Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin

Article abstract of DOI:10.1016/j.steroids.2014.05.025

Diosgenin has been modified to furostane derivatives after opening the F-spiroacetal ring. The aldehyde group at C26 in derivative 8 was unexpectedly transformed to the ketone 9. The structure of ketone 9 was confirmed by spectroscopy and finally by X-ray crystallography. Five of the diosgenin derivatives showed significant anticancer activity against human cancer cell lines. The most potent molecule of this series i.e. compound 7, inhibited cellular growth by arresting the population at G₀/G₁ phase of cell division cycle. Cells undergo apoptosis after exposure to the derivative 7 which was evident by increase in sub G₀ population in cell cycle analysis. Docking experiments showed caspase-3 and caspase-9 as possible molecular targets for these compounds. This was further validated by cleavage of PARP, a caspase target in apoptotic pathway. Compound 7 was found non-toxic up to 1000 mg/kg dose in acute oral toxicity in Swiss albino mice.

Full text of DOI:10.1016/j.steroids.2014.05.025

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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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Synthesis of novel anticancer agents through opening of spiroacetal ring
of diosgenin
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A.A. Hamid a,c, Mohammad Hasnain b, Arjun Singh a, Balakishan Bhukya a, Omprakash a,
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Prema G. Vasudev a, Jayanta Sarkar b, Debabrata Chanda a, Feroz Khan a, O.O. Aiyelaagbe d,
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Arvind S. Negi a,
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a CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow 226015, India
b CSIR-Central Drug Research Institute (CSIR-CDRI), B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
c Department of Chemistry, University of Ilorin, Ilorin, Nigeria
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d Organic Chemistry Unit, Department of Chemistry, University of Ibadan, Ibadan, Nigeria
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Diosgenin has been modified to furostane derivatives after opening the F-spiroacetal ring. The aldehyde
group at C26 in derivative 8 was unexpectedly transformed to the ketone 9. The structure of ketone 9 was
confirmed by spectroscopy and finally by X-ray crystallography. Five of the diosgenin derivatives showed
significant anticancer activity against human cancer cell lines. The most potent molecule of this series i.e.
compound 7, inhibited cellular growth by arresting the population at G0/G1 phase of cell division cycle.
Cells undergo apoptosis after exposure to the derivative 7 which was evident by increase in sub G0 pop-
ulation in cell cycle analysis. Docking experiments showed caspase-3 and caspase-9 as possible molecular
targets for these compounds. This was further validated by cleavage of PARP, a caspase target in apoptotic
pathway. Compound 7 was found non-toxic up to 1000 mg/kg dose in acute oral toxicity in Swiss albino
mice.
Received 10 December 2013
Received in revised form 26 May 2014
Accepted 29 May 2014
Available online xxxx
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Keywords:
Diosgenin
X-ray crystallography
Anticancer
Cell cycle
Ó 2014 Published by Elsevier Inc.
Caspase
Acute oral toxicity
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1. Introduction
Development of cancer therapeutics from steroids has been an
attractive choice for medicinal chemists and many active
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Cancer is a major heath menace. Over the period, cancer has
become a challenge to public healthcare system. The morbidity
and mortality of cancer is so high that it is an economic concern
to the society nowadays. There are more than 100 types of cancers.
Worldwide lung, stomach, liver, colon and breast cancer cause the
most deaths each year. About 70% of all cancer deaths occur in low-
and middle-income countries. Tobacco use is the single largest pre-
ventable cause of cancer in the world causing 20% of cancer deaths.
Cancers of major public health relevance such as breast, cervical
and colorectal cancer can be cured if detected early and treated
adequately. One fifth of all cancers worldwide are caused by a
chronic infection, for example human papillomavirus (HPV) causes
cervical cancer and hepatitis B virus (HBV) causes liver cancer [1].
Despite continued efforts of researchers to combat cancer, this dis-
ease presents about 13% of total deaths.
molecules have emerged. Steroids have been developed either as
antiproliferative or cytotoxic agents. Withaferin A (1), Gymnasterol
(2), 24-hydroxyperoxide desmosterol (3), timosaponin A-III (4) etc.
are some of the notable plant based cytotoxic steroidal leads [2,3].
Semisynthetic modification of some these natural products have
yielded better cytotoxic analogs. Several synthetic analogs of with-
aferin are much better cytotoxic compounds [2].
Diosgenin (5) is a C27 spiroacetal steroidal sapogenin abun-
dantly available in nature. It is obtained mainly in saponin form
from Smilex spp., Dioscorea spp., Costus speciosus etc. The molecule
exhibits significant activity against colon and leukemia cells by
inducing apoptosis [4a–c]. In the present communication, we mod-
ified diosgenin at spiroketal position to get few anticancer analogs.
While transforming C29 aldehyde to Schiff’s bases a C28 ketone was
formed which was confirmed by spectroscopy. All the derivatives
were evaluated for cytotoxicity by Sulphorhodamine assay against
five human cancer cell lines. The best analog of the series was fur-
ther evaluated for cell cycle analysis and in-vivo acute oral toxicity
in Swiss-albino mice.
⇑
Corresponding author. Tel.: +91 522 2718583; fax: +91 522 2342666.
E-mail address: arvindcimap@rediffmail.com (A.S. Negi).
http://dx.doi.org/10.1016/j.steroids.2014.05.025
0039-128X/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Hamid AA et al. Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin. Steroids (2014),
http://dx.doi.org/10.1016/j.steroids.2014.05.025

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481.3294; IR (KBr, cmꢁ1): 3423, 2934, 1731, 1456, 1376, 1248,
1035.
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2. Experimental
2.1. General
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2.2.3. Synthesis of (22b,25R)-3b-hydroxy,26-formyl-furost-5-en-3b-
acetate (8)
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Melting points were determined in open capillaries using E-Z
Melt automated melting point apparatus, Stanford Research Sys-
tem, USA and were uncorrected. The starting substrate diosgenin
was procured for Sigma, USA. Dry solvents were prepared as per
standard methods. Reactions were monitored on aluminum thin
layer chromatography (TLC, UV254nm plates), E. Merck Germany.
Further, visualization was accomplished by spraying with a solu-
tion of 2% ceric sulfate in 10% aqueous sulfuric acid and charring
at 80–100 °C. Column chromatography was carried out on silica
gel (100–200 mesh, Avra Chemicals, India). NMR spectra were
obtained on Bruker Avance-300 MHz instrument with tetrameth-
ylsilane (TMS, chemical shifts in d ppm) as an internal standard.
ESI mass spectra were recorded on API 3000 LC-MS-MS, Applied
Biosystem, USA after dissolving the compounds in methanol or
acetonitrile. The best compound 7 was analyzed for high resolution
mass (ESI-HRMS) also in Agilent 6520 Q-TOF. FT-IR spectra were
recorded on Perkin-Elmer SpectrumBX. X-ray diffraction data were
collected on a Bruker AXS SMART APEX CCD diffractometer using
Alcohol 7 (200 mg, 0.43 mmol) was dissolved in dry dichloro-
methane (10 mL) and stirred at room temperature. To this pyridi-
nium chlorochromate (PCC) (200 mg, 0.93 mmol) was added and
further stirred for an hour. Solvent was evaporated and residue
was dissolved in ethyl acetate (30 mL). It was acidified with dil.
