Synthesis of diosgenin p-nitrobenzoate by Steglich method, its crystal structure and quantum chemical studies

Article abstract of DOI:10.1016/j.molstruc.2012.05.057

In the present study, a novel one pot synthetic route for the synthesis of diosgenin p-nitrobenzoate (2) is described from cheap, commercially available naturally occurring sapogenin-diosgenin. The molecular geometry, IR frequencies, Gauge-including atomic orbital (GIAO), ¹H and ¹³C NMR chemical shifts of compound 2 has been calculated in the ground state by using the Hartree-Fock (HF) and density functional method (DFT/B3LYP) using 6-31G(d,p) basis set. The structure of diosgenin p-nitrobenzoate (2) has been confirmed by single crystal X-ray diffraction. The compound crystallizes in monoclinic form having space group P21 with cell parameters a = 7.719(2) ?, b = 8.425(2) ? and c = 22.578(6) ?, α = 90.00, β = 98.46 and γ = 90.00. The oxygen atoms O5 and O4 of the nitro and carbonyl ester, respectively display weak intermolecular N1O5?H7′ and C1′O4? H4′ interactions having dimensions of 2.61 and 2.59 ?, respectively to form intricate 1D network. The study of the electronic properties such as HOMO and LUMO energy were performed using time dependent DFT (TD-DFT) calculations. The calculated HOMO and LUMO energy values indicate that charge transfer takes place within the molecule. The compound was screened for cytotoxicity and anti-adipogenic activity.

Full text of DOI:10.1016/j.molstruc.2012.05.057

Journal of Molecular Structure 1028 (2012) 88–96
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis of diosgenin p-nitrobenzoate by Steglich method, its crystal structure
and quantum chemical studies
a
a
a
b
a
Arun Sethi a, , Akriti Bhatia , Dolly Shukla , Abhinav Kumar , Ravi Sonker , Rohit Prakash ,
⇑
Gitika Bhatia b
a Department of Chemistry, University of Lucknow, Lucknow 226 007, UP, India
b Biochemistry Division, C.D.R.I., Lucknow 226 001, UP, India
h i g h l i g h t s
"
"
"
"
"
One pot Steglich esterification of diosgenin, without opening of spiro ring in high yield.
Stereochemistry assigned to the title compound using X-ray crystallography.
Theoretical calculations made using DFT/B3LYP/6-31G(d,p) method.
Chemical reactivity explained with the aid of molecular elestroscostatic potential surface.
The title compound was screened for cytotoxicity and anti-adipogenic activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 April 2012
Received in revised form 21 May 2012
Accepted 23 May 2012
Available online 13 June 2012
In the present study, a novel one pot synthetic route for the synthesis of diosgenin p-nitrobenzoate (2) is
described from cheap, commercially available naturally occurring sapogenin–diosgenin. The molecular
geometry, IR frequencies, Gauge-including atomic orbital (GIAO), 1H and 13C NMR chemical shifts of com-
pound 2 has been calculated in the ground state by using the Hartree–Fock (HF) and density functional
method (DFT/B3LYP) using 6-31G(d,p) basis set. The structure of diosgenin p-nitrobenzoate (2) has been
confirmed by single crystal X-ray diffraction. The compound crystallizes in monoclinic form having space
Keywords:
DFT
X-ray crystallography
Anti-adipogenic activity
HOMO and LUMO energy
GIAO method
group P21 with cell parameters a = 7.719(2) Å, b = 8.425(2) Å and c = 22.578(6) Å,
a = 90.00, b = 98.46 and
c
= 90.00. The oxygen atoms O5 and O4 of the nitro and carbonyl ester, respectively display weak inter-
molecular N1AO5ꢀ ꢀ ꢀH70 and C10@O4ꢀ ꢀ ꢀH40 interactions having dimensions of 2.61 and 2.59 Å, respec-
tively to form intricate 1D network. The study of the electronic properties such as HOMO and LUMO
energy were performed using time dependent DFT (TD-DFT) calculations. The calculated HOMO and
LUMO energy values indicate that charge transfer takes place within the molecule. The compound was
screened for cytotoxicity and anti-adipogenic activity.
Steglich esterification
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
breast [11], and liver [12]. Studies have proved that diosgenin
can be absorbed through the gut and plays an important role in
Diosgenin (25R-Spirost-en-3b-ol) is one of the most important
steroidal sapogenin, which is widely used in the pharmaceutical
industry as a precursor for the synthesis of a number of steroidal
drugs like progesterone [1] and cortisone [2]. In recent years, there
have been many reports on the important pharmacological attri-
butes of diosgenin such as anti-cancer activity, antagonistic effect
on rheumatoid arthritis, anti-malarial and anti-proliferative prop-
erty [3–6]. Several reports have shown that diosgenin inhibits pro-
liferation and induces apoptosis in a wide variety of tumor cells of
colon [7], osteosarcoma [8], leukemia [9], erythroleukemia [10],
the control of cholesterol metabolism [13]. Preclinical studies have
also demonstrated diosgenin efficacy against hyperglycemia,
hypercholesterolemia and hypertriacylglycerolemia [14–16]. As
part of our programme for synthesis of some biologically active
steroidal derivatives [17,18], one pot synthesis of a new biologi-
cally active diosgenin derivative (2) is herein reported. A typical
procedure to synthesize esters is the Fischer esterification, where
in a carboxylic acid is treated with an alcohol in the presence of
a mineral acid catalyst. Diosgenin when subjected to Fishers ester-
ification usually leads to the formation of three compounds (as ver-
ified by TLC) which probably may be due to Spiro ring cleavage,
resulting in the formation of side products, as the spiroketal ring
is highly sensitive towards acidic conditions and readily undergoes
⇑
Corresponding author. Tel.: +91 9415396239.
