Ring A structural modified derivatives of withaferin A and the evaluation of their cytotoxic potential

Article abstract of DOI:10.1016/j.steroids.2011.05.012

Regio-/stereoselective Michael addition to ring A of withaferin-A was performed using an optimized reaction procedure to synthesise a library of 2,3-dihydro,3-β-substituted withaferin-A derivatives. The analogues thus obtained were evaluated for in vitro

Full text of DOI:10.1016/j.steroids.2011.05.012

Steroids 76 (2011) 1213–1222
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Ring A structural modified derivatives of withaferin A and the evaluation of their
cytotoxic potential
Syed Khalid Yousufa, Rabiya Majeedb, Mudassier Ahmadb, Payare lal Sangwana, Basant Purnimaa,
A.K. Saxsenab, K.A. Suria, Debaraj Mukherjeea,∗, Subhash Chandra Tanejaa
a Natural Product Chemistry, Indian Institute of Integrative Medicine, Canal Road Jammu, 180001, India
b Cancer Pharmaclogy, Indian Institute of Integrative Medicine, Canal Road Jammu, 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2011
Received in revised form 24 May 2011
Accepted 26 May 2011
Available online 12 June 2011
Regio-/stereoselective Michael addition to ring A of withaferin-A was performed using an optimized
reaction procedure to synthesise a library of 2,3-dihydro,3-␤-substituted withaferin-A derivatives. The
analogues thus obtained were evaluated for in vitro cytotoxicity against various human cancer cell lines.
3-Azido analogue exhibited 35-fold increase (IC50 = 0.02–1.9 ␮M) in cytotoxicity against almost the entire
cell lines tested when compared to the parent molecule. However, further modifications of 3-azido
analogue with various alkynes under Husigen’s cycloaddition conditions generated a variety of triazole
derivatives with reduced cytotoxicity.
Keywords:
Withaferin A
Michael addition
3+2 Cycloaddition
␣,␤-Unsaturated group
Cytotoxicity
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
covery, trapping of the nucleophiles like thiols by covalent coupling
represents an important mechanism of biological activity [4]. This
The presence of steroidal structural framework in natural prod-
ucts has opened new vistas in medicinal and biological chemistry.
They are part of the cell membrane and their significance can be
attributed to essential biological functions including adrenal and
sex hormones, bile acids, precursors of certain vitamins as well as
their roles in steroid based chemotherapeutics such as in inflam-
mations, and metabolic diseases. The steroidal framework provides
a template for structural modifications as these are generally func-
tional group rich chemical entities. Moreover, these biologically
significant motifs are reported to be non-toxic, less susceptible to
multidrug resistance (MDR) and highly bio-available owing to their
capability to penetrate the biological membranes [1]. This is also
true to the skeleton with an ␣,␤-unsaturated function whereby the
assimilation of heteroatom (S, N & O) has been reported to augment
or modulate the biological activities of newly generated chemi-
cal entities [2]. Literature is full of examples underscoring natural
products with ␣,␤-unsaturated carbonyl moiety exhibiting cancer
chemo-preventive and chemo-protective activities (Fig. 1) [2].
The main characteristic related to biological profile of ␣,␤-
unsaturated carbonyl compounds is their ability to act as Michael
acceptors [3]. Although, traditionally shunned in modern drug dis-
has lead to the discovery of many biologically relevant pathways to
understand the mechanism of action of the particular drug can-
didate [2]. Research on Michael acceptors, long confined to the
realm of toxicology [5] was rekindled by the development of the
antioxidant inflammation modulator homoterpenoid bardoxolone
methyl (RTA402) [6], which was given the orphan drug status by
the FDA for the treatment of pancreatic cancer. Currently, irre-
versible binding at active sites has been demonstrated to be one
of the reasons to drug resistance in cancer treatment [2]. Thus,
in order to include the ␣,␤-unsaturated carbonyl group in drug
development strategies, the potentially adverse effects must be
circumvented. This can be achieved by fine tuning the reactiv-
ity of the ␣,␤-unsaturated carbonyl unit leading to a high degree
of specificity. Different approaches for comparing similar natural
products and synthetic molecules have been followed in order to
predict the reactivity of ␣,␤-unsaturated carbonyl compounds. The
␣,␤-unsaturated carbonyl moiety permits the direct adjustment of
electrophilicity and reduction potential through modification of the
peripheral substituents [2].
In this regard, withaferin A (KK-1), a steroidal lactone from
Withania somnifera (Aswagandha) has attracted the attention of
chemists as well as biologists due to its interesting structure
and anti-cancer [7,8], adaptogenic, anti-stress, anti-convulsant,
immunomodulatory and neurological effects [9,10]. It is known to
be a natural proteosome inhibitor [11], an agent inducing apoptosis
∗
Corresponding author. Tel.: +91 191 2569000 006; fax: +91 191 2569111 333.
E-mail address: dmukherjee@iiim.res.in (D. Mukherjee).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.05.012

1214
S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
O
O
O
O
O
N
H
O
OH
H
HO
O
O
O
OH
O
O
HOOC
OCONH2
O
HO
HO
O
Herbimycin A (1)
15d-PGJ2 (2)
Kaempferol (3)
Helenalin (4)
Fig. 1. Natural products with ␣,␤-unsaturated carbonyl functionality.
by inhibiting topoisomerase-I DNA complex [12] and a mitotic poi-
son with antiangiogenic potential [13]. Even as the actual mode
of action of withaferin A remains ambiguous and may be spe-
cific to cell-types. Recently, it has been reported to induce actin
microfilament aggregation mediated by annexin-II [14]. The chem-
istry of Withania species has been extensively studied and several
groups of chemical constituents such as steroidal lactones, alka-
loids, flavonoids, and tannin have been identified, extracted, and
isolated [15]. At present, more than 12 alkaloids, 40 withano-
lides, and several sitoindosides (a withanolide containing a glucose
molecule at carbon 27) have been isolated and reported from aerial
parts, roots and berries of Withania species [15]. Withaferin A
(Fig. 2) was the first member of withanolide group to be isolated
from the well-known South-Asian medicinal plant, W. somnifera.
Lavie’s group [16] elucidated the structure of withaferin A in leaves
of this plant, which is mainly valued for its anti-cancerous proper-
ties.
Chemically it represents an interesting natural polyfunctional
lactone with 28 carbon atoms based on ergostane framework
wherein C (22) and C (26) are oxidized to form delta lactone with
5 major functionalities (i) ␣,␤-unsaturated ketone group in ring A,
(ii) a secondary hydroxyl group at C-4, (iii) a labile epoxide ring
between C-5 and C-6, (iv) a primary hydroxyl group at C-27 and (v)
6-membered lactone ring (E) with ␣,␤-unsaturated carbonyl group.
There are only a few reports available on the structural mod-
ification of withaferin-A owing to presence of multiple reactive
sites. Much emphasis has been given to the hydroxyl groups at C-
27 and 5␤,6␤-epoxide group for antiproliferative activity [17–19].
The only report available in the literature showed that ring A ana-
logue of withaferin-A exhibiting activity against sarcoma-180 in
mice; however, the conflicting results has made it difficult to ascer-
tain the importance of ring A. Thus, a detailed investigation of the
importance of ␣,␤-unsaturated group in ring A is much desirable.
Recently, it has been reported that 2,3-dihydrowithaferin A-3␤-O-
sulphate (5) [20] acts as a potent prodrug of withaferin-A. In the
light of promising therapeutic prospective of withaferin A as an
anticancer molecule reported by our institute [21,22], we became
interested in the study of modifications of the ring A. We envis-
aged that substitution of the ␣,␤-unsaturated carbonyl system of
withaferin A at the ␤-position may be a practical approach to
find new activities of the modified compounds as this will obviate
irreversible covalent binding in active sites by biological nucle-
ophiles, a reason behind drug resistance in cancer treatment. Thus,
in the present communication, we disclose regio- and stereose-
lective nucleophilic addition to ring A in the presence of multiple
electrophilic sites, evaluation of cytotoxicity of the so generated
library and further modification of the most active derivatives for
the pursuit of anticancer lead molecules.