HCl (5%, 10 mL) and washed with water. The organic layer was
dried over anhydrous sodium sulfate and dried in vacuo. The crude
mass was recrystallised with chloroform-hexane (1:3) to get alde-
hyde 8 as brown crystalline solid.
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8: Yield = 182 mg (91%), mp = 119–123 °C; 1H NMR (CDCl3):
d0.80 (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): d 13.78 (C21), 16.79
(C18), 19.22 (C19), 19.71 (C27), 21.03 (C11), 21.77 (acetate CH3),
28.15 (C24), 30.07 (C2), 31.11 (C23), 31.96 (C20), 32.37 (C7),
32.59 (C15), 37.09 (C10), 37.39 (C1), 38.26 (CH), 38.48 (C12),
39.77 (C4), 41.08 (C13), 46.72 (C25), 50.41 (C9), 57.29 (C14),
65.44 (C17), 74.28 (C3), 83.28 (C16), 90.11 (C22), 122.73 (C6),
140.09 (C5), 170.91 (acetate ester), 205.54 (C26); ESI Mass
(MeOH): 457.3 [M+H]+, 479.3 [M+Na]+, 495.4 [M+K]+; IR (KBr,
cmꢁ1): 2833, 1739, 1254.
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MoK radiation (k = 0.71073 Å). Nomenclature of steroid deriva-
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tives has been given as per the recommendations published by
the Joint Commission on the Biochemical Nomenclature (JCBN) of
IUPAC [5].
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2.2. Chemical synthesis
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2.2.1. Synthesis of (22b,25R)-spirost-5-en-3b-yl-3-acetate (6)
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2.2.4. Synthesis of (22b)-3b-hydroxy,25-oxo-27-nor-furost-5-en-3b-
acetate (9)
Acetylation of diosgenin (5) was done as per reported method
[6a] with a little modification using dry chloroform as a co-solvent.
6: Yield = 1.01 g (91%), mp = 193–96 °C [195 °C, 6b]; 1H NMR
(CDCl3), d 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); d 14.89 (C21), 16.64 (C18), 17.51 (C11), 19.69 (C19),
21.20 (C11), 21.74 (acetate CH3), 28.12 (C24), 29.19 (C2), 30.66
(C25), 31.78 (C23), 31.80 (C8), 32.21 (C7), 32.41 (C15), 37.10
(C10), 37.34 (C1), 38.47 (C12), 40.10 (C4), 40.63 (C13), 42.00
(C20), 42.68, 50.35 (C9), 56.82 (C14), 62.52 (C17), 67.19 (C26),
74.26 (C3), 81.16 (C16), 109.60 (C22), 122.72 (C6), 140.05 (C5),
170.82 (acetate ester). ESI Mass (MeOH): 457.3 [M+H]+, 479.3
[M+Na]+, 495.4 [M+K]+. IR (KBr, cmꢁ1): 2907, 1724, 1451, 1231.
Aldehyde 8 (200 mg, 0.44 mmol) was taken in ethanol (10 mL)
and stirred at ambient temperature (30–35 °C). To this 3,4,5-trime-
thoxyaniline (200 mg, 1.09 mmol) was added and further stirred
for 2 h. The solvent was evaporated, residue was dissolved in ethyl
acetate (30 mL) and washed with water. The organic phase was
dried over anhydrous sodium sulfate and dried in vacuo. The crude
mass was purified through silica gel column eluting with ethyl ace-
tate:hexane. The ketone 9 was obtained at 8–10% ethyl acetate
hexane as creamish white solid.
9: Yield = 163 mg (84%), mp = 138–40 °C; 1H NMR (CDCl3): d
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): d 16.80 (C18), 19.01 (C19), 19.71
(C21), 21.02 (C11), 21.80 (acetate CH3), 27.44 (C23), 28.13 (C1),
30.33 (C26), 31.95 (C8), 32.37 (C7), 32.55 (C15), 37.10 (C10),
37.38 (C2), 38.24 (C20), 38.48 (C12), 39.75 (C4), 41.09 (C13),
41.28 (C24), 50.38 (C9), 57.27 (C14), 65.39 (C17), 74.28 (C3),
83.69 (C16), 89.53 (C22), 122.72 (C6), 140.12 (C5), 170.95 (Acetate
ester), 209.24 (C25); ESI Mass (MeOH): 443.3 [M+H]+, 465.4
[M+Na]+, 481.3 [M+K]+; IR (KBr, cmꢁ1): 2927, 1724, 1453, 1372,
1241.
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2.2.2. Synthesis of (22b,25R)-3b,26-dihydroxyfurost-5-en-3b-acetate
(7)
Compound 7 was synthesized as per reported method [6a].
7: Yield = 164 mg (81%), mp = 108–110 °C [6a]. 1H NMR (CDCl3):
d 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): d 16.80 (C18),
17.00 (C20), 19.30 (C27), 19.69 (C19), 21.03 (C11), 21.77 (acetate
CH3), 28.13 (C24), 30.46 (C2), 30.82 (C25), 31.94 (C8), 32.36 (C7),
32.59 (C15), 36.08 (C23), 37.07 (C10), 37.37 (C1), 38.28 (C4),
38.46 (C12), 39.78 (C13), 41.07 (C20), 50.39 (C9), 57.28 (C14),
65.48 (C17), 68.27 (C26), 74.28 (C3), 83.57 (C16), 90.71 (C22),
122.74 (C6), 140.04 (C5), 170.91 (acetate ester); ESI Mass (MeOH):
459.4 [M+H]+, 481.3 [M+Na]+, 497.4 [M+K]+; ESI-HRMS: 459.3467
for C29H47O4, cal: 459.3474; 481.3282 for C29H46O6Na, cal:
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2.2.5. Wittig reaction on aldehyde 8
Synthesis of (22b)-(E)-26-Benzylidene-3b-yl-furost-5-en-3-acetate
(10): Benzyltriphenylphosphonium bromide (Wittig salt, 200 mg)
was taken in dry toluene (10 mL). To this stirred solution pre-
washed sodium hydride (200 mg, 8.33 mmol) added and stirred
for 20 min. Aldehyde 8 (100 mg, 0.22 mmol) was added and the
reaction mixture was further stirred for 2 h. Toluene was evapo-
rated under vacuum and residue was taken in ethyl acetate (20
Q3 198
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2.2.8. (22b)-(E)-26-(30,4050-Trimethoxybenzylidene)-3b-yl-furost-5-
en-3-acetate (13)
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mL ꢀ 3), washed with water and dried over anhydrous sodium
sulfate. 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.
Yield = 142 mg (52%), oil; 1H NMR (CDCl3): d 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, ace-
tate), 2.29 (bd, 2H, 7-CH2), 3.84 (s, 9H, 3XOCH3), 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 & 7.8 Hz), 6.22 (d,
1H, 27-CH, J = 15.9 Hz), 6.54 (s, 2H, 20 & 60-CH of phenyl ring).
