Synthesis of diosgenin analogues as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.jsbmb.2014.04.006

We herein report the synthesis of diosgenin analogues from commercially available diosgenin as the starting material. The structures of newly synthesised compounds were confirmed by ¹H NMR, ¹³C NMR and mass spectrometry. All analogues were evaluated for in-vitro anti-inflammatory profile against LPS-induced inflammation in primary peritoneal macrophages isolated from mice by quantification of pro-inflammatory (TNF-α, IL-6 and IL-1β) cytokines in cell culture supernatant using the ELISA technique followed by in-vitro cytotoxicity study. Among the synthesised analogues, analogue 15 [(E) 26-(3′,4′,5′- trimethoxybenzylidene)-furost-5en-3β-acetate)] showed significant anti-inflammatory activity by inhibiting LPS-induced pro-inflammatory cytokines in a dose-dependent manner without any cytotoxicity. Efficacy and safety of analogue 15 were further validated in an in-vivo system using LPS-induced sepsis model and acute oral toxicity in mice. Oral administration of analogue 15 inhibited the pro-inflammatory cytokines in serum, attenuated the liver and lung injury and reduced the mortality rate in sepsis mice. Acute oral toxicity study showed that analogue 15 is non-toxic at higher dose in BALB/c mice. Molecular docking study revealed the strong binding affinity of diosgenin analogues to the active site of the pro-inflammatory proteins. These findings suggested that analogue 15 may be a useful therapeutic candidate for the treatment of inflammatory diseases.

Full text of DOI:10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
1
2
Synthesis of diosgenin analogues as potential anti-inflammatory
agents
Monika Singha,1, A.A. Hamidb,d,1, Anil K. Mauryaa, Om Prakashc, Feroz Khanc,
Q1
3
4
Anant Kumara, O.O. Aiyelaagbee, Arvind S. Negib,∗∗, Dnyaneshwar U. Bawankulea,∗
5
6
7
8
9
a Molecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India
b Medicinal Chemistry Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India
c Molecular and Structural Biology Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India
d Department of Chemistry, University of Ilorin, Ilorin, Nigeria
e Organic Chemistry Unit, Department of Chemistry, University of Ibadan, Ibadan, Nigeria
10
1217
12
a r t i c l e i n f o
a b s t r a c t
13
Article history:
Q3
We herein report the synthesis of diosgenin analogues from commercially available diosgenin as
the starting material. The structures of newly synthesised compounds were confirmed by 1H NMR,
13C NMR and mass spectrometry. All analogues were evaluated for in-vitro cytotoxicity followed
by anti-inflammatory profile against LPS-induced inflammation in primary peritoneal macrophages
isolated from mice by quantification of pro-inflammatory (TNF-␣, IL-6 and IL-1␤) cytokines in cell
culture supernatant using the ELISA technique. Among the synthesised analogues, analogue 15 [(E) 26-
(3ꢀ,4ꢀ,5ꢀ-trimethoxybenzylidene)-furost-5en-3␤-acetate)] showed significant anti-inflammatory activity
by inhibiting LPS-induced pro-inflammatory cytokines in a dose-dependent manner without any cyto-
toxicity. Efficacy and safety of analogue 15 were further validated in an in-vivo system using LPS-induced
sepsis model and acute oral toxicity in mice. Oral administration of analogue 15 inhibited the pro-
inflammatory cytokines in serum, attenuated the liver and lung injury and reduced the mortality rate in
sepsis mice. Acute oral toxicity study showed that analogue 15 is non-toxic at higher dose in BALB/c mice.
Molecular docking study revealed the strong binding affinity of diosgenin analogues to the active site of
the pro-inflammatory proteins. These findings suggested that analogue 15 may be a useful therapeutic
candidate for the treatment of inflammatory diseases.
14
15
16
17
Received 29 December 2013
Received in revised form 2 April 2014
Accepted 5 April 2014
Available online xxx
18
19
20
21
22
23
24
25
26
Keywords:
Diosgenin
Inflammation
Macrophage
Lipopolysaccharide
Sepsis
Docking
Mice
© 2014 Elsevier Ltd. All rights reserved.
28
1. Introduction
inflammation. Inflammation is linked to a wide range of progressive
diseases, including sepsis, auto-immune disorders, neurological
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
29
30
31
32
33
34
35
36
37
38
Inflammation is a complex and an important host defence mech-
anism in response to different stimuli, such as pathogens, physical
injury and chemical injury. At a damaged site, inflammation is
initiated by migration of immune cells from blood vessels and
release of mediators, followed by recruitment of inflammatory cells
and release of reactive oxygen species, reactive nitrogen species
and pro-inflammatory cytokines to eliminate foreign pathogens,
resolving infection and repairing injured tissues [1]. In general, nor-
mal inflammation is rapid and self-limiting, but aberrant resolution
and prolonged release of inflammatory mediators lead to chronic
diseases, metabolic disorder and cardiovascular disease [2,3] which
impose severe social and financial burdens including poor quality
of life, high health-care costs and substantial loss of productiv-
ity. Sepsis, a life-threatening disease with a high mortality rate,
is accompanied by systemic inflammation with excessive pro-
duction of pro-inflammatory cytokines including tumour necrosis
factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤) [4]. Lipopolysaccha-
ride (LPS), an endotoxin and the outer membrane component of
Gram-negative bacteria, is a major pathogenic factor in sepsis [5].
LPS has been established for inflammatory research because LPS
induces systemic inflammation mimicking the initial clinical fea-
tures of sepsis [6].
Steroids are very important class of anti-inflammatory agents
(SAIA). They suppress immune response through inhibition of NF-
␬B and by suppression of pro-inflammatory cytokines. They also
inhibit production of prostaglandins and leukotrienes. Some of
the notable steroidal anti-inflammatory drugs are dexamethasone,
∗
Corresponding author. Tel.: +91 5184455452.
Corresponding author.
Q2
∗∗
E-mail addresses: as.negi@cimap.res.in (A.S. Negi), du.bawankule@cimap.res.in,
bawankuledu@gmail.com (D.U. Bawankule).
1
Equal contribution.
http://dx.doi.org/10.1016/j.jsbmb.2014.04.006
0960-0760/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
2
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
O
OH
OH
OH
O
O
O
O
O
S
O
O
O
O
O
OH
O
O
HO
HO
O
O
HO
HO
O
H
HO
O
O
O
HO
O
H
F
Cl
F
F
Cl
O
Cl
O
O
5
O
4
2
3
7
O
6
Fig. 1. Structures of standard anti-inflammatory steroid drugs; dexamethasone (2), beclometasone dipropionate (3), fluticasone (4), budesonide (5), mometasone (6) and
ciclesonide (7).
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
beclometasone dipropionate, fluticasone, budesonide, mometa-
sone, ciclesonide, etc. (Fig. 1). These are used for the management
of various human disease conditions linked with inflammation [7].
The majority of natural molecules and their analogues
showed potent anti-inflammatory activities and some established
themselves as clinical agents for chronic inflammatory disease
conditions. Therefore, plant-based natural compounds play a sig-
nificant role in the development of anti-inflammatory drugs in the
pharmaceutical industry which can serve as a good source of lead
molecules suitable for further modification during the drug devel-
opment process.
Diosgenin is a C27 spiroketal steroidal sapogenin abundantly
available in nature. It is mainly present in the form of saponin
in the plants including Trigonella, Dioscorea [8], Costus [9] and
Smilax species [10]. In traditional medicine, it is used as an anti-
hypercholesterolemia, antihypertriacylglycerolemia, antidiabetes
and antihyperglycemia agent [11]. Several pharmacological reports
reveal that diosgenin improves vascular function by increasing
aortic eNOS expression in chronic renal failure model in rat [12],
inhibits proliferation and induces apoptosis in a wide variety of
cancer cell lines [13,14]. The antiproliferative and apoptotic prop-
erties are due to its ability to arrest the cell cycle, activate p53,
release apoptosis-inducing factor, modulate caspase-3 activity [15]
and due to inhibition of the ERK, JNK and phosphoinositide 3-
kinase signalling pathways and nuclear factor kappa B activity
(NF-␬B) [16]. The NF-␬B family of transcription factors has been
increasingly recognised as a crucial player in many steps of cancer
and inflammation [17]. Recently, it has been reported that dios-
genin exhibits anti-inflammatory activity due to down-regulation
of ICAM-1 expression through NF␬B pathway [18]. In the present
study, we have synthesised several analogues of diosgenin by
modifying at spiroketal ring (Fig. 2) to examine its influences on
inflammatory response; we validated the hypothesis using in-vitro
and in-vivo bioassay techniques and it was further confirmed using
in-silico study.
per standard methods. Reagents were used as such without any fur-
ther purification. Reactions were monitored in Merck aluminium
sheet silica gel thin layer plates (TLC, 60F254), visualised in UV-
cabinet (ꢀmax = 254 and 365 nm) and further charred with 2% ceric
sulphate in 10% aqueous sulphuric acid with subsequent heating
at 80–100 ◦C. Melting points were determined in open capillar-
ies in E-Z Melt melting point apparatus, Stanford Research System,
USA. Solvents were evaporated under reduced pressure at 50 ◦C in
Buchi Rotavapor. Compounds were purified through column chro-
matography over silica gel (Avra Chemicals, India, 100–200 mesh).
