(22β,25R)-3β-Hydroxy-spirost-5-en-7-iminoxy-heptanoic acid exhibits anti-prostate cancer activity through caspase pathway

Article abstract of DOI:10.1016/j.steroids.2017.01.001

Prostate cancer is one of the most common cancers in men. Diosgenin and related compounds are potential cytotoxic agents. Twelve diverse analogues of long chain fatty acid/ester of diosgenin-7-ketoxime have been prepared. Six of the analogues exhibited significant anticancer activity against a panel of human cancer cell lines with IC₅₀ranging from 12 to 35?μM. Compound 16, the best representative of the series exerted S phase arrest in DU145 prostate cancer cells and induced apoptosis through caspase pathway. Additionally, these analogues inhibited lipopolysaccharide induced pro-inflammatory cytokines (TNF-α and IL-6) up to 47.7% and 23.3% respectively. Compound 16 was found to be safe in acute oral toxicity in Swiss albino mice up to 300?mg/kg dose. The anticancer and antiinflammatory properties of compound 16 are important and can further be optimized for a better anti-prostate cancer candidate.

Full text of DOI:10.1016/j.steroids.2017.01.001

Accepted Manuscript
(
22β,25R)- 3β-Hydroxy-spirost-5-en-7-iminoxy-heptanoic acid exhibits anti-
prostate cancer activity through caspase pathway
A.A. Hamid, Tanu Kaushal, Raghib Ashraf, Arjun Singh, Amit Chand Gupta,
Om Prakash, Jayanta Sarkar, Debabrata Chanda, D.U. Bawankule, Feroz Khan,
Karuna Shanker, O.O. Aiyelaagbe, Arvind S. Negi
PII:
S0039-128X(17)30011-9
http://dx.doi.org/10.1016/j.steroids.2017.01.001
STE 8072
DOI:
Reference:
To appear in:
Steroids
Received Date:
Revised Date:
Accepted Date:
16 September 2016
16 December 2016
12 January 2017
Please cite this article as: Hamid, A.A., Kaushal, T., Ashraf, R., Singh, A., Chand Gupta, A., Prakash, O., Sarkar,
J., Chanda, D., Bawankule, D.U., Khan, F., Shanker, K., Aiyelaagbe, O.O., Negi, A.S., (22β,25R)- 3β-Hydroxy-
spirost-5-en-7-iminoxy-heptanoic acid exhibits anti-prostate cancer activity through caspase pathway, Steroids
(
2017), doi: http://dx.doi.org/10.1016/j.steroids.2017.01.001
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(22β,25R)- 3β-Hydroxy-spirost-5-en-7-iminoxy-heptanoic acid exhibits anti-
prostate cancer activity through caspase pathway
a,,c
a
b
a
a
a
A. A. Hamid , Tanu Kaushal , Raghib Ashraf , Arjun Singh , Amit Chand Gupta , Om Prakash , Jayanta
b
a
a
a
a
d
Sarkar , Debabrata Chanda , D.U. Bawankule , Feroz Khan , Karuna Shanker , O.O. Aiyelaagbe , Arvind S.
a*
Negi
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-
2
26031, India.
Department of Chemistry, University of Ilorin, Ilorin, Nigeria
Organic Chemistry Unit, Department of Chemistry, University of Ibadan, Ibadan, Nigeria.
c
d
Abstract
Prostate cancer is one of the most common cancers in men. Diosgenin and related compounds are
potential cytotoxic agents. Twelve diverse analogues of long chain fatty acid/ester of diosgenin-7-
ketoxime have been prepared. Six of the analogues exhibited significant anticancer activity against a
panel of human cancer cell lines with IC50 ranging from 12-35µM. Compound 16, the best
representative of the series exerted S phase arrest in DU145 prostate cancer cells and induced apoptosis
through caspase pathway. Additionally, these analogues inhibited lipopolysaccharide induced pro-
inflammatory cytokines (TNF-α and IL-6) up to 47.7% and 23.3% respectively. Compound 16 was
found to be safe in acute oral toxicity in Swiss albino mice up to 300mg/kg dose. The anticancer and
antiiflammatory properties of compound 16 are important and can further be optimized for a better
anti-prostate cancer candidate.
Keywords: Diosgenin, oxime derivatives, anticancer, caspase pathway, antiinflammatory, acute oral toxicity.
1
. Introduction
Steroids are pharmacologically important class of compounds which are biosynthesized through
mevalonate pathway in the plants. Due to their crucial role in human physiology and interesting
biochemistry, they have attained importance among the researchers [1]. Steroids have an excellent
ability to pass through cell membranes and bind to the nuclear and membrane receptors. A small
modification in steroid moiety elicits an extensive biological response. Due to their fascinating
structural framework and wide range of pharmacological response, steroids have tempted medicinal
chemists and biochemists to explore suitable modifications to induce various pharmacological
activities. Steroids have been strategically modified as antihormones like antiestrogens, antiprogestins,
anti-androgens etc. [2]. Their role as antiinflammatory agents is well established. Steroids have also
been modified as anticancer agents where their role as antiproliferative (cytostatic) and cytotoxic is
well distinguished [2].
…
………………………………………………………………………………………………….
*
Corresponding author. Tel.:+91 522 2718583; Fax: +91 522 2342666;
E. mail: arvindcimap@rediffmail.com (as negi) ; CIMAP Communication No. CIMAP/PUB/2016/45
1

Diosgenin (1, Fig. 1) is a steroidal sapogenin present mainly in Dioscorea spp. It also occurs in
Costus speciosus, Anemarrhena asphodeloides and in several species of Smilax. Owing to its high
occurrence (~3-5%) in some plants, it is used as a main precursor for the synthesis of sex hormones,
corticosteroids and other steroid products [3a,b]. Diosgenin has also been modified to various
pharmacologically active analogues. Diosgenin exhibits anticancer [4a,b], antiinflammatory [5] anti-
thrombosis [6], enhancing regulatory T-cell immunity [7], antiviral [8], antidiabetic [9], and
antimicrobial activities [10] etc. In the recent past, diosgenin analogues have attained a special attention
for inducing apoptosis in several human carcinomas. The antiproliferative and apoptotic properties of
diosgenin are due to its ability to arrest the cell cycle, activate p53, release apoptosis-inducing factor
and modulate caspase-3 activity [11]. Diosgenin, its saponins and other analogues induce apoptosis
mainly prostate cancer cells and colon carcinoma cells [12a-e]. Some of the notable anticancer
derivatives of diosgenin are Diocin, Timosaponin A-III [13], 17-deoxy-26-hydroxy-22-oxo derivative,
and several others [14].
The present study describes synthesis of some novel analogues of diosgenin at C7-position.
Two of the analogues exhibited potential anticancer activity against DU145 prostate cancer cell line.
The effect of both the analogues was also checked on production of LPS induced proinflammatory
cytokines (TNF-α and IL-6). Compound 16, the best analogue of the series was further evaluated for
cell cycle analysis and mechanistic studies. Finally, in-vivo acute oral toxicity of compound 16 was
evaluated in Swiss-albino mice.
[
Figure 1]
2
. Experimental
2
.1 General
The starting substrate diosgenin was procured from Sigma-Aldrich USA. All the reagents were
analytical grade and were used without any purification. Solvents were dried as per standard
procedures and stored under 4Å molecular sieves. Reactions were monitored on Merck precoated silica
gel aluminium sheets (TLC, UV254) and compounds were detected under UV light (254nm and
o
3
65nm), spraying with 2% ceric sulphate-10% sulphuric acid and heated to 100 C. Purification was
done over column silica gel (100-200 mesh, Avra Chemicals, India) eluting with organic solvents.
Melting points were determined on E-Z Melt MP apparatus in open capillaries (Stanford Research
System, USA) and were uncorrected. NMR experiments were performed on Bruker Avance
(
300MHz/500MHz) spectrometer with TMS as internal standard (δ, 0.00 ppm). Electrospray Ionisation
mass spectra were recorded on API3000 LC-MS-MS (Applied Biosystem, USA) after dissolving the
compounds in methanol or acetonitrile. High Resolution Mass (HRMS) were recorded on Agilent
6
520Q-TOF after dissolving the compounds in methanol. FT-IR spectra were recorded on Perkin–
Elmer SpectrumBX. Purity of compounds was determined on Waters ACQUITY UPLC BEH130Å
system. Nomenclature of steroid derivatives has been given as per the recommendations by the Joint
Commission on the Biochemical Nomenclature (JCBN) of IUPAC [15].
2