13C NMR (CDCl3, 75 MHz): d 16.83 (C18), 19.41 (C19), 19.70
(C21), 21.01 (C11), 21.77 (acetate CH3), 28.12 (C1), 30.07, 31.81,
31.96, 32.36 (C7), 32.65 (C15), 34.45, 37.08, 37.37 (C2), 37.96,
38.27, 38.47 (C12), 39.77 (C4), 41.06, 50.37 (C9), 56.41, 57.26
(C14), 61.26, 65.55 (C17), 74.24 (C3), 83.55 (C16), 90.72 (C22),
103.39 (20 & 60 of phenyl ring), 122.74 (C6), 128.64 (C27), 134.05
(C5), 136.50 (C26), 137.61 (40 of phenyl ring), 140.06 (C26),
153.63 (30 & 50 of phenyl ring), 170.85 (acetate ester); ESI Mass
(MeOH): 621.5 [M+H]+, 643.5 [M+Na]+, 659.4 [M+K]+;
10: Yield = 158 mg (68%), oil; 1H NMR (CDCl3): d 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, ace-
tate), 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): d 16.83 (C18), 19.39 (C19), 19.72
(C21), 21.06 (C11), 21.79 (acetate CH3), 28.16 (C1), 30.06 (C28),
31.85, 31.99, 32.40 (C7), 32.66 (C15), 34.48 (C24), 37.12 (C10),
37.41 (C2), 38.04, 38.31 (C20), 38.51 (C12), 39.82 (C4), 41.10,
50.44 (C9), 57.31 (C14), 65.60 (C17), 74.33 (C3), 83.58 (C16),
90.82 (C22), 122.77 (C6), 126.39 (C30 & C50 of Phenyl ring),
127.15 (C26), 128.73 (C20 & C60 of phenyl ring), 128.83 (C27),
137.05 (C40 of phenyl ring), 138.33 (C10 of phenyl ring), 140.09
(C5), 170.97 (acetate ester); ESI Mass (MeOH): 531.5 [M+H]+,
553.5 [M+K]+, 569.6 [M+K]+,; IR (KBr, cmꢁ1): 2946, 1734, 1456,
1372, 1244.
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2.2.9. Synthesis of (22b)-3b-Acetoxy-furost-5-en-26-aldoxime (14)
Aldehyde 8 (100 mg, 0.22 mmol) was taken in ethanol (10 mL).
To this dry pyridine (0.5 mL) and hydroxyaminehydrochloride
(100 mg, 1.43 mmol) was added and refluxed for 2 h. On comple-
tion ethanol was evaporated and dil HCl (10 mL) was added to it,
extracted with ethyl acetate (20 mLx3), washed with water and
dry in vacuo. The residue thus obtained was purified through silica
gel coumn using hexane-ethyl acetate as eluants. The desired
aldoxime 14 was obtained as creamish white solid.
2.2.6. (22b)-(Z)-26-(40-Nitrobenzylidene)-3b-yl-furost-5-en-3-acetate
(11): procedure same as for 10, Wittig salt (200mg) was 4-
nitrobenzyltriphenylphosphonium bromide
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14: Yield = 73 mg (70%), mp = 130–33 °C; 1H NMR (CDCl3): d
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): d 16.81 (C18),
18.44, 19.26 (C19), 19.70 (C27), 21.03 (C11), 21.78 (acetate CH3),
28.12 (C24), 30.06 (C2), 31.32 (C23), 31.95 (C20), 32.36 (C7),
32.56 (C15), 35.06, 37.08 (C10), 37.38 (C1), 38.24 (C8), 38.47
(C12), 39.78 (C4), 41.07 (C13), 50.39 (C9), 57.28 (C14), 65.46
(C17), 74.32 (C3), 83.62 (C16), 90.39 (C22), 122.75 (C6), 140.06
(C5), 156.46 (C26), 170.01 (acetate ester); ESI Mass (MeOH):
472.4 [M+H]+, 494.5 [M+Na]+, 510.3 [M+K]+; IR (KBr, cmꢁ1):
3405, 2948, 1735, 1455, 1371, 1256, 1043.
Yield = 157 mg (62%), oil; 1H NMR (CDCl3): d 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, ace-
tate), 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): d 16.80 (C18), 19.29 (C19), 19.69 (C21), 20.62, 21.01
(C11), 21.81 (acetate CH3), 28.11 (C1), 29.74, 31.38, 31.95, 32.35
(C7), 32.99 (C15), 34.01 (C23), 37.09, 37.36 (C2), 38.22, 38.45
(C12), 39.74 (C4), 41.10, 50.37 (C9), 57.27 (C14), 65.40 (C17),
74.50 (C3), 83.69 (C16), 90.54 (C22), 122.77 (C6), 123.92, 124.33,
126.80, 130.53 (C27), 134.03 (C26), 140.04 (C5), 142.28 (20 & 60
of phenyl ring), 143.26 (30 & 50 of phenyl ring), 162.83 (40 of phenyl
ring), 171.85 (acetate ester); ESI Mass (MeOH): 576.6 [M+H]+,
574.6 [MꢁH]+, 598.6 [M+Na]+, 614.5 [M+K]+; IR (KBr, cmꢁ1):
2940, 1728, 1595, 1516, 1340, 1246.
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
2.2.10. Synthesis of (22b)-3b-Acetoxy-27-nor-furost-5-en-25-
ketoxime (16)
The ketoxime 16 was prepared from 9 using the same method
as for aldoxime 14.
2.2.7. (22b)-(Z)-26-(30,40,50-Trimethoxybenzylidene)-3b-yl-furost-5-
en-3-acetate (12): procedure same as for 10 Wittig salt was 4-
nitrobenzyltriphenylphosphonium bromide (200 mg) to get 12 and 13
Yield = 79 mg (29%), oil; 1H NMR (CDCl3): d 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, ace-
tate), 2.29 (bd, 2H, 7-CH2), 3.85 (s, 9H, 3XOCH3), 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, 20 & 60-CH of phe-
nyl ring). 13C NMR (CDCl3, 75 MHz): d 16.83 (C18), 19.41 (C19),
19.71 (C21), 21.03 (C11), 21.82 (acetate CH3), 28.14 (C1), 30.06,
31.97, 32.37 (C7), 32.61 (C15), 33.39, 35.13, 37.10, 37.39 (C2),
38.29, 38.48 (C12), 39.77 (C4), 41.06, 50.38 (C9), 56.42, 57.28
(C14), 61.30, 65.51 (C17), 74.30 (C3), 83.58 (C16), 90.71 (C22),
106.17 (20 & 60 of phenyl ring), 122.77 (C6), 128.05 (C27), 133.94
(40 of phenyl ring), 139.38 (C26), 140.09 (C5), 153.27 (30 & 50 of
phenyl ring), 170.97 (acetate ester); ESI Mass (MeOH): 621.5
[M+H]+, 643.4 [M+K]+, 659.4 [M+K]+,; IR (KBr, cmꢁ1): 2945, 1733,
1582, 1504, 1456, 1243.