NMR experiments were performed on Bruker Avance DRX 300 MHz
spectrometer using tetramethylsilane (TMS, ␦ scale, 0.00 ppm) as
internal standard. Splitting of peaks are abbreviated as s for singlet,
d for doublet, t for triplet, q for quartet, bs for broad singlet and m
for multiplet. 1H and 13C spectra are reported. Electrospray mass
analysis was done on API 3000 Triple Quad LC–MS–MS (Applied
Biosystem, USA) mass spectrometer after dissolving samples in
methanol or acetonitrile. Nomenclature of diosgenin analogues has
been done as per recommendations published by the Joint Commis-
sion on the Biochemical Nomenclature (JCBN) of IUPAC [19].
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
2.2. Chemical synthesis
117
2.2.1. Synthesis of analogues 8 and 17
118
119
Acetylation was done as per reported method [20]. Substrate
was taken in dry chloroform and pyridine at room temperature
and acetic anhydride was added to it. Usual work-up was done after
pouring into water.
120
121
Q4 122
123
Analogue
8
(3ˇ-Hydroxy-(25R)-Spirost-5-en-3ˇ-acetate):
Yield = 91%, mp = 193–196 ◦C; 1H NMR (CDCl3), ı 0.77 (s, 3H,
18-CH3), 0.96 (d, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 1.11–2.31 (m,
25H, rest of the 1×CH3, 8×CH2 and 6×CH of steroidal ring), 2.01
(s, 3H, CH3COO, acetate), 2.24–2.31 (bd, 2H, 7-CH2), 3.38 (m, 2H,
26-CH2), 4.37 (bs, 1H, 3-CH), 4.42 (bd, 1H, 16-CH), 5.36 (s, 1H,
6-CH). 13C NMR (CDCl3, 75 MHz); ı 14.89, 16.64, 17.51, 19.69,
21.20, 21.74, 28.12, 29.19, 30.66, 31.78, 31.80, 32.21, 32.41, 37.10,
37.34, 38.47, 40.10, 40.63, 42.00, 42.68, 50.35, 56.82, 62.52, 67.19,
74.26, 81.16, 109.60, 122.72, 140.05, 170.82; ESI mass (MeOH):
457.3 [M + H]+, 479.3 [M + Na]+, 495.4 [M + K]+.
124
125
126
127
128
93
94
2. Materials and methods
129
130
2.1. General procedures
131
132
95
96
The starting substrate diosgenin (purity ∼93%) was procured
Q5 133
134
from Sigma Chemicals, USA. All the dry solvents were prepared as
Analogue 17 (Furost-5-en-3ˇ, 26N-diacetoxy-26-aldoxime):
Yield = 92%, mp = 90–93 ◦C, 1H NMR (CDCl3): ı 0.79 (s, 3H, 18-
CH3), 0.99 (s, 3H, 19-CH3), 1.02–1.97 (m, 29H, rest of the 2×CH3,
8×CH2 and 7×CH of steroidal ring), 2.02 (s, 6H, 2×CH3COO, 3 and
26-oxime acetates), 2.30 (bd, 2H, 7-CH2, J = 6.9 Hz), 3.29 (bs, 1H,
22-CH), 4.29 (bs, 1H, 3-CH), 4.60 (bd, 1H, 16-CH), 5.36 (s, 1H, 6-CH),
7.50-7.53 (d, 1H, 26-CH, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz): ı
16.81, 19.20, 19.72, 19.91, 21.03, 21.81, 26.33, 28.14, 30.08, 31.52,
31.96, 32.38, 32.59, 37.11, 37.39, 38.06, 38.28, 38.49, 39.77, 41.09,
50.40, 57.29, 65.32, 74.29, 83.83, 90.14, 122.74, 140.12, 163.46,
169.16, 170.95; ESI mass (MeOH): 551 [M + K−1]+.
135
O
136
137
R
O
138
O
139
140
HO
141
AcO
1
142
Planned analogues
143
Fig. 2. Structures of diosgenin (1) and planned analogues.
144
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
3
481.3 [M + K]+; ESI-HRMS: 443.31616 (calculated) and 443.3139
(observed) for C28H43O4.
207
208
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
2.2.2. Synthesis of (25R) furost-5-en-, 3ˇ-acetoxy, 26-ol (9)
Diosgenin 3-acetate (200 mg, 0.44 mmol) was stirred in a 5 mL
acetic acid. To this solution, sodium cyanoborohydride (200 mg,
3.17 mmol) was added in portions over a period of 30 min. After 2 h,
when the reaction was complete, the reaction mixture was poured
in ice-cool water, extracted with ethyl acetate (3 × 30 mL), washed
with water and dried over anhydrous sodium sulphate. The organic
layer was dried in-vacuo to get a crude mass, which was purified
through column chromatography over silica gel using hexane-ethyl
acetate as eluants. The desired alcohol 9 was obtained at 10–12%
ethyl acetate–hexane as white crystalline solid.
2.2.5. Synthesis of Wittig products 12–15
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
2.2.5.1. Synthesis of (22ˇ)-(E)-26-benzylidene-3ˇ-yl-furost-5-en-3-
acetate (12). Sodium hydride (200 mg, 8.3 mmol) was washed with
dry hexane and taken in 10 mL dry toluene. To this Wittig salt
(150 mg, 0.34 mmol) was added and refluxed for 20 min. To this
aldehyde 10 (100 mg, 0.22 mmol) was added and further refluxed
for 4 h. Toluene was evaporated under vacuum and residue was
taken in ethyl acetate, washed with water and dried over anhy-
drous sodium sulphate. The organic layer was dried in-vacuo to get
a crude mass, which was purified through silica gel column eluting
with ethyl acetate–hexane. The desired product was obtained as
yellowish viscous liquid.
Analogue 9: Yield = 81%, mp = 121–122 ◦C; 1H NMR (CDCl3): ı
0.83 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.03–1.90 (m, 28H, rest of
the 2×CH3, 8×CH2 and 6×CH of steroidal ring), 2.05 (s, 3H, CH3COO,
acetate), 2.35 (bd, 2H, 7-CH2), 3.36 (bs, 1H, 22-CH), 3.48 (m, 2H,
27-CH2OH), 4.34 (bs, 1H, 3-CH), 4.63 (bs, 1H, 16-CH), 5.40 (s, 1H, 6-
CH). 13C NMR (CDCl3, 75 MHz): ␦ 16.80, 17.00, 19.30, 19.69, 21.03,
21.77, 28.13, 30.46, 30.82, 31.94, 32.36, 32.59, 36.08, 37.07, 37.37,
38.28, 38.46, 39.78, 41.07, 50.39, 57.28, 65.48, 68.27, 74.28, 83.57,
90.71, 122.74, 140.04, 170.91; ESI mass (MeOH): 459.4 [M + H]+,
481.3 [M + Na]+, 497.4 [M + K]+.
Analogue 12: Yield = 68%, oil; 1H NMR (CDCl3): ı 0.73 (s, 3H, 18-
CH3), 0.95 (s, 3H, 19-CH3), 1.02–1.95 (m, 26H, rest of the 2×CH3,
7×CH2 and 6×CH of steroidal ring), 1.99 (s, 3H, CH3COO, acetate),
2.23 (d, 2H, 4-CH2 J = 5.4 Hz), 2.30 (bd, 2H, 7-CH2), 3.23 (bd, 1H,
22-CH), 4.22 (bs, 1H, 16-CH), 4.51 (bs, 1H, 3-CH), 5.28 (s, 1H, 6-
CH), 5.96 (dd, 1H, 26-CH, J = 15.6 Hz and 7.8 Hz), 6.26 (d, 1H, 28-CH,
J = 15.6 Hz), 7.12 (m, 5H, aromatic protons of phenyl ring). 13C NMR
(CDCl3, 75 MHz): ı 16.83, 19.39, 19.72, 21.06, 21.79, 28.16, 30.06,
31.85, 31.99, 32.40, 32.66, 34.48, 37.12, 37.41, 38.04, 38.31, 38.51,
39.82, 41.10, 50.44, 57.31, 65.60, 74.33, 83.58, 90.82, 122.77, 126.39,
127.15, 128.73, 128.83, 137.05, 138.33, 140.09, 170.97; ESI mass
(MeOH): 531.5 [M + H]+, 553.5 [M + K]+, 569.6 [M + K]+.