2. Experimental
2.1. General methods
1H and 13C NMR spectra were recorded on 200 and 500 MHz
spectrometers with TMS as the internal standard. Chemical shifts
are expressed in parts per million (ı ppm). MS were recorded on LC
Mass spectrometer. IR spectra (KBr) discs were recorded on Bruker
Vector 22 instrument. Silica gel coated aluminium plates were used
for TLC. Elemental analyses were performed on Elementar instru-
ment. Reagents and solvents used were mostly of LR grade. The
chromatograms were visualised under UV-254–366 nm and cerric
ammonium sulphate spray.
2.2. Plant material, extraction and isolation of withaferin-A
W. somnifera leaves were collected from Jammu, India and its
identification was done by the taxonomists at IIIM Jammu. The
accession (herbarium accession no. 22004) is being maintained in
the institute farm. The shade-dried leaves (1 kg) were ground and
defatted three times with n-hexane (3 × 1.5 l) by soaking overnight
at room temperature. The spent material was further extracted
with MeOH (3 × 1 l) overnight at room temperature. The desired
28
27
25
24
23
21
OH
OH
E
22
18
14
O
O
20
17
16
O 26
O
11
O
O 19
12
13
D
C
1
9
2
3
15
10
8
A
B
5
4
7
HO3SO
6
O
O
OH
2,3-Dihydrowithaferin A-3β-O-sulphate (5)
OH
Withaferin A (KK-1)
Fig. 2. Structure of withaferin-A (KK-1) and its 3␤-O-sulphate analogue (6).

S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
1215
compound withaferin A was isolated by using the known procedure
[23].
(C-Ar), 128.4 (CH-Ar), 128.3 (CH-Ar), 128.2 (CH-Ar), 126.5 (CH-Ar),
126.4 (CH-Ar); ESI MS (m/z): 603 [M+Na]. Anal. Cal. for C34H44O6S:
C, 70.39, H, 7.70; Found: 70.31, H, 7.64.
2.3. General reaction procedure for Michael type addition [24]
KK-4: Prepared by the general procedure 1 using 1:1 mmol
(470 mg) of KK-1 and p-chlorothiophenol, purified on silica gel
(EtOAc/PE: 45/55) to obtain the product KK-4 (82%) as white solid:
[␣]D20 = +24 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 7.46 (d,
J = 8.1 Hz, 2H, Ar), 7.12 (d, J = 8.3 Hz, 2H, Ar), 4.41 (dt, J = 12.0, 5.5,
3.5 Hz, 2H, H-22), 4.27 (brs, 2H, H-27), 3.72–3.69 (m, 1H, H-3), 3.47
(d, J = 4.4 Hz, 1H, H-4), 3.23 (bs, 1H, H-6), 2.45 (dd, J = 4.3, 10.9 Hz,
2H, H-2), 2.43–2.40 (m, 2H), 2.35–2.32 (m, 1H), 2.01 (s, 3H, H-28),
1.95–1.94 (m, 3H), 1.68–1.66 (m, 2H), 1.39–1.37 (m, 2H), 1.36–1.34
(m, 2H), 1.21 (s, 3H, CH3-19), 1.15–1.14 (m, 2H), 1.12–1.10 (m, 2H),
1.01 (d, J = 6.9 Hz, 3H, H-21), 0.60 (s, 3H, CH3-18). 13C NMR (CDCl3,
50 MHz): 207.9 (qC, C-1), 29.1 (CH2, C-2), 43.2 (CH, C-3), 75.6 (CH,
C-4), 65.12 (qC, C-5), 60.4 (CH, C-6), 29.7 (CH2, C-7), 29.78 (CH, C-
8), 44.18 (CH, C-9), 50.51 (qC, C-10), 21.50 (CH2, C-11), 27.51 (CH2,
C-12), 42.71 (qC, C-13), 56.00 (CH, C-14), 24.28 (CH2, C-15), 39.09
(CH2, C-16), 51.99 (CH, C-17), 9.6 (CH3, C-18), 14.2 (CH3, C-19), 39.0
(CH, C-20), 13.7 (CH3, C-21), 77.7 (CH, C-22), 29.68 (CH2, C-23),
125.61 (qC, C-24), 153.18 (qC, C-25), 167.68 (qC, C-26), 55.7 (CH2,
C-27), 20.0 (CH3, C-28), 134.2 (qC, Ar), 132.6 (qC, Ar), 128.9, 128.8,
128.3, 128.2 (4 × CH, Ar); ESI MS (m/z): 638 [M+Na]. Anal. Cal. for
Triethylamine was added to a solution of nucleophile (1.2 equiv.)
in dry methanol (3 ml) at room temperature to maintain the pH of
8.5. Withaferin A (1 equiv., 470 mg, 1 mmol) was separately dis-
solved in dry methanol (2 ml) and the solution was added to the
methanolic solution of nucleophile and kept for the required time.
The progress of the reaction was monitored through TLC. After the
completion, the reaction mixture was dried completely, dissolved
in water (5 ml) and extracted with CHCl3 (10 ml) three times to
obtain the product.
2.4. General procedure for Husigen’s (3+2) cycloaddition [25]
CuI (10 mol%) was added to a solution of 2,3-dihydro,3-␤-azido
withaferin-A (KK-2) (513 mg, 1 mmol) and alkyne (1 mmol) in
acetonitrile (5 ml). The reaction mixture was stirred for 6–7 h at
room temperature and the progress of the reaction was monitored
through TLC. The reaction mixture was filtered and the filtrate was
concentrated over rotary evaporator, passed through silica gel col-
umn to yield the desired product (KK-11–15).
C34H43ClO6S: C, 66.45, H, 7.04; Found: C, 66.45, H, 7.11.
KK-6: Prepared by the general procedure 1 using 1:1 mmol
2.5. Spectral analysis of compounds
(470 mg) of KK-1 and p-methoxythiophenol, purified on silica gel
(EtOAc/PE: 43/57) to obtain the product KK-6 (85%) as white
solid: [␣]D20 = +30 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 7.44
(d, J = 8.5 Hz 2H, Ar), 6.8 (d, J = 8.34 Hz, 2H, Ar), 4.39 (dt, J = 12.0,
5.5, 3.5 Hz, 2H, H-22), 4.26 (brs, 2H, H-27), 3.8 (s, 3H, OMe-Ar),
3.68–3.66 (m, 1H, H-4), 3.4 (d, J = 4.2, 1H, H-3), 3.21 (bs, 1H, H-
6), 2.52 (dd, J = 4.2, 11.3 Hz, 2H, H-2), 2.43–2.41 (m, 2H), 2.35–2.33
(m, 1H), 2.06 (bs, 3H, H-28), 1.98–1.96 (m, 3H), 1.65–1.63 (m,
2H), 1.37–1.36 (m, 2H), 1.34–1.33 (m, 2H), 1.28 (s, 3H, CH3-19),
1.16–1.14 (m, 2H), 1.12–1.10 (m, 2H), 0.99 (d, J = 6.6 Hz, 3H, CH3-
21), 0.61 (s, 3H, CH3-18). 13C NMR (CDCl3, 50 MHz): 209.59 (qC,
C-1), 29.7 (CH2, C-2), 41.1 (CH, C-3), 75.8 (CH, C-4), 65.10 (qC, C-
5), 59.5 (CH, C-6), 29.6 (CH2, C-7), 29.4 (CH, C-8), 44.10 (CH, C-9),
50.30 (qC, C-10), 21.30 (CH2, C-11), 26.99 (CH2, C-12), 42.63 (qC,
C-13), 56.99 (CH, C-14), 24.01 (CH2, C-15), 39.13 (CH2, C-16), 51.95
(CH, C-17), 10.5 (CH3, C-18), 14.7 (CH3, C-19), 39.0 (CH, C-20), 13.1
(CH3, C-21), 78.3 (CH, C-22), 29.73 (CH2, C-23), 125.82 (qC, C-24),
153.56 (qC, C-25), 167.74 (qC, C-26), 56.7 (CH2, C-27), 19.6 (CH3, C-
28), 159.9 (qC, Ar), 132.6 (qC, Ar), 114.7, 114.6, 128.4, 128.3, 128.2
(5 × CH, Ar), 55.3 (OMe, Ar); ESI MS (m/z): 633 [M+Na]. Anal. Cal.
for C35H46O7S: C, 68.82, H, 7.59; Found: 68.95, H, 7.67.