2
2
.2. Chemical synthesis
.2.1. Synthesis of (22β,25R)-spirost-5-en-3β-yl-3-acetate (2)
Diosgenin (1g, 2.4mmol) was taken in dry chloroform (10mL). To this stirred solution, DMAP
(
50mg) and acetic anhydride (1mL, 10.5mmol) were added and further stirred for 2h at RT. The
reaction mixture was diluted with water and extracted with chloroform, washed with dil. HCl (5%,
0mL) and washed with water. Organic layer was dried over anhydrous sodium sulphate and
1
evaporated to dryness. Residue was recrystallised with chloroform-hexane (1:3) to get compound 2 as
white amorphous solid.
o
o
1
2
: Yield=93%, mp=193-95 C [195 C, [16]; H NMR (CDCl
CH , J=6.9Hz), 1.02 (s, 3H, 19-CH ), 1.11-2.31 (m, 25H, rest of the 1xCH
steroidal ring), 2.01 (s, 3H, CH COO, Acetate), 2.24-2.31 (bd, 2H, 7-CH ), 3.38 (m, 2H, 26-CH
bs, 1H, 3-CH), 4.42 (bd, 1H, 16-CH), 5.36 (s, 1H, 6-CH). C NMR (CDCl , 75 MHz); δ 14.89 (C21),
), 28.12 (C24), 29.19 (C2), 30.66 (C25),
3
), δ 0.77 (s, 3H,18-CH
3
), 0.96 (d, 3H, 27-
and 6xCH of
), 4.37
3
3
3
, 8xCH
2
3
2
2
1
3
(
3
1
3
4
6.64 (C18), 19.69 (C19), 21.20 (C11), 21.74 (acetate CH
3
1.78 (C23), 31.80 (C8), 32.21 (C7), 32.41 (C15), 37.10 (C10), 37.34 (C1), 38.47 (C12), 40.10 (C4),
0.63 (C13), 42.00 (C20), 42.68, 50.35 (C9), 56.82 (C14), 62.52 (C17), 67.19 (C26), 74.26 (C3), 81.16
(
C
C16), 109.60 (C22), 122.72 (C6), 140.05 (C5), 170.82 (acetate ester). ESI Mass (MeOH) for
+
+
+
-1
29
H
44
4
O : 457.3 [M+H] , 479.3 [M+Na] , 495.4 [M+K] . IR (KBr, cm ): 2907, 1724, 1451, 1231.
2
.2.2. Synthesis of (22β,25R)-spirost-5-en-7-oxo-3β-yl-3-acetate (3)
A mixture of dry methylene chloride (25mL) and dry pyridine (10mL) was stirred with cooling (5-
o
1
0 C). To this crushed chromium trioxide (1g) was added and further stirred for 10 min. To this
reaction mixture diosgenin 3-acetate (2, 500mg, 1.09mmol) was added as a solution in methylene
chloride (3-5mL) drop-wise. It was stirred for 30min in ice-bath and then at room temperature for 4
hours. On completion methanol (2mL) was added to decompose the excess of chromic acid. Solvent
was evaporated and water was added to it. The reaction mixture was extracted with ethyl acetate,
organic layer was washed with water, dries over anhydrous sodium sulphate and evaporated. The crude
mass was purified through silica gel column eluting with hexane-ethyl acetate to get the desired 7-
ketone derivative (3) as creamish white solid at 8% ethyl acetate-hexane.
o
1
3
: Yield= 351g (68%), mp=179-182 C; H NMR (CDCl
J=6.6Hz), 1.14 (s, 3H, 19-CH ), 1.16-2.84 (m, 23H, rest of the 1xCH
ring), 2.04 (s, 3H, CH COO, 3-acetate), 3.39 (m, 2H, 26-CH ), 4.46 (bs, 1H, 3-CH), 4.71 (bs, 1H, 16-
CH), 5.71 (s, 1H, 6-CH). C NMR (CDCl , 75 MHz); δ 15.02 (C21), 16.79 (C18), 17.50 (C27), 17.64
), 27.72 (C24), 29.20 (C2), 30.70 (C23), 31.84 (C23), 34.09
3
), δ 0.78 (s, 3H,18-CH
3
), 0.97 (d, 3H, 27-CH
and 6xCH of steroidal
3
,
3
3
, 7xCH
2
3
2
1
3
3
(
(
(
1
4
C19), 21.29 (C11), 21.59 (acetate CH
3
C15), 36.39 (C1), 38.18 (C12), 38.84 (C10), 39.05 (C4), 41.36 (C13), 41.98 (C20), 45.29 (C8), 49.87
C14), 50.07 (C9), 61.57 (C17), 67.17 (C26), 72.53 (C3), 81.13 (C16), 109.55 (C22), 126.87 (C6),
64.43 (C5), 170.61 (acetate ester), 201.65 (C7). ESI Mass (MeOH) for C29
93.4 [M+Na] , 509.3 [M+K] . IR (KBr, cm ): 2907, 1724, 1451, 1231.
+
H
42
O
5
: 471.4 [M+H] ,
+
+
-1
3

2
.2.3. Synthesis of (22β,25R)-7-(hydroxyimino)-spirost-5-en-3β-yl-3-acetate (4)
Diosgenin 7-ketone (3, 500mg, 1.06mmol) was taken in dry pyridine (6mL). To this hydroxylamine
hydrochloride (150mg, 2.16mmol) was added and refluxed for 2h. On completion, water was added to
it, acidified with HCl (5%, 10mL), extracted with ethyl acetate, washed with water and dried over
anhydrous sodium sulphate. The organic layer was dried in vacuo to get oxime 4 as oil.
1
4
: Yield=494mg (96%), oil; H NMR (CDCl
3
), δ 0.77 (s, 3H,18-CH
, 7xCH
COO, 3-acetate), 2.41-2.43 (m, 3H), 2.82 (bs, 1H), 3.44 (m, 2H, 26-CH
3
), 0.97 (d, 3H, 27-CH
and 6xCH of steroidal ring), 2.01
), 4.56 -4.68 (m,
, 75 MHz); δ 14.97 (C21), 17.08 (C18),
), 27.85 (C24), 29.61 (C2), 30.64 (C25),
3
, J=6.6Hz),
1
(
.11 (s, 3H, 19-CH
3
), 1.20-2.41 (m, 21H, rest of the 1xCH
3
2
s, 3H, CH
H, 3-CH & 16-CH), 6.57 (s, 1H, 6-CH). C NMR (CDCl
7.46 (C27), 18.28 (C19), 20.88 (C11), 21.67 (C3-acetate CH
1.71 (C23), 34.88 (C15), 35.16 (C1), 38.43 (C12), 38.85 (C10), 39.00 (C4), 36.66 (C8), 41.01 (C13),
1.96 (C20), 49.80 (C14), 50.26 (C9), 60.98 (C17), 67.11 (C26), 73.38 (C3), 81.28 (C16), 114.21 (C6),
09.96 (C22), 151.60 (C5), 156.74 (C7), 170.73 (C3, acetate ester). ESI Mass (MeOH) for C29
86 [M+H] , 508 [M+Na] ; Negative mode: 484 [M-H] . ESI-HRMS for C29
86.3219, found 486.3197.
3
2
1
3
2
1
3
4
1
4
4
3
3
H
43NO :
5
+
+
-
+
H44NO [M+H] , calc.
5
2
.2.4. General synthesis of fatty acid esters (5-10)
Synthesis of (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-ethylacetate (5)
Diosgenin 7-keto oxime (4, 200mg, 0.41mmol) was taken in dry acetone (15mL) and anhydrous
potassium carbonate (1g). To this ethyl iodoacetate (0.1mL, 181mg, 0.84mmol) was added and the
reaction mixture was refluxed for 4h. On completion, potassium carbonate was filtered out and filtrate
was evaporated. The residue was dissolved in ethyl acetate and washed with water. The organic layer
was dried over anhydrous sodium sulphate and evaporated. The residue thus obtained was purified
through filter column of silica gel eluting with ethyl acetate-hexane. The desired ester 4 was obtained at
6
% ethyl acetate-hexane as white amorphous solid.
o
1
5
: Yield= 210mg (89%), mp=146-148 C; H NMR (CDCl
, J=6.9Hz), 1.12 (s, 3H, 19-CH ), 1.33 (t, 3H, CH , ethyl, J=8.0Hz), 1.20-2.51 (m, 24H, rest of the
xCH , 8xCH and 5xCH of steroidal ring), 2.02 (s, 3H, CH COO, 3-acetate), 3.42 (m, 2H, 26-CH ),
.20 (q, 2H, OCH , ethyl, J=7.1Hz) 4.54 (s, 2H, OCH ), 4.56 (m, 2H, 3-CH & 16-CH), 6.54 (s, 1H, 6-
, 75 MHz); δ 14.65 (C21), 15.05 (CH , ethyl), 16.99 (C18), 17.51 (C27), 18.28
C19), 20.94 (C11), 21.68 (C3-acetate CH ), 27.82 (C24), 29.21 (C2), 30.70 (C25), 31.82 (C23), 34.92
3
), δ 0.77 (s, 3H,18-CH
3
), 0.98 (d, 3H, 27-
CH
1
4
3
3
3
3
2
3
2
2
2
1
3
CH). C NMR (CDCl
3
3
(
(
(
3
C15), 36.61 (C1), 38.42 (C12), 38.81 (C10), 39.11 (C4), 36.66 (C8), 41.11 (C13), 41.96 (C20), 49.85
C14), 50.50 (C9), 61.51 (C17), 61.13 (CH2, ethyl), (C26), 71.01 (2’-CH2), 73.20 (C3), 81.08 (C16),
1
14.48 (C6), 109.58 (C22), 153.33 (C5), 157.70 (C7), 170.66 (C3, acetate ester), 170.66 (ester). ESI
+
+
+
-1
Mass (MeOH) for C33
H
49NO : 572.8 [M+H] , 595.7 [M+Na] , 611.6 [M+K] ; IR (KBr, cm ): 2947,
7
1
2
737, 1638, 1451, 1241, .
.2.5. (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-ethylpropionate (6)
1
3
There were some isomeric CH peaks in C indicating isomeric mixture.
4