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
2.2.3. Synthesis of furost-5-en-3ˇ-acetoxy 26-al (10)
Alcohol 9 (200 mg, 0.43 mmol) was taken in 10 ml methylene
chloride. To this stirred solution, pyridinium chlorochromate (PCC,
200 mg, 0.93 mmol) was added and further stirred for an hour. On
completion, solvent was evaporated and residue was dissolved in
ethyl acetate. It was acidified with dil. HCl and washed with water.
The organic layer was dried over anhydrous sodium sulphate and
dried in-vacuo. The residue thus obtained was recrystallised with
chloroform–hexane (1:3) to get aldehyde 10 as brown coloured
solid.
Analogue 13 (z) 26-(4ꢀ-nitrobenzylidene)-furost-5en-3ˇ-acetate:
Yield = 62%, oil; 1H NMR (CDCl3): ı 0.71 (s, 3H, 18-CH3), 0.96 (s, 3H,
19-CH3), 1.05–1.88 (m, 26H, rest of the 2×CH3, 7×CH2 and 6×CH
of steroidal ring), 2.02 (s, 3H, CH3COO, acetate), 2.21 (bs, 2H, 4-
CH2), 2.30 (bd, 2H, 7-CH2), 3.20 (bd, 1H, 22-CH), 4.20 (bs, 1H, 16-
CH), 4.50 (bs, 1H, 3-CH), 5.26 (bs, 1H, 6-CH), 6.29 (m, 1H, 26-CH),
6.79 (d, 1H, 28-CH, J = 9.0 Hz), 7.33–8.05 (m, 4H, aromatic protons
of phenyl ring). 13C NMR (CDCl3, 75 MHz): ı 16.80, 19.29, 19.69,
20.62, 21.01, 21.81, 28.11, 29.74, 31.38, 31.95, 32.35, 32.99, 34.01,
37.09, 37.36, 38.22, 38.45, 39.74, 41.10, 50.37, 57.27, 65.40, 74.50,
83.69, 90.54, 122.77, 123.92, 124.33, 126.80, 130.53, 134.03, 140.04,
142.28, 143.26,162.83, 171.85; ESI mass (MeOH): 576.6 [M + H]+,
574.6 [M–H]+, 598.6 [M + Na]+, 614.5 [M + K]+.
Analogue 10: Yield = 91%, mp = 119–123 ◦C; 1H NMR (CDCl3): ı
0.80 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.16–1.97 (m, 28H, rest of
the 2×CH3, 8×CH2 and 6×CH of steroidal ring), 2.16 (s, 3H, CH3COO,
Acetate), 2.46 (bd, 2H, 7-CH2), 3.45 (bs, 1H, 22-CH), 4.44 (bs, 1H, 3-
CH), 4.73 (bd, 1H, 16-CH), 5.50 (s, 1H, 6-CH), 9.75 (s, 1H, 26-CHO).
13C NMR (CDCl3, 75 MHz): ı 13.78, 16.79, 19.22, 19.71, 21.03, 21.77,
28.15, 30.07, 31.11, 31.96, 32.37, 32.59, 37.09, 37.39, 38.26, 38.48,
39.77, 41.08, 46.72, 50.41, 57.29, 65.44, 74.28, 83.28, 90.11, 122.73,
140.09, 170.91, 205.54; ESI mass (MeOH): 457.3 [M + H]+, 479.3
[M + Na]+, 495.4 [M + K]+; ESI-HRMS: 457.33181 (calculated), and
457.3311 (observed) for C29H45O4.
Analogue 14 (z) 26-(3ꢀ,4ꢀ,5ꢀ-trimethoxybenzylidene)-furost-5en-
3ˇ-acetate: Yield = 29%, oil; 1H NMR (CDCl3): ı 0.79 (s, 3H, 18-CH3),
0.95 (s, 3H, 19-CH3), 1.04–1.87 (m, 29H, rest of the 2×CH3, 8×CH2
and 7×CH of steroidal ring), 2.02 (s, 3H, CH3COO, acetate), 2.29 (bd,
2H, 7-CH2), 3.85 (s, 9H, 3×OCH3), 4.28 (bd, 1H, 22-CH), 4.61 (bs,
1H, 3-CH), 5.39 (t, 1H, 6-CH), 6.28 (d, 1H, 27-CH, J = 11.4 Hz), 6.32
(bd, 1H, 26-CH), 6.50 (d, 2H, 2ꢀ and 6ꢀ-CH of phenyl ring). 13C NMR
(CDCl3, 75 MHz): ı 16.83, 19.41, 19.71, 21.03, 21.82, 28.14, 30.06,
31.97, 32.37, 32.61, 33.39, 35.13, 37.10, 37.39, 38.29, 38.48, 39.77,
41.06, 50.38, 56.42, 57.28, 61.30, 65.51, 74.30, 83.58, 90.71, 106.17,
122.77, 128.05, 133.94, 139.38, 140.09, 153.27, 170.97; ESI mass
(MeOH): 621.5 [M + H]+, 643.4 [M + K]+, 659.4 [M + K]+.
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
2.2.4. Synthesis of 27-nor- furost-5-en- 3ˇ-acetoxy-25-one (11)
Aldehyde10 (200 mg, 0.44 mmol)wastakenin 10 mL ethanol. To
this stirred solution, 3,4,5-trimethoxyaniline (200 mg, 1.09 mmol)
was added and further stirred for 2 h at room temperature. The
solvent was evaporated and the residue was dissolved in ethyl
acetate and washed with water. The organic phase was dried
over anhydrous sodium sulphate and dried in-vacuo to get a
residue. It was purified through silica gel column eluting with ethyl
acetate:hexane. The desired ketone 11 was obtained at 8–10% ethyl
acetate–hexane as creamish white solid.
Analogue 15 (E) 26-(3ꢀ,4ꢀ,5ꢀ-trimethoxybenzylidene)-furost-5en-
3ˇ-acetate: Yield = 52%, oil; 1H NMR (CDCl3): ı 0.77 (s, 3H, 18-CH3),
0.94 (s, 3H, 19-CH3), 1.04–1.84 (m, 30H, rest of the 2×CH3, 9×CH2
and 6×CH of steroidal ring), 1.99 (s, 3H, CH3COO, acetate), 2.29 (bd,
2H, 7-CH2), 3.84 (s, 9H, 3×OCH3), 4.10 (bd, 1H, 22-CH, J = 7.1 Hz),
4.27 (bs, 1H, 16-CH), 4.54 (bs, 1H, 3-CH), 5.33 (s, 1H, 6-CH), 5.98 (dd,
1H, 26-CH, J = 15.6 Hz and 7.8 Hz), 6.22 (d, 1H, 27-CH, J = 15.9 Hz),
6.54 (s, 2H, 2ꢀ and 6ꢀ-CH of phenyl ring). 13C NMR (CDCl3, 75 MHz):
ı 16.83, 19.41, 19.70, 21.01, 21.77, 28.12, 30.07, 31.81, 31.96, 32.36,
32.65, 34.45, 37.08, 37.37, 37.96, 38.27, 38.47, 39.77, 41.06, 50.37,
56.41, 57.26, 61.26, 65.55, 74.24, 83.55, 90.72, 103.39, 122.74,
128.64, 134.05, 136.50, 137.61, 140.06, 153.63, 170.85; ESI mass
(MeOH): 621.5 [M + H]+, 643.5 [M + Na]+, 659.4 [M + K]+.
Analogue 11: Yield = 84%, mp = 138–140 ◦C; 1H NMR (CDCl3):
ı 0.79 (s, 3H, 18-CH3), 0.98 (s, 3H, 19-CH3), 1.02–1.87 (m, 23H,
rest of the 1×CH3, 8×CH2 and 4×CH of steroidal ring), 1.95 (s,
3H, CH3COO, acetate), 2.13 (s, 3H, 26-CH3CO), 2.32 (d, 1H, 7-CH2,
J = 6.3 Hz) 2.51–2.63 (bd, 2H, 24-CH2), 3.26–3.29 (bs, 1H, 22-CH),
4.24–4.29 (bs, 1H, 16-CH), 4.57 (bd, 1H, 3-CH), 5.35 (s, 1H, 6-CH).