KK-2: Prepared by the general procedure 1 using 1.8 mmol
(878 mg) of KK-1 and purified on silica gel (EtOAc/PE: 1/1) to obtain
the product KK-2 (73%) as white solid: [␣]D20 = +55 (c 0.1, CHCl3);
IR (KBr), 2105 cm−1 1H NMR (CDCl3, 500 MHz): ı 4.40 (dt, J = 12.0,
,
3.5 Hz, 2H, H-22), 4.30 (brs, 2H, H-27), 4.08 (ddd, J = 15.7, 4.1, 3.3 Hz,
1H, H-3), 3.46 (d, J = 4.1 Hz, 1H, H-4), 3.26 (brs, 1H, H-6), 3.07
(dd, J = 7.3,15.7,Hz, 2H, H-2), 2.54–2.53 (m, 2H, H-23), 2.35–2.32
(m, 1H), 2.05 (s, 3H, CH3-28), 1.92–1.90 (m, 3H), 1.67–1.65 (m,
2H), 1.39–1.37 (m, 2H), 1.36–1.34 (m, 2H), 1.24 (s, 3H, CH3-19),
1.14–1.13 (m, 2H), 1.11–1.10 (m, 2H), 0.93 (d, J = 6.9, 3H, CH3-21),
0.67 (s, 3H, CH3-18). 13C NMR (CDCl3, 50 MHz): ı 208.49 (qC, C-1),
38.75 (CH2, C-2), 58.87 (CH, C-3), 75.15 (CH, C-4), 65.10 (qC, C-5),
59.85 (CH, C-6), 29.7 (CH2, C-7), 29.78 (CH, C-8), 44.3 (CH, C-9),
50.6 (qC, C-10), 21.49 (CH2, C-11), 27.07 (CH2, C-12), 42.7 (qC, C-
13), 56.10 (CH, C-14), 24.4 (CH2, C-15), 39.13 (CH2, C-16), 51.1 (CH,
C-17), 9.9 (CH3, C-18), 14.8 (CH3, C-19), 39.0 (CH, C-20), 13.34 (CH3,
C-21), 78.73 (CH, C-22), 29.68 (CH2, C-23), 125.7 (qC, C-24), 153.18
(qC, C-25), 167.68 (qC, C-26), 57.33 (CH2, C-27), 20.04 (CH3, C-28);
ESI MS (m/z): 536 [M+Na]. Anal. Cal. for C28H39N3O6: C, 65.51, H,
7.60, N3, 8.10; Found: C, 65.47, H, 7.67, N3, 8.18.
KK-7: Prepared by the general procedure 1 using 1:1 mmol
(470 mg) of KK-1 and furan-2-methanethiol, purified on silica gel
(EtOAc/PE: 45/55) to obtain the product KK-7 (84%) as white solid:
[␣]D20 = +41 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 7.3 (d,
J = 9.3 Hz, 1H, H-6ꢀ), 6.31–6.20 (m, 2H, H-4ꢀ, H-5ꢀ), 4.4 (dt, J = 12.0,
5.5, 3.5 Hz, 2H, H-22), 4.28 (brs, 2H, H-27), 3.71 (bs, 1H, H-4), 3.58 (d,
J = 13.1 Hz, 1H, H-1ꢀa/b), 3.48 (d, J = 13.11 Hz, 1H, H-1ꢀa/b), 3.45–3.43
(m, 1H, H-3), 3.22 (bs, 1H, H-6), 2.54–2.53 (m, 2H), 2.51 (dd, J = 4.5,
11.0 Hz, 2H, H-2), 2.48–2.44 (m, 2H, H-2ꢀ), 2.35 (m, 1H), 2.00 (s,
3H, CH3-28), 1.94–1.93 (m, 3H), 1.69–1.67 (m, 2H), 1.41–1.39 (m,
2H), 1.35–1.33 (m, 2H), 1.26 (s, 3H, CH3-19), 1.14–1.13 (m, 2H),
1.12–1.11 (m, 2H), 1.02 (d, J = 7.0 Hz, 3H, CH3-21), 0.62 (s, 3H, CH3-
18), 13C NMR (CDCl3, 50 MHz): ı 209.8 (qC, C-1), 29.9 (CH2, C-2),
42.2 (CH, C-3), 75.4 (CH, C-4), 65.3 (qC, C-5), 60.0 (CH, C-6), 29.71
(CH2, C-7), 29.78 (CH, C-8), 44.0 (CH, C-9), 50.3 (qC, C-10), 22.12
(CH2, C-11), 27.32 (CH2, C-12), 42.81 (qC, C-13), 56.11 (CH, C-14),
24.79 (CH2, C-15), 39.35 (CH2, C-16), 51.96 (CH, C-17), 10.3 (CH3,
C-18), 14.5 (CH3, C-19), 39.8 (CH, C-20), 13.6 (CH3, C-21), 78.9
(CH, C-22), 29.68 (CH2, C-23), 125.91 (qC, C-24), 153.00 (qC, C-25),
167.83 (qC, C-26). 56.01 (CH2, C-27), 20.2 (CH3, C-28), 151 (qC, C-
KK-3: Prepared by the general procedure 1 using 1:1 mmol
(470 mg) of KK-1 and thiophenol, purified on silica gel (EtOAc/PE:
45/55) to obtain the product KK-3 (85%) as white solid:
[␣]D20 = +43.1 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 7.50
(d, J = 7.5 Hz, 2H, Ar), 7.3 (d, J = 7.2 Hz, 2H, Ar), 7.24 (m, 1H, Ar),
4.40 (dt, J = 12.0, 5.5, 3.5 Hz, 2H, H-22), 4.29 (brs, 2H, H-27), 3.45
(bd, J = 3.8 Hz, 1H, H-4), 3.74–3.68 (m, 1H, H-3), 3.22 (bs, 1H, H-
6), 2.56 (dd, J = 4.5, 11.7 Hz, 2H, H-2), 2.50–2.48 (m, 2H, H-23),
2.35–2.33 (m, 1H), 1.99 (s, 3H, H-28), 1.94–1.91 (m, 3H), 1.65–1.63
(m, 2H), 1.40–1.38 (m, 2H), 1.35–1.33 (m, 2H), 1.27 (s, 3H, CH3-19),
1.16–1.14 (m, 2H), 1.11–1.09 (m, 2H), 1.02 (d, J = 6.9, 3H, H-21), 0.64
(s, 3H, CH3-18). 13C NMR (CDCl3, 50 MHz): ı 208.0 (qC, C-1), 28.4
(CH2, C-2), 42.19 (CH, C-3), 70.0 (CH, C-4), 65.10 (qC, C-5), 59.3 (CH,
C-6), 29.7 (CH2, C-7), 29.78 (CH, C-8), 44.14 (CH, C-9), 50.45 (qC,
C-10), 21.55 (CH2, C-11), 27.04 (CH2, C-12), 42.63 (qC, C-13), 56.01
(CH, C-14), 24.24 (CH2, C-15), 39.03 (CH2, C-16), 51.90 (CH, C-17),
10.5 (CH3, C-18), 14.6 (CH3, C-19), 39.0 (CH, C-20), 13.3 (CH3, C-21),
77.7 (CH, C-22), 29.68 (CH2, C-23), 125.61 (qC, C-24), 153.18 (qC,
C-25), 167.68 (qC, C-26), 55.7 (CH2, C-27), 19.0 (CH3, C-28), 132

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S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
3ꢀ), 142 (CH, C-6ꢀ), 110.6 (CH, C-5ꢀ), 107.9 (CH, C-4ꢀ), 31.1 (CH2,
C-12), 42.63 (qC, C-13), 56.01 (CH, C-14), 24.42 (CH2, C-15), 39.03
(CH2, C-16), 51.90 (CH, C-17), 11.6 (CH3, C-18), 14.4 (CH3, C-19),
39.0 (CH, C-20), 13.6 (CH3, C-21), 78.9 (CH, C-22), 29.68 (CH2, C-
23), 125.61 (qC, C-24), 153.18 (qC, C-25), 167.68 (qC, C-26), 56.8
(CH2, C-27), 20.6 (CH3, C-28), 172.9 (qC, COOMe), 54.0 (CH3, OMe),
60.12 (CH, C-3;), 37.8 (CH2, C-2ꢀꢀ); ESI MS (m/z): 628 [M+Na]. Anal.
Cal. for C32H47NO8S: C, 63.45, H, 7.82, N, 2.31; Found: 63.52, H, 7.90,
N, 2.39.