E-mail address: alkaarunsethi@rediffmail.com (A. Sethi).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.05.057

A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
89
O
O
O
COOH
O
DCC
DMAP
O
C
+
O
O2N
HO
NO2
2
1
Scheme 1.
ring opening process. However, in our newly adopted experimental
protocol, Steglich esterification of diosgenin was carried out using
N,N0-Dicylcohexylcarbodiimide (DCC) as a coupling reagent and 4-
Dimethylaminopyridine (DMAP) as a catalyst (Scheme 1) [19]. The
structure of the desired compound diosgenin p-nitrobenzoate (2)
in which the Spiro ring is intact, is well interpreted with the help
of 1H, 13C, 2D (1HA1H COSY) NMR, IR and UV–Vis spectroscopy.
In order to provide additional proof, and to investigate the stereo-
chemistry and crystal packing of molecule, single crystal X-ray dif-
fraction analysis was also performed. Further in order to get a
better knowledge of the reactivity of the synthesized compound,
theoretical studies by Hartree–Fock (HF) and density functional
method (DFT/B3LYP) were also carried out. In addition, HOMO
and LUMO analysis have been carried out to elucidate the informa-
tion regarding charge transfer within the molecule.
150.44(C-50), 139.28(C-5), 136.18(C-20), 130.65(C-30 and C-70),
123.45(C40 and C-60), 122.909(C-6), 109.283(C-22), 80.787(C-3),
5.705(C-26), 66.848(C-16), 62.087(C-17), 56.426(C-9), 49.941(C-
14), 41.617(C-4), 40.266(C-10), 39.709(C-20), 38.069(C-13),
36.925(C-23), 36.772(C-2), 32.065(C-1), 31.843(C-12), 31.408(C-
7), 31.386(C-25), 30.295(C-8), 28.807(C-15), 27.769(C-24),
20.842(C-11),
14.524(C-21).
19.369(C-18),
17.134(C-19),
16.287(C-27),
2.2.1. Crystal structure determination and refinement
Intensity data for the colorless crystals of the compound 2 was
collected at 100(2) K on a Bruker SMART diffractometer system
equipped with graphite monochromated Mo
Ka radiation
k = 0.71073 Å. The final unit cell determination, scaling of the data,
and corrections for Lorentz and polarization effects were per-
formed with Bruker SAINT [20]. A symmetry-related (multi-scan)
absorption correction has been applied. Structure solution, fol-
lowed by full-matrix least squares refinement was performed
using the WINGX-1.70 suite [21] of programs throughout. The
structure was solved by direct methods SHELXS97 [22] and crystal
refinement was done using SHELXL97 [22]. All non-hydrogen
atoms were refined anisotropically; hydrogen atoms were located
at calculated positions and refined using a riding model with iso-
tropic thermal parameters fixed at 1.2 times the Ueq value of the
appropriate carrier atom. The conformations of the ring were cal-
culated using PLATON [23]. The perspective view of the molecule
2. Experimental section
2.1. Material and measurements
All reagents used for synthesis were purchased from Sigma Al-
drich (St. Louis, MO) and used without further purification. IR spec-
tra was recorded in KBr disk on
a Nicolet MX-1 FTIR
spectrophotometer, 1H NMR spectra was recorded in CDCl3 solvent
on a Bruker DRX-300 MHz spectrometer, 2D (1HA1H COSY) NMR
spectra was recorded on 600 MHz Varian Inova spectrometer,
whereas 13C NMR was recorded on 150 MHz Varian Inova spec-
trometer using CDCl3 as the solvent where the chemical shifts were
reported in parts per million (ppm) units with respect to TMS as
internal standard. Melting point was determined using open capil-
lary tube method and is uncorrected. The compound was purified
by column chromatography and the purity of the compound was
checked by TLC. Cell culture media and supplements were pur-
chased from Invitrogen (Carlsbad, CA).
Table 1
Crystallographic data and structure refinement for compound 2.
CCDC no.