13C NMR (CDCl3, 75 MHz): ı 16.80, 19.01, 19.71, 21.02, 21.80, 27.44,
28.13, 30.33, 31.95, 32.37, 32.55, 37.10, 37.38, 38.24, 38.48, 39.75,
41.09, 41.28, 50.38, 57.27, 65.39, 74.28, 83.69, 89.53, 122.72, 140.12,
170.95, 209.24; ESI mass (MeOH): 443.3 [M + H]+, 465.4 [M + Na]+,
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
4
271
272
273
274
275
276
277
278
279
280
281
282
2.2.6. General synthesis of aldoxime 16
2.3.3. Cytotoxicity evaluation
333
334
335
336
337
338
339
340
341
342
343
Analogue 16 Furost-5-en-3ˇ-acetoxy 26-aldoxime: Yield = 70%,
mp = 130–133 ◦C; 1H NMR (CDCl3): ı 0.78 (s, 3H, 18-CH3), 0.97 (s,
3H, 19-CH3), 1.07–1.86 (m, 26H, rest of the 2×CH3, 8×CH2 and
4×CH of steroidal ring), 2.01 (s, 3H, CH3COO, acetate), 2.31 (bd,
2H, 7-CH2), 3.29 (bs, 1H, 22-CH), 4.29 (bs, 1H, 3-CH), 4.59 (bd, 1H,
16-CH), 5.36 (s, 1H, 6-CH), 7.29 (bd, 1H, 26-CH, J = 6.3 Hz). 13C NMR
(CDCl3, 75 MHz): ı 16.81, 18.44, 19.26, 19.70, 21.03, 21.78, 28.12,
30.06, 31.32, 31.95, 32.36, 32.56, 35.06, 37.08, 37.38, 38.24, 38.47,
39.78, 41.07, 50.39, 57.28, 65.46, 74.32, 83.62, 90.39, 122.75, 140.06,
156.46, 170.01; ESI mass (MeOH): 472.4 [M + H]+, 494.5 [M + Na]+,
510.3 [M + K]+.
Effect of diosgenin analogues on cytotoxicity was carried out
in peritoneal macrophage cells using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium (MTT) assay. Peritoneal macrophage
cells (0.5 × 106 cells/well) isolated from mice were suspended
in DMEM medium (Sigma, USA) containing 10% heat-inactivated
foetal bovine serum (Gibco, USA) and incubated in a 96-well culture
plate at 37 ◦C in 5% CO2 in an incubator and left overnight to attach.
Cells were treated (1, 10 and 30 ␮g/mL) and incubated for 24 h at
37 ◦C in 5% CO2. After treating the incubated cells, 20 ␮L aliquots of
MTT solution (5 mg/mL in PBS) were added to each well and left for
4 h . Then, the MTT-containing medium was carefully removed and Q6 344
the formazan crystals formed were solubilised in DMSO (100 ␮L) for
10 min. The culture plate was placed on a micro-plate reader (Spec-
tramax; Molecular Devices, USA) and the absorbance was measured
at 550 nm. The amount of colour produced is directly proportional
to the number of viable cells. Cell cytotoxicity was calculated as the
percentage of MTT absorption as percentage (%) of survival = (mean
experimental absorbance/mean control absorbance × 100).
345
346
347
348
349
350
351
283
2.3. Pharmacology
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
2.3.1. Cell culture
Primary cell culture was carried out as described previously [21].
In brief, the macrophage cells were collected from the peritoneal
cavities of mice (8-week-old female Swiss albino mice) after an
intra peritoneal injection of 1.0 mL of 1% peptone (BD Biosciences,
USA) 3 days before harvesting. Mice were euthanised by cervical
dislocation under ether anaesthesia and peritoneal macrophages
were obtained by intra-peritoneal injection of phosphate buffer
saline (PBS; pH 7.4). Membrane debris was removed by filter-
ing the cell suspensions through sterile gauze. The viability of
the cells was determined by trypan blue exclusion and the viable
macrophage cells at the concentration of 0.5 × 106 live cells/mL
were used for the experimentation. Cells were grown in tissue cul-
ture plates in DMEM (Dulbecco modified Eagle medium, Sigma)
supplemented with 10% foetal bovine serum with 1× stabilised
antibiotic–antimycotic solution (Sigma) in a CO2 incubator at 37 ◦C
with 5% CO2 and 90% relative humidity.
2.3.4. In-vivo study
352
353
354
355
356
357
Animal experiments were carried out as per the approved proto-
col by the Institutional Animal Ethics Committee (IAEC) followed by
the Committee for the Purpose of Control and Supervision of Exper-
imental Animals (CPCSEA), Government of India (Registration no.:
400/01/AB/CPCSEA).
2.3.4.1. LPS-induced sepsis model in mice. BALB/c mice, 6–8 weeks
of age, and weighing 18–22 g, were procured from the institute’s
animal house and acclimatised to the animal room for a week prior
to experiment. The mice were fed with the standard mice feed and
ad libitum drinking water under standard environmental conditions
of 22 3 ◦C, 12:12 dark-to-light cycle. After 1 week of adaptation,
the mice were randomly divided into six groups of six mice each.
Sepsis was induced in the experimental mice except normal group
of mice by intra-peritoneal (i.p.) injection of LPS (5 mg/kg) in normal
saline. Analogue 15 was dissolved in 0.7% carboxymethyl cellulose
(CMC) to obtain a uniform suspension and administered orally 24 h
and 2 h before LPS injection.
358
359
360
361
362
363
364
365
366
367
368
369
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
2.3.2. Quantification of pro-inflammatory cytokines
Cells were pretreated with 1 and 10 ␮g/mL of diosgenin ana-
logues and standard anti-inflammatory drug dexamethasone
(Sigma, USA), respectively, for 30 min. The cells were stimulated
with lipopolysaccharide (LPS, E. coli 055:B5 Sigma, USA; 1 ␮g/mL).
After incubation with LPS for 24 h, supernatants were collected and
immediately frozen at −80 ◦C. Harvested supernatants were tested
for quantification of pro-inflammatory cytokines using mouse-
specific enzyme immuno assay (EIA) kits (BD Biosciences, USA)
following the manufacturer’s protocol. Briefly, the ELISA plates
were coated (100 ␮L per well) with specific mouse TNF-␣, IL-1␤
and IL-6 capture antibody and incubated overnight at 4 ◦C. The
plate was blocked with 200 ␮L/well assay diluents. Culture super-
natant and standard (100 ␮L) were added into the appropriate
coated wells and incubated for 2 h at room temperature (20–25 ◦C).
After incubation, the plates were washed thoroughly 5 times with
wash buffer. 100 ␮L of detecting solution (detection antibody
and streptavidin HRP) was added into each well. The plates were
sealed and incubated for 1 h at RT and then washed thoroughly
5 times with wash buffer. 100 ␮L of tetramethylbenzidine (TMB)
substrate solution was added to each well and the plate was
incubated (without plate sealer) for 30 min at room temperature
in the dark. Finally, 50 ␮L of stop solution (2 N H2SO4) was added
to each well. The colour density was measured at 450 and 570 nm
using a microplate reader (Spectramax; Molecular Devices, USA).
Subtracted absorbance was measured at 570 nm from absorbance
450 nm. The values of TNF-␣, IL-1␤ and IL-6 were expressed
as pg/mL. The percentage (%) inhibition of pro-inflammatory
cytokine production was calculated as follows:%inhibition =
100 × (concentration of vehicle control−concentration of test treatment) where
vehicle control inOdDicoaftceosncceentlrlastitorneoaftveedhicwleictohntvroelhicle in LPS-induced
inflammation.
Group 1: Normal control administered CMC
Group 2: Vehicle control administered CMC + LPS
Group 3: Analogue 15 (3 mg/kg) + LPS
Group 4: Analogue 15 (10 mg/kg) + LPS
Group 5: Analogue 15 (30 mg/kg) + LPS
370
371
372
373
374
Blood was collected 2 h after LPS administration from orbital
plexus and serum was separated and stored at −80 ◦C until anal-
ysis. Pro-inflammatory cytokines (IL-1␤, IL-6 and TNF-␣) from
serum were quantified using commercially available mouse-
specific enzyme immune assay kits (BD Biosciences, USA) by
following the manufacturer’s instruction.
375
376
377
378
379
380
2.3.4.2. Histopathology examination. Liver and lung samples were
obtained 24 h after LPS challenge. Tissues were collected and
fixed in 10% buffered formalin. After fixation, tissues were rinsed
with water, dehydrated with graded concentration of ethanol and
embedded in paraffin wax. The samples were sectioned into 5 ␮m
thick and mounted on glass slides. The sections were then de-
waxed using xylene and ethanol, and stained with haematoxylin
and eosin (H&E stain). A representative area was selected for qual-
itative light microscopic analysis under 100× magnification.
381
382
383
384
385
386
387
388
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
5
A.
O
O
O
O
CH2OH
i
O
CHO
AcO
O
iii
ii
O
HO
O
iv
1
AcO
8
AcO
9
AcO
11
10
B.
H3C
R1
H3C
H3C
H3C
C
C
CHO
CH=NOR
R2
O
H
O
O
vi
v
AcO
AcO
AcO
12 (trans): R1=H, R2=Phenyl;
10
16: R=H;
17: R=Ac
vii
13 (cis): R1=4-nitrophenyl, R2=H;
14 (cis): R1=3,4,5-trimethoxyphenyl, R2=H;
15 (trans):R1=H, R2=3,4,5-trimethoxyphenyl.