o
1
6
: Yield=198mg (82%), mp=141-143 C; H NMR (CDCl
CH , J=6.9Hz), 1.13 (s, 3H, 19-CH ), 1.30 (t, 3H, CH , ethyl ester, J=7.8Hz), 1.22-2.82 (m, 27H, rest
of the 1xCH , 9xCH and 6xCH of steroidal ring), 2.00 (s, 3H, CH COO, 3-acetate), 3.37-3.42 (m, 2H,
6-CH ), 4.15 (q, 2H, OCH , ethyl ester, J=4.7Hz) 4.19 (m, 2H, -N-OCH ), 4.64 (m, 2H, 3-CH & 16-
CH), 6.52 (s, 1H, 6-CH). C NMR (CDCl , 75 MHz); δ 14.69 (C21), 15.04 (CH , ethyl), 16.95 (C18),
7.50 (C27), 18.23 (C19), 20.91 (C11), 21.64 (C3-acetate CH ), 27.81 (C24), 29.21 (C2), 30.67 (C25),
3
), δ 0.75 (s, 3H,18-CH
3
), 0.95 (d, 3H, 27-
3
3
3
3
2
3
2
2
2
2
13
3
3
1
3
4
7
1
3
1.80 (C23), 34.98 (C15), 36.55 (C2’), 36.66 (C1), 38.40 (C12), 38.83 (C10), 39.09 (C4), 36.66 (C8),
1.14 (C13), 41.92 (C20), 49.85 (C14), 50.42 (C9), 61.57 (C17), 61.03 (CH2, ethyl), 67.13 (C26),
3.14 and 73.24 (C3), 81.05 and 81.09 (C16), 114.46 and 114.74 (C6), 109.48 (C22), 153.08 (C5),
56.90 (C7), 173.31 (C3, acetate ester), 170.66 (ester). ESI Mass (MeOH) for C34
M+H] , 608 [M+Na] , 644 [M+K] ; IR (KBr, cm ): 2948, 1738, 1638, 1458, 1241.
H
51NO : 586
7
+
+
+
-1
[
2
.2.6. (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-ethylbutyrate (7)
o
1
7
: Yield=205mg (83%), mp=145-148 C; H NMR (CDCl
, J=6.9Hz), 1.12 (s, 3H, 19-CH ), 1.32 (bs, 3H, CH , ethyl ester), 1.21-2.90 (m, 29H, rest of the
xCH , 10xCH and 6xCH of steroidal ring), 2.03 (s, 3H, CH COO, Acetate), 3.46 (m, 2H, 26-CH ),
.12 (q, 2H, OCH , J=7.2Hz), 4.42 (m, 1H, 3-CH), 4.60 (m, 1H,
6-CH), 6.45 (s, 1H, 6-CH). ESI Mass (MeOH) C35
3
), δ 0.77 (s, 3H,18-CH
3
), 0.97 (d, 3H, 27-
CH
3
3
3
1
4
1
3
2
3
2
2
, ethyl, J=7.1Hz) 4.05 (t, 2H, -N-OCH
2
+
+
H
53NO : 600.9 [M+H] , 622.8 [M+Na] , 638.7
7
+
-1
[
M+K] ; IR (KBr, cm ): 2928, 1737, 1637, 1459, 1241.
2
.2.7. (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-ethylpentanoate (8)
o
1
8
: Yield= 176mg (70%), mp=199-202 C; H NMR (CDCl
, J=6.9Hz), 1.19 (s, 3H, 19-CH ), 1.23 (t, 3H, CH , ethyl ester, J=7.1Hz), 1.21-2.84 (m, 31H, rest
of the 1xCH , 8xCH and 6xCH), 2.02 (s, 3H, CH COO, 3-acetate), 3.44 (m, 2H, 26-CH ), 4.06-4.13
m, 4H, OCH of ethyl and N-O-CH ), 4.54 (m, 1H, 3-CH), 4.66 (m, 1H,16-CH), 6.56 (s, 1H, 6-CH).
C NMR (CDCl , 75 MHz); δ 14.55 (C21), 14.98 (CH , ethyl), 17.04 (C18), 17.45 (C27), 18.28 (C19),
0.88 (C11), 21.65 (C3-acetate CH ), 27.85 (C24), 29.12 (C2), 30.66 (C25), 31.75 (C23), 34.88 (C15),
3
), δ 0.76 (s, 3H,18-CH
3
), 0.97 (d, 3H, 27-
CH
3
3
3
3
2
3
2
(
2
2
1
3
3
3
2
3
3
4
7
6.67 (C2’), 37.57 (C1), 38.45 (C12), 38.87 (C10), 39.02 (C4), 36.66 (C8), 30.66 (C25), 41.04 (C13),
1.96 (C20), 49.84 (C14), 50.30 (C9), 61.15 (C17), 60.76 (CH2, ethyl), 60.76 (5’CH2), 67.13 (C26),
3.34 (C3), 81.22 (C16), 114.08 (C6), 109.84 (C22), 151.85 (C5), 156.99 (C7), 171.54 (C3, acetate
+
+
ester), 170.67 (ester). ESI Mass (MeOH) for C36
H
55NO : 636.7 [M+Na] ; 652.9 [M+K] .
7
2
.2.8. (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-ethylhexanoate (9)
o
1
9
: Yield=217mg (84%), mp=89-91 C; H NMR (CDCl
), 1.19 (t, 3H, CH , ethyl ester, J=7.1Hz), 1.21-2.93 (m, 33H, rest of the
and 6xCH), 2.01 (s, 3H, CH COO, 3-acetate), 3.34 (m, 2H, 26-CH ), 4.04-4.09 (q,
of ethyl and N-O-CH , J=7.1Hz), 4.46 (m, 1H, 3-CH), 4.60 (m, 1H,16-CH), 6.52 (s, 1H, 6-
, 75 MHz); δ 14.47, 14.97, 16.95, 17.41, 18.19, 20.80, 21.32, 21.59, 27.76,
8.82, 31.67, 32.10, 34.63, 36.61, 37.00, 37.20, 37.62, 38.40, 38.88, 38.95, 40.96, 41.89, 49.80, 49.88,
3
), δ 0.72 (s, 3H,18-CH
3
), 0.92 (d, 3H, 27-CH
3
,
J=6.9Hz), 1.11 (s, 3H, 19-CH
1
4
CH). C NMR (CDCl
2
3
3
xCH
3
, 12xCH
2
3
2
H, OCH
2
2
1
3
3
5

5
4.12, 60.84, 61.15, 67.11, 70.36, 73.40, 81.08, 109.89, 114.33, 152.32, 157.16, 170.91, 171.80. ESI
+
Mass (MeOH) for C37
H
57NO
7
: 628.3 [M+H] ;
2
1
1
.2.9. (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-ethylheptanoate (10)
1
0: Yield=243mg (92%), oil; H NMR (CDCl
.11 (s, 3H, 19-CH ), 1.23 (t, 3H, CH , ethyl, J=7.5Hz), 1.23-2.39 (m, 35H, rest of the 1xCH
COO, 3-acetate), 3.39 (m, 2H, 26-CH ), 4.08-4.13 (m, 4H, OCH
, J=8.0Hz, 5.3Hz), 4.46 (m, 1H, 3-CH), 4.60 (m, 1H,16-CH), 6.45 (s, 1H, 6-CH); C
, 75 MHz); δ14.26, 14.68, 16.63, 17.12, 17.95, 20.57, ESI Mass (MeOH) for
calc:642.4370,
3
), δ 0.78 (s, 3H,18-CH
3
), 0.96 (d, 3H, 27-CH
3
, J=7.0Hz),
, 13xCH
of ethyl
3
3
3
2
and 6xCH), 2.02 (s, 3H, CH
and N-O-CH
NMR (CDCl
3
2
2
1
3
2
3
+
+
C
38
H59NO
7
:
664.7 [M+Na] ; 681.3 [M+K] ; ESI-HRMS for
C
38
H60NO
7
-1
observed:642.4360; IR (KBr, cm ): 2929, 1735, 1460, 1176.
2
.2.10. Saponification of esters (5-10) to get acid derivatives (11-16)
Synthesis of (22β,25R)- 3β-hydroxy-spirost-5-en-7-iminoxy-ethanoic acid (11)
Ester 6 (100mg, 0.17mmol) was taken in 1.2% aqueous methanolic alkali (water:MeOH=1:9). The
reaction mixture was stirred at room temperature for 2h. Solvent was removed in vacuo and water was
added, acidified with dil. HCl (5%, 10mL), extracted with ethyl acetate. Organic layer was washed with
water, dried over sodium sulphate and evaporated. The residue thus obtained was recrystallised with
chloroform:hexane (1:2) to get 11 as solid.
o
1
1
1: Yield=87mg (91%), mp=145-146 C; H NMR (CDCl
CH , J=6.9Hz), 1.14 (s, 3H, 19-CH ), 1.19-2.62 (m, 25H, rest of the 1xCH
steroidal ring), 3.42 (m, 2H, 26-CH ), 4.54 (s, 2H, N-OCH ), 4.56 (m, 2H, 3-CH & 16-CH), 6.54 (s,
H, 6-CH). ESI Mass (MeOH) for C29 : 502.4 [M+H] , 524.3 [M+Na] , 540.4 [M+K] ; IR
3
), δ 0.78 (s, 3H,18-CH
3
), 0.95 (d, 3H, 27-
3
3
3
, 8xCH
2
and 6xCH of
2
2
+
+
+
1
H
43NO
6
-1
(
KBr, cm ): 2947, 1638, 1451, 1241, .
2
.2.11. (22β,25R)- 3β-acetoxy-spirost-5-en-7-iminoxy-propanoic acid (12)
o
1
1
2: Yield=82 (86%), mp=114-116 C; H NMR (CDCl
), 1.27-2.66 (m, 27H, rest of the 1xCH
COO, Acetate), 3.38 (m, 2H, 26-CH
3
), δ 0.72 (s, 3H,18-CH
3
), 0.92 (d, 3H, 27-CH
and 6xCH of steroidal
), 4.13 (m, 1H, 3-
, 75 MHz); δ 14.63 (C21), 16.91
), 29.15 (C24), 29.58 (C2), 30.62
2’-CH2), 30.62 (C25), 31.50 (C8), 31.75 (C23), 32.06 (C15), 37.59 (C1), 38.78 (C12), 40.98 (C4),
1.10 (C13), 41.89 (C20), 49.99 (C9), 54.24 (C14), 61.05 (3’-CH2), 62.31 (C17), 67.09 (C26), (2’-
3
,
J=6.6Hz), 1.22 (s, 3H, 19-CH
ring), 2.11 (s, 3H, CH
CH), 4.32 (m, 1H, 16-CH), 6.46 (s, 1 H, 6-CH). C NMR (CDCl
3
3
, 9xCH
2
3
2
), 3.79 (bs, 2H, N-O-CH
2
1
3
3
(
(
C18), 17.46 (C27), 18.28 (C19), 20.92 (C11), 21.96 (C3-acetate CH
3
4
CH2), 71.12 (C3), 81.05 (C16), 113.54 (C6), 109.49 (C22), 156.9 (C5), 160 (C7), 171 (C3, acetate
+
ester), 173.51 (COOH); ESI Mass (MeOH) for C32
H47NO
7
: 580.2 [M-Na] .
2
.2.12. (22β,25R)- 3β-hydroxy-spirost-5-en-7-iminoxy-butanoic acid (13)
6