854729
Crystal description
Empirical formula
Formula weight (g molꢁ1
Temperature (K)
Crystal system
Space group
a (Å)
Colorless, plate
C
34H45NO6
)
563.71
100(2)
Monoclinic
P2(1)
7.719(2)
2.2. Synthesis of Diosgenin p-nitrobenzoate (DPNB)
A
solution of p-nitrobenzoic acid (0.25 g, 1.5 mmol), DCC
b (Å)
c (Å)
8.425(2)
22.578(6)
90.00
98.46
90.00
1452.3(7)
2
1.289
0.087
608
0.35 ꢂ 0.20 ꢂ 0.15
1.82–28.34
9548
5600 [Rint = 0.0525]
5600/0/370
R1 = 0.0641, wR2 = 0.1327
R1 = 0.0961, wR2 = 0.1667
1.061
(0.20 g, 1.0 mmol), DMAP (0.18 g, 1.5 mmol) and diosgenin
(0.62 g, 1.5 mmol) in toluene (20 mL) was stirred mechanically at
room temperature until reaction was complete (progress of reac-
tion was monitored by TLC). N,N0-dicyclohexylurea formed during
the reaction was filtered off and the filtrate washed successively
with water, 5% HCl and water, and then dried over anhydrous so-
dium sulfate. Toluene was evaporated under reduced pressure
and the crude product obtained was purified by column chroma-
tography using ethyl acetate–hexane (5%) as eluent (Yield 0.58 g,
95%). Slow evaporation of ethyl acetate during recrystallization
yielded colorless needle-shaped crystals of compound 2. m.p:
218 °C. 1H NMR (CDCl3, 300 MHz) d (ppm) 8.272 (2H, d, Ar AH,
J = 6 Hz), 8.221 (2H, d, ArAH, J = 6 Hz), 5.447 (1H, m, H-6), 4.933–
4.885 (1H, m, H-3), 4.460–4.389 (1H, m, H-16), 3.499–3.345 (2H,
m, H-26), 2.503 (2H, d, H-4, J = 6 Hz), 1.313 (3H, s, CH3-19), 1.293
(3H, s, CH3-18), 0.96 (3H, d, CH3-21,J = 6 Hz), 0.78 (3H, d, CH3-27,
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dcalc (g cmꢁ3
)
Absorption coefficient (mmꢁ1
F(000)
)
Crystal size (mm3)
h (min–max) (°)
Reflections collected
Independent reflections
Data/restraints/parameters
Final R indices [I > 2
r(I)]
R indices (all data)
Goodness-of-fit on F2
Absolute structure parameter
ꢁ0.4(15)
Largest difference peak and hole (e Åꢁ3
Absolute structure parameter
)
0.359 and ꢁ0.368
ꢁ0.05(5)
J = 6 Hz); 13C NMR (150 MHz, CDCl3)
d
(ppm) 164.06(C-10),

90
A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
was prepared using ORTEP [24]. The crystallographic data are sum-
marized in Table 1.
2.2.2.4. Oil red O staining. 3T3-L1 adipocytes were fixed in 4% para-
formaldehyde for 20 min, washed with PBS and stained with 0.34
Oil red O (Sigma, Saint Louis, MO) in 60% isopropanol for 15 min.
It was washed with PBS thrice and stain was extracted with 80%
isopropanol by keeping it at room temperature for 30 min and
absorbance was measured at 520 nm.
2.2.2. Anti-adipogenic activity
2.2.2.1. Cell culture. Rat 3T3-L1 preadipocytes were cultured in
DMEM (Dulbecco0s modified Eagle0s medium) supplemented with
10% (v/v) fetal bovine serum (FBS) so as to aid them to grow to con-
fluency. Two days postconfluency, 3T3-L1 preadipocytes (day 0)
2.2.3. Computational details
All calculations of the title compound 2 were carried out using
Gaussian 03 program package [27] for predicting the molecular
structure, vibrational wavenumbers, 1H and 13C NMR chemical
shifts. The geometry of the molecule was optimized by density
functional theory (DFT) as well as Hartree Fock (HF) level of theory
using B3LYP functional and 6-31G(d,p) basis set [28]. In the vibra-
tional frequency calculations, all stationary points were assigned
minimum values using the optimized geometrical parameters
and their harmonic vibrational wave numbers are positive. The
vibrational frequencies in the harmonic approximation were calcu-
lated using B3LYP/6-31G(d,p). Since the calculated vibrational fre-
quencies were higher than the experimental frequencies, they
were scaled down by the single scaling factor 0.9608 [29]. The elec-
tronic properties were calculated using TD-DFT/B3LYP/6-31G(d,p)
method. All molecular structures were visualized using CHEM-
CRAFT [30] and Gauss View [31] softwares.
were stimulated for 72 h by adding 500
methylxantine), 1 M dexamethasone and 1
the DMEM/10%FBS culture medium in presence of compound 1
and 2 at concentrations of 5 M and 10 M (treatment group) as
well as 0.1% DMSO (control or vehicle group) to induce differenti-
ation of preadipocytes to adipocytes. Subsequently, on day 3, the
l
M IBMX (3-isobutyl-1-
l
lM insulin [MDI] to
l
l
MDI medium was replaced with DMEM/10%FBS containing 1 lM
insulin. On day 5, the MDI medium was replaced with DMEM/
10%FBS and refreshed at 2 days intervals thereafter until analysis
was performed on day 7–10. Between day 3 and induction of dif-
ferentiation (day 7–10) cells maintained in culture medium were
observed daily.