Scheme 1. (i) Ac2O, dry pyridine, CHCl3, RT, 2 h, 91%; (ii) AcOH, NaCNBH4, RT, 3 h, 81%; (iii) dry DCM, PCC, reflux, 1 h, 91%; (iv) ethanol, 3,4,5-trimethoxyaniline, RT, 2 h, 84%;
(v) Wittig salt, NaH, toluene, reflux, 3–4 h, 29–68%; (vi) NH2OH·HCl, EtOH, reflux, 2 h, 70%; (vii) Ac2O, dry pyridine, dry CHCl3, RT, 92%.
389
390
391
392
393
394
395
396
397
2.3.4.3. Survival study. The second set of experiments was per-
formed to examine the effect of analogue 15 on LPS-induced
lethality in mice. The female BALB/c mice were divided into five
groups (n = 6). The treatment group received analogue 15 orally at
doses of 3, 10 and 30 mg/kg, 24 and 2 h before injection of LPS,
the LPS group received an intraperitoneal injection at a dose of
15 mg/kg, and the control group received the equal amount of vehi-
cle instead of analogue 15. Survival of mice was monitored every
12 h for 5 days.
2.5. Statistical analysis
426
Results were presented as the means SE and analysed using
GraphPad Prism 4. The ANOVA followed by Tukey’s multiple
comparison test was used to assess the statistical significance
of vehicle vs treatment groups. Results are presented as the
means SE. Differences with a p value < 0.05 were considered
significant.
427
428
429
430
431
432
3. Results
433
434
398
399
400
401
402
403
404
405
406
2.3.5. Acute oral toxicity study
Acute oral toxicity study of the analogue 15 was done on BALB/c
male mice as described previously [22]. For this study, mice admin-
istered orally with analogue 15 (300 mg/kg body weight) were
considered as the test group and mice treated with corresponding
volume of vehicle (0.7% carboxymethyl cellulose [CMC]) were con-
sidered the control group. Mice were observed individually, after
dosing, at least once during the first 30 min, periodically during the
first 24 h and daily thereafter for a total of 7 days.
3.1. Chemistry
The synthetic strategy was depicted as Scheme 1. Diosgenin (1)
was acetylated with acetic anhydride–pyridine system in dry chlo-
roform to get diosgenin 3-acetate (8) at room temperature. The
spiroketal bond was reduced with sodium cyanoborohydride in
acetic acid to get a primary alcohol (9). The alcohol 9 was oxidised
to its corresponding aldehyde (10) by treating it with pyridinium
chlorochromate (PCC) in dichloromethane.
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
407
2.4. Molecular docking
Further, while preparing Schiff’s bases of aldehyde 10 with
an aromatic amine, ketone 11 was obtained unexpectedly.
Compound 10 was reacted with an aromatic amine (aniline/3,4-
methylenedioxyaniline/3,4,5-trimethoxyaniline) in ethanol and
each time it was transformed to ketone 11. We did not do any
mechanistic study to establish this transformation. However, it is
assumed that after the formation of a Schiff’s base, it was converted
to an enamine. Further decomposition of enamine by water might
have converted it to ketone 11. The structure of 11 was established
by 1D, 2D NMR, ESI-HRMS and finally by X-ray crystallography [25].
Diverse analogues were prepared on aldehyde 10.
Three different Wittig salts (benzyltriphenylphosphonium
bromide, 4-nitrobenzyltriphenylphosphonium bromide, 3,4,5-
trimethoxybenzyltriphenylphosphonium bromide) were treated
with sodium hydride–toluene system to get styrene type ana-
logues (12–15) at C27 of steroidal framework of aldehyde 10.
Aldoxime (16) and oxime-acetate (17) were also prepared on 10
by treating with hydroxylamine hydrochloride and then acety-
lating. All theses diosgenin analogues were confirmed through
spectroscopy.
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
Two-dimensional molecular structures were drawn with the
ChemDraw Ultra and energy minimization was performed with
MM2/MM3 molecular mechanics parameter until achieving the
lowest stable energy state. This energy minimization pro-
cess was performed until the energy change was less than
0.001 kcal mol−1 or the molecules had been updated almost
300 times [23]. The 3D chemical structure of known drug
was collected from the PubChem compound database of NCBI,
USA (http://www.pubchem.ncbi.nlm.nih.gov). Crystallographic 3D
structures of target proteins TNF-␣ (PDB: 2AZ5), IL-6 (PDB: 1ALU)
and IL-1␤ (PDB: 9ILB) were retrieved from the Brookhaven Pro-
tein Databank (PDB) (http://www.pdb.org). Hydrogen atoms were
added to the protein targets to achieve the correct ionisation and
tautomeric states of amino acid residues such as His, Asp, Ser and
Glu. Molecular docking of the compounds against selected tar-
get was achieved using the ‘AutoDock Vina’ software. To perform
the automated docking of ligands into the active sites, we used a
Lamarckian genetic algorithm [24].
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
6
Table 1
Q11
Effect of diosgenin analogues on production of pro-inflammatory cytokines in LPS-induced inflammation in macrophage cells..
Pro-inflammatory cytokine (pg/mL)
Analogues
Dose (␮g/mL)
TNF-␣
IL-6
IL-1␤
Basal
–
–
68.17 9.67
93.52 5.14
22.65 4.09
Vehicle
Diosgenin
1
1301.07 93.68#
912.63 21.71#
103.08 8.72#
1
1138.74 77.05ns
1052.72 93.68*
1192.86 79.42ns
794.84 13.09*
1077.04 66.6ns
801.94 53.08*
1037.36 43.22ns
810.96 24.37*
1227.5 58.02ns
1155.38 45.14ns
1049.54 73.35ns
548.76 58.51*
1013.42 46.32*
623.78 63.25*
971.20 75.12*
458.94 55.30*
950.220 33.90*
307.56 65.03*
1215.66 54.18ns
1178.92 61.79ns
1067.20 63.58ns
493.38 45.40*
439.48 57.49*
177.04 35.18*
792.58 49.25ns
693.69 16.10*
777.13 15.17ns
718.24 19.70*
841.72 61.28ns
774.53 61.50ns
822.39 30.99ns
781.33 34.57ns
740.96 57.40ns
727.53 52.43*
599.58 13.33*
569.58 32.86*
551.97 27.30*
540.14 33.92*
557.82 16.70*
465.60 12.43*
517.61 25.54*
387.61 16.01*
758.15 52.4ns
687.64 34.12*
667.26 57.24*
591.70 16.89*
313.85 50.57*
163.74 10.18*
96.50 3.18ns
81.33 7.23ns
98.00 6.18ns
87.33 4.98ns
92.46 7.47ns
82.46 6.06ns
98.67 6.94ns
93.45 4.09ns
88.67 10.27ns
83.61 6.13ns
98.60 4.70ns
82.67 8.42ns
98.65 4.45ns
95.33 7.54ns
97.00 1.77ns
84.67 2.34ns
93.50 5.18ns
69.00 8.15*
96.0 6.24ns
94.44 5.26ns
92.50 7.23ns
70.67 8.02*
88.50 2.73ns
57.66 8.85*
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
8
9
10
11
12
13
14
15
16
17
Dexamethasone
n = 3.
#
Normal vs vehicle.
p < 0.05, vehicle vs treatment.
*
Fig. 3. Effect of analogue 15 and dexamathasone on percent (%) inhibition of pro-inflammatory cytokine production in LPS-induced inflammation in macrophage cells. (A)
TNF-␣; (B) IL-6; (C) IL-1␤.
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
Table 2
7
462
3.2. Pharmacology
Effect of diosgenin analogues on percent (%) cell viability in macrophage cells using
MTT assay.
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
3.2.1. Effect of diosgenin analogues on LPS-induced
pro-inflammatory cytokines production in macrophages
Analogues
Dose (␮g/mL)
% Cell viability
All the synthesised diosgenin analogues were evaluated for
their anti-inflammatory status against the production of pro-
inflammatory cytokines (TNF-␣, IL-6 and IL-1␤) using ELISA
technique in LPS-induced inflammation in macrophage cells at the
concentrations of 1 and 10 ␮g/mL.
Production of pro-inflammatory cytokines was significantly
(p < 0.05) increased in LPS-stimulated cells when compared with
normal un-stimulated cells. All the analogues including parent
diosgenin exhibited inhibition of pro-inflammatory cytokines pro-
duction when compared with vehicle treated LPS-stimulated cells
(Table 1).