o
1
1
3: Yield=73 (83%), mp=147-149 C; H NMR (CDCl
J=6.9Hz), 1.11 (s, 3H, 19-CH ), 1.15-2.57 (m, 29H, rest of the 1xCH
ring), 3.42 (t, 2H, 26-CH , J=6.0Hz), 4.05 (t, 2H, N-OCH , J=6.3Hz), 4.56 (m, 2H, 3-CH & 16-CH),
.54 (s, 1H, 6-CH). C NMR (CDCl , 75 MHz); δ 14.65 (C21), 15.05 (CH , ethyl), 16.99 (C18),
7.51 (C27), 18.28 (C19), 20.94 (C11), 27.82 (C24), 29.21 (C2), 30.70 (C25), 31.82 (C23), 34.92
3
), δ 0.68 (s, 3H,18-CH
3
), 0.90 (d, 3H, 27-CH
3
,
3
3
, 10xCH
2
and 6xCH of steroidal
2
2
13
6
1
3
3
(
(
1
C15), 36.61 (C1), 38.42 (C12), 38.81 (C10), 39.11 (C4), 36.66 (C8), 41.11 (C13), 41.96 (C20), 49.85
C14), 50.50 (C9), 61.51 (C17), 61.13 (CH2, ethyl), (C26), 71.01 (2’-CH ), 73.20 (C3), 81.08 (C16),
14.48 (C6), 109.58 (C22), 153.33 (C5), 157.70 (C7), 174.81 (Acetate) 178.47 (COOH). ESI Mass
2
+
(
MeOH) for C31
H
47NO
6
: 529.4 [M ] .
2
.2.13. (22β,25R)- 3β-hydroxy-spirost-5-en-7-iminoxy-pentanoic acid (14)
o
1
1
4: Yield=79mg (89%), mp=159-162 C; H NMR (CDCl
, J=5.4Hz), 1.11 (s, 3H, 19-CH ), 1.15-2.57 (m, 31H, rest of the 1xCH
steroidal ring), 3.42 (t, 2H, 26-CH , J=6.3Hz), 4.54 (s, 2H, N-OCH ), 4.56 (m, 2H, 3-CH & 16-CH),
.54 (s, 1H, 6-CH). C NMR (CDCl , 75 MHz); δ 14.65 (C21), 15.05 (CH3, ethyl), 16.99 (C18),
7.51 (C27), 18.28 (C19), 20.94 (C11), 27.82 (C24), 29.21 (C2), 30.70 (C25), 31.82 (C23), 34.92
3
), δ 0.68 (s, 3H,18-CH
3
), 0.90 (d, 3H, 27-
CH
3
3
3
, 11xCH
2
and 6xCH of
2
2
1
3
6
1
3
(
(
1
C15), 36.61 (C1), 38.42 (C12), 38.81 (C10), 39.11 (C4), 36.66 (C8), 41.11 (C13), 41.96 (C20), 49.85
C14), 50.50 (C9), 61.51 (C17), 61.13 (CH , ethyl), (C26), 71.01 (2’-CH ), 73.20 (C3), 81.08 (C16),
14.48 (C6), 109.58 (C22), 153.33 (C5), 157.70 (C7), 178.47 (COOH). ESI Mass (MeOH) for
2
2
+
-1
C
34
H
51NO : 544.3 [M+H] ; IR (KBr, cm ): 2947, 1737, 1638, 1451, 1241, .
7
2
.2.14. (22β,25R)-3β-hydroxy-spirost-5-en-7-iminoxy-hexanoic acid (15)
o
1
1
5: Yield=87mg (91%), mp=155-157 C; H NMR (CDCl
CH , J=6.6Hz), 1.13 (s, 3H, 19-CH ), 1.18-2.78 (m, 24H, rest of the 1xCH
steroidal ring), 3.42 (m, 2H, 26-CH ), 3.44 (t, 2H, N-OCH , J=6.3Hz), 4.33 (m, 2H, 3-CH & 16-CH),
.41 (s, 1H, 6-CH). C NMR (CDCl , 75 MHz); δ13.72, 15.53, 16.16, 16.97, 19.39, 23.64, 24.33,
3
), δ 0.72 (s, 3H,18-CH
3
), 0.93 (d, 3H, 27-
3
3
3
, 8xCH
2
and 6xCH of
2
2
1
3
6
2
5
3
8.46, 29.13, 30.16, 30.30, 31.27, 32.86, 33.49, 35.70, 36.12, 37.33, 37.62, 37.99, 41.25, 48.72, 48.99,
0.27, 60.21, 60.46, 65.52, 69.20, 79.55, 107.93, 111.81, 151.61, 155.01, 172.84; ESI Mass (MeOH)
+
for C33
H51NO
6
: 558.3 [M+H] .
2
.2.15. (22β,25R)- 3β-hydroxy-spirost-5-en-7-iminoxy-heptanoic acid (16)
o
1
1
6: Yield=80mg (84%), mp=237-239 C; H NMR (CDCl
, J=7.0Hz), 1.14 (s, 3H, 19-CH ), 1.24-2.45 (m, 35H, rest of the 1xCH
steroidal ring), 3.46 (bs, 2H, 26-CH ), 4.02 (t, 2H, N-OCH , J=6.5Hz), 4.46 (m, 2H, 3-CH & 16-CH),
.43 (s, 1H, 6-CH). C NMR (CDCl , 100MHz); δ 14.67, 16.64, 18.06, 20.65, 22.69, 24.77, 25.62,
3
), δ 0.77 (s, 3H,18-CH
3
), 0.97 (d, 3H, 27-
CH
3
3
3
, 13xCH
2
and 6xCH of
2
2
1
3
6
2
4
3
8.81, 28.88, 29.15, 29.36, 30.30, 31.22, 34.03, 34.60, 36.59, 37.22, 38.34, 38.87, 40.73, 41.60, 42.14,
9.73, 50.31, 61.09, 66.84, 71.04, 73.52, 80.84, 109.35, 113.43, 153.06, 155.71, 178.77. ESI Mass
+
+
-
(
MeOH) for C34
H
53NO : 594.7 [M+Na] , 610.5[M+K] ; Negative mode: 570.9 [M-H] . ESI-HRMS
6
-1
calc for C34
H
54NO : 572.3914, found, 572.3912; IR (KBr, cm ): 2947, 1737, 1638, 1451, 1241,
6
7