2.2.2.2. Cell viability. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide] cell proliferation and viability assay is a
safe, in vitro assay for the measurement of cell proliferation and
reduction in cell viability. The tetrazolium compound MTT was re-
duced by metabolically active cells to insoluble purple formazan
dye crystals. Preconfluent 3T3-L1 preadipocytes were seeded in
96-well culture plates at a density of 10,000 cells/well. Compounds
1 and 2 (treatment group) as well as control group (0.1% DMSO)
were added to culture medium at time of plating. After 48 h fol-
lowing plating, the cells were incubated with 0.5 mg/mL MTT for
45 min. The medium was aspirated and the insoluble formazan
3. Results and discussion
3.1. Optimized geometry and energies
Initial geometry of compound 2 was taken from the single crys-
tal X-ray diffraction data and was optimized. The ground state
optimized structure of compound 2 is presented in Fig. 1. The opti-
mized structure and experimental structure (from single crystal X-
ray) of compound 2 were compared by superimposing them using
a least squares algorithm that minimizes the distances of the cor-
responding non-hydrogen atoms as shown in Supplementary
Fig. 1. The agreement between the optimized and experimental
crystal structure was quite good showing that the geometry opti-
mization almost exactly reproduces the experimental conforma-
tion. The relative energies of the molecule were calculated
employing ab-initio functions, HF and DFT functional (B3LYP)
methods. The energy for the optimized compound 2 structure is
ꢁ1817.34376 and ꢁ1828.81778 a.u. as calculated by HF and DFT
level of theory respectively. However the optimized energy of the
title compound at HF level of theory was found to be
11.4740 a.u. higher than the DFT level of theory.
product was dissolved in DMSO (150 lL) for at least 2 h in the dark.
MTT reduction was quantified by measuring the absorbance at
550 nm. Both control/treatment groups were assayed in triplicate
and each experiment was repeated at least three times.
2.2.2.3. TG quantification. On day 8 following differentiation, TG
concentration was determined by extracting the cells with CHCl3/
MEOH (2:1 v/v) as described earlier [25], separating the chloroform
and methanol–water phases, removing phospholipids, and further
processing the cultured cells using modified method adopted by
Frayn and Maycock [26]. Triglycerides were then quantified spec-
trophotometrically as glycerol using an enzymatic assay kit (Sigma
chemical).
Fig. 1. Optimized geometry of compound 2 at B3LYP/6-31G(d,p) level of theory.

A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
91
Table 2
presented in Fig. 2. The bond distances, bond angles and dihedral
angles of DPNB are in good agreement with those reported for sim-
ilar type of steroid molecules [33,34]. The C1AC10 bond distance in
ring A is 1.555(5) Å and C9AC10 bond distance in ring B is
1.550(5) Å both corresponding to values slightly greater than other
CAC bond lengths in ring A and ring B. This may be due to the pres-
ence of bridgehead methyl group at the junction of ring A and B,
which relieves the strain and thus causes lengthening of C1AC10
and C9AC10 bonds.
The C22AO1 bond length 1.423 Å(4) is shorter than C16AO1
bond length 1.433 Å(4). This is probably due to the presence of
two electronegative atoms, O1 and O2, with non-bonded electrons
adjacent to C22. This feature has been observed in similar type of
steroidal molecules [33,34].
Comparison between few selected experimental and optimized geometry parameters
of compound 2 using B3LYP/6-31G(d,p).
Bonds
Bond lengths
(X-ray)
Optimized bond
(DFT)
Optimized bond
(HF)
C3AC4
C9AC8
1.520(5)
1.537(5)
1.524(5)
1.550(5)
1.494(5)
1.540(5)
1.542(4)
1.550(5)
1.423(4)
1.424(4)
1.536(5)
1.522(6)
1.487(5)
1.530
1.553
1.534
1.569
1.500
1.548
1.553
1.560
1.424
1.424
1.554
1.536
1.498
1.522
1.547
1.533
1.562
1.500
1.545
1.544
1.550
1.397
1.398
1.544
1.531
1.499
C
C
10AC5
10AC9
C6AC7
C9AC11
C
C
14AC13
17AC16
O1AC22
C
C
C
C
22AO2
20AC22
24AC25
The six membered ring A adopts a chair conformation with C1,
C2, C4 and C5 atoms displaying equal deviations of 0.013 Å from
their plane. However, slight deviation from the ideal chair form
can be expressed by the loss of rotational symmetry through bonds
0
0
1 AC2
Bond angle
0
C
1 AO3AC3
118.7(3)
107.2(3)
108.8(3)
114.4(3)
107.5(3)
110.3(3)
117.3
107.9
109.8
115.0
107.7
110.7
119.0
107.9
109.7
115.1
107.7
110.4
C5AC10AC1
C7AC8AC9
C8AC14AC13
C1AC2 and C4AC5 [
retention of the perpendicular mirror symmetry
D
C2 (C1AC2) = 1.6(4), (C4AC5) = 1.6(4)] with
Cs (C3) =
[
D
2.4(4)]. In ring B the C5AC6 bond length of 1.329 Å(5) confirms
the location of double bond, with the environment at C5 atom
being planer. Due to the presence of double bond, ring B adopts
C
C
15AC16AC17
23AC22AO2
Dihedral angle
0
C
1 AO3AC3AC4
ꢁ73.6(4)
ꢁ168.6(3)
ꢁ168.6(3)
ꢁ60.9(4)
48.8(3)
85.0
166.1
170.7
59.5
ꢁ46.8
ꢁ138.1
173.9
83.4
165.9
170.6
59.3
ꢁ47.2
ꢁ137.3
174.5
an 8b-9
a
half-chair conformation with the rotational axis bisecting
C2
C4AC5AC10AC9
C7AC8AC9AC11
C8AC14AC13AC12
C5AC6 and C8AC9 bonds and with asymmetry parameter [
D
(C5AC6) = 0.7(4)]. Ring C on the other hand has approximately
chair conformation typical for totally saturated ring. The atoms
C12, C11 and C8 are deviated by 0.003 Å whereas atom C14 shows
0.004 Å deviation from the plane passing through these atoms.