Diosgenin
1
1
104.31 5.97
101.18 1.97
97.73 1.78
104.25 9.05
99.62 1.77
97.80 3.08
99.42 2.66
96.87 3.71
93.34 1.39
105.40 2.41
101.81 4.85
95.17 5.81
102.22 2.66
99.63 4.48
94.47 6.09
101.62 3.56
97.92 2.64
96.62 5.34
100.25 2.87
98.20 0.90
97.13 1.18
101.33 3.55
99.65 1.96
98.25 2.78
103.28 6.28
100.78 2.67
96.73 2.11
104.21 4.19
100.94 3.67
94.51 4.40
102.83 3.30
97.26 2.69
97.57 4.17
10
30
1
10
30
1
10
30
1
10
30
1
10
30
1
10
30
1
10
30
1
10
30
1
8
9
10
Among the all analogues, analogue 15 [(E) 26-(3ꢀ,4ꢀ,5ꢀ-
trimethoxybenzylidene)-furost-5en-3␤-acetate] possessed signif-
icant inhibition of TNF-␣, IL-1␤ and IL-6 at both the concentration
(Table 1). The percent inhibition of pro-inflammatory cytokines
by analogue 15 in comparison to dexamethasone, a standard
anti-inflammatory steroid drug is depicted in Fig. 3. These pro-
inflammatory cytokines are the mediators of various acute and
chronic inflammation linked diseases. Based on this study, analogue
15 was chosen for its further validation for evaluation of efficacy
and safety in in-vivo system using LPS-induced sepsis as a systemic
inflammation model and acute oral toxicity respectively in mice.
11
12
13
14
15
10
30
1
10
30
1
16
487
488
489
490
491
492
493
3.2.2. In-vitro cytotoxicity profile of diosgenin analogues
We first examined the in-vitro cytotoxicity profile of all syn-
thesised diosgenin analogues using MTT assay in peritoneal
macrophage cells isolated from mice. The significant change in
percent live cell population was not observed (p < 0.05) at any con-
centration of diosgenin analogues treatment when compared with
normal cells. Results are summarised in Table 2.
17
10
30
Dexamethasone
1
10
30
99.87 3.92
96.11 5.55
93.78 8.71
n = 3.
494
495
496
497
498
499
500
501
502
503
504
505
506
3.2.3. Effect of analogue 15 on pro-inflammatory cytokine
production in sepsis model of mice
dose-dependent manner at 5 days and these animals are still sur-
vived (Fig. 6).
521
522
To substantiate the physiological function of analogue 15 in in-
vivo system, we examine the therapeutic anti-inflammatory effect
of analogue 15 in a mouse model of sepsis, a systemic inflammatory
condition. Serum level of pro-inflammatory cytokines (TNF-␣, IL-6
and IL-1␤) was significantly increased in LPS-challenged vehicle-
treated mice when compared to normal mice. Oral administration
of analogue 15 before LPS challenge significantly (p < 0.05) inhibited
TNF-␣ and IL-6 production in a dose-dependent manner at dose
rates of 3, 10 and 30 mg/kg body weight. The higher pretreatment
dose (10 and 30 mg/kg) could also inhibit the IL-1␤ production in
serum (Fig. 4).
3.3. Acute oral toxicity study
523
The acute toxicity study showed that a single oral administra-
tion of analogue 15 (300 mg/kg) did not produce any mortality,
behavioural changes (gait, posture, fur, depression and panting) in
the mice as compared to the control group. Similarly, no signifi-
cant changes were recorded in body weight, organ weight, serum
biochemical (total bilirubin, creatinine, triglycerides, SGOT and glu-
cose) as well as haematology parameters (total RBCs and total
WBCs) of the treated group when compared to the control group.
The representative results are depicted in Table 3 and Fig. 7.
524
525
526
527
528
529
530
531
532
507
508
509
510
511
512
513
514
3.2.4. Histopathological changes in lung and liver tissues
To elucidate the effect of analogue 15 on lung and liver injuries
in sepsis model of mice, histopathological examination of liver
and lung tissue section was performed. Significant damage was
observed in liver and lung tissues as evidenced by inflammatory
cell infiltration in LPS-challenged vehicle-treated mice when com-
pared with normal mice. Pretreatment of analogue 15 ameliorated
the tissue damage in a dose-dependent manner (Fig. 5).
3.3.1. Molecular interaction study of diosgenin analogues
through docking
533
534
535
536
537
538
539
540
541
542
543
544
545
In-vitro and in-vivo anti-inflammatory profiles of diosgenin
analogues were further confirmed by molecular docking exper-
iments. The aim of the molecular interaction study was to
explore the molecular interaction of diosgenin analogues with
pro-inflammatory cytokine receptors. The interaction study
was compared with dexamethasone, a standard steroidal anti-
inflammatory drug (Table 4).
The studied molecules show molecular interaction with TNF-
␣ (PDB: 2AZ5), IL-6 (PDB: 1ALU) and IL-1␤ (PDB: 9ILB). The
representative molecular interaction results of analogue 15 and
dexamethasone are depicted in Fig. 8.
515
516
517
518
519
520
3.2.5. Effect of analogue 15 on LPS-induced mortality in mice
The survival study was conducted to assess the protective effect
of analogue 15 on LPS-induced mortality in mice. We monitored
its effect on survival of mice for 5 days at 12 h. All the mice in
LPS-challenged vehicle-treated group died within 48 h. Pretreat-
ment of analogue 15 resulted in a markedly improved survival in
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
8
Fig. 4. Effect of analogue 15 on pro-inflammatory cytokine production in sepsis model of mice. (A) TNF-␣; (B) IL-6; (C) IL-1␤. Data are expressed as mean SEM: *p < 0.05;
vehicle vs treatment; # vehicle vs normal; (Tukey’s multiple comparison test); n = 6.
546
547
548
549
550
551
552
553
554
555
556
TNF-␣ interaction study found that residues Tyr-119, Leu-
120, Gly-121, Leu-57, Tyr-59, Ser-60, Gly-122 and Tyr-151 were
commonly shared by dexamethasone and diosgenin analogue 15.
Similarly analysis of binding pocket residues of IL-6 interacted with
the diosgenin analogue 15, and dexamethasone shared Asn-63,
Leu-64, Pro-65, Glu-93, Val-96, Tyr-97, Pro-139, Thr-143, Asn-
144, Leu-147 and Lys-150 residues. IL-1␤ interaction with the
diosgenin analogue 15 and dexamethasone revealed the com-
mon residues as Leu-57, Ile-58, Tyr-59, Ser-60, Gln-61, Asn-63,
Leu-64, Pro-65, Gly-122, Tyr-151, Ile-155, Tyr-119, Leu-120 and
Gly-121.
4. Discussion
557
There has been increasing interest in the discovery and devel-
opment of novel pharmaceuticals from natural origin that have
the same or better efficacy accompanied by less side effects. The
majority of natural molecules and their analogues showed potent
anti-inflammatory activities and some established themselves as
clinical agents for chronic inflammatory disease conditions. There-
fore, plant-based natural compounds play a significant role in
the development of anti-inflammatory drugs in the pharmaceu-
tical industry which can serve as good lead molecules suitable
558
559
560
561
562
563
564
565
566
Fig. 5. Representative microphotograph of lung and liver (H&E stain, 100×) sections from the mice. Arrows indicate leukocyte infiltration.
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
9
production of pro-inflammatory cytokines (TNF-␣, IL-6 and
IL-1␤). Synthesised analogues along with parent diosgenin exhib-
ited inhibition of pro-inflammatory cytokines productions in
LPS-stimulated cells without any cytotoxic effect as assessed by
MTT assay. Several previous studies reported that diosgenin, a
parent molecule, reduces the production of inflammatory medi-
ators by inhibiting CK2, JNK, NF-␬B and AP-1 activation [18,27].
This study, to our knowledge, provides the first evidence that
synthesised analogues of diosgenin modifies at spiroketal ring
exhibiting anti-inflammatory activity by inhibiting the production
of pro-inflammatory cytokines in LPS-induced inflammation in
macrophage cells. Pro-inflammatory cytokines, namely, TNF-
␣, IL-1␤ and IL-6, in LPS-induced macrophages, are known to
have profound effects on the regulation of immune reactions,
haematopoiesis and inflammation [28]. Overproduction of these
cytokines has been implicated in the pathogenesis of many disease
processes. The control of macrophage overproduction of these
mediators should greatly facilitate the treatment of many inflam-
mation-linked diseases such as sepsis, rheumatoid arthritis and
autoimmune diabetes [16,17].
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
Fig. 6. Effect of analogue 15 on LPS-induced mortality in mice.
Among all analogues, analogue 15 [(E) 26-(3ꢀ,4ꢀ,5ꢀ-
567
568
569
570
571
572
573
574
575
576
577
578
for further modification during the drug development process.