C-1ꢀ), 30.86 (CH2, C-2ꢀ),; ESI MS (m/z): 607 [M+Na]. Anal. Cal. for
C33H44O7S: C, 67.78, H, 7.58; Found: 67.86, H, 7.47.
KK-8: Prepared by the general procedure 1 using 1:1 mmol
(470 mg) of KK-1 and methyl ester of l-phenylalanine, purified on
silica gel (EtOAc/PE: 60/40) to obtain the product KK-8 (78%) as
white solid: [␣]D20 = +19 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz):
ı 7.46–7.25 (m, 5H, Ar-H), 4.41 (dt, J = 11.9.0, 5.5, 3.5 Hz, 2H, H-22),
4.27 (brs, 2H, H-27), 3.71 (bs, 3H, COOCH3), 3.60 (dd, J = 5.0, 10.7 Hz,
1H, H-2ꢀ), 3.30 (d, J = 3.1 Hz, 1H, H-4), 3.1 (bs, 1H, H-6), 3.03–3.01
(m, 1H, H-3), 3.01 (d, J = 5.1, 2H, H-3ꢀ), 2.67 (bd, J = 14.7 Hz, 2H, H-2),
2.54 (m, 2H), 2.35 (m, 1H), 2.03 (bs, 3H, CH3-28), 1.98–1.96 (m, 3H),
1.70–1.68 (m, 2H), 1.42–1.40 (m, 2H), 1.39–1.38 (m, 2H), 1.28 (s, 3H,
CH3-19), 1.17–1.15 (m, 2H), 1.13–1.12 (m, 2H), 0.98 (d, J = 6.1 Hz, 3H,
CH3-21), 0.68 (s, 3H, CH3-18), 13C NMR (CDCl3, 50 MHz): ı 209.5
(qC, C-1), 39.7 (CH2, C-2), 42.99 (CH, C-3), 76.5 (CH, C-4), 65.10 (qC,
C-5), 61.8 (CH, C-6), 31.1 (CH2, C-7), 29.62 (CH, C-8), 44.41 (CH, C-
9), 50.45 (qC, C-10), 21.25 (CH2, C-11), 27.64 (CH2, C-12), 42.51 (qC,
C-13), 56.44 (CH, C-14), 23.99 (CH2, C-15), 39.21 (CH2, C-16), 51.99
(CH, C-17), 11.6 (CH3, C-18), 17.05 (CH3, C-19), 39.24 (CH, C-20),
13.3 (CH3, C-21), 78.9 (CH, C-22), 29.68 (CH2, C-23), 125.23 (qC, C-
24), 153.49 (qC, C-25), 167.58 (qC, C-26), 56.9 (CH2, C-27), 20.08
(CH3, C-28), 175 (qC, COOMe), 138.1 (qC, Ar), 63. 4 (CH, C-2ꢀ)), 38.6
(CH2, C-2ꢀ), 126.3, 128.9, 128.4, 128.25, 128.22 (5 × CH, Ar),; ESI MS
(m/z): 672 [M+Na]. Anal. Cal. for C38H51NO8: C, 70.29, H, 7.91, N,
2.16; Found: 70.31, H, 7.99, N, 2.21.
KK-9: Prepared by the general procedure 1 using 1:1 mmol
(470 mg) of KK-1 and methylester of l-valine purified on silica
gel (EtOAc/PE: 60/40) to obtain the product KK-9 (81%) as white
solid: [␣]D20 = +16 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı
4.40 (dt, J = 12.0, 5.5, 3.5 Hz, 2H, H-22), 4.26 (brs, 2H, H-27), 3.76
(bs, 3H, COOCH3), 3.40 (d, J = 3.4 Hz, 1H, H-4), 3.31 (bs, 1H, H-6),
3.14 (d, J = 4.4 Hz, 1H, H-1ꢀ), 3.05 (dd, J = 5.1, 14.7 Hz, 1H, H-3), 3.02
(bd, J = 6.5 Hz, 1H, H-3ꢀ), 2.54 (m, 2H), 2.35–2.34 (m, 1H), 2.32 (dd,
J = 4.1, 14.1 Hz, 2H, H-2), 2.00 (bs, 3H, CH3-28), 1.96–1.95 (m, 3H),
1.83–1.81 (m, 1H, H-4ꢀ), 1.69–1.68 (m, 2H), 1.65–1.62 (m, 2H, H-
2ꢀ) 1.39–1.38 (m, 2H), 1.37–1.35 (m, 2H), 1.23 (s, 3H, CH3-19),
1.15–1.13 (m, 2H), 1.10–1.1.08 (m, 2H), 0.99 (d, J = 6.5 Hz, 3H, CH3-
18), 0.90 (d, J = 6.5 Hz, 3H, CH3), 0.88 (d, J = 6.5 Hz, 3H, CH3), 0.69 (s,
3H, CH3-21), 13C NMR (CDCl3, 50 MHz): ı 210.1 (qC, C-1), 37.7 (CH2,
C-2), 42.93 (CH, C-3), 78.7 (CH, C-4), 65.4 (qC, C-5), 63.0 (CH, C-6),
30.9 (CH2, C-7), 28.99 (CH, C-8), 44.34 (CH, C-9), 50.40 (qC, C-10),
21.01 (CH2, C-11), 27.21 (CH2, C-12), 42.01 (qC, C-13), 56.78 (CH,
C-14), 24.74 (CH2, C-15), 39.13 (CH2, C-16), 51.72 (CH, C-17), 10.5
(CH3, C-18), 14.2 (CH3, C-19), 39.50 (CH, C-20), 13.9 (CH3, C-21),
75.5 (CH, C-22), 29.68 (CH2, C-23), 125.99 (qC, C-24), 153.08 (qC, C-
25), 167.10 (qC, C-26), 55.7 (CH2, C-27), 20.3 (CH3, C-28), 175.4 (qC,
COOCH3), 61.3 (CH, C-2ꢀ), 55.7 (CH3, OMe), 44.20 (CH2, C-3ꢀ), 24.1
(CH, C-4ꢀ), 22.6, 22.5 (2 × CH3); ESI MS (m/z): 624 [M+Na]. Anal. Cal.
for C34H52NO8: C, 67.75, H, 8.70, N, 2.39; Found: 67.85, H, 8.79, N,
2.39.
KK-11: Prepared by the general procedure 2 using 1mmol
(513 mg) of KK-2 and methyl propiolate purified on silica gel
(EtOAc/PE: 65/35) to obtain the product KK-11 (99%) as white solid:
[␣]D20 = +27 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 8.6 (bs, 1H,
Triazole), 4.40 (dt, J = 12.0, 5.5, 3.5 Hz, 2H, H-22), 4.29 (brs, 2H, 2H,
H-27), 3.9 (bs, 3H, CO2Me), 3.7 (dd, J = 4.5, 16.5 Hz, 1H, H-4), 3.4 (d,
J = 3.0 Hz, 1H, H-3), 3.20 (bs, 1H, H-6), 3.11 (dd, J = 9.6, 16.5 Hz, 2H,
H-2), 2.54–2.52 (m, 2H), 2.35–2.33 (m, 1H), 2.10 (s, 3H, CH3-28),
1.99–1.97 (m, 3H), 1.67–1.65 (m, 2H), 1.37–1.35 (m, 2H), 1.34–1.32
(m, 2H), 1.27 (s, 3H, CH3-19), 1.14–1.12 (m, 2H), 1.11–1.10 (m,
2H),1.02 (d, J = 6.7 Hz, 3H, CH3-21), 1.31 (s, 3H, CH3-19), 0.72 (bs,3H,
CH3-18), 13C NMR (CDCl3, 50 MHz): ı 209.5 (qC, C-1), 39.78 (CH2,
C-2), 57.9 (CH, C-3), 65.10 (qC, C-5), 76.5 (CH, C-4), 59.4 (CH, C-6),
32.01 (CH2, C-7), 30.18 (CH, C-8), 43.99 (CH, C-9), 50.50 (qC, C-10),
22.99 (CH2, C-11), 27.89 (CH2, C-12), 42.02 (qC, C-13), 56.04 (CH,
C-14), 24.98 (CH2, C-15), 39.23 (CH2, C-16), 51.98 (CH, C-17), 11.6
(CH3, C-18), 17.05 (CH3, C-19), 39.0 (CH, C-20), 13.3 (CH3, C-21),
78.7 (CH, C-22), 29.88 (CH2, C-23), 125.0 (qC, C-24), 156.48 (qC, C-
25), 167.91 (qC, C-26). 56.9 (CH2, C-27), 20.08 (CH3, C-28), 160.8
(qC, COOMe) 139.4 (qC, triazole), 128.9 (CH, triazole), 55.8 (CH3,
COOMe),; ESI MS (m/z): 620 [M+Na]. Anal. Cal. for C32H43N3O8: C,
64.30, H, 7.25, N, 7.03; Found: 64.38, H, 7.33, N, 7.11.