2
.2.16. (22β,25R)-7-Oxo-spirosta-3,5-diene (17)
Benzyltriphenylphosphonium bromide (Wittig salt, 1g, 2.1mmol) was stirred in dry toluene (15mL). To
this sodium hydride (100mg, 4mmol) was added and further stirred for 10 min. 7-Keto derivative 3
(
(
250mg, 0.53mmol) was added to it and reaction mixture was refluxed for 2h. On completion water
20mL) was added to it and reaction mixture was extracted with ethyl acetate, organic layer was
washed with water, dries over anhydrous sodium sulphate and evaporated. The residue thus obtained
was recrystallised with chloroform-hexane (1:4) mixture to get an exclusive product (17) was obtained
as creamish white solid. Reaction was done with several other wittig salts of benzyl bromides ( 4-nitro;
3
,4,5-trimethoxy; 3,4-methylenedioxy) yielding always the same product 17.
o
1
1
7: Yield=78mg (89%), mp=182-185 C; H NMR (CDCl
, J=6.9Hz), 1.14 (s, 3H, 19-CH ), 1.19-2.29 (m, 19H, rest of the 1xCH
steroidal ring), 2.52 (t, 1H, 2-CH , J=6.3Hz), 2.87 (t, 1H, 2-CH ), 3.38 (m, 2H, 26-CH
3
), δ 0.76 (s, 3H,18-CH
3
), 0.95 (d, 3H, 27-
CH
3
3
3
, 5xCH
2
and 6xCH of
), 4.41 (q, 1H,
2
2
2
1
3
1
6-CH, J=4.6Hz), 5.58 (s, 1H, 6-CH), 6.14 (m, 2H, 3-CH and 4-CH). C NMR (CDCl
δ15.02, 16.86, 16.97, 17.50, 21.31, 23.76, 29.19, 30.69, 31.83, 33.20, 34.27, 36.80, 39.31, 41.79, 42.01,
5.96, 49.94, 50.61, 61.60, 67.15, 81.41, 109.51, 124.32, 128.05, 137.13, 161.59, 202.14 ; ESI Mass
3
, 75 MHz);
4
+
+
(
MeOH) for C27
H
38
O
3
: 411.2 [M+H] ; ESI-HRMS for C27
H
39
O [M+H] , calc. 411.2899, found
3
4
11.2872.
2
.3. Purity profile of analogues 9, 10, 11, 13, 14 and 16
The ACQUITY UPLC BEH130Å (2.1×100mm, 1.7µm) C18 column (Waters, USA) thermostated at
5±0.1°C and fitted in ACQUITY UPLC H-Class Bio System was used for purity determination of the
compounds prior to in-vitro & in-vivo assay. UPLC analysis was performed with a two-solvent system
using isocratic elution of acetonitrile/methanol and water (mobile phase-I: Water: CH CN- 70:30;
mobile phase-II: Water: CH OH- 80:20; run time-12min; flow rate- 0.3 ml/min). The injection volume
was 2µl. The column elution was monitored in the range of 190-400nm using photodiode array detector
A16UPD0038, ACQUITY Waters, Cell volume- 500nl). Purity of the compounds was calculated
2
3
3
(
using UPLC peak area normalization method. UPLC provides improved resolution and higher
throughput as compared to traditional reverse phase-HPLC system. In the absence of primary
standards, peak area normalization method was adopted to calculate the chromatographic purity of
respective compounds [17].
2
2
.4. Pharmacology
.4.1. Cell Culture
Human cancer cell lines, FaDU (Hypopharyngeal carcinoma), A549 (lung carcinoma), DLD1
(
(
colorectal adenocarcinoma), MCF-7 (Breast adenocarcinoma), DU145 (Prostate carcinoma) and PC3
Prostate carcinoma) were originally obtained from American type of cell culture collection (ATCC),
USA and grown at 37˚C in DMEM supplemented with 10% FBS and Ab-Am (antibiotic-antimitotic)
solution in a CO
2
incubator (New Brunswick/Eppendorff, Germany) under 5% CO and 95% relative
2
8

humidity. Tamoxifen, doxorubicin, and podophyllotoxin were used as standard anticancer drugs
positive control) for cytotoxicity.
(
2
.4.2. Cytotoxicity evaluation by Sulphorhodamine assay
Cytotoxicity of diosgenin derivatives was evaluated by Sulphorhodamine B assay [18]. In brief, in
4
9
6‐well culture plates, 10 cells/well were added and incubated overnight at 37ºC in 5% CO
2
. Next day
(
at 80% confluency), serial dilutions of test compound were added to the wells. Untreated cells served
as control. After 48h, cells were fixed with ice‐cold 50% (w/v) trichloroacetic acid (100µL/well) for 1h
at 4°C. Cells were then stained with SRB dye (0.4% w/v in 1% acetic acid, 50µL/well), washed and
air‐dried. Bound dye was solubilised with 10mM Tris base (150µL/well) and absorbance was read at
5
%
40nm on a plate reader (Biotek, USA). The cytotoxic effect was calculated as;
inhibition in cell growth= [1- (Absorbance of treated cells/Absorbance of untreated cells) x100].
Determination of 50% inhibitory concentration (IC50) was based on dose-response curves.
2
.4.3. Cell cycle analysis
The effect of most potent compound 16 on cell division cycle was assessed by flow cytometry with PI-
5
stained cellular DNA, as described earlier [19]. Briefly, in 6-well culture plate, 4 x10 cells per well
were seeded and grown overnight at 37ºC in 5% CO
time points, cells were harvested by trypsinization and fixed with ice-cold 70% ethanol for 30min at
°C. The pellets were washed with PBS and re-suspended in a solution containing propidium iodide
20mg/mL), TritonX100 (0.1%) and RNase (1mg/mL) in PBS. After distribution of cells in different
phases of cell cycle,“Cell Quest” software was used for calculations.
2
. After treating with compound 16 for different
4
(
2
.4.4. Western blot assay
Compound 16 treated cells were lysed (30min, in ice) with cold M-PER (mammalian protein extraction
reagent) supplemented with protease inhibitor cocktail. Equal amount of proteins (25µg) extracted from
the cells treated at different time intervals were separated by 10% SDS-polyacrylamide gel
electrophoresis and transferred electrophoretically onto PVDF membranes. The membranes were
blocked with 5% nonfat dry milk powder dissolved in Tris-buffer saline (TBS; 20mM Tris-HCl, pH
o
7
.6, 137mM NaCl) containing 0.1% Tween20 (TBS-T) for 1h at RT (30 C) and subsequently incubated
overnight with anti-PARP antibody (cat# 9542, Cell Signaling Technology, USA) at 4ºC. After 3
washes with TBS-T, the membranes were incubated with HRP conjugated anti-rabbit antibody for 1h at
RT and washed thrice with TBS-T. Proteins were detected with an enhanced chemiluminescence (ECL)
reagent and visualized by a chemiluminescence detector (Bio-Rad Laboratories, USA). The
densitometry analysis of blots was done by using Bio-Rad Image Lab 4.0 software.
2
.4.5. Antiinflammatory activity
As per protocol [20a,b], primary macrophage cells were isolated from the peritoneal cavities of mice
(
8-week-old female Swiss albino mice) after an intra-peritoneal injection of 1.0mL of 1% peptone (BD
9

Biosciences, USA) 3 days before harvesting. Mice were euthanized by cervical dislocation under ether
anesthesia and peritoneal macrophages were obtained by intra-peritoneal injection of PBS (pH, 7.4).
Membrane debris was removed by filtering the cell suspensions through sterile gauze. The viability of
cells was determined by trypan blue exclusion and the viable macrophage cells at the concentration of
6
0
.5×10 live cells/mL were used for the experimentation. Cells were grown in DMEM (Dulbecco
modified Eagle medium, Sigma) supplemented with 10% FBS with 1X stabilized antibiotic-
antimycotic solution (Sigma) in a CO incubator at 37ºC with 5% CO and 90% relative humidity. The
2
2
effect of diosgenin analogues 10, 13, 14, and 16 was evaluated on production of pro-inflammatory
cytokines in LPS induced inflammation in primary macrophage cells isolated from peritoneal cavity of
Swiss albino mice.
Cells were pretreated with 1µg/mL and 10µg/mL of diosgenin analogues 10, 13, 14, and 16 and
standard anti-inflammatory drug, dexamethasone (Sigma Aldrich, USA) for 30 min. The cells were
stimulated with lipopolysaccharide (LPS; 0.5µg/mL). After incubation with LPS for 24h, supernatants
were collected and immediately frozen at −80ºC. 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
(
100µL/well) with specific mouse TNF-α and IL-6 capture antibody respectively and incubated
overnight at 4ºC. The plate was blocked with 200µL/well assay diluents. Culture supernatant and
standard (100µL) were added into the appropriate coated wells and incubated for 2h at room
temperature (20–25ºC). After incubation, the plates were washed thoroughly 5 times with wash buffer.
1
00µL of detecting solution (detection antibody and streptavidin HRP) were added into each well.
Plates were sealed and incubated for 1h at RT and then the plates were washed thoroughly 5 times with
wash buffer. 100µL of tetramethylbenzidine (TMB) substrate solution were added to each well and
incubated (without plate sealer) for 30 min at room temperature in the dark. 50µL of stop solution (2N
H
2
SO ) was added to each well. The color density was measured at 450 and 570 nm using a microplate
4
reader (Molecular Devices, USA). Subtract absorbance at 570 nm from absorbance 450 nm. The values
of TNF-α and IL-6 were expressed as pg/mL. The % inhibition of pro-inflammatory cytokine
production was calculated as follows:
%
inhibition=100 x (concentration of vehicle control-concentration of test treatment)/ OD of concentration of vehicle
control, where vehicle control indicates cells treated with vehicle in LPS-induced inflammation.
2
.4.6. In-vivo acute oral toxicity for safety studies
In view of significant anti-cancer activity of compound 16 in in-vitro model, acute oral toxicity of the
same was carried out in Swiss albino mice. Experiment was conducted in accordance with the
Organization for Economic Co-operation and Development (OECD) test guideline No 423 (1987). For
this study, 30 mice (15 male and 15 female) were taken and divided into five groups comprising 3 male
and 3 female mice in each group (n=6) weighing between 20-25 g. The animals were maintained at
0
2
2±5 C with humidity control and also on an automatic dark and light cycle of 12h. The animals were
fed with the standard mice feed and provided ad libitum drinking water. Mice of Group I was kept as
10