Yield = 87 mg (84%), mp = 151–53 °C; 1H NMR (CDCl3): d 0.79 (s,
3H, 18-CH3), 0.99 (s, 3H, 19-CH3), 1.02–1.95 (m, 28H, rest of the
1 ꢀ CH3, 8 ꢀ CH2 and 5 ꢀ CH of steroidal ring), 1.97 (s, 3H, CH3COO,
acetate), 2.03 (s, 3H, 25-CH3C@NAO), 2.34 (bd, 2H, 7-CH2), 3.33
(bm, 1H, 22-CH), 4.29 (bm, 1H, 16-CH), 4.53 (bm, 1H, 3-CH), 5.36
(s, 1H, 6-CH). 13C NMR (CDCl3, 75 MHz): d 16.80 (C18), 19.20
(C19), 19.72 (C21), 21.04 (C11), 21.81 (acetate CH3), 28.14 (C1),
30.08 (C26), 30.19 (C23), 31.96 (C8), 32.38 (C7), 32.59 (C15),
33.58 (C24), 37.10 (C10), 37.39 (C2), 38.20 (C20), 38.48 (C12),
39.77 (C4), 41.10 (C13), 50.39 (C9), 57.29 (C14), 65.49 (C17),
74.30 (C3), 83.70 (C16), 89.70 (C22), 122.76 (C6), 140.09 (C5),
158.98 (C25), 170.97 (acetate ester); ESI Mass (MeOH): 458.3
[M+H]+, 480.3 [M+Na]+, 496.4 [M+K]+; IR (KBr, cmꢁ1): 3382,
2941, 1721, 1448, 1374, 1248.
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
325
326
327
328
2.2.11. Synthesis of (22b)-25-Hydroxy-3b-yl-27-nor-furost-5-en-3-
acetate (18)
Ketone 9 (100 mg, 0.23 mmol) was stirred in methanol (5 mL).
To this stirred solution sodium borohydride (30 mg, 0.79 mmol)
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333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
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407
408
409
410
411
412
413
414
was added and reaction mixture was further stirred for 30 min. On
completion, solvent was evaporated in vacuo and residue was
taken in ethyl acetate (2 ꢀ 20 mL). It was acidified with dil. HCl
(5%, 5 mL), washed with water (2 ꢀ 10 mL) and organic layer was
dried over anhydrous sodium sulfate. The solvent was evaporated
in vacuo and residue thus obtained was recrystallised with chloro-
The unit cell dimensions were determined to be a = 6.0194 (6) Å,
b = 11.7762 (14) Å, c = 35.229 (4) Å, Z = 4, V = 2497.2 (5) Å3,
Dcalc = 1.177 gm/cm3, F(000) = 968. X-ray diffraction data were
collected on a Bruker AXS SMART APEX CCD diffractometer using
Mo Ka radiation (k = 0.71073 Å). Data were acquired using x scan
mode at room temperature (293 K). The structure was solved by
direct methods using SHELXS [7a] and was refined against F2 with
full-matrix least squares method by using SHELXL [7b]. All the non
hydrogen atoms were refined anisotropically. Hydrogen atoms
were fixed geometrically in idealized positions and were refined
as riding over the atoms to which they were bonded. 10149 reflec-
tions were measured (5107 independent reflections) with
Rint = 0.060. The final R-value was 0.055 (wR = 0.1239) for 2040
observed reflections with [I > 2sigI] and for 286 parameters. The
goodness-of-fit was 0.902. The largest difference peak was
0.28 eÅꢁ3 and the largest difference hole was ꢁ0.25 eÅꢁ3. The
absolute configuration was chosen based on the known configura-
tion of the starting diosgenin molecule. Crystallographic data
(excluding structure factors) have been deposited with the Cam-
bridge Crystallographic Data Centre as supplementary publication
number CCDC 967022. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK, (fax:+44-(0)1223-336033 or e-mail: deposit@ccdc.cam.
ac.uk)
form-hexane (1:4) to get 18 as mixture of both 25a/b-hydroxy
isomers.
18: Yield = 86 mg (86%), mp = 159–62 °C; 1H NMR (CDCl3): d
0.80 (s, 3H, 18-CH3), 0.95 (s, 3H, 19-CH3), 1.02–1.95 (m, 25H, rest
of the 2 ꢀ CH3, 8 ꢀ CH2 and 3 ꢀ CH of steroidal ring), 2.03 (s, 3H,
CH3COO, Acetate), 2.30 (s, 1H, 20-CH), 2.32 (bd, 2H, 7-CH2), 3.35
(bs, 1H, 22-CH), 3.79 (m, 1H, 25-CHOH), 4.33 (bd, 1H, 16-CH),
4.59 (bs, 1H, 3-CH), 5.36 (s, 1H, 6-CH). 13C NMR (CDCl3, 75 MHz):
d 16.80 (C18), 19.08 (C19), 19.71 (C21), 21.03 (C11), 21.81(acetate
CH3), 23.62 (CH), 24.01 (CH), 28.14 (C1), 30.08 (C23), 31.93 (C8),
32.36 (C7), 32.49 (C15), 36.69 (C20), 37.10 (C10), 37.38 (C2),
38.28 (C24), 38.47 (C12), 39.81 (C4), 41.10 (C13), 50.38 (C9),
57.26 (C14), 65.17 C17), 67.91/68.75 (C25a/b) 74.28 (C3), 83.80
(C16), 90.60 (C22), 122.75 (C6), 140.08 (C5), 170.94 (acetate ester);
ESI Mass (MeOH): 445.3 [M+H]+, 467.4 [M+Na]+, 483.2 [M+K]+; IR
(KBr, cmꢁ1): 3423, 2935, 1732, 1245,
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
2.2.12. (22b)-3b-Acetoxy-furost-5-en-26-aldoxime acetate (15)
The procedure is same as for 6 starting substrate was 14.
Yield = 1.0 g (92%), mp = 90–93 °C; 1H NMR (CDCl3): d 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 ꢀ CH3-
COO, 3 & 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): d 16.81 (C18), 19.20 (C19), 19.72 (C27), 19.91 (C21),
21.03 (C11), 21.81 (acetate CH3), 26.33 (Oxime acetate CH3),
28.14 (C1), 30.08 (C23), 31.52 (C20), 31.96, 32.38 (C7), 32.59
(C15), 37.11 (C10), 37.39 (C2), 38.06, 38.28 (C24), 38.49 (C12),
39.77 (C4), 41.09 (C13), 50.40 (C9), 57.29 (C14), 65.32 (C17),
74.29 (C3), 83.83 (C16), 90.14 (C22), 122.74 (C6), 140.12 (C5),
163.46 (C26), 169.16 (Oxime acetate), 170.95 (3-acetate ester);
ESI Mass (MeOH): 514.4 [M+H]+, 536.3 [M+Na]+; IR (KBr, cmꢁ1):
2934, 1734, 1454, 1372, 1244.
415
2.4. Biological evaluation
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
2.4.1. Cytotoxicity evaluation by Sulphorhodamine assay
Cytotoxic activity of the compounds was assessed by Sulpho-
rhodamine B dye based plate assay [8]. In brief, 104 cells/well were
added in 96-well culture plates and incubated overnight at 37 °C in
5% CO2. Next day (at 80% confluency), serial dilutions of test com-
pound were added to the wells. Untreated cells served as control.
After 48 h, cells were fixed with ice-cold 50% (w/v) Tri-chloro ace-
tic acid (100
SRB (0.4% w/v in 1% acetic a&&&cid, 50
dried. Bound dye was solubilized with 10 mM Tris base (150
L/well) and absorbance was read at 540 nm on a plate reader
l
L/well) for 1 h at 4 °C. Cells were then stained with
lL/well), washed and air-
l
(Biotek, USA). The cytotoxic effect of compound was calculated
as% inhibition in cell growth as per formula: [1- (Absorbance of
drug treated cells/Absorbance of untreated cells) x100]. Determi-
nation of 50% inhibitory concentration (IC50) was based on dose-
response curves.