Marginal increase in the deviation from plane for C14 (0.004 Å)
as compared to C12, C11 and C8 (0.003 Å) is because of the fact that
C14 is a member of five membered ring D. Ring C therefore due to
the strain at junction with five-membered D ring shows slight
C
C
C
15AC14AC13AC17
15AC16AC17AC20
20AC22AC23AC24 ꢁ175.4(3)
140.5(3)
The optimized molecular structure of compound 2 possesses C1
point group symmetry. The optimized structural parameters (bond
lengths, bond angles, dihedral angle) of compound 2 have been
compared with those obtained experimentally from the single
crystal X-ray diffraction data as shown in Table 2. It was seen that
the DFT and HF methods gave comparable geometries, which differ
from each other by not more than 0.037 Å/0.046 Å (DFT/HF) in
bond length and 2.0°/2.0° (DFT/HF) in bond angles. The bond
lengths of hydrogen atoms (CAH) show variation in our DFT and
HF calculations from single crystal data (CAH). The elongated
CAH bond is not only observed in our DFT and HF calculations
but similar observations have been reported earlier [32].
distortion from ideal chair form [DC2 (C8AC9) = 5.1(4)], [DCs
(C8) = 8.0(4)].
Table 3
Endocyclic torsion angles (°) about the ring junctions of compound 2.
Junction
A/B
Atoms
Torsion angle
Characteristics
Quasi-trans
C4AC5AC10AC1
C6AC5AC10AC9
ꢁ50.27
11.74
B/C
C/D
D/E
C7AC8AC9AC10
C14AC8AC9AC11
63.19
ꢁ47.15
Trans
Trans
Cis
C12AC13AC14AC8
C17AC13AC14AC15
ꢁ60.87
3.2. X-ray single crystal structure
48.74
C13AC17AC16ACA15
C20AC17AC16AO1
14.67
20.85
Compound 2 crystallizes in P21 space group having two
molecules per unit cell. The ORTEP diagram of the compound 2 is
Fig. 2. ORTEP view of compound 2 with displacement ellipsoids drawn at 30% probability level.

92
A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
Ring
D
adopts
a
13b-14
a
half chair conformation
[DC2
(C13AC14) = 1.6(4)]. The five membered ring E[
D
Cs (O1) = 4.1(4)]
has an exo O1 envelope conformation with the O1 atom showing
a deviation of 0.550 from the C22AC20AC17AC16 plane and the
dihedral angle between the planes C22AC20AC17AC16 and
C22AO1AC16 corresponds to 39.64°. The six membered ring F ac-
quires chair conformation with asymmetry parameters
[DC2
(C23AC24) = 0.5(4)], [ Cs (C24) = 2.2(3)], where the atoms C26,
D
O2, C23, C24 display equal deviations of 0.009 Å from the
C26AO2AC23AC24 plane. The ring junctions B/C and C/D are trans,
whereas the ring junction A/B is quasi trans while D/E is cis. A list
of endocyclic torsion angles given in Table 3 and Newman projec-
tions along the bonds involved in ring fusion are shown in Fig. 3.
The crystal structure of the molecule is stabilized by weak inter-
molecular interactions. The oxygen atoms O5 and O4 of the nitro
and carbonyl ester, respectively display weak intermolecular
N1AO5ꢀ ꢀ ꢀH70 and C10@O4ꢀ ꢀ ꢀH40 interactions to form intricate 1D
network (Fig. 4) The N1AO5ꢀ ꢀ ꢀH70 interaction have dimension of
2.61 Å and \N1AO5ꢀ ꢀ ꢀH70 is 159.35°. While in the case of
C10@O4ꢀ ꢀ ꢀH40 interaction the length is 2.59 Å and \C10@O4ꢀ ꢀ ꢀH40
Fig. 3. Newman projections along C10AC5 (A), C9AC8 (B), C14AC13 (C) and
C16AC17 (D) bonds.
Fig. 4. Molecular chain of compound 2 showing weak intermolecular interactions.
Fig. 5. Molecular diagram for unit cell of compound 2.

A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
93
Table 4
is 155.90°. Molecules in the unit cell are packed together to form
well-defined layers. Molecules within the layers are arranged in
an antiparallel manner (Fig. 5).
Experimental and selected theoretical vibrational wavenumbers (cmꢁ1) of compound
2.