control and animals of Groups II, III, IV and V were kept as experimental. The animals were
acclimatized for 7 days in the experimental environment prior to the actual experimentation.
Compound 16, suspended in carboxymethylcellulose (CMC, 0.7%) was given at 5, 50, 300 and
1
000mg/kg body weight to animals of Groups II, III, IV and V 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 compound 16 and thereafter 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 lachrymation, pilo-erection respiratory patterns,
changes in gait, posture and response to handling were also recorded [21]. In addition to observational
study, body weights were recorded and blood and serum samples were collected from all the animals
th
on 7 day of the experiment. The samples were analysed for total RBC, WBC, differential leucocytes
count, haemoglobin percentage and biochemical parameters like ALP, SGPT, SGOT, total cholesterol,
triglycerides, creatinine, bilirubin, serum protein and tissue protein activity. The animals were then
sacrificed and were necropsed for any gross pathological changes. Weights of vital organs like liver,
heart, kidney etc. were also recorded [22].
2
.4.7. Statistical analysis
Statistical analysis for cytotoxicity was carried out in Microsoft Excel. Antiinflammatory results were
presented as the means ± SE and analyzed using GraphPad Prism 4. The ANOVA followed by Tukeys
multiple comparison test was used to assess the statistical significance of vehicle verses treatment
groups. Results are presented as the means ± SE. Differences with a P value <0.05 were considered
significant.
3
. Results
3
.1. Chemistry
Scheme-1 denotes the synthetic strategy of these derivatives starting with diosgenin (1). The 3-alcohol
of diosgenin (1) was protected as acetyl derivative by treating it with acetic anhydride-pyridine in dry
chloroform at room temperature to get diosgenin 3-acetate (2) in 93% yields. Derivative 2 was oxidized
at 7-position by treating with Collins reagent i.e. Dipyridine-CrO
diosgenin 7-oxo-3-acetate (3) in 68% yield. The Collins reagent was prepared in-situ from dry pyridine
and CrO in dichloromethane during the reaction and subsequently substrate 2 was added. 7-Keto
3
in dry dichloromethane to afford
3
derivative (3) was converted to keto-oxime (4) by treating it with hydroxylamine hydrochloride in dry
pyridine under reflux in 96% yield. The oxime hydroxyl was used as a handle to hook-up various
ethylbromo fatty acid esters. Oxime 4 was refluxed with various ethylbromo fatty acid esters in THF-
sodium hydride to get fatty acid esters of diosgenin-7-oxime (5-10) in quantitative yields (68-87%).
All these esters were carefully hydrolysed by treating with 5% aqueous-alcoholic (1:9) KOH to give
corresponding free fatty acids (11-16). Wittig reaction on 7-keto derivative (3) with various benzylic
Wittig salts (Ph
3
P-CH -Ar, 4-nitro, 3,4,5-trimethoxy, 3,4-methylenedioxy) was unsuccessful and
2
11

always yielded unexpected 3,5-dienone derivative (17) as an exclusive product. All these products were
purified through column chromatography and confirmed by spectroscopy [Supporting information].
[
Scheme 1]
Compound 17 was obtained as white amorphous powder. Its structure was established by spectroscopy.
1
3
Its molecular formulae C27
H
38
1
O
3
was determined by its C NMR and ESI-HR-MS at m/z 411.2872
+
13
[
M+H] (Calcd. 411.2899). In H NMR acetyl protons were absent which was further confirmed in C
NMR (δ21.6 and 170.6 both absent). C NMR showed total 27 distinct carbons (four CH
1
3
3
, eight CH ,
2
ten CH and five quaternary) indicating two less carbons as compared to substrate 3 which was for 3-
acetyl group. There were six resonances at δ 109.51 (C22), 124.32 (C6), 128.05 (C4), 137.13 (C3),
1
61.59 (C5) and 202.14 (C7) in the transformed product 17. Absence of acetyl group and introduction
3
,4
of one more double bond (∆ ) showed possibility of dehydroacetoxylation of 3-acetoxy group as
acetic acid. Rest of the carbon resonances were similar to substrate 3 exhibiting intact rings C, D, E and
F. With all these observations the structure of transformed product 17 was confirmed as (22β,25R)-7-
oxo-spirosta-3,5-diene.
The purity of compounds 9, 19, 11, 13, 14 and 16 was determined following high-low
chromatographic approach by changing hydrophobic interactions without any acid additives unless
otherwise mentioned. The purity of the compounds was 96.0-99.4%. As expected, mobile phase-I (70%
acetonitrile and water) produced more symmetrical peak shapes than mobile phase composition –II and
earlier retention times (mobile phase-I: 9.937, 3.261, 3.268, 9.067, 6.662 and 5.811min; mobile phase-
II: 12.208, 4.935, 4.971, 8.847, 6.730 and 6.607 min) for all the compounds analyzed except compound
1
6. Broad peak shape of compound 16 was observed when chromatographed with mobile composition-
II due structural characteristic (presence of heptanoid free acid chain) which became symmetrical on
introducing acetic acid (1%, v/v) as ion-paring reagent by reducing charge-charge interaction.
3
3
.2. Pharmacology
.2.1. Cytotoxicity
All the semi-synthetic analogues were evaluated for anticancer activity against a panel of human cancer
cell lines viz. FaDu (hypopharyngeal carcinoma), A549 (Lung carcinoma), DLD1 (colorectal
adenocarcinoma), MCF-7 (Breast adenocarcinoma), DU145 (prostate carcinoma) and PC3 (Prostate
carcinoma) (Table 1). Out of thirteen analogues only eight analogues exhibited significant cytotoxicity
(
IC50<50µM). Rest of the analogues was declared as inactive. However, analogues 13 and 16 exhibited
good activity. Most of the fatty acids derivatives of diosgenin were active. Fatty acid ester derivatives
with six (9) and seven (10) carbon chain were active. Diosgenin 7-ketoxime analogue with longest fatty
acid chain was the best analogue of the series. Analogue 16 was the best representative from the series
exhibiting potent anticancer activity (IC50=12.8µM) against prostate cancer cell line DU-145.
[
Table 1]
12

3
.2.2. Cell cycle analysis
Cell cycle is an important phenomenon in cells for their proliferation and multiplication. It is controlled
by a complex series of signaling pathways by which cell grows, replicates its DNA and divides. In cell
cycle analysis, diosgenin analogue 16 significantly induced S phase arrest in DU-145 prostate cancer
cells at 13µM concentration. In 24h incubation S phase arrest was quite significant but there was no
such effect at 48h incubation. (Figure 2).
[
Figure 2]
3
.2.3. Western blot experiment
In the present study, we found cleavage of PARP in DU-145 cells at 24h exposure to the diosgenin
analogue 16 at 13µM concentration implying activation of caspases by the molecule to trigger apoptosis
(Figure 3).
[
Figure 3]
3
.2.4. Antiinflammatory activity of diosgenin derivatives
Cancer and inflammation are associated with each other. The antiinflammatory effect of theses
cytotoxic compounds was assessed against lipopolysaccharide (LPS) induced proinflammatory
cytokines (TNF-α and IL-6) (Table 2). All the four diosgenin derivatives 10, 13, 14 and 16 exhibited
moderate level of antiiflammatory activity through inhibition of TNF-α (31.8-47.7%) at 1µg/mL and
1
0µg/mL doses. However, inhibition of IL-6 was very low (13.8-23.3%) as compared to diosgenin and
dexamethasone. Anticancer agent with antiinflammatory property is rare and more desirable.
[
Table 2]
3
.2.5. Safety studies
There were no observational changes, morbidity and mortality during the experimental period in all the
group of experimental animals up to the tested dose levels of 1000mg/kg. Blood and serum samples did
not show any significant changes in the parameters studied like haemoglobin level, RBC count, WBC
count, differential leucocytes count, SGPT, ALP, creatinine, triglycerides, cholesterol, albumin, serum
protein (Table 3 & Fig. 5) except serum albumin and SGOT levels. Serum albumin level was found to
be significantly higher (Table 4) in the group of the animals treated with the test compound at
1
000mg/kg body weight compare to the control while SGOT level was found to be significantly higher
(
Table 3) in the group of animals treated with the test compound at 1000mg/kg body weight compare to
control and all other treatment groups. Animals on gross pathological study did not show any change in
any of the organs studied including their absolute and relative weight (Figure 4). Since, rise in serum
albumin level indicates damage of liver function and burden on vital function of kidney and immune
system and additional significant rise in the level of SGOT indicate that the test compound 16 found to
be toxic at 1000mg/kg body weight. Therefore, the experiment showed that compound 16 is well
tolerated by the Swiss albino mice up to the dose level of 300mg/kg body weight as a single acute oral
13