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
2.2.13. (22b)-3b-Hydroxy-27-nor-furost-5-en-25-ketoxime-3b,25-
diacetate (17)
Procedure as for 6.
Yield = 57 mg (52%), mp = 113–16 °C; 1H NMR (CDCl3): d 0.80 (s,
3H, 18-CH3), 0.96 (s, 3H, 19-CH3), 1.08–1.88 (m, 24H, rest of the
1 ꢀ CH3, 8 ꢀ CH2 and 5 ꢀ CH of steroidal ring), 1.98 (s, 3H, CH3COO,
Acetate), 2.03 (s, 3H, 25-CH3COO-, acetate), 2.16 (t, 3H, CH3CO),
2.36 (bd, 2H, 7-CH2), 3.30 (bs, 1H, 22-CH), 4.32 (bd, 1H, 16-CH,
J = 5.4 Hz), 4.60 (bs, 1H, 3-CH), 5.38 (s, 1H, 6-CH). 13C NMR (CDCl3,
75 MHz): d 16.82 (C18), 19.13 (C19), 19.72 (C21), 20.09, 21.03
(C11), 21.80 (acetate CH3), 28.04 (C1), 29.89 (C26), 30.08 (C2),
31.97 (C8), 32.37 (C7), 32.57 (C15), 33.76 (C24), 37.10, 37.39
(C23), 38.07, 38.48 (C12), 39.76 (C4), 41.12 (C13), 50.40 (C9),
57.28 (C14), 65.46 (C17), 74.27 (C3), 83.73 (C16), 89.61 (C22),
122.71 (C6), 140.12 (C5), 166.81 (C25), 169.26 (Oxime acetate),
170.90 (3-acetate ester); ESI Mass (MeOH): 500.4 [M+H]+, 538.3
[M+K]+; IR (KBr, cmꢁ1): 2924, 2369, 1735, 1458, 1371, 1245.
2.4.2. Cell cycle analysis
432
433
434
435
436
437
438
439
440
441
442
443
The effect of most potent compound on cell division cycle was
assessed by flow cytometry with PI-stained cellular DNA, as
described earlier [9]. Briefly, cells (4 x105 per well) were seeded
in 6-well culture plate and grown overnight at 37 °C in 5% CO2.
After exposure to the compound for different time points, cells
were harvested by trypsinization and fixed with ice-cold 70% eth-
anol for 30 min at 4 °C. The pellets were washed with PBS and re-
suspended in a solution containing PI (20 mg/ml), Triton X100
(0.1%) and RNase (1 mg/ml) in PBS. After distribution of cells in dif-
ferent phases of cell cycle was calculated using ‘‘Cell Quest’’
software.
444
445
446
447
448
449
450
451
452
2.4.3. Western blot assay
Compound treated cells were lysed (30 min, in ice) with cold M-
PER (mammalian protein extraction reagent) supplemented with
385
386
2.3. Crystal structure determination by X-ray diffraction
crystallography
protease inhibitor cocktail. Equal amount of proteins (25 lg)
extracted from the cells treated for different time intervals were
separated by 10% SDS-polyacrylamide gel electrophoresis and
transferred electrophoretically onto PVDF membranes. The mem-
branes were blocked with 5% nonfat dry milk powder dissolved
in Tris-buffered saline (TBS; 20 mM Tris-HCl pH 7.6, 137 mM NaCl)
387
388
389
390
Single crystals of compound 9 (C28H42O4, M = 442.6) were
obtained by slow evaporation from a 50:50 mixture of chloroform
and methanol at 4 °C. The compound crystallized in orthorhombic
space group P212121 with one molecule in the asymmetric unit.
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5
O
O
O
O
CH2OH
AcO
a
CHO
O
c
b
HO
O
5
AcO
6
AcO
7
d
8
O
O
AcO
9
Scheme 1. (a) Ac2O, dry pyridine, dry CHCl3, RT, 2 h, 91%; (b) AcOH, NaCNBH3, RT, 5 h, 81%; (c) Dry DCM,PCC, reflux, 1 h, 91%; (d) Ethanol, substituted aniline, RT, 2 h, 66–84%.
25
O
21
26
OH
OOH
O
27
20
18
24
O
O
12
23
O
17 22
11
19
O
O
13
1
4
14
16
15
2
3
9
8
OH
OH
O
O
HO
HO
H
7
5
HO
HO
6
O
HO
O
OH
1
5
2
OH
3
O
4
O
HO
HO
OH
Fig. 1. Some of the plant based potent cytotoxic molecules on steroidal framework; 1: withaferin A, 2: gymnasterol 3: 24-hydroxyperoxide desmosterol, 4: timosaponin A-III,
5: diosgenin.
Table 1
Effect of various amines on transformation of 8–9.
Entry
Aldehyde
Amine
Solvent
Reaction time (h)
%Yield of 9
1
2
3
4
5
6
7
8
9
8
8
8
8
8
8
8
8
8
No amine
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
16
2
2
No reaction
66
84
3,4,-Dimethoxyaniline
3,4,5-Trimethoxyaniline
3,4-Methylenedioxyaniline
Benzyl amine
3,4-Methylenedioxybenzyl amine
Ammonia
2
72
16
16
16
16
16
No reaction
No reaction
No reaction
No reaction
No reaction
Methylamine
Ethylamine
R
R
C
H
O
R
H
C
H3C
H
N
O
N
H
N
H
H
H
O
AcO
H
O
O
8
O
H
H2O
Aldehyde
Schiff's base
Enamine
O
NHCH3
O
R
+
9
Ketone
Fig. 2. Plausible mechanism of conversion of aldehyde 8 to ketone 9.
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O
CH3
O
AcO
Fig. 3. HMBC correlations of side chain of ketone 9.
453
454
455
456
457
458
459
460
461
462
containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature and
subsequently incubated overnight with anti-PARP antibody (cat#
9542, Cell Signaling Technology, USA) at 4 °C. After 3 washes with
TBS-T, the membranes were incubated with HRP conjugated anti-
rabbit antibody for 1 h at room temperature and washed again
with TBS-T (x3 times). Proteins were detected with an enhanced
chemiluminescence (ECL) reagent and visualized by a chemilumi-
nescence detector (Bio-Rad Laboratories, USA). The densitometry
analysis of blots was done by using Bio-Rad Image Lab 4.0
software.
Fig. 5. Cell cycle analysis of compound 7.
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
2.4.4. Docking studies
The two dimensional structures of the molecules were con-
structed with the ChemDraw Ultra. Energy minimization of the
compounds was performed with ‘ChemBio office’ considering
MM2/MM3 molecular mechanics parameter up to its lowest stable
energy state. This energy minimization process was performed
until the energy change was less than 0.001 kcal molꢁ1 or the mol-
ecules had been updated almost 300 times. The 3D chemical struc-
ture of doxorubicin (Positive control) was retrieved from the
PubChem compound database at NCBI (http://www.pub-
chem.ncbi.nlm.nih.gov). Crystallographic 3D structures of target
proteins (caspase-9: (PDB:1NW9- chain B) and caspase-3:
(PDB:3KJF) were retrieved from the Brookhaven Protein Databank
(http://www.pdb.org) [10,11]. Hydrogen atoms were added to
the protein targets to achieve the correct ionization and tautomeric
states of amino acid residues such as His, Asp, Ser, and Glu. Molec-
ular docking of the compounds against selected target was
achieved using the ‘AutoDock Vina’. To perform the automated
docking of ligands into the active sites, we used a Lamarkian
genetic algorithm [12].
