 7, 8, 9, 10 along
with theoretical wavelength (nm) and energy (eV) at TD-DFT/B3LYP/6-31G(d,p).
trum of compound 10 showed
a strong intensity bands at
2924.5 cmꢁ1 and 2851.9 cmꢁ1 corresponding to asymmetric and
symmetric CAH stretch along with a medium intensity band at
1726.7 cmꢁ1 for ester C@O respectively. These experimental val-
ues showed good correlation with the calculated values. The
Compound
Experimental
k (nm)
TD-DFT/B3LYP/6-31G(d,p)
E (eV)
k (nm)
E (eV)
4
239 and 276
5.1876
4.4922
5.8483
5.6356
5.6614
5.7667
5.1876
234 and 250
4.9575
5.2884
5.9484
6.1356
6.1882
6.0612
5.0644
vibrational bands observed at 1180.0 cmꢁ1 1040.1 cmꢁ1 and
,
5
7
8
9
10
212
220
219
215
239
208
202
200
204
244
661.8 cmꢁ1 corresponds to CAOAC stretch, symmetric CAO
stretch and OACAO deformation for acetate groups of the glyco-
side with the calculated values being 1169.8 cmꢁ1, 1051.8 cmꢁ1
and 667.8 cmꢁ1 respectively [46].

122
A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
Fig. 3. HOMO–LUMO molecular orbitals depicting the band gap for compound 4 (3a), compound 5 (3b), compound 7 (3c), compound 8 (3d), compound 9 (3e), and compound
10 (3f).
Table 7
Calculated eHOMO
reactants 2, 3, 6 and products 4, 5, 7, 8, 9, 10 in eV.
,
e
LUMO, energy band gap (eH
ꢁ
e
L), chemical potential (
l
), electronegativity (
v
), global hardness (g), global softness (S) and global electrophilicity index (x) for
Comp.
eH
eL
eH
ꢁ
eL
v
l
g
S
x
2
3
6
4
5
7
8
9
10
ꢁ6.3213
ꢁ5.8987
ꢁ6.2606
ꢁ5.9057
ꢁ6.1602
ꢁ6.1234
ꢁ6.3294
ꢁ6.1232
ꢁ6.2157
ꢁ1.2515
ꢁ0.6721
ꢁ0.7725
ꢁ0.6789
ꢁ0.4904
ꢁ0.5257
ꢁ0.6101
ꢁ0.5766
ꢁ1.3451
5.0698
5.2265
5.488
5.2268
5.6698
5.5977
5.7193
5.5465
4.8706
3.7864
3.2854
3.5166
3.2923
3.3253
3.3246
3.4698
3.3499
3.7804
ꢁ3.7864
ꢁ3.2854
ꢁ3.5166
ꢁ3.2923
ꢁ3.3253
ꢁ3.3246
ꢁ3.4698
ꢁ3.3499
ꢁ3.7804
2.5349
2.6133
2.744
2.6134
2.8349
2.7988
2.8597
2.7733
2.4353
0.19725
0.19133
0.18221
0.19132
0.17637
0.17864
0.17485
0.18029
0.20531
2.8278
2.0652
2.2533
2.0738
1.9502
1.9745
2.1050
2.0232
2.9342
3.5. Electronic absorption
239 nm, which is obviously due to
band was observed at 276 nm, which arises due to n–
p
–
p* transitions, another weak
* transition.
p
The experimental absorption wavelengths (k), excitation ener-
gies (E), along with the computed values of absorption wavelength
(k) and excitation energies (E) are tabulated in Table 6. The com-
puted values of absorption wavelength (k), excitation energies (E)
and absorbance (A) were studied with the help of TD-DFT ap-
proach. Since the FMO’s are composed mainly of p-atomic orbitals,
Highest occupied molecular orbital (HOMO) and Lowest unoc-
cupied molecular orbital (LUMO) are one of the important param-
eters used for predicting the reactivity of compounds. HOMO
characterizes the electron donating ability whereas LUMO charac-
terizes the electron accepting ability. The energy gap between
HOMO and LUMO called the ‘band gap’ helps in explaining the
eventful charge transfer interaction within the molecule. The 3-D
plots of HOMO–LUMO molecular orbitals depicting the band gap
therefore these compounds show chiefly
However in case of compound 4, in addition to a strong band at
p–
p* type transitions.

A. Sethi et al. / Journal of Molecular Structure 1052 (2013) 112–124
123
Table 8
synthesized pregnane derivatives 4, 5, 7, 8, 9 and 10, compound
10 with high values of chemical potential ( ) = 3.780, global elec-
trophilicity index ( ) = 2.934 and softness (S) = 0.2053 also shows
its strong electrophilic nature, as a result compound 10 can further
be used for the introduction of newer substituent’s.
Selected electrophilic reactivity descriptors (fkþ; skþ; xkþ) and nucleophilic reactivity
descriptors (fkꢁ; Skꢁ; xꢁk ) of reactant 2 and reactant 6 using Hirshfeld atomic charges.
l
x
Skꢁ
xkꢁ
fkþ
xkþ
Skþ
fkꢁ
Atom no.