dose. However, it is advisable to conduct sub-acute and or chronic experiment with the test drug to
observe for any adverse effect on continuous exposure of compound 16 in view of significant increase
in serum albumin and SGOT level in animals treated with the test drug at 1000mg/kg.
[
Table 3]
[
Figure 4] [Figure 5]
4
. Discussion
Both dioscin and diosgenin exhibit cancer chemopreventive and cytotoxic effects [23a,b]. Diosgenin
suppresses fatty acid synthase expression through modulating Akt, mTOR and JNK phosphorylation
[
24]. While, dioscin induces apoptosis by the activation of p38 MAPK and JNK through the caspase
dependent mitochondrial death pathway in HL-60 cells [25]. The long chain fatty acid/ester analogues
of diosgenin have shown moderate cytotoxic activity against DU-145 prostate cancer cell line.
Compound 16 exhibited good anticancer activity (IC50=12µM).
Prostate cancer is the second most frequently diagnosed cancer in men accounting about 15% of
all male cancers [26]. However, its prevalence is higher and prognoses is poorer in developed countries
than the rest of the world. A complete understanding of the causes of prostate cancer remains unclear
[
27]. The treatment includes surgery, radiation therapy, brachytherapy, high intensity focused
ultrasound, chemotherapy, hormonal therapy or some combinations [28a,b]. But, most of the hormone
dependent cancers has become resistant to treatment after one to three years and regain growth against
hormone therapy [29]. There is still a need for suitable anti-prostate cancer drug.
The cytotoxic effect of compound 16 was found to be through activation of caspase pathway. In
our western blot experiments compound 16 cleaved PARP (Poly ADP-ribose polymerase), a family of
cysteine-aspartic protease proteins involved in a number of cellular processes like DNA repair and
programmed cell death. Caspase family plays an important role in the activation of apoptosis. Caspase-
3
is a critical promoter of apoptosis. It is responsible for proteolytic degradation of PARP which
facilitates cellular disassembly serving as a marker cells leading to apoptosis [30]. Both caspase-3 and
caspase-9 are crucial to initiate caspase cascade leading to apoptosis. Caspase-9 is an initiator caspase
while caspase-3 is an effecter (executioner) caspase [31]. PARP is involved in DNA repair when cell
experiences environmental stress [32a]. During apoptosis, PARP is cleaved by caspases and inactivated
[
32b,c]. Hence, cleavage of PARP is a remarkable marker for detection of apoptosis. In the present
study, we found cleavage of PARP in DU-145 cells at 24h exposure to the compound 16 implying
activation of caspases by the molecule to trigger apoptosis. In some of the cancer cells diosgenin
induces apoptosis through caspase pathway [33].
Cell cycle regulation ensures the precision in genomic replication and cell division [34]. The
two major checkpoints G
1
/S and G /M transitions allow the cells to control any modification in DNA
2
content. ‘Cell cycle arrest’ is a stopping point in the cell cycle, where it is no longer involved in the
processes related to duplication and division. Diosgenin analogue 16 caused S phase arrest in 24h in
DU-145 cells and induced apoptosis. Induction of cell cycle arrest in cancer cell lines constitutes one of
the most prevalent strategies to stop or limit cancer spreading [35].
14

Cancer and inflammation have been linked with each other [36a,b]. Sometimes prolonged
inflammation becomes cause to cancer. Inflammation is associated to cancer even after development of
cancer. Role of NF-κB and many other inflammatory mediators like cytokines (TNFα etc.) in tumor
promotion have been linked [37]. These pro-inflammatory factors are over-expressed during the
progression of cancer. Diosgenin analogues 14 and 16 inhibited TNF-α and IL-6 expressions
moderately at 1µg/mL and 10µg/mL concentrations. This additional property is very important and
desirable in anticancer agents. Diosgenin and its analogues have been reported to possess
antiinflammatory property previously [38].
In acute oral toxicity compound 16 was found to be safe up to 300mg/kg oral dose. But at a
higher dose of 1000mg/kg, there were some indications of liver damage. There was concentration
dependent liver toxicity only in two parameters of liver i.e. SGOT and albumin, rest other parameters
were normal. In case of anticancer drug through oral administration, it is possible. Soft drug design
approach should be applied to tackle this problem. However, therapeutic index will be important to
monitor the dose and route of administration of this compound.
4
. Conclusion
Present study has provided several novel analogues of diosgenin at C7 position. Two of the analogues
possessed significant anticancer and antiinflammatory activities simultaneously, which is rare and
desirable. Analogue 16, the best representative of the series, exerted cell cycle arrest at S phase and
induced apoptosis. The mechanism of antiproliferative action of compound 16 is through caspase
dependent apoptosis pathway. Compound 16 is a safe anticancer derivative of diosgenin. The lead
obtained in this study can further be optimized for better activity in future.
Supporting Information
Spectral data of compounds are available on online version.
Acknowledgement
Authors are thankful to the Directors of CSIR-CDRI and CSIR-CIMAP for constant encouragement
and support. Mrs. Namita Gupta is acknowledged for help in spectral data. The financial support from
NMPB through GAP-333 is duly acknowledged. TWAS-CSIR fellowship to Mr. A. A. Hamid is duly
acknowledged.
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19

Figures, Tables and Scheme
Figure 1: Various bioactive diosgenin related compounds and present prototype.
Figure 2: Cell cycle analysis of analogue 16 in DU-145 cells
Figure 3: Cleavage of PARP, a caspase target by compound 16 in DU-145 cell lines.
Figure 4A. In- silico molecular docking studies elucidating the possible mechanisms of diosgenin (1),
its analogues 14 and 16, and positive control doxorubicin with Caspase-9 (PDB: 1NW9). The docking
studies were carried out using ‘AutoDock Vina’.
Figure 4B. In- silico molecular docking studies elucidating the possible mechanisms of diosgenin (1),
its analogues 14 and 16, and positive control doxorubicin with Caspase-3: (PDB: 3KJF). The docking
studies were carried out using ‘AutoDock Vina’.
Figure 5. Effect of compound 16 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).
Figure 6. Effect of compound 16 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).
Table 1. Cytotoxicity activity of diosgenin analogues against human cancer cell line by
Sulphorhodamine assay (incubation period=48h);
Table 2: Effect of Diosgenin analogues on production of LPS-induced pro-inflammatory cytokines in
primary macrophage cells
Table 3. Details about docking studies carried out for finding of molecular interactions of compounds
under study
Table 4. Effect of compound 16 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on body
weight, haematological and serum biochemical parameters in Swiss albino mice (Mean±SE; n=6; *
P<0.05 compared to control; a P<0.05 compared to control, 5, 50, 300, 1000 mg/kg).
Scheme 1: Reagents and conditions: (a) Ac
dry Pyridine, CrO , 4h, 68%; (c) dry pyridine, NH
ethylbromo ester, RT, 0.5h-2h, 70-92%; (e) 1.2%KOH in water:MeOH (1:9), RT, 1-2h, 84-91%; (f) dry
toluene, PPh .CH -Ar, NaH, reflux, 2-4h, 84-91%.
2
O, dry pyridine, dry CHCl
3
, RT, 2h, 91%; (b) Dry DCM,
3
2
OH-HCl, reflux, 2h, 96%; (d) dry THF, NaH,
3
2
20

Figure 1
O
O
O
O
O
O
O
O
HO
OH
OH
OH
OH
O
O
HO
HO
H
HO
O
O
-
2 2
N-O (CH )n-COOR
O
O
R
1
O
H
3
C
OH
H
HO
O
Diosgenin
O
Timosaponin A-III
H
3
C
O
O
Prototypes
HO HO OH
Dioscin
HO
HO
OH
Figure 2
8
6
4
2
0
Compound 16
0
Control
0
0
0
2
4 h
8 h
4
G0/G1
S
G2/M
Figure 3
Figure 4A
21

Diosgenin
Analogue 14
Analogue 16
Doxorubicin
Figure 4B:
Diosgenin
Analogue 14
Analogue 16
Doxorubicin
Figure 5
1
1
1
.60
.40
.20
Absolute Organ Wt.(g)
7
.00
.00
Relative Organ Wt.(% Body
weight)
6
Control
Control
5mg/kg
5
4
3
2
1
0
.00
.00
.00
.00
.00
.00
5
5
3
1
mg/kg
1.00
0.80
5
3
1
0mg/kg
0mg/kg
00mg/kg
000mg/kg
00mg/kg
000mg/kg
0
0
0
0
.60
.40
.20
.00
Liver Lungs Brain KidneySpleen Heart
Major Body Organ
Major Body Organ
Figure 6.
22

Differential Leucocyte Count
Control
7
6
5
4
3
2
1
0
5
5
3
1
mg/kg
0
0
0
0
0
0
0
0 mg/kg
00 mg/kg
000 mg/kg
Table 1:
a
IC50(µM) (Mean±SE)
Compound Name
9
FaDu
A549
DLD1
MCF-7
35.10±3.54
DU145
PC3
33.30±5.89
26.95±7.19
31.64±6.02
21.95±3.06
16.56±1.57
15.21±0.90
>50
35.49±2.66
31.52±2.56
‐‐‐
1
0
1
21.92±4.18
29.04±3.45
26.20±3.99
17.05±3.61
40.20±2.32
31.22±160
22.70±3.13
16.01±1.88
14.55±0.64
2.19±0.34
0.104±0.013
16.34±
29.43±5.63
33.34±0.98
22.73±4.59
15.59±1.89
35.43±0.48
33.03±0.60
21.19±2.14
14.67±1.26
‐‐‐
1
‐‐‐
‐‐‐
13
14
21.85±0.53
16
15.77±2.22
15.77±1.98
12.82±1.26
25.39±1.70
<1.25
<1.25
<1.25
<1.25
<1.25
Doxorubicin
Podophyllotoxin
Tamoxifen
<1.25
<1.25
<1.25
<1.25
ND
10.07±0.46
8.29±
15.68±
19.82±
‐‐‐
a
a=mean of three independent experiments (n=3); IC50>50µM inactive.
Table 2:
Compound
LPS
Dose
TNF-α
(pg/mL)
TNF-α IL-6
% inhibition (pg/mL)
IL-6
(
0.5µg/mL)
(µg/mL)
% Inhibition
Normal
Vehicle
-
-
-
24.66±0.72
NA
288.33±85.11
NA
#
#
0.5µg/mL
0.5µg/mL
153±6.17
00
2733±29.40
00
*
**
**
1
1
1
1
0
3
4
6
1
104.33±7.21
31.80±4.71
38.45±1.58
33.76±1.44
35.83±2.92
34.20±6.50
47.71±2.74
36.60±4.71
41.72±7.83
23.20±3.77
2354±61.69
13.86±2.25
16.02±2.01
13.02±0.74
17.24±3.10
14.41±1.48
23.33±3.24
17.63±2.88
19.73±1.66
12.26±2.76
***
***
1
0
0
94.16±2.42
2295±55.01
*
**
**
0.5µg/mL
0.5µg/mL
0.5µg/mL
0.5µg/mL
1
101.33±2.20
2353.66±20.40
***
***
1
1
98.16±4.47
2261.66±84.84
*
**
**
1
100.66±9.95
2339±40.46
*
**
***
0
80±4.19
97±7.21
2095.33±88.64
***
1
2251±78.83***
*
**
***
1
0
89.16±11.98
117.50±5.77
2193.66±45.60
2397.66±75.51*
*
Diosgenin
1
23