Fig. 6. Cleavage of PARP, a caspase target by compound 7.
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
an automatic dark and light cycle of 12 hours. The animals were
fed with the standard mice feed and provided ad libitum drinking
water. Mice of group 1 were kept as control and animals of groups
2, 3, 4 and 5 were kept as experimental. The animals were acclima-
tized for 7 days in the experimental environment prior to the
actual experimentation. The test compound was solubilized in
dimethyl sulphoxide with few drops of tween 80 and then sus-
pended in caboxymethyl cellulose (0.7%) and was given at 5, 50,
300 and 1000 mg/kg body weight to animals of groups 2, 3, 4
and 5 respectively once orally. Control animals received only
vehicle.
The animals were checked for mortality and any signs of ill
health at hourly interval on the day of administration of drug
and there after a daily general case side clinical examination was
carried out including changes in skin, mucous membrane, eyes,
occurrence of secretion and excretion and also responses like lach-
rymation, piloerection respiratory patterns etc. Also changes in
gait, posture and response to handling were also recorded [13].
In addition to observational study, body weights were recorded
and blood and serum samples were collected from all the animals
on 7th day of the experiment in acute oral toxicity. The samples
were analyzed for total RBC, WBC, differential leucocytes count,
hemoglobin percentage and biochemical parameters like ALKP,
SGPT, SGOT, total cholesterol, triglycerides, creatinine, bilirubin,
serum protein, tissue protein, malonaldehyde and reduced GSH
activity. The animals were then sacrificed and were necropsed
483
484
485
486
487
488
489
490
491
492
493
2.4.5. In-vivo acute oral toxicity
In view of potent anti-cancer activity of compound 7 in in-vitro
model, acute and sub-acute oral toxicity of the same was carried
out in Swiss albino mice for its further development into drug
product. Experiment was conducted in accordance with the Orga-
nization for Economic Co-operation and Development (OECD) test
guideline No 423 (1987).
For this experiment, 30 mice (15 male and 15 female) were
taken and divided into four groups comprising 3 male and 3 female
mice in each group weighing between 20 and 25 g. The animals
were maintained at 22 50C with humidity control and also on
Fig. 4. Molecular conformation of 9 in crystals. Thermal ellipsoids are shown at 50% probability level.
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531
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533
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536
537
538
539
540
541
542
543
for any gross pathological changes. Weights of vital organs like
liver, heart, kidney etc. were recorded [14].
F of spiroketal linkage was done by using sodium cyanoborohy-
dride in AcOH at room temperature to get the alcohol 7 that pos-
sesses furostenic system. Oxidation of alcohol 7 with pyridinium
chlorochromate (PCC) yielded aldehyde 8 (Fig. 1).
522
523
2.4.6. Statistical analysis
We tried to prepare some Schiff’s bases onto C29 aldehyde (8),
but unexpectedly, we ended with a C28 ketone (9). Aldehyde 8 on
treatment with an aromatic amine in ethanol at room temperature
afforded product 9 as a ketone in 2 h. Various aromatic amines
(3,4,-dimethoxyaniline, 3,4,5-trimethoxyaniline 3,4-methylenedi-
oxyaniline) were tried and every time the same product 9 was
obtained in varied yields (66–84%). The reaction of aldehyde 8 with
aliphatic amines (MeNH2 and EtNH2) and even with ammonia was
unsuccessful. Benzyl amines (BnNH2 and 3,4-methylenedioxyben-
zylamine) also did not yield the product 9 (Table 1). It will be
Statistical analysis was carried out in Microsoft Excel.
524
3. Results and discussion
525
526
527
528
529
Scheme 1 denotes the synthetic strategy of these derivatives
starting with diosgenin (5). Diosgenin 5 was converted to dios-
genin acetate 6 by acetylation with acetic anhydride-pyridine in
dry chloroform at room temperature. The spectral data of acetate
6 was alike the earlier reports [6a]. The reductive cleavage of ring
Fig. 7a. In-silico molecular docking studies elucidating the possible mechanisms of diosgenin (5), its derivatives 7, and positive control doxorubicin with Caspase-9 (PDB:
1NW9-B). The docking studies were carried out using ‘AutoDock Vina’, using Lamarkian genetic algorithm.
Fig. 7b. In-silico molecular docking studies elucidating the possible mechanisms of diosgenin (5), its derivatives 7, and positive control doxorubicin with Caspase-3: (PDB:
3KJF). The docking studies were carried out using ‘AutoDock Vina’, using Lamarkian genetic algorithm.
Table 2
Effect of solvent polarity on transformation of compound 8–9.
Entry
Aldehyde
Amine
Solvent
Reaction time (t) h
% Yield of 9
1
2
3
4
5
8
8
8
8
8
3,4,5-Trimethoxyaniline
3,4,5-Trimethoxyaniline
3,4,5-Trimethoxyaniline
3,4,5-Trimethoxyaniline
3,4,5-Trimethoxyaniline
EtOH
MeOH
THF
Dichloromethane
Toluene
2
2
2
2
2
84
73
44
49
28
Table 3
In-vitro cytotoxicities of diosgenin analogs.
S. no.
Compound no./name
IC50 (uM) (Mean SE)a
KB
C-33A
DU145
A549
MCF-7
1
2
3
4
5
6
7
7
8
14
15
11.43 1.47
17.55 1.25
15.97 1.96
13.96 0.39
17.52
7.54 0.93
11.54 0.10
11.18 0.63
12.27 0.38
13.15
>20
16.826
>20
>20
10.95 0.73
15.17 1.36
14.53 1.07
14.28 0.20
18.67
17.08 0.09
13.27 0.25
>20
12.24 0.28
<1.25
15.27 1.18
12.56 0.18
16.29
10.07 0.46
<1.25
17
Tamoxifen
Podophyllotoxin
10.25 1.21
<1.25
6.94 0.09
<1.25
8.54 1.01
<1.25
a
Rest of the compounds have IC50 > 20
lM, considered inactive.
Please cite this article in press as: Hamid AA et al. Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin. Steroids (2014),
http://dx.doi.org/10.1016/j.steroids.2014.05.025

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worth mentioning that without aromatic amine this transforma-
tion does not take place. Aldehyde 8 did not form Schiff’s base with
any of these amines. The structure of the ketone 9 was confirmed
by 1D NMR (1H & 13C), 2D NMR (COSY, HSQC, HMBC), mass and IR
spectroscopy. A plausible mechanism is given in Fig. 2.
Compound 9 was obtained as yellow amorphous powder. Its
structure was established by spectroscopy. Its molecular formulae
C28H42O4 was determined by its HR-ESI-MS at m/z 443.3139
[M+H]+ (Calcd 443.3161). 13C NMR showed total 28 distinct car-
bons indicating loss of one carbon as compared to aldehyde 8.