Diosgenin (DG) is a C27 spiroketal steroidal sapogenin abun-
dantly available in nature. It is mainly present in the form of
saponin in the plants [26]. Previous investigations have shown
that diosgenin plays an important pharmacological role including
anti-inflammatory activity [18]. In present study, we have reported
the influence of synthesised analogues of diosgenin by modify-
ing at spiroketal ring on LPS-induced inflammatory response; an
in-vitro, in-vivo study and it was further confirmed using in-silico
study.
trimethoxybenzylidene)-furost-5en-3␤-acetate]
possessed
significant (p < 0.05) inhibition of pro-inflammatory cytokines
(TNF-␣, IL-6 and IL-1␤) at both the concentrations. It would also
be interesting to evaluate its therapeutic effect in in-vivo systemic
inflammation model. To substantiate the physiological function of
most potent analogue, we have further evaluated the therapeutic
efficacy and safety profile of analogue 15 in in-vivo system using
LPS-induced sepsis and acute oral toxicity respectively in mice.
Sepsis is marked by a systemic inflammatory response. Persistent
or inappropriate or overproduction of multiple pro-inflammatory
mediators such as TNF-␣ and IL-6 leads to severe injury and
We have evaluated the in-vitro anti-inflammatory sta-
tus of the synthesised diosgenin analogues against the
Table 3
Effect of analogue 15 on acute toxicity at 300 mg/kg as a single oral dose in BALB/c mice.
0th day
Control
7th day
Parameters studied
Experimental
Control
Experimental
Body weight (g)
SGOT (U/L)
SGPT (U/L)
Cholesterol (mg/dL)
Triglycerides (mg/dL)
Haemoglobin (g/dL)
RBC (million/mm3)
WBC (thousands/mm3)
21.85 0.36
32.30 4.34
23.46 4.01
101.03 1.33
147.09 7.28
14.81 2.11
7.31 1.17
21.80 0.40
31.56 3.21
22.45 3.02
105 2.87
128.47 5.71
14.96 1.34
7.80 1.33
4.35 0.75
22.53 0.49
31.50 5.34
23.83 7.53
103.0 1.69
106.93 7.14
13.42 1.51
7.58 0.38
22.6 0.361
31.06 8.42
21.96 3.28
100.28 1.58
108.69 4.67
14.44 0.86
7.21 0.41
4.09 0.12
5.51 0.19
4.32 0.34
Data are expressed as mean S.E.M., n = 6.
Fig. 7. Effect of analogue 15 as a single acute oral dose at 300 mg/kg on (A) absolute and (B) relative organ weight in BALB/c albino mice (n = 6, non significant changes were
found compared to control).
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
10
Table 4
Binding affinity (kcal mol−1) and interacting residual information of diosgenin derivatives with respect to the anti-inflammatory targets TNF-␣ (PDB: 2AZ5), IL-6 (PDB: 1ALU)
and IL-1␤ (PDB: 9ILB).
Protein
Ligands
Binding affinity (kcal mol−1
)
Pocket residues
Analogue 15
−9
Gln(C)-61, Gly(C)-121, Gly(C)-122, Ile(C)-58, Ile(C)-155, Leu(C)-57, Leu(C)-120,
Ser(C)-60, Tyr(C)-59, Tyr(C)-119, Tyr(C)-151, Gly(D)-121, Leu(D)-57,
Leu(D)-120, Ser(D)-60, Tyr(D)-59, Tyr(D)-119, Tyr(D)-151
Gln(B)-125, Leu(B)-55, Leu(B)-157, Gly(C)-121, Gly(C)-122, Leu(C)-57,
Leu(C)-120, Tyr(C)-59, Tyr(C)-119, Gly(D)-121, Leu(D)-120, Ser(D)-60,
Tyr(D)-59, Tyr(D)-119, Tyr(D)-151
TNF-␣ (2AZ5)
Dexamethasone
−7.9
Analogue 15
−6
Asn-63, Leu-64, Pro-65, Lys-66, Lys-86, Glu-93, Val-96, Tyr-97, Pro-139,
Asp-140, Thr-143, Asn-144, Leu-147, Lys-150
IL-6 (1ALU)
Dexamethasone
−6.4
Asn-61, Asn-63, Asn-144, Glu-93, Leu-64, Leu-147, Lys-150, Pro-65, Pro-139,
Thr-143, Tyr-97, Val-96
Analogue 15
−5.7
−7.4
Arg-4, Asn-108, Glu-51, Glu-105, Ile-56, Leu-6, Leu-110, Lys-103, Met-44,
Met-148, Phe-46, Phe-150, Ser-5
Asn-7, Asn-66, Asp-86, Glu-64, Leu-62, Lys-63, Lys-65, Pro-87, Pro-91, Ser-43,
Ser-153, Tyr-68, Tyr-90, Val-85
IL-1␤ (9ILB)
Dexamethasone
Fig. 8. Analogue 15 and dexamethasone docked on target protein; TNF-␣ (PDB: 2AZ5), IL-6 (PDB: 1ALU) and IL-1␤ (PDB: 9ILB).
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
increased mortality in sepsis [29,30]. Thus, regulation of multiple
mediators could be more beneficial than suppression of single
mediator. In fact, the clinical trials targeting single inflamma-
tory cytokine have been proved ineffective in the treatment of
sepsis [31,32]. In LPS-induced sepsis model study in mice, we
demonstrated that the oral administration of analogue 15 before
LPS challenge significantly (p < 0.05) inhibited TNF-␣ and IL-6
production in dose-dependent manner at the dose rate of 3, 10
and 30 mg/kg body weight and higher dose (30 mg/kg) is required
to inhibit (p < 0.05) the IL-1␤ production in serum. Pretreatment of
analogue 15 also ameliorated the lung and liver injuries in sepsis
model of mice as an evidence of histopathological examination
as well as it helps to improve the survival of mice in lethal sepsis
model. Overproduction of TNF-␣ and IL-1␤ leads to tissue damage
[33,34], multiple organ failure and finally causes lethal sepsis [21].
Therefore, agents attenuating the production of pro-inflammatory
cytokines may have potential as treatments for prevention of
lethal sepsis [22].
5. Conclusion
635
Collectively, we demonstrated that diosgenin analogues inhibit
the production of pro-inflammatory cytokines in both in-vitro and
in-vivo condition, it was further confirmed with docking study.
This finding confirms the suitability of diosgenin analogues as
candidates for further investigation towards the management of
inflammation related diseases.
636
637
638
639
640
641
Conflict of interest
642
643
There are no conflicts of interest.
Acknowledgements
644
We acknowledge the Council of Scientific and Industrial Q7 645
Research (CSIR), New Delhi, India for financial support through XII Q8 646
Molecular interaction study of diosgenin analogue 15 with pro-
inflammatory targets (TNF-␣, IL-6 and IL-1␤) through docking
showed high binding affinity i.e. low docking energy. Sev-
eral previous reports also concluded that the molecules having
high binding affinity with targeted protein exhibited therapeutic
efficacy [35,36].
FYP networking projects BSC0121 and BSC0203. We are thankful to
Director, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow, India for rendering essential research facilities and sup-
port. CSIR-Senior Research Fellowship to Ms. Monika Singh and
TWAS-CSIR fellowship to Mr. A.A. Hamid are duly acknowledged.
647
648
649
650
651
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006

G Model
SBMB41871–11
ARTICLE IN PRESS
M. Singh et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
11
652
References
[20] B.S. Furniss, A.J. Hannaford, P.W.G. Smith, A.R. Tatchell, Vogel’s textbook of prac-
tical organic chemistry, fifth edition, Addison Wesley Longman Limited Essex
CM20 2JE, England, UK, 1989.
[21] D.U. Bawankule, S.K. Chattopadhyay, A. Pal, K. Saxena, S. Yadav, U. Faridi, M.P.
Darokar, A.K. Gupta, S.P. Khanuja, Modulation of inflammatory mediators by
coumarinolignoids from Cleome viscosa in female swiss albino mice, Inflam-
mopharmacology 6 (2008) 272–277.
[22] D. Chanda, K. Shanker, A. Pal, S. Luqman, D.U. Bawankule, D. Mani, M.P. Darokar,
Safety evaluation of Trikatu, a generic Ayurvedic medicine in Charles Foster
rats, J. Toxicol. Sci. 34 (2009) 99–108.
[23] S. Parihar, A. Kumar, A.K. Chaturvedi, N.K. Sachan, S. Luqman, B. Changkija, M.
Manohar, O. Prakash, D. Chanda, F. Khan, C.S. Chanotiya, K. Shanker, A. Dwivedi,
R. Konwar, A.S. Negi, Synthesis of combretastatin A4 analogues on steroidal
framework and their anti-breast cancer activity, J. Steroid Biochem. Mol. Biol.
137 (2013) 332–344.
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
[1] R. Medzhitov, Origin and physiological roles of inflammation, Nature 454
(2008) 428–435.