Reactant 2
C17
C16
In order to define a particular reactive site within the molecule,
local reactivity descriptors such as local softness (Ski), Fukui Func-
0.0712
0.1831
0.0179
0.0513
0.0140
0.0361
0.0035
0.0101
0.2013
0.5177
0.0506
0.1450
tion (FF) and local electrophilicity index (xk) [50,51] are used. In
Reactant 6
C17
C16
0.0203
0.0606
0.0121
0.0318
0.0037
0.0110
0.0022
0.0058
0.0457
0.1365
0.0273
0.0718
DFT theory of chemical reactivity, Fukui function f(r) is considered
as one of the most fundamental indicator for defining the site
selectivity in a given molecular species and for soft–soft type of
interactions, the preferred reactive site in a molecule is the one
of compounds 4, 5, 7, 8, 9, and 10 are best illustrated in Fig. 3. In
Fig. 3a one clearly sees that the electron density of HOMO is cen-
tered on C16AC17 double bond, C@N group and bromine atom
while in LUMO the electron density is located around the
C16AC17 double bond and the CA20 @NAO group for compound
4. Similarly, the HOMO orbitals are distributed over C5AC6 double
bond and the oxygen of the hydroxyl group at C-3 position, while
in LUMO the electron density is mainly located on the phenyl ring
in case of compound 5 as seen in Fig. 3b. In compound 7, electron
density of HOMO is mainly located over the two oxygen of the side
chain and oxygen of the carbonyl group at C-20, whereas LUMO is
centered on C-17 carbon and oxygen of COCH3 group seen in
Fig. 3c. In Fig. 3d, the HOMO in case of compound 8 is chiefly dis-
tributed over C-15, C-16 and C-17 carbon as well as oxygen’s pres-
ent in the side chain OACH2ACH2AOACH2ACH2AOAc, while the
LUMO orbitals can be seen distributed chiefly over C-17 and CO
of COCH3. In compound 9, one sees the HOMO and LUMO orbitals
located on C16, C-17 carbons and C@O of COCH3 group as shown in
Fig. 3e. In Fig. 3f, the HOMO orbitals in compound 10, are centered
on oxygen atom of the acetyl groups (OACOACH3) besides car-
bonyl oxygen of all acetyl groups except that of 60 position CH2OAc
in compound 10. LUMO is however mainly concentrated over C16-
C17 double bond and C@O of COCH3. The energies of HOMO LUMO
orbitals as well as the values for HOMO LUMO band gap energy are
given in Table 7.
with maximum values of (fk, sk,
xk) [52]. Using Hirshfeld atomic
charges of neutral, cation and anion state of initial reactant 2 and
6, the condensed Fukui functions (fkþ; fkꢁ; fk0), local softness
(Skþ; Skꢁ; S0k ) and local electrophilicity indices (xþk xkꢁ 0k ) for atomic
; ; x
sites C-16 and C-17 were calculatedþand are listed in Table 8. In
case of reactant 2 the values of fkþ; Sk ; xkþ for two atomic sites C-
16 and C-17 are 0.1831, 0.0361, 0.1450 and 0.0712, 0.0140,
0.2013 respectively. Similarly for reactant 6, the values of local
reactivity descriptors fkþ; Skþ; xþk for atomic sites C-16 and C-17
are 0.0606, 0.0110, 0.1365 and 0.0203, 0.0037, 0.0457 respectively.
Maxiþmum values of all the three local reactivity descriptors
(fkþ; Sk ; xþk ) in case of both reactant 2 and reactant 6 indicate that
C-16 site is more prone for nucleophilic attack in comparison to
C-17 atomic site. Therefore, these local reactivity descriptors calcu-
lated in case of both reactant 2 and reactant 6, confirms the favor-
able site of attack at C-16 position which further leads to the
formation of the desired products.
4. Conclusion
Findings from the above study suggest that the reactant 2 was
more electrophilic in comparison to other two reactants 3 and 6
and it is for this reason that even after the formation of glycoside
10, the electrophilicity of the basic pregnane moiety was retained
and thus further reaction can be carried out so as to generate more
potent biologically active product. Compound 9 showed potent
anti-dyslipidemic and anti-oxidant activity. Since this compound
possessed an alkyl chain, hence it could be concluded that for fur-
ther studying the anti-dyslipidemic and anti-oxidant activity, dif-
ferent types of alkyl groups-straight as well branched chain can
be introduced at C-16 position.
3.6. Reactivity descriptors
DFT based global and local reactivity descriptors have been
extensively used for rationalization and interpretation of diverse
aspects of chemical bonding, reaction mechanism and reactive
centers. These quantum chemical descriptors are related to elec-
tronic structure of compounds and to the mechanism that is in-
volved in the covalent bond formation between the nucleophile
and the electrophile. DFT makes it possible to define and well jus-
tify different concepts of chemical reactivity. Chemical hardness
Acknowledgment
The authors are grateful to the RSIC division of the Central Drug
Research Institute, Lucknow for the analytical data.
(g), chemical potential (l), polarizability (a) and softness (S) [47–
51], known as the global reactivity descriptors are used to define
the properties of a molecule as a whole, particularly its good elec-
trophilic/nucleophilic nature. Global reactivity descriptors like
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