There were 5 methyls (d16.80, 19.01, 19.71, 21.80, 30.33), 9 meth-
ylenes (d21.02, 27.44, 28.13, 32.37, 32.55, 37.38, 38.48, 39.75,
41.28), 9 methines (d31.95, 38.24, 50.38, 57.27, 65.39, 74.28,
83.69, 89.53, 122.72) and 5 quaternary carbons (d 37.10, 41.09,
140.12, 170.95, 209.24). There was no aldehyde group and a car-
bonyl group was generated as ketone at d209.24 ppm. Least change
in the chemical shifts of C1 to C22 suggested possibility of no
change in the ring system of the ketone 9. From HSQC spectrum,
chemical shifts of 22-CH (d3.26 m, 89.53), 23-CH2 (d1.72, 27.44),
24-CH2 (d2.49-2.61, 41.28) and 26-CH3 (d2.13, 30.33) were estab-
lished. In 1H-1H COSY spectrum, 22-CH (d3.26) showed coupling
with 23-CH2 (d1.72) and 24-CH2 (d2.49-2.61) showed coupling
with 23-CH2 (d1.72). In HMBC correlations, 26-CH3 protons
(d2.13 ppm) showed correlations with C24 (d41.28) and C25
ketone (d209.24). 24-CH2 protons (d2.49-2.61) exhibited correla-
tions with C26 (d30.33) and C23 (d27.44) carbons. C23- CH2 pro-
tons (d1.72) showed correlations with C22 (d89.53), C24 (41.28)
and C25 (d209.24) carbons. HMBC correlations of side chain are
NO2
C
C
C
C
H
H
O
H
H
O
11
AcO
10
e
AcO
e
CHO
O
8
AcO
f
e
H3CO
OCH3
OCH3
C
N-OH
H
O
g
C
C
14
AcO
H
H
O
12
AcO
AcO
+
H
C
H
N-OAc
C
C
O
H
O
OCH3
OCH3
15
H3CO
13
AcO
Scheme 2. (e) Wittig salt, NaH, dry Toluene, RT, 3–4 h, 62–81%; (f) NH2OH.HCl, EtOH, reflux, 2 h, 84%; (g) Ac2O, dry pyridine, dry CHCl3, RT, 3 h, 92%.
O
OAc
OH
N
N
O
O
O
f
g
AcO
AcO
AcO
17
9
16
OH
h
O
AcO
Mixture of isomers
18
Scheme 3. (f) NH2OH.HCl, EtOH, reflux, 2 h, 79%; (g) Ac2O, dry pyridine, dry CHCl3, RT, 3 h, 89%; (h) NaBH4-MeOH, RT, 30 min, 86% (Mix of isomers).
Please cite this article in press as: Hamid AA et al. Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin. Steroids (2014),
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shown in Fig. 3. The structure of 29 was finally authenticated by
single crystal X-ray crystallography (Fig. 4).
Cell cycle regulation ensures the fidelity of genomic replication
and cell division. G1/S and G2/M transitions are two major check-
points to allow the cells to control any modification in DNA con-
tent. Checkpoint loss results in genomic instability and induce
carcinogenesis. Induction of cell cycle arrest in cancer cell lines
constitutes one of the most prevalent strategies to stop or limit
cancer spreading [16]. Our data demonstrate that Compound 7
arrests cells at G0/G1 phase of division cycle which is associated
with decrease in G2/M population. We also observed accumulation
of cells in sub-G0 phase after treatment confirming induction of
apoptosis by the molecule.
Diosgenin and related compounds have been good cytotoxic
agents against various human cancer cell lines [17a–f]. Diosgenin
and its semisynthetic derivatives induced apoptosis through
increased expression of caspase-3 [18a–e]. Caspases are a family
of cystein-aspartic proteases, which are crucial mediators of apop-
tosis. Among these caspase-3 encoded as CAS3 gene is identified in
numerous mammals. It remains as zymogens (procaspase) unless
activated biochemically. It cleaves and activates caspases 6, 7
and 9, and the protein itself is processed by caspases 8, 9 and 10.
Caspase-3 is necessary for its typical role in apoptosis, where it is
responsible for chromatin condensation and DNA fragmentation
[19]. PARP is an enzyme involved in DNA repair when cell are
exposed to environmental stress [20]. During apoptosis, PARP is
cleaved and inactivated by caspases [21,22]. Therefore, cleavage
of PARP is a well established marker for detection of apoptosis.
In the present study we found cleavage of PARP in MCF-7 cells at
24 h exposure to the Compound 7 implying activation of caspases
by the molecule to trigger apoptosis.
The binding affinity obtained in the docking experiment
allowed the activity of the diosgenin derivative 7 to be compared
to that of the standard anticancer compound doxorubicin (Table 4).
The diosgenin and its active derivatives ‘7’ showed high binding
affinity (high negative docking energy) against known human
caspases 9 (-7.3 kcal/mol) and caspase 3 (-7.8 kcal/mol) (PDB:
1NW9-B; PDB: 3KJF). When we compared how the binding site
pocket amino acid residues interacted with the derivatives, we
found that the in case of CASPASE 9, ‘PHE, PRO, THR, GLY, ARG,
and HIS’ amino acids were in share with positive control doxorubu-
cin. While, in case of CASPASE 3, ‘GLN, PHE, TYR, TRP, SER, ALA, GLY
and ARG’ were in share with doxorubicin.
Further, the molecular conformation of compound 9 in crystals
is depicted in Fig. 4. All the bond lengths and bond angles are
within the accepted range. The crystal packing is stabilized by
van der Waals interactions, in the absence of strong hydrogen bond
donors in this molecule. However, the ketonic O atom and the
methylene group (C24) adjacent to the ketone functionality of
symmetry-related molecules are at 3.22Å apart, suggesting a weak
C-H...O interaction (O3...H = 2.55Å, C24...O3 = 3.22Å, \C24-
H...O3 = 125.8°). The ring conformations of compound 9 are very
similar to that observed in other diosgenin derivatives and solvates
(Figs. 5, 6, 7A, 7B).
The effect of various solvents was also observed, as shown in
Table 2. Ethanol was found to be the best solvent. However, meth-
anol was equally good. In case of less polar solvents like THF,
dichloromethane and toluene the yield of the product 9 was rela-
tively low (Table 3).
Further, several styrene derivatives were prepared on to 8 alde-
hyde using Wittig reaction. Various Wittig salts (Benzylidenetri-
phenylphosphonium
bromide)
were
prepared
using
triphenylphosphine and corresponding benzylbromide in toluene.
Finally the styrene derivatives of diosgenin were obtained by treat-
ing wittig salt with aldehyde 8 in sodium hydride/toluene under
reflux conditions in 29–68% yields. 8 was transformed to aldoxime
(14) also and then oxime acetate (15) by usual methodologies [15].
All these transformations are depicted in Scheme 2.
Similarily, Scheme 3 shows modification of ketone 9 to various
other derivatives. 9 was modified to ketoxime (16) and its corre-
sponding acetate (17) same as described earlier for aldehyde 8.
The ketone group of 9 was reduced to alcohol (18) with sodium
borohydride in methanol. Both C25a-OH and C25b-OH isomers
were formed in equal ratio as evident from 13C NMR at d67.91
and d68.75 respectively.