*
**
***
1
0
0
95.16±5.08
37.79±3.32
61.11±4.89
76.57±6.23
2048±36.75
25.06±1.34
66.13±2.74
74.60±3.16
***
***
Dexamethasone
0.5µg/mL
1
59.5±7.48
925.66±75.13
***
***
1
35.83±9.53
694 ±86.49
≠
ns
*
Normal vs Vehicle; P<0.05 ; Vehicle vs Treatment; ‐not significant, P<0.05; n=3
Table 3.
24

Compd
code
Caspase-3 (PDB entry 3kjf)
Binding
Caspase-9 (PDB entry 1nw9)
Binding
affinity
Kcal/mol)
Binding pocket amino acids
affinity
Binding pocket amino acids
(
(kcal/mol)
GLY[A]-304, GLY[A]-305, GLY[A]-
306, TYR[A]-324, ASN[B]-265,
ASN[B]-268, GLN[B]-245, GLN[B]-
320, GLU[B]-261, ILE[B]-266, ILE[B]-
ALA[A]-162, ARG[A]-64, CYS[A]-163,
GLN[A]-161, GLU[A]-123, GLY[A]-
1
6
6
2
2
2
22, GLY[A]-165, HIS[A]-121, MET[A]-
1, PHE[A]-128, SER[A]-120, THR[A]-
2, THR[A]-166, ARG[B]-207, ASP[B]-
53, PHE[B]-250, PHE[B]-252, PHE[B]-
56, SER[B]-205, SER[B]-251, TRP[B]-
06, TYR[B]-204
341,
LYS[B]-262, LYS[B]-280, PHE[B]-
246, PHE[B]-267, PHE[B]-319,
PRO[B]-318, PRO[B]-338, SER[B]-
271, SER[B]-339, THR[B]-337,
VAL[B]-264
GLY[A]-304, GLY[A]-305, GLY[A]-
06, TYR[A]-324, ASN[B]-265,
ASN[B]-268, ASP[B]-340, GLN[B]-
245, GLN[B]-320, GLU[B]-261,
LEU[B]-217,
LEU[B]-244,
1
4
-10.2
-7.4
3
ALA[A]-162, ARG[A]-64, CYS[A]-163,
GLN[A]-161, HIS[A]-121, SER[A]-63,
SER[A]-65, SER[A]-120, THR[A]-62,
ARG[B]-207, ASN[B]-208, ASP[B]-253, -7.7
PHE[B]-250, PHE[B]-252, PHE[B]-256,
SER[B]-205, SER[B]-209, SER[B]-251,
TRP[B]-206
GLY[B]-269, GLY[B]-276, GLY[B]-
277, ILE[B]-266, ILE[B]-341, LEU[B]-
1
6
-8.3
217,
LYS[B]-280, PHE[B]-246, PHE[B]-
267, PHE[B]-319, PRO[B]-318,
PRO[B]-338, SER[B]-271, SER[B]-
39, THR[B]-337, VAL[B]-264
ARG[A]-258, GLY[A]-304, GLY[A]-
05, GLY[A]-306, LYS[A]-299,
TYR[A]-324, ASN[B]-265, ASN[B]-
68, GLN[B]-245, GLN[B]-320,
GLU[B]-261, GLY[B]-269, ILE[B]-
41, LEU[B]-244, PHE[B]-246,
PHE[B]-267, PHE[B]-319, PRO[B]-
18, PRO[B]-338, SER[B]-339,
THR[B]-337
ARG[A]-258, CYS[A]-303, CYS[A]-
327, GLY[A]-304, GLY[A]-305,
GLY[A]-306, GLY[A]-326, HIS[A]-
302, LEU[A]-256, LYS[A]-299,
LEU[B]-244,
LEU[B]-275,
3
3
CYS[A]-163, GLU[A]-123, GLY[A]-122,
GLY[A]-165, HIS[A]-121, MET[A]-61,
2
PHE[A]-128, THR[A]-62, THR[A]-166,
-8.5
-
8.4
Diosgenin
ARG[B]-207, PHE[B]-250, PHE[B]-256,
3
SER[B]-205, SER[B]-251, TRP[B]-206,
TYR[B]-204
3
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,
-8.2
Doxorubicin -8.2
ASN[B]-208, ASP[B]-253, PHE[B]-250,
PHE[B]-252, PHE[B]-256, SER[B]-205,
SER[B]-251, TRP[B]-206, TYR[B]-204
LYS[A]-328, GLU[B]-261, HIS[B]-
243, LEU[B]-244, SER[B]-242
25

Table 4:
Dose of compound 16 at mg/kg body weight as a single oral dose
Parameters
Control
24.18±1.82
11.19±.39
6.29±0.26
11.27±0.61
5 mg/kg
21.35±1.85
12.07±0.50
6.17±0.29
12.49±1.07
50 mg/kg
23.0±0.74
12.51±0.63
6.28±0.03
10.84±0.35
300 mg/kg
22.41±1.53
11.80±0.47
5.93±0.32
11.32±0.64
1000 mg/kg
23.26±0.91
11.36±0.41
5.54±0.34
th
Body wt. 7 day (g)
Haemoglobin (g/dL)
3
RBC (millions/mm )
WBC
11.68±0.39
3
(
thousands/mm )
ALP (U/L)
264.1±20.6
29.77±2.07
22.41±1.06
5.03±0.25
0.51±0.04
123.2±11.56
0.95±0.02
167.3±16.5
33.79±2.19
24.24±0.86
6.45±1.17
0.63±0.10
139.5±8.99
0.97±0.08
191.6±27.6
31.86±2.13
25.93±1.30
7.20±0.85
0.55±0.13
145.8±18.47
1.00±0.07
224.0±16.70
33.05±2.35
24.47±1.45
6.47±0.18
234.3±24.5
a
SGOT (U/L)
SGPT (U/L)
43.80±1.19
25.21±1.32
8.48±1.07*
0.50±0.05
Albumin (g/dL)
Creatinine (mg/dL)
Triglycerides (mg/dL)
Serum Protein
0.74±0.08
135.2±16.08
0.90±0.05
133.1±13.45
0.85±0.05
(
mg/ml)
Cholesterol (mg/dL)
277.80±18.9
0.06±0.01
295.97±21.7
0.14±0.07
270.90±23.4
0.09±0.01
337.96±14.7
0.08±0.02
281.37±18.6
0.12±0.04
Bilirubin (mg/dL)
Scheme 1:
O
O
O
O
O
O
O
b
O
c
a
HO
AcO
AcO
O
2
3
AcO
N-OH
1
4
d
O
O
O
e
O
AcO
N-OR
HO
N-OR
1
1-16
5-10
-COOCH
1
1
1
1
1
1
1: R1=H, R2= -CH
2: R1== -CH -CH
3: R= -CH -CH -CH
4: R= -CH
5: R= -CH
6: R= -CH
2
-COOH;
-COOH;
COOH;
-CH -COOH;
5: R= -CH
2
2
-CH
-COOCH
-CH COOCH
-CH -COOCH
CH -CH -COOCH
-CH -CH -CH COOCH
3
;
2
2
6: R= -CH
7: R= -CH
2
-CH
-CH
2
2 3
-CH ;
2
2
2
2
2
2
2
-CH
3
;
2
-CH
-CH
-CH
2
-CH
-CH
-CH
2
2
8: R= -CH
9: R= -CH
2
-CH
-CH
2
-CH
-CH
2
2
2 3
-CH ;
2
2
2
2
2
2
CH
CH
2
-CH
-CH
2
-COOH;
-CH -COOH;
2
2
2
2
2
2 3
-CH ;
2
2
2
10: R= -CH
2
-CH
2
-CH
2
2
2
2
2
-CH
3
;
O
O
O
O
f
O
AcO
O
3
17
26

…
………………..
Graphical Abstract
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(22β,25R)-3β-hydroxy-spirost-5-en-7-iminoxy-heptanoic
Leave this area blank for abstract info.
acid exhibits anti-prostate cancer activity through
caspase pathway
A.A. Hamid, Tanu Kaushal, Raghib Ashraf, Arjun Singh,
Amit Chand Gupta, Om Prakash, Jayanta Sarkar,
Debabrata Chanda, D.U. Bawankule, Feroz Khan, Karuna Shanker,
*
O.O. Aiyelaagbe, Arvind S. Negi
Cell cycle arrest
DU145)
S-Phase arrest
Cytotoxicity
(
DU145 (Prostate cancer)
IC50=12.8µM
O
O
Apoptosis induction
27

Highlights
*
*
*
*
*
*
Diverse long chain fatty acid/esters of diosgenin‐7‐ketoxime have been prepared.
Analogue 16, cytotoxicity IC50=12µM prostate cancer, cell cycle arrest at S phase.
Induces apoptosis through caspase pathway, shows affinity to caspase‐3/9.
Simultaneously antiiflammatory property by inhibition of TNF‐α and IL‐6.
Safe up to 1000mg/kg dose in Swiss‐albino mice.
Simultaneous, antiinflammatory property in an anticancer molecule is favourable.
28