KK-12: Prepared by the general procedure 2 using 1mmol
(513 mg) of KK-2 and 1-methoxy-4-prop-2-ynyloxy benzene, puri-
fied on silica gel (EtOAc/PE: 70/30) to obtain the product KK-12
(99%) as white solid: [␣]D20 = +38 (c 0.1, CHCl3); 1H NMR (CDCl3,
500 MHz): ı 8.1 (bs, 1H, triazole), 7.28 (d, J = 8.9 Hz, 2H, Ar), 6.7
(d, J = 9.1 Hz, 2H, Ar), 6.84–6.82 (m, 1H, Ar), 4.63 (bs, 2H, OCH2),
4.41 (dt, J = 12.0, 5.5, 3.5 Hz, 2H, H-22), 4.27 (brs, 2H, H-27), 3.73 (s,
3H, OMe-Ar), 3.64 (d, J = 3.2 Hz, 1H, H-4), 3.42 (d, J = 3.2 Hz, 1H, H-
3), 3.30 (bs, 1H, H-6), 3.09 (dd, J = 9.6, 16.6 Hz, 2H, H-2), 2.53–2.51
(m, 2H), 2.31–2.30 (m, 1H), 2.05 (s, 3H, CH3-28), 1.96–1.95 (m, 3H),
1.69–1.68 (m, 2H), 1.39–1.38 (m, 2H), 1.37–1.35 (m, 2H), 1.23 (s, 3H,
CH3-19), 1.15–1.13 (m, 2H), 1.10–1.1.0 (m, 2H),1.02 (d, J = 6.4 Hz,
3H, CH3-21), 0.69 (s, 3H, CH3-18), 13C NMR (CDCl3, 50 MHz): ı 209.8
(qC, C-1), 39.91 (CH2, C-2), 63.0 (CH, C-3), 76.5 (CH, C-4), 65.10 (qC,
C-5), 61.5 (CH, C-6), 31.6 (CH2, C-7), 30.08 (CH, C-8), 44.94 (CH, C-9),
50.33 (qC, C-10), 21.52 (CH2, C-11), 28..04 (CH2, C-12), 43.00 (qC,
C-13), 56.77 (CH, C-14), 24.90 (CH2, C-15), 39.93 (CH2, C-16), 51.12
(CH, C-17), 11.6 (CH3, C-18), 14.4 (CH3, C-19), 39.09 (CH, C-20), 13.6
(CH3, C-21), 78.9 (CH, C-22), 29.68 (CH2, C-23), 125.88 (qC, C-24),
153.48 (qC, C-25), 167.78 (qC, C-26), 56.8 (CH2, C-27), 20.06 (CH3,
C-28), 154, 152 (qC, C-Ar), 150.8 (CH, triazole), 115.8, 115.8, 114.38,
114.33 (4 × CH, Ar), 127.8 (CH, Triazloe), 70.4 (CH2, OCH2); ESI MS
(m/z): 698 [M+Na]. Anal. Cal. for C38H49N3O8: C, 67.53, H, 7.31, N,
6.22; Found: 67.62, H, 7.39, N, 6.29.
KK-10: Prepared by the general procedure 1 using 1:1 mmol
(470 mg) of KK-1 and l-cysteine methyl ester, purified on silica
gel (EtOAc/PE: 60/40) to obtain the product KK-3 (83%) as white
solid: [␣]D20 = +10 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 4.40
(dt, J = 12.0, 5.5, 3.5 Hz, 2H, H-22), 4.29 (brs, 2H, H-27), 3.80 (s, 3H,
COOCH3), 3.79 (dd, J = 4.1, 15.2 Hz, 1H, H-3ꢀ), 3.4 (d, J = 4.0 Hz, 1H, H-
4), 3.35 (d, J = 4.1 Hz, 2H, H-2ꢀ), 3.2 (bs, 1H, H-6), 3.0 (ddd, J = 6.4, 5.1,
4.5 Hz, 1H, H-3), 2.57–2.55 (m, 1H, H-1ꢀ), 2.54–2.52 (m, 2H), 2.35
(dd, J = 4.5, 11.7 Hz, 2H, H-2), 2.29 (m, 1H), 1.99 (bs, 3H, CH3-28),
1.95–1.93 (m, 3H), 1.70–1.68 (m, 2H), 1.43–1.41 (m, 2H), 1.38–1.37
(m, 2H), 1.26 (s, 3H, CH3-19), 1.16–1.15 (m, 2H), 1.10–1.08 (m,
2H), 0.98 (d, J = 6.5 Hz, 3H), 0.69 (s, 3H, CH3-21), 13C NMR (CDCl3,
50 MHz): ı 209.8 (qC, C-1), 39.9 (CH2, C-2), 43.2 (CH, C-3), 76.5 (CH,
C-4), 65.10 (qC, C-5), 61.8 (CH, C-6), 31.09 (CH2, C-7), 29.87 (CH, C-
8), 44.24 (CH, C-9), 49.52 (qC, C-10), 21.59 (CH2, C-11), 27.71 (CH2,
KK-13: Prepared by the general procedure 2 using 1mmol
(513 mg) of KK-2 and 5-methoxy-2-prop-2-ynyloxy benzaldehyde,
purified on silica gel (EtOAc/PE: 72/28) to obtain the product KK-13
(99%) as white solid: [␣]D20 = +14 (c 0.1, CHCl3); 1H NMR (CDCl3,
500 MHz): ı 9.86 (bs, 1H, CHO), 7.66 (bs, 1H, triazole), 7.44 (d,
J = 7.0 Hz 2H, Ar), 7.16 (d, J = 8.1 Hz, 1H, Ar), 4.99 (bs, 2H), 4.38 (dt,
J = 12.0, 5.5, 3.5 Hz, 2H, H-22), 4.26 (brs, 2H, H-27), 3.8 (s, 3H, OMe-
Ar), 3.71 (dd, J = 4.6, 16.2 Hz, 1H, H-4), 3.40 (d, J = 4.2, 1H, H-3),
3.31 (bs, 1H, H-6), 2.55–2.54 (dd, J = 4.5, 11.7 Hz, 1H), 2.51–2.50 (m,
2H), 2.34–2.33 (m, 1H), 2.03 (bs, 3H, CH3-28), 1.98–1.96 (m, 3H),
1.70–1.68 (m, 2H), 1.42–1.40 (m, 2H), 1.39–1.38 (m, 2H), 1.28 (s,
3H, CH3-19), 1.17–1.15 (m, 2H), 1.13–1.12 (m, 2H), 0.98 (d, J = 6.1 Hz,

S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
1217
OH
OH
O
O
O
O
O
O
TMSN3
MeOH, rt,
N3
O
O
OH
OH
KK-1
KK-2
Scheme 1. Reaction of withaferin A (KK-1) with TMSN3.
3H, CH3-21), 0.68 (s, 3H, CH3-18), 13C NMR (CDCl3, 50 MHz): ı 210.1
(qC, C-1), 37.6 (CH2, C-2), 58.8 (CH, C-3), 78.7 (CH, C-4), 65.0 (qC,
C-5), 59.9 (CH, C-6), 30.9 (CH2, C-7), 29.98 (CH, C-8), 45.0 (CH, C-9),
50.61 (qC, C-10), 22.05 (CH2, C-11), 27.19 (CH2, C-12), 42.87 (qC,
C-13), 56.61 (CH, C-14), 24.64 (CH2, C-15), 39.23 (CH2, C-16), 51.63
(CH, C-17), 10.5 (CH3, C-18), 14.1 (CH3, C-19), 38.99 (CH, C-20),
13.9 (CH3, C-21), 75.5 (CH, C-22), 29.68 (CH2, C-23), 125.68 (qC,
C-25), 153.69 (qC, C-24), 167.97 (qC, C-26), 55.7 (CH2, C-27), 20.3
(CH3, C-28), 199.0 (qC, CO), 153.1, 152.2, 122.5 (3 × qC, Ar), 139.2
(CH, triazole), 121.8, 118.3, 117.9 (4 × CH, Ar), 70.9 (OCH2); ESI MS
(m/z): 603 [M+Na]. Anal. Cal. for C39H49N3O9: C, 66.55, H, 7.02, N,
5.97; Found: 66.63, H, 7.12, N, 5.90.