Experimental Calculated Assignment
3076.8
2922.6
2874.7
2363.5
3099.5
2997.1
2759.3
Aromatic CAH stretching
Aliphatic CAH stretching
Aliphatic CAH stretching
Overtone of CAO stretching 6-membered ring
3.3. Anti-adipogenic activity
The anti-adipogenic activity is shown in Fig. 6. Cell viability was
measured after 2 days of incubation using MTT assay. As illustrated
in Fig. 6a. compounds 1 and 2 (treatment group) in the concentra-
2215.8
2163.8
Overtone of CAH in plane bending vibration of
1055.2 cmꢁ1
C@O stretching
Aliphatic C@C stretching
Asymmetric NO2 stretching
Aromatic C@C stretching
tion range tested (5 lM/10 lM) did not exhibit cytotoxicity, and
1724.7
1608.0
1528.6
1451.9
1726.9
1612.7
1534.3
1445.9
1409.0
1341.4
1331.1
1285.9
1247.5
1231.3
1157.8
1019.4
975.7
moreover they did not influence the cell viability and proliferation
during the preadipocytic stage. Also, treatment using control group
(0.1% DMSO) showed similar results. The compounds 1 and 2 were
also investigated for their effect on 3T3-L1 preadipocytes differen-
tiation as shown in Fig. 6b. Both the compounds showed mild
reduction of adipogenesis of 3T3-L1 preadipocytes. As displayed
by Oil red O staining in Fig. 6c, cells of control group/vehicle group
had accumulated lipid droplets by end of 3T3-L1 preadipocytes dif-
ferentiation. On addition of compounds 1 and 2 at concentrations
1345.9
1278.9
Symmetric NO2 stretching
CAO stretching
1217.7
1174.9
1055.2
978.6
CAO stretching 5-membered ring
CAO stretching 6-membered ring
CAH in plane bending vibration of aromatic
CAH out of plane bending vibration
p-Disubstituted (aromatic)
5 lM/10 lM decrease in lipid accumulation was observed with
fewer number of lipid cells visible. The compounds 1 and 2 (treat-
841.0
843.8
ment group), were also tested for dose dependent inhibitory effect
764.8
723.7
CH2 rocking
on TG concentration in adipocytes, both the compounds at 10 lM
concentration showed a decrease in TG levels as compared to the
control group Fig. 6d.
with their assignments are given in Table 4. The total number of
atoms in compound 2 is 86; hence it should give 252 (3n ꢁ 6) nor-
mal vibrational modes. The calculated vibrational wavenumbers
are however much higher than the experimental wavenumbers
due to discard of anharmonicity present in real system. The aro-
matic CAH stretching is observed at 3076.8 cmꢁ1 in experimental
IR, which closely relates to the calculated value 3050 cmꢁ1. The
CH3 stretching vibration is observed at 2922.6 cmꢁ1 while the cal-
culated is observed at 2997 cmꢁ1 [35]. The C@C stretching of the
olefin and aromatic are observed at 1608 and 1451.9 cmꢁ1 which
is in close agreement with calculated values at 1612 and
1446 cm1. Ester carbonyl stretching was calculated at 1726 cmꢁ1
and the corresponding absorption band was observed at
3.4. Spectral analysis
The molecular conformation obtained from crystalline struc-
ture, as well as the one yielded by geometry optimization, exhibits
no special symmetries and hence the molecule belongs to C1 point
group. As a consequence, all the fundamental vibrations of free
molecule are both IR and Raman active.
3.4.1. IR spectrum assignments
The correlation graph between experimental and theoretical IR
wavenumbers is given in Supplementary Fig. 2. Few selected (cal-
culated and experimental) vibrational wavenumbers (in cmꢁ1
)
Fig. 6. Graphical representation of cytotoxicity and anti-adipogenic activity of compounds 1 and 2.

94
A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
Table 5
Table 6
Experimental and calculated GIAO 1H NMR chemical shifts (ppm) for compound 2
Experimental and calculated 13C NMR chemical shifts (ppm) for compound 2 using
using DFT/B3LYP/6-31G(d,p).
DFT/B3LYP/6-31G(d,p).
Atom no.
dexp.
dcalcd.
C10
164.06
136.18
130.65
152.84
132.38
127.15 (C70)
125.03 (C30)
143.72
119.81
39.53
30.12
73.70
40.25
135.96
121.87
34.81
34.81
52.79
42.54
24.33
42.21
45.15
58.72
34.52
80.57
64.85
18.13
21.10
45.57
17.27
106.68
33.59
30.69
32.86
65.68
C20
C30 + C70
C50
C60 + C40
C1
C2
C3
C4
C5
C6
C7
150.44
122.90
32.06
27.76
75.70
36.92
139.28
123.45
32.06
31.84
49.94
39.70
20.84
38.06
40.26
56.42
31.40
80.78
62.08
16.28
19.36
41.61
14.52
109.28
31.38
28.80
30.29
66.84
17.13
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
19.02
the use of this level of theory in the prediction of the chemical
shifts of the investigated compound 2. The structure of the title
compound 2 was further confirmed by 2D NMR.