All synthesized compounds were evaluated against C33A
(Cervical carcinoma), A549 (Lung carcinoma), KB (HeLa contam-
inant of mouth epidermal carcinoma), MCF-7 (Breast adenocar-
cinoma), DU145 (prostate carcinoma) human cancer cell lines
by Sulphorhodamine assay [7]. Only five of the analogs exhib-
ited significant anticancer activity, rest were inactive at 20
concentration.
lM
Table 4
Molecular interactions of diosgenin (5) and its derivative 7 docked with Caspase-9: (PDB: 1NW9-B) and Caspase-3: (PDB: 3KJF) and binding pocket residues (amino acids).
Compound code
Caspase-3 (PDB entry 1pau)
Caspase-9 (PDB entry 1nw9)
Binding
affinity
(Kcal/
mol)
Binding pocket amino acids
Binding
affinity
(kcal/
Binding pocket amino acids
mol)
ꢁ7.9
ARG(B)-207, ASN(B)-208, GLU(B)-248, PHE(B)-
247, PHE(B)-250, PHE(B)-256, SER(B)-209,
SER(B)-249, SER(B)-251, TRP(B)-206, TRP(B)-214
ꢁ8.7
ALA-316, ARG-408, GLN-245, LEU-244,
PHE-406, PRO-318, PRO-336, THR-337
O
O
HO
5
ꢁ7.8
CYS(A)-163, GLU(A)-123, GLY(A)-122, HIS(A)-
121, MET(A)-61, PHE(A)-128, THR(A)-166,
ARG(B)-207, ASN(B)-208, PHE(B)-250, PHE(B)-
256, SER(B)-209, SER(B)-249, SER(B)-251,
TRP(B)-206, TRP(B)-214, TYR(B)-204
ꢁ7.3
GLN-240, GLY-288, GLY-350, GLY-395,
ILE-396, LYS-394, LYS-398, PHE-348,
PHE-351, PRO-349, THR-347, TRP-354,
TYR-397
CH2OH
O
O
O
7
Doxorubicin
ꢁ8.2
ALA(A)-162, ARG(A)-64, CYS(A)-163, GLN(A)-
161, GLY(A)-122, HIS(A)-121, MET(A)-61,
SER(A)-120, ARG(B)-207, ASP(B)-253, PHE(B)-
256, SER(B)-205, SER(B)-251, TRP(B)-206,
TYR(B)-204
ꢁ8.2
ARG-178, ARG-180, ASP-186, CYS-287,
GLY-182, GLY-288, GLY-360, HIS-237,
PHE-351, PRO-349, PRO-357, SER-183,
SER-361, THR-179, THR-181
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Table 5
A.A. Hamid et al. / Steroids xxx (2014) xxx–xxx
Effect of compound 7 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on body weight, haemogram and serum biochemical parameters in Swiss albino mice
(Mean SD; n = 6) compared to control, 5, 50, 300 and 1000 mg/kg).a significant (P < 0.05).
Parameters
Dose of compound 7 at mg/kg body weight as a single oral dose
Control
5 mg/kg
50 mg/kg
300 mg/kg
1000 mg/kg
Body weight (gm)
Hemoglobin (gm/dL)
RBC (million/mm3)
WBC (1000⁄/mm3)
ALKP (U/L)
SGOT (U/L)
SGPT (U/L)
Albumin (g/dL)
Creatinine (mg/dL)
Triglycerides (mg/dL)
Serum protein (mg/mL)
Cholesterol (mg/dL)
23.32 0.60
11.23 0.92
5.82 0.19
10.68 2.14
222.1 14.4
24.44 2.37
12.96 1.96
3.60 0.45
23.31 0.48
11.44 0.50
6.55 0.30
11.03 2.24
259.4 6.9
32.49 3.51
18.76 1.79
3.08 0.15
0.48 0.09
23.95 0.45
10.94 0.58
5.85 0.31
10.48 1.76
263.6 16.2
31.50 2.98
18.07 3.20
2.98 0.13
21.92 0.47
11.51 1.02
5.42 0.34
10.71 2.52
239.4 13.7
36.04 3.33
19.96 4.63
2.87 0.24
23.10 0.55
12.10 0.40
6.19 0.30
12.53 2.77
139.7 17.4
32.54 3.50
18.33 2.27
3.27 0.11
0.63 0.11
172.21 17.57
1.15 0.13
0.50 0.07
172.73 19.15
0.94 0.08
0.44 0.05
171.74 19.80
1.00 0.08
0.47 0.08
166.89 17.97
1.23 0.09
180.55 19.70
1.02 0.08
114.3 21.2
156.4 35.8
115.9 23.4
123.8 25.6
156.4 28.3
Fig. 8. Effect of compound 7 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on absolute and relative organ weight in Swiss albino mice (n = 6, Non significant changes
were found compared to control).
654
655
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667
In acute oral toxicity studies of compound 7, no observational
changes, morbidity and mortality were observed throughout the
experimental period up to the dose level of 1000 mg/kg body
weight. No morbidity or any other gross observation changes could
be noticed in the group of animals treated with the test drug at
1000 mg/kg. Blood and serum samples upon analysis showed
non-significant changes in all the parameters studied like total
hemoglobin level, RBC count, WBC count, differential leucocytes
count, SGPT, creatinine, triglycerides, cholesterol, albumin, serum
protein (Table 5 and Fig. 8) except significant changes in ALKP
activities in groups of animals treated with the compound at
1000 mg/kg; wherein ALKP activity was decreased significantly
compared to control. Animals on gross pathological study showed
no changes in any of the organs studied including their absolute
and relative weight (Figs. 7a and 7b). Therefore, the experiment
showed that compound 7 is well tolerated by the Swiss albino mice
up to the dose level of 1000 mg/kg body weight as a single acute
oral dose except significant decrease in ALKP activities in groups
treated with compound 7 at 1000 mg/kg. However, sub-acute
and or chronic experiment with the test drug needs to be carried
out to look for any adverse effect on repeated exposure to com-
pound 7 for its future development [23] Fig. 10.
Q4 668
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4. Conclusion
677
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685
Diosgenin was modified to obtain novel derivatives. The unu-
sual transformation was observed and product has been confirmed.
Compound 7 is the most potent derivative of this series which
inhibits cell proliferation by arresting the population in G0/G1
phase of the cell cycle. The mechanism of antiproliferative action
of this molecule is through induction of caspase depended apopto-
sis pathway. Compound 7 is a non-toxic anticancer furostane
derivative of diosgenin. The lead obtained in this study can further
be optimized for better activity in future.
686
Acknowledgements
687
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691
Authors are thankful to the Directors of CSIR-CDRI and CSIR-
CIMAP for constant encouragement and support. Spectral data
received by spectral The financial support from CSIR is duly
acknowledged. TWAS-CSIR fellowship to Mr. A. A. Hamid is duly
acknowledged.
Fig. 9. Effect of compound 7 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg
body weight on differential leucocytes counts in Swiss albino mice (n = 6, Non
significant changes were found compared to control).
Please cite this article in press as: Hamid AA et al. Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin. Steroids (2014),
http://dx.doi.org/10.1016/j.steroids.2014.05.025

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(d) Perez-Diaz JOH, Rarova L, Ocampo JPM, Magaria-Vergara NE, Farfan N,
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Please cite this article in press as: Hamid AA et al. Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin. Steroids (2014),
http://dx.doi.org/10.1016/j.steroids.2014.05.025