1.13–1.12 (m, 2H), 0.98 (d, J = 6.1 Hz, 3H, CH3-21), 0.68 (s, 3H, CH3-
18), 13C NMR (CDCl3, 50 MHz): ı 208.9 (qC, C-1), 39.7 (CH2, C-2),
57.3 (CH, C-3), 76.1 (CH, C-4), 64.98 (qC, C-5), 61.2 (CH, C-6), 31.9
(CH2, C-7), 29.87 (CH, C-8), 44.34 (CH, C-9), 50.15 (qC, C-10), 21.71
(CH2, C-11), 27.84 (CH2, C-12), 42.92 (qC, C-13), 55.99 (CH, C-14),
24.64 (CH2, C-15), 39.95 (CH2, C-16), 52.01 (CH, C-17), 11.5 (CH3,
C-18), 14.1 (CH3, C-19), 38.98 (CH, C-20), 13.1 (CH3, C-21), 79.6
(CH, C-22), 29.61 (CH2, C-23), 153.38 (qC, C-24), 124.61 (qC, C-25),
167.94 (qC, C-26), 55.58 (CH2, C-27), 20.42 (CH3, C-28), 110.5, 70.7,
70.6, 68.6, 66.8 (4 × CH, C-1ꢀ, C-2ꢀ, C-3ꢀ, C-4ꢀ, C-5ꢀ), 62.0 (CH2, C-
6ꢀ), 61.0 (CH2, OCH2), 170.2, 170.1, 169.6, 167.1 (4 × OAc), 139.2
(CH, triazole), 125.5 (qC); ESI MS (m/z): 922 [M+Na]. Anal. Cal. for
C
4.73.
45H61N3O16: C, 60.06, H, 6.83, N, 4.67; Found: C, 60.11, H, 6.92, N,
KK-14: Prepared by the general procedure 2 using 1 mmol
(513 mg) of KK-2 and 3-propargyl diacetonide glucoside, purified
on silica gel (EtOAc/PE: 75/25) to obtain the product KK-14 (73%) as
white solid: [␣]D20 = +35 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz):
ı 7.7 (bs, 1H, triazole), 5.87 (d, J = 3.7 Hz, 1H), 4.83 (bs, 2H, –OCH2),
4.59 (d, J = 3.7, 1H), 4.4 (dt, J = 12.0, 5.5, 3.5 Hz, H-22), 4.30–4.29 (m,
2H), 4.28 (brs, 2H, H-27), 4.10–4.07 (m, 2H), 4.0 (dd, J = 5.3, 8.2 Hz,
1H), 3.65 (dd, J = 5.4, 15.9 Hz, 1H, H-4), 3.46 (d, J = 4.1 Hz,1H, H-3),
3.26 (bs, 1H, H-6), 2.55–2.54 (dd, J = 4.5, 11.7 Hz, 2H, H-2), 2.52–2.50
(m, 2H), 2.32–2.30 (m, 1H), 2.00 (bs, 3H, CH3-28), 1.95–1.93 (m, 3H),
1.71–1.69 (m, 2H), 1.49 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.39–1.38 (m,
2H), 1.37–1.36 (m, 2H), 1.34 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.26 (s,
3H, CH3-19), 1.15–1.14 (m, 2H), 1.12–1.11 (m, 2H), 0.98 (d, J = 6.1 Hz,
3H, CH3-21), 0.68 (s, 3H, CH3-18),0.66 (s, 3H), 0.99 (d, J = 6.5 Hz, 3H,
CH3-21), 13C NMR (CDCl3, 50 MHz): ı 209.5 (qC, C-1), 38.9 (CH2,
C-2), 58.7 (CH, C-3), 78.7 (CH, C-4), 65.87 (qC, C-5), 60.0 (CH, C-6),
30.5 (CH2, C-7), 29.86 (CH, C-8), 43.99 (CH, C-9), 49.98 (qC, C-10),
21.95 (CH2, C-11), 27.94 (CH2, C-12), 42.36 (qC, C-13), 56.18 (CH,
C-14), 24.46 (CH2, C-15), 39.67 (CH2, C-16), 51.98 (CH, C-17), 11.5
(CH3, C-18), 15.3 (CH3, C-19), 39.06 (CH, C-20), 13.3 (CH3, C-21),
76.7 (CH, C-22), 29.48 (CH2, C-23), 153.83 (qC, C-24), 125.81 (qC, C-
25), 167.88 (qC, C-26), 55.61 (CH2, C-27), 20.91 (CH3, C-28), 114.05,
111.98 (2 × qC), 105.21 (CH), 82.59, 81.70, 81.01, 72.2 (5 × CH), 67.4,
63.8 (2 × CH2), 26.89, 26.80, 26.24, 25.5 (4 × CH3); .ESI MS (m/z):
834 [M+Na]. Anal. Cal. for C43H61N3O12: C, 63.61, H, 7.57, N, 5.18;
Found: C, 63.69, H, 7.64, N, 5.26.
KK-15: Prepared by the general procedure 2 using 1 mmol
(513 mg) of KK-2 and ı-propargyl galactoside purified, on silica gel
(EtOAc/PE: 80/20) to obtain the product KK-15 (99%) as white solid:
[␣]D20 = +33 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): ı 7.6 (bs,
1H), 5.34 (d, J = 3.4, 1H, H-1ꢀ), 5.16 (dd, J = 8.0, 10.0 Hz, 1H, H-4ꢀ),
4.98 (dd, J = 3.4, 10 Hz, 1H, H-2ꢀ), 4.90 (bs, 2H, OCH2), 4.59 (t, J = 8.0,
17.2 Hz, 1H, H-3ꢀ), 4.41 (dt, J = 12.0, 5.5, 3.5 Hz, H-22), 4.25 (bs, 2H,
H-27), 4.14 (dd, J = 6.5, 11.0 Hz, 1H, H-6a/H-6b), 4.07–4.04 (m, 1H,
H-6a/H-6b), 3.91 (dd, J = 6.4, 10 Hz, 1H, H-5ꢀ), 3.44 (d, J = 5.0 Hz, 1H,
H-4), 3.25 (bs, 1H, H-6), 3.11 (dd, J = 9.2, 16.0 Hz, 1H, H-3), 2.57–2.55
(dd, J = 4.5, 9.2 Hz, 1H), 2.54–2.52 (m, 2H), 2.35 (m, 1H), 2.09 (s, 3H,
-OAc), 2.05 (s, 3H, CH3-28), 1.99 (s, 3H, -OAc), 1.98 (s, 3H, -OAc),
1.96 (s, 3H, -OAc), 1.95–1.94 (m, 3), 1.67–1.66 (m, 2H), 1.42–1.40
(m, 2H), 1.39–1.38 (m, 2H), 1.28 (s, 3H, CH3-19), 1.17–1.15 (m, 2H),
3. Result and discussion
In order to comprehend the role of double bond in the ring A of
withaferin A for anticancer activity, we envisaged its functionaliza-
tion through Michael addition [24]. In our initial studies we have
chosen TMSN3 as the nucleophile. Thus, a solution of withaferin-A
in methanol was treated with a basic solution of nucleophile TMSN3
[26] at room temperature (Scheme 1). For optimization of the reac-
tion conditions, the addition reaction was carried out at varying pH
using different organic and inorganic bases (Table 1).
It was observed that a pH of 8–8.5 is optimal for the addition
reaction. Among the various bases used, the most favourable was
triethylamine in terms of yield, reaction time and minimal side
product formation. After the formation of the product and process-
ing, it was purified through silica gel (60–120 mesh size) column
chromatography. The structure of the product KK-2 was elucidated
by spectroscopic methods (Table 2). Its 1H NMR spectrum showed
Table 1
Standardisation of reaction conditions for Michael addition.