3.4.3. UV–Vis absorption spectra
Compound 2 shows one strong absorption in the UV–Vis spec-
trum due to
p ?
p⁄ transition (Supplementary Fig. 3). On the basis
of fully optimized ground state structure, TD-DFT/B3LYP/6-
31G(d,p) calculation have been used to determine the lowering ex-
cited state of compound 2. The TD-DFT calculation predicts two
electronic transitions at kmax = 336 nm, f = 0.0082, kmax = 284 nm,
f = 0.0002 compared to the experimental values kmax 352 and
312 nm respectively. Observed and calculated electronic transi-
tions of high oscillatory strengths are given in Table 7. The molec-
ular orbital plot of compound 2 is shown in Fig. 7. Highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbi-
tal (LUMO) are very popular quantum chemical parameters for pre-
dicting the chemical reactivity of the compounds. The HOMO
energy characterizes the electron donating ability whereas the
LUMO characterizes the electron accepting ability, and the gap be-
tween HOMO and LUMO thus characterizes the molecular chemi-
cal stability index [36].
The energy gap between HOMO and LUMO is a critical parame-
ter in determining the molecular electrical transport property be-
cause it is a measure of electron conductivity. The HOMO of
compound 2 presents a charge density localized on nitro group,
whereas LUMO is characterized by charge distribution on all the
carbon atoms of aromatic ring including the nitro group as well
as on carbonyl group of ester. HOMO-6 however show charge den-
sity distribution around carbon atoms of aromatic ring as well as
carbon–carbon double bond of ring B.
1727.7 cmꢁ1. The CAO stretching vibration of ester group was ob-
served at 1278.9 cmꢁ1 which correspond to calculated value at
1285 cmꢁ1. The CAO stretching vibrations of five and six mem-
bered ring are observed in FT-IR spectrum at 1217.6 and
1174.9 cmꢁ1 which closely correlate with the calculated values at
1231 and 1157 cmꢁ1. An asymmetric stretching and symmetric
vibrations of the nitro function of compound 2 was observed at
1528.6 cmꢁ1 and 1346 cmꢁ1 respectively, which is in well agree-
ment with theoretically computed value observed at 1534 cmꢁ1
and 1331 cmꢁ1 respectively.
3.4.2. NMR spectroscopy
The experimental and calculated values of 1H and 13C NMR
chemical shifts of the title compound 2 are given in Tables 5 and
6 respectively. The carbon NMR spectrum shows only 33 peaks
of different intensities, while 35 are present in the molecular for-
mula. This suggests the presence of symmetry, as a result of which
30, 70 and 40, 60 carbon atoms are equivalent. Thirty three carbon
peaks in the molecule are observed from 164.06 to 14.23 ppm in
the 13C NMR spectra whereas the calculated values are from
152.84 to 17.27 ppm. The agreement between calculated [GIAO
B3LYP/6-31G(d,p)] and experimental data is reasonable, justifying

A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
95
Table 7
Experimental and theoretical UV spectral parameters of compound 2.
No.
Electronic transitions
E (eV)
Oscillatory strength (f)
Calculated (kmax
)
Observed (kmax
)
Assignment
1.
2.
H ? L
H-6 ? L
3.6857
4.3572
0.0082
0.0002
336.39
284.55
352
312
p
?
pꢃ
n ? pꢃ
Fig. 7. HOMO–LUMO molecular plot of compound 2.
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.molstruc.2012.05.057.
4. Conclusion
A new synthetic compound diosgenin p-nitro benzoate (2) has
been synthesized in quantitative yield by Steglich esterification
in which the spiro ring remains intact. The title compound, which
was characterized and studied by X-ray single crystallography, FT-
IR, 1H, 13C NMR, 2D NMR and UV–Vis spectroscopic analysis,
showed anti-adipogenic activity.
References
[1] M. Aradhana, A.C. Rao, R.K. Kale, Int. J. Exp. Biol. 30 (1992) 367–370.
[2] R.E. Marker, J. Krueger, J. Am. Chem. Soc. 62 (1940) 3349–3350.
[3] C. Djerassi, Steroids 57 (1992) 631–641.
[4] P. Trouillas, C. Corbiere, B. Liagre, J.L. Duroux, J.L. Banaytout, Bioog. Med. Chem.
13 (2005) 1141–1149.
[5] B. Liagre, P. Vergne-Salle, C. Corbiere, J.L. Charissoux, J.L. Beneytout, Arthritis.
Res. Ther. 6 (2004) R373–R383.
[6] G. Alvarez, A. Pabon, J. Carmona, S. Blair, Pytother. Res. 18 (2004) 845–848.
[7] M.J. Liu, Z. Wang, Y. Zu, R. Wong, Q.Y. Wu, Cancer Chemother. Pharmacol. 55
(2005) 79–90.
[8] J. Raju, J.M. Patlolla, M.V. Swamy, C.V. Rao, Cancer Epidemiol. Biomark. Prev. 13
(2004) 1392–1398.
[9] C. Corbiere, B. Liagre, A. Bianchi, K. Bordji, M. Dauca, Int. J. Oncol. 22 (2003)
899–905.
Acknowledgement
Financial assistance in the form of fellowship from Jawaharlal
Nehru Fellowship program is gratefully acknowledged by Ms. Akri-
ti Bhatia.