[2] P.C. Calder, R. Albers, J.M. Antoine, S. Blum, R. Bourdet-Sicard, G.A. Ferns, G. Folk-
erts, P.S. Friedmann, G.S. Frost, F. Guarner, M. Lovik, S. Macfarlane, P.D. Meyer,
L. M’Rabet, M. Serafini, E.W. Van, L.J. Van, D.W. Vas, S. Vidry, B.M. Winklhofer-
Roob, J. Zhao, Inflammatory disease processes and interactions with nutrition,
Br. J. Nutr. 101 (2009) S1–S45.
[3] M. Guha, N. Mackman, LPS induction of gene expression in human monocytes,
Cell. Signal. 13 (2001) 85–94.
[4] H.K. de Jong, T. van der Poll, W.J. Wiersinga, The systemic proinflammatory
response in sepsis, J. Innate Immun. 2 (2010) 422–430.
[5] B. Beutler, E.T. Rietschel, Innate immune sensing and its roots: the story of
endotoxin, Nat. Rev. Immunol. 3 (2003) 169–176.
[6] K. Doi, A. Leelahavanichkul, P.S. Yuen, R.A. Star, Animal models of sepsis and
sepsis-induced kidney injury, J. Clin. Invest. 119 (2009) 2868–2878.
[7] P.J. Barnes, How corticosteroids control inflammation: Quintiles Prize Lecture
2005, Br. J. Pharmacol. 148 (2006) 245–254.
[24] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of dock-
ing with a new scoring function, efficient optimization and multithreading, J.
Comput. Chem. 31 (2010) 455–461.
[25] A.A. Hamid, M. Hasnain, A. Singh, Omprakash, P.G. Vasudev, J. Sarkar, D. Chanda,
F. Khan, O.O. Aiyelaagbe, A.S. Negi, Synthesis of novel anticancer agents through Q9 726
[8] J. Raju, R. Mehta, Cancer chemopreventive and therapeutic effects of diosgenin,
a food saponin, Nutr. Cancer 61 (2009) 27–35.
opening of spiroketal ring of diosgenin, Steroids (2014), under revision.
[26] M. Gao, L. Chen, H. Yu, Q. Sun, J. Kou, B. Yu, Diosgenin down-regulates NF-␬B
p65/p50 and p38MAPK pathways and attenuates acute lung injury induced by
lipopolysaccharide in mice, Int. Immunopharmacol. 15 (2013) 240–245.
[27] D.H. Jung, H.J. Park, H.E. Byun, Y.M. Park, T.W. Kim, B.O. Kim, S.H. Um, S.
Pyo, Diosgenin inhibits macrophage-derived inflammatory mediators through
downregulation of CK2, JNK, NF-kappaB and AP-1 activation, Int. Immunophar-
macol. 10 (2010) 1047–1054.
Q10727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
[9] M.M. Gupta, S.U. Farooqui, R.N. Lal, Distribution and variation of diosgenin in
different parts of Costus speciosus, J. Nat. Prod. 44 (1981) 486–489.
[10] B. Shao, H. Guo, Y. Cui, M. Ye, J. Han, D. Guo, Steroidal saponins from Smilax china
and their anti-inflammatory activities, Phytochemistry 68 (2007) 623–630.
[11] M.A. McAnuff, W.W. Harding, F.O. Omoruyi, H. Jacobs, E.Y. Morrison, H.N. Ase-
mota, Hypoglycemic effects of steroidal sapogenins isolated from Jamaican
bitter yam, Dioscorea polygonoides, Food Chem. Toxicol. 43 (2005) 1667–
1672.
[12] J. Manivannan, E. Balamurugan, T. Silambarasan, B. Raja, Diosgenin improves
vascularfunctionby increasingaorticeNOSexpression, normalizedyslipidemia
and ACE activity in chronic renal failure rats, Mol. Cell. Biochem. 384 (2013)
113–120.
[13] P.S. Chen, Y.W. Shih, H.C. Huang, H. Cheng, W. Diosgenin, A steroidal saponin,
inhibits migration and invasion of human prostate cancer PC-3 cells by reducing
matrix metalloproteinase expression, PLoS ONE 6 (2011) e20164.
[14] F. Li, P.P. Fernandez, P. Rajendran, K.M. Hui, G. Sethi, Diosgenin a steroidal
saponin, inhibits STAT3 signaling pathway, leading to suppression of prolifera-
tion and chemosensitization of human hepatocellular carcinoma cells, Cancer
Lett. 292 (2010) 197–207.
[15] C. Corbiere, B. Liagre, F. Terro, J.L. Beneytout, Induction of antiproliferative effect
by diosgenin through activation of p53, release of apoptosis-inducing factor
(AIF) and modulation of caspase-3 activity in different human cancer cells, Cell
Res. 14 (2004) 188–196.
[16] S. Shishodia, B.B. Aggarwal, Diosgenin inhibits osteoclastogenesis, invasion,
and proliferation through the downregulation of Akt, I kappa B kinase acti-
vation and NF-kappa B-regulated gene expression, Oncogene 25 (2006) 1463–
1473.
[17] B. Hoesel, J.A. Schmid, The complexity of NF-␬B signaling in inflammation and
cancer, Mol. Cancer 12 (2013) 1–15.
[18] J.X. Song, L. Ma, J.P. Kou, B.Y. Yu, Diosgenin reduces leukocytes adhesion and
migration linked with inhibition of intercellular adhesion molecule-1 expres-
sion and NF-kb p65 activation in endothelial cells, Chin. J. Nat. Med. 10 (2012)
142–149.
[19] Nomenclature of Steroids, IUPAC and International Union of Biochemistry-Joint
commission on Biochemical Nomenclature, Pure and Applied Chemistry, vol.
61, No. 10, 1989, pp. 1783–1822.
[28] I. Christiaens, D.B. Zaragoza, L. Guilbert, S.A. Robertson, B.F. Mitchell, D.M. Olson,
Inflammatory processes in preterm and term parturition, J. Reprod. Immunol.
79 (2008) 50–57.
[29] M.G. Netea, J.W. van der Meer, M. van Deuren, B.J. Kullberg, Proinflammatory
cytokines and sepsis syndrome: not enough, or too much of a good thing?
Trends Immunol. 24 (2003) 254–258.
[30] C. Gerard, B.J. Rollins, Chemokines disease, Nat. Immunol. 2 (2001) 108–115.
[31] E. Abraham, K. Reinhart, S. Opal, I. Demeyer, C. Doig, A.L. Rodriguez, R. Beale,
P. Svoboda, P.F. Laterre, S. Simon, B. Light, H. Spapen, J. Stone, A. Seibert, C.
Peckelsen, C. De Deyne, R. Postier, V. Pettilä, A. Artigas, S.R. Percell, V. Shu,
C. Zwingelstein, J. Tobias, L. Poole, J.C. Stolzenbach, A.A. Creasey, Efficacy and
safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sep-
sis: a randomized controlled trial, J. Am. Med. Assoc. 290 (2003) 238–247.
[32] P. Qiu, X. Cui, A. Barochia, Y. Li, C. Natanson, P.Q. Eichacker, The evolving expe-
rience with therapeutic TNF inhibition in sepsis: considering the potential
influence of risk of death, Expert Opin. Invest. Drugs 20 (2011) 1555–1564.
[33] T. Yan, Q. Li, H. Zhou, Y. Zhao, S. Yu, G. Xu, Z. Yin, Z. Li, Z. Zhao, Gu-4 suppresses
affinity and avidity modulation of CD11b and improves the outcome of mice
with endotoxemia and sepsis, PLoS ONE 7 (2012) e30110.
[34] H. Li, Y. Wang, H. Zhang, B. Jia, D. Wang, H. Li, D. Lu, R. Qi, Y. Yan, Hu. Wang,
Yohimbine enhances protection of berberine against LPS-Induced mouse
lethality through multiple mechanisms, PLoS ONE 7 (2012) e52863.
[35] S. Sharma, S.K. Chattopadhyay, D.K. Yadav, F. Khan, S. Mohanty, A. Maurya, D.U.
Bawankule, QSAR, docking and in vitro studies for anti-inflammatory activity of
cleomiscosin A methyl ether derivatives, Eur. J. Pharm. Sci. 47 (2012) 952–964.
[36] D.K. Yadav, V. Mudgal, J. Agrawal, A.K. Maurya, D.U. Bawankule, C.S. Chanotiya,
F. Khan, S.T. Thul., Molecular docking and ADME studies of natural compounds
of Agarwood oil for topical anti-inflammatory activity, Curr. Comput. Aided
Drug Des. 9 (2013) 360–370.
Please cite this article in press as: M. Singh, et al., Synthesis of diosgenin analogues as potential anti-inflammatory agents, J. Steroid
Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.006