S. No.
1
Base used
NaOAc
pH
Time (h)
Yield%a
7.5
8
6
–
–
6
–
–
3.5
–
–
–
6
–
–
6
–
–
35
40
40
45
50
53
75
80
88
65*
40
40
40
35
40
40
8.5
7.5
8
8.5
7.5
8
2
3
DIPEA
Et3N
8.5
9.5
7.5
8
8.5
7.5
8
4
5
C5H5N
Pyrrolidine
8.5
*aSome degradation of starting material along with the desired product was noticed.

1218
S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
Table 2
1H NMR (500 MHz, CDCl3) and 13C NMR (50 MHz, CDCl3) of withaferin A (KK-1) and the Michael adduct (KK-2); a comparison.
Position
KK-1
KK-2
1H NMR (CDCl3, 500 MHz)
13C NMR
1HNMR
13C NMR
1
2
3
4
5
–
202.48
132.42
142.47
69.85
63.99
61.80
13.31
78.78
28.82
153.71
125.57
167.23
56.97
20.08
–
208.49
38.75
58.87
75.15
65.10
59.85
13.34
78.73
29.68
153.18
125.61
167.68
57.33
20.04
6.2 (d, J = 10 Hz)
6.98 (dd, J = 10.0, 6.0 Hz)
3.76 (d, 1H, J = 6.0 Hz)
–
3.24 (brs, 1H)
1.02 (d, J = 7.0 Hz, 3H)
4.35 (brs, 2H)
2.50 (brs, 2H)
3.07 (dd, J = 7.3, 15.7, Hz, 2H)
4.08 (ddd, J = 15.7, 4.1, 3.3 Hz, 1H)
3.46 (d, J = 4.1 Hz, 1H)
–
6
3.26 (brs, 1H)
21
22
23
24
25
26
27
28
1.01 (d, J = 6.9, 3H)
4.40 (dt, J = 12.0, 3.5, Hz)
2.54 (m, 2H)
–
–
–
4.40 (dt, J = 12.0, 3.5 Hz)
2.02 (s, 3H)
4.30 (brs, 2H)
2.05 (s, 3H)
sets of signals similar to those in the spectrum of the reactant except
the two additional signals as dd at ı 3.07 for H-2 and ddd at ı 4.08
for H-3. The disappearance of characteristic signals at ı 6.17 (d,
J = 10 Hz), and ı 6.96 (dd, J = 10 Hz, 6 Hz) for H-2 and H-3 respec-
ner. Thus, methyl propiolate in equimolar ratio was added to a
solution of KK-2 in acetonitrile, followed by the addition of copper
iodide (CuI) as [Cu(I)] source (Scheme 5).
The reaction mixture was allowed to stir at room temperature,
and after 6 h the consumption of the starting material indicated
the completion of reaction. Accordingly, other triazole derivatives
were also synthesised applying the same reaction procedure. Fur-
thermore, the carbohydrate based alkyne partners were also found
to react smoothly under these reaction conditions to afford the
triazole glycoconjugates KK-14, 15 (Scheme 6).
tively corresponding to ␣,␤-unsaturated carbonyl group in the 1
H
NMR of KK-1 was the clear indication of formation of the Michael
adduct KK-2. The signal for H-4 d 3.46 (d, J = 4.1 Hz, 1H) was shifted
slightly upfield in the 1H spectrum of KK-2 as compared to KK-1 as
expected. These assignments which clearly differed from those of
KK-1, were confirmed by using techniques of 2D NMR. In the 1H–1H
COSY plot of KK-2, H-3 at ı 4.08 showed correlations with H-4
and H-2a/b at ı 3.46 and 3.07, respectively. Further strong correla-
tions were found between H-2 and H-4. In the 13C NMR spectrum,
two additional signals appeared at 38.75 and 58.87 for C-2 (CH2)
and C-3 (CH) respectively along with the downfield shift of C-4 to
ı 75.15. These interpretations were supported by the HMBC and
HSQC spectrum of KK-2 also.
In order to study the influence of free hydroxyls on the cytotox-
icity, the triazole derivative KK-15 was deacetylated using NaOMe
in methanol to synthesise KK-16.
4. Biology
4.1. Sulphorhodamine B assay for % growth inhibition
The experimental procedure developed for the TMSN3 was then
applied to thio-nucleophiles that included thiophenol, chloroth-
iophenol, p-methoxy thiophenol and furan-2-methanethiol and
amino acids such as phenyl alanine, valine and cysteine. Thus,
thiophenol when allowed to react with withaferin-A under the
standardised reaction procedure, the complete conversion of start-
ing material occurred within 1.5 h. The product was characterised
through spectroscopic analysis and was assigned the structure KK-
3. The other thiol nucleophiles too reacted smoothly to yield the
corresponding Michael adducts (KK-4–7) in good to excellent yields
(Scheme 2).
The sulphorhodamine B (SRB) [27] assay was used to screen the
semi-synthetic library of withaferin-A for cytotoxicity. The assay
relies on the ability of SRB to bind to protein components of cells
that have been fixed to tissue culture plates by trichloroacetic acid
(TCA). SRB is a bright-pink aminoxanthene dye with two sulphonic
groups that bind to basic amino-acid residues under mild acidic
OH
Owing to their role in many metabolic processes, next we
thought of using amino acids in our reaction system. Since,
in this reaction the amino group is nucleophilic, whereas the
carboxylic group is electrophilic which needs to be protected.
Therefore, during the process of using them as nucleophiles, the
methyl esters of amino acids were used. Thus, amino esters of l-
phenylalanine, l-valine and l-cysteine were subjected to Michael
addition with withaferin A under the standardised reaction condi-
tions to obtain the corresponding Michael adducts (KK-8, 9) within
3.5 h (Scheme 3).
O
O
O
RSH, Et3N
KK-1
MeOH, rt, pH = 8-8.5
RS
O
OH
KK-3-KK-7
In the case of l-cystein, wherein the nucleophilicity of thio
centre is more compared to the amino group, the thio addition
product KK-10 was obtained that was fully characterised by 1H
NMR (Scheme 4).
After observing significant cytotoxicity in KK-2, we decided for
its further derivatization using Husigen’s [3+2] cycloaddition reac-
tion [25], due to the biological importance of triazoles, besides
the simplicity of the reaction conditions. These derivatives are
expected to have more hydrophilic character than the parent
molecule. Initially, methyl propiolate was used as the alkyne part-
R =
Cl
Br
KK-5, 82%
KK-3, 85%
KK-4, 82%
O
H3CO
KK-6, 85%
KK-7, 84%
Scheme 2. Reaction of thiol nucleophiles with KK-1.

S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
1219
OH
O
O
O
R
NH2
O
Et3N
OCH3
KK-1
+
O
MeOH, rt, pH = 8-8.5
MeO
N
H
O
L-Amino acid methyl ester
R
OH
KK-8-9
R = CH2Ph,
KK-8, 78%
-CH(CH3)2
KK-9, 81%
Scheme 3. Reaction of methyl esters of amino acids with KK-1.
OH
O
O
O
NH2
HS
Et3N
OMe
S
O
+
KK-1
MeOH, rt, pH = 8-8.5
O
MeO
H2N
O
L-Cysteine methyl ester
OH
KK-10, 83%
Scheme 4. Reaction of cysteine methyl ester with KK-1.
conditions, and dissociate under basic conditions. As the binding
of SRB is stoichiometric, the amount of dye extracted from stained
cells is directly proportional to the cell mass.
all the 10 molecules are further screened at lower micro molar
concentrations (Table 4).
The cytotoxicity result in Table 4 indicated that the compounds
KK-1, KK-2, KK-3 and KK-15 have maintained their cytotoxicity at
lower micro molar concentrations on all the four cancer cell lines
screened i.e. prostate, ovary, breast and leukemia, where as the
cervical cancer cell line was fairly resistant to the cytotoxic effect
of these compounds. Prostate and ovarian cancer cell line became
resistant to the action of the compounds KK-4, KK-10, KK-11, KK-12,
KK-14 and KK-16, respectively, whereas these compounds main-
tained their cytotoxicity on breast and leukemia cancer cell lines.
Since the compounds KK-1, KK-2, KK-3 and KK-15 are the most
active on all the cancer cell lines, therefore these four molecules
are further evaluated at still lower micro molar conc (Table 5).
The IC50 values of the above four compounds on the various
human cancer cell lines like lung, ovary, CNS, colon and leukemia
are given in Table 6.