[10] M.J. Liu, Z. Wang, Y. Ju, R.N. Wong, Q.Y. Wu, Cancer Chemother. Pharmacol. 55
(2005) 79–90.
[11] D.Y. Leger, B. Liagre, P.J. Cardot, J.L. Beneytout, S. Battu, Anal. Biochem. 335
(2004) 267–278.
Appendix A. Supplementary material
[12] S. Srinivasan, S. Koduru, R. Kumar, G. Venguswamy, N. Kyprianou, Int. J. Cancer
125 (2009) 961–967.
[13] F. Li, P.P. Fernandez, P. Rajendran, K.M. Hui, G. Sethi, Cancer Lett. 292 (2010)
197–207.
[14] J. Laguna, A. Gomez-Puyou, A. Pena, J. Guzman-Garcia, J. Atheroscler. Res. 2
(1962) 459–470.
[15] M.A. Mcanuff, W. W Harding, F.O. Omuruyi, H. Jacobs, E. Y Morrison, Food
Chem. Toxicol. 43 (2005) 1667–1672.
[16] M.A. Juarez-Oropeza, J.C. Diaz-Zagoya, J.L. Rabinowitz, Int. J. Biochem. 19
(1987) 679–683.
[17] C.S. Kwon, H.Y. Sohn, S.H. Kim, J.H. Kim, K.H. Son, Biosci. Biotechnol. Biochem.
67 (2003) 1451–1456.
[18] A. Sethi, G. Bhatia, A.K. Khanna, M.M. Khan, A. Bishnoi, A.K. Pandey, A. Maurya,
Med. Chem. Res. 20 (2011) 36–46.
[19] B. Neises, W. Steglich, Angew. Chem. Int. Ed. 17 (1978) 522–524.
The supplementary information contains images of 1H (Supple-
mentary Fig. 5), 13C NMR (Supplementary Fig. 6), 2D spectra (Sup-
plementary Fig. 7) and UV spectra (Supplementary Fig. 3) of the
compound 2. Also figures showing comparison of experimental
and optimized structure, IR correlation graph, and molecular elec-
trostatic potential diagram has been included as supplementary
data. The values for calculated Cartesian atomic coordinates is also
given. All other information about the crystal of the title compound
2 can be obtained in the form of supplementary crystallographic
data deposited with the Cambridge Crystallographic Centre under
the deposition number 854729. Supplementary data associated

96
A. Sethi et al. / Journal of Molecular Structure 1028 (2012) 88–96
[20] SAINT+ 6.02 ed.; Bruker AXS; Madison, WI, 1999.
[21] L.J. Farrugia, J. Appl. Crystallogr. 32 (1999) 837–838.
[22] G.M. Sheldric, Acta Crystallogr. A64 (2008) 112–122.
[23] A.L. Spek, J. Appl. Crystallogr. 36 (2003) 7–13.
Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L.
Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M.
Challacombe, P.M.W. Gill, B. Johnson, W. Chen, Wong, M.W. Wong, C.
Gonzalez, J.A. Pople, Gaussian 03, Revision C.02, Gaussian Inc., Wallingford,
CT, 2004.
[24] M.N. Burnett, C.K. Johnson, ORTEP-III: Oak Ridge Thermal Ellipsoid Plot 318
Program for Crystal Structure Illustrations, Report ORNL-6895, Oak Ridge 319
National Laboratory, Oak Ridge, TN, USA, 1996.
[25] J. Folch, M. Lees, G.H.S. Stanley, J. Biol. Chem. 226 (1957) 497.
[26] K.N. Frayn, P.F. Maycock, J. Lipid Res. 21 (1980) 139–144.
[27] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox,
H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E.
Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y.
Ayala, K. Voth, G.A. Morokuma, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S.
Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K.
Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J.
[28] A.D. Becke, J. Chem. Phys. 98 (1993) 5648–5652.
[29] N. Sundaraganesan, E. Kavitha, S. Sebastian, J.P. Cornard, M. Martel,
Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 74 (2009) 788–797.
[30] G.A. Zhurko, D.A. Zhurko, Chemcraft: Lite Version Build 08 (Freeware), 2005.
[31] Computer program Gauss View 3.09, Ver. 2, Gaussian, Inc., PA. Pittsburgh.
[32] S.J. Grabowski, A.T. Dubis, J. Phys. Chem. A 108 (2004) 5815–5822.
[33] P.E. Oscar, C.E. Edurado, Z.T.E. Gloria, T.B. Silvia, B.J. Enrique, Z. Naturforsch.
57c (2002) 947–950.
[34] H.N. De Armas, N.M. Blaton, O.M. Peeters, C.J. De Ranter, R.P. Hernandez, M.A.I.
Arteagas, R.P. Gil, F.C. Manchado, Acta Crystallogr. C55 (1999) 601.
[35] G. Varsanyi, Assignments for Vibrational Spectra of Seven Hundred Benzene
Derivatives, vol. 1–2, Adam Hilger, 1974.
[36] S.W. Xia, X. Xu, Y.L. Sun, Y.L. Fan, Y.H. Fan, C.F. Bi, D.M. Zhang, L.R. Yang, China J.
Struct. Chem. 25 (2006) 849–853.