To determine the effect of the synthesised compounds on cell
number over time, SRB assays were performed as described. Var-
ious human cancer cell lines which include lung, ovary, colon and
prostate were seeded in flat-bottomed 96-well plates. The cells
were allowed to adhere overnight, and then media containing
samples at different concentrations were added. The plates were
assayed for 48 h. The cells were fixed by adding 50 ml per well of ice-
cold 50% TCA to each well for 60 min. The plates were washed five
times in running tap water and stained with 100 ml per well SRB
reagent (0.4%, w/v, SRB in 1% acetic acid for 30 min. The plates were
washed five times in 1% acetic acid and allowed to dry overnight.
SRB was solubilised with 100 ml per well 10 mM Tris-base, shaken
for 5 min and the OD was measured at 570 nm with reference wave-
length of 620 nm. The results are given in Table 3.
The cytotoxicity data in Table 3 clearly showed that out of 16
compounds 10 molecules are found active during primary screen-
ing. These 10 molecules which include KK-1 – KK-4, KK-8, KK-11,
KK-12, KK-14 and KK-16 are the most active on all the four can-
cer cell lines that is lung, ovary, colon and prostate. Subsequently
It is apparent from the bioactivity data above that the major-
ity of the compounds are able to induce the growth inhibition in
the cell lines tested and the preliminary structure–activity rela-
tionships (SAR) can be deduced. The most active product KK-2
(IC50 = 0.02–1.9 ␮M) has an azide group at C-3 combined with
OH
O
O
O
Cu(I)
+
CO2Me
KK-2
CH3CN, rt, 6hrs
N
N
MeO2C
O
OH
N
KK-11
Scheme 5. Cu(I) catalysed 1,3-dipolar cycloaddition reaction of KK-2 and methyl propiolate.

1220
S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
OH
O
O
O
Cu(I)
+
R
KK-2
CH3CN, rt, 6hrs
N
N
R
O
OH
N
KK-12-15
MeO
O
MeO
O
CHO
KK-13, 98%
KK-12, 98%
OAc
O
OAc
O
O
O
O
O
O
AcO
O
OAc
KK-15, 99%
KK-14, 99%
OH
OH
O
O
HO
OH
KK-16, 99%
Scheme 6. Cu(I) catalysed 1,3-dipolar cycloaddition reaction of KK-2.
Table 3
In vitro determination of cytotoxicity of structurally modified derivatives of withaferin-A against panel of human cancer cell lines.
Tissue type
Lung
Ovary
Colon
Prostate
PC-3
Cell line type
A-549
IGR-OV-1
CoLo-205
S. No.
1
Code
KK-1
Conc.(␮M)
% Growth inhibition
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
95
91
98
96
95
90
95
62
94
17
57
1
90
83
98
92
96
88
95
88
77
60
55
33
76
60
69
48
79
51
90
85
88
83
86
79
67
52
90
88
86
82
97
90
98
96
99
91
96
92
94
88
67
0
93
13
99
35
99
21
99
98
99
97
88
84
21
17
99
95
98
98
97
91
86
62
61
76
94
91
99
97
98
96
98
80
90
89
33
8
2
KK-2
3
KK-3
4
KK-4
5
KK-5
6
KK-6
7
KK-7
54
0
57
9
8
KK-8
81
15
86
17
94
61
96
62
90
60
44
5
88
36
94
23
98
97
98
80
86
55
34
3
98
85
97
91
94
60
27
45
90
68
9
KK-9
10
11
12
13
14
15
16
KK-10
KK-11
KK-12
KK-13
KK-14
KK-15
KK-16
95
65
95
82
100
100
52
60
96
76
83
81
92
79
60
85
17
18
19
20
Paclitaxel
Mitomycin
5-Flurouracil
Adriyamycin

S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
1221
Table 4
Cytotoxicity of compounds KK-1, KK-2, KK-3, KK-4, KK-8, KK-11, KK-12, KK-14, KK-15 and KK-16 on five cancer cell lines.
Tissue type
Prostate
Ovary
Breast
MCF-7
Cervix
HeLa
Leukemia
THP-1
Cell line type
S. No.
PC-3
IGR-OV-1
Inst. code
Conc. (␮M)
% Growth inhibition
1
2
3
4
5
6
7
8
KK-1
KK-2
KK-3
KK-4
KK-10
KK-11
KK-12
KK-14
KK-15
KK-16
5-Flurouracil
Mitomycin
Paclitaxel
1
1
1
1
1
1
1
1
1
1
1
1
1
64
54
85
66
68
82
58
34
49
68
68
–
71
38
0
0
16
0
6
37
2
9
9
–
90
65
62
58
60
54
14
56
65
37
54
–
79
75
66
63
71
54
60
80
64
–
95
91
90
83
86
43
77
94
87
–
9
10
11
12
13
56
–
–
–
Table 5
Cytotoxicity of compounds KK-1, KK-2, KK-3 and KK-15 on 6 cancer cell lines.
Tissue type
Lung
Ovary
Prostate
CNS
Colon
CoLo-205
Leukemia
THP-1
Cell line type
A-549
IGR-OV-1
PC-3
IMR-32
S. No.
1
Inst. Code
KK-1
Conc. (␮M)
% Growth inhibition
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
1
9
47
36
38
30
40
20
32
77
–
3
20
44
60
3
20
13
34
65
–
8
37
40
58
31
40
10
30
18
28
–
24
44
43
40
51
23
40
30
48
–
60
48
62
40
58
30
60
–
2
3
4
KK-2
KK-3
KK-15
32
50
40
50
15
26
–
5
6
7
Paclitaxel
Mitomycin
Adriyamycin
1
1
54
–
–
73
–
–
–
–
–
–
Table 6
IC50 values of KK-1, KK-2, KK-3 and KK-15 on various cell lines.
Tissue type
Cell line type
Lung
A-549
Ovary
IGROV-1
Prostate
PC-3
CNS
IMR-32
Colon
CoLo-205
Leukemia
THP-1
S. No.
Inst. Code
IC50 values (␮M)
1
2
3
4
KK-1
KK-2
KK-3
KK-15
2.7
0.8
0.9
1.8
2.8
0.08
0.6
2.7
0.7
0.38
2.0
0.1
1.9
2.2
2.4
1
0.06
0.02
0.13
0.4
0.07
1.4
0.2
2.3
hydroxyl at C-4 and epoxide ring at C-5, 6 intact. The presence
of azide group at C-3 led to a 35-fold increase in activity as com-
pared to the parent molecule in case of IGROV-1cell line (KK-1
vs. KK-2, Table 6). Further, it was also observed that the activ-
ity of the compounds depended upon the side chain with KK-15
bearing a galactose moiety showing comparable cytotoxicity to
the parent molecule. Increasing the hydrophilicity by deacetylating
KK-15 to KK-16 resulted in deleterious effect on growth inhibi-
tion. Among the thiol group bearing compounds KK-3 – KK-7, the
molecule with thiophenol substituent (KK-3) displayed the better
activity (IC50 = 0.13–2.2) as compared to other thiol derivatives.
In this series the electron-withdrawing groups showed a posi-
tive effect on the biological activity with chloro being more active
than bromo, while the electron-donating groups tend to decrease
the cytotoxicity. On the other hand, the compounds KK-8–KK-10
bearing amino acid functionality at C-3 showed no significant cyto-
toxicity compared to the parent molecule. Among this group, KK-10
with cysteine methyl ester as the substituent was found to be
better.
5. Conclusions
A small library of 2,3-dihydro,3-␤-substituted withaferin A
analogues was synthesised using Michael addition and Husigen’s
(3+2) cycloaddition reactions and screened to study their cytotoxic
potential against various human cancer cell lines. It was observed
that the fine tuning in ring A of the ␣,␤-unsaturated group in with-
aferin A can facilitate the development of the more potent lead
molecules. The nature of the substituent at C-3 rather than the
␣,␤-unsaturated carbonyl group is essential for the cytotoxicity. A
detailed study to understand the mode of action of the most active
derivatives i.e. KK-2, KK-3, KK-15 is underway and will be published
in due course of time.
Acknowledgements
The authors are thankful to the director IIIM Jammu for his inter-
est and support and CSIR for the project grant SIP0027.

1222
S.K. Yousuf et al. / Steroids 76 (2011) 1213–1222
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