Synthesis and cytotoxicity of semisynthetic withalongolide a analogues

Article abstract of DOI:10.1021/ml400267q

The natural product withaferin A exhibits potent antitumor activity and other diverse pharmacological activities. The recently discovered withalongolide A, a C-19 hydroxylated congener of withaferin A, was recently reported to possess cytotoxic activity against head and neck squamous cell carcinomas. Semisynthetic acetylated analogues of withalongolide A were shown to be considerably more cytotoxic than the parent compound. To further explore the structure-activity relationships, 20 new semisynthetic analogues of withalongolide A were synthesized and evaluated for cytotoxic activity against four different cancer cell lines. A number of derivatives were found to be more potent than the parent compound and withaferin A.

Full text of DOI:10.1021/ml400267q

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Cytotoxicity of Semisynthetic Withalongolide A
Analogues
Hashim F. Motiwala,† Joseph Bazzill,‡ Abbas Samadi,§ Huaping Zhang,∥ Barbara N. Timmermann,∥
Mark S. Cohen,‡ and Jeffrey Aube*,†
́
†Department of Medicinal Chemistry, University of Kansas, Del Shankel Structural Biology Center, 2034 Becker Drive, West
Campus, Lawrence, Kansas 66047, United States
‡Division of Endocrine Surgery, Department of Surgery, University of Michigan Hospital and Health Systems, Ann Arbor, Michigan
48109, United States
§Department of Pharmacology, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160, United States
∥Department of Medicinal Chemistry, School of Pharmacy, University of Kansas, Lawrence, Kansas 66045, United States
S
* Supporting Information
ABSTRACT: The natural product withaferin A exhibits potent antitumor
activity and other diverse pharmacological activities. The recently discovered
withalongolide A, a C-19 hydroxylated congener of withaferin A, was
recently reported to possess cytotoxic activity against head and neck
squamous cell carcinomas. Semisynthetic acetylated analogues of with-
alongolide A were shown to be considerably more cytotoxic than the parent
compound. To further explore the structure−activity relationships, 20 new
semisynthetic analogues of withalongolide A were synthesized and evaluated
for cytotoxic activity against four different cancer cell lines. A number of
derivatives were found to be more potent than the parent compound and withaferin A.
KEYWORDS: Withalongolide A, withaferin A, triacetate, jaborosalactone, macrocycle, DRO81-1
ithanolides are a group of naturally occurring C28
steroidal lactones assembled on an ergostane skeleton.
semisynthetic derivatives withalongolide A 4,27-diacetate 7 and
withalongolide A 4,19,27-triacetate 8 (Scheme 1) showed
improved potency and selectivity against the melanoma cell line
B16F10 over both the parent compounds 2 and 1.12 This led us
to initiate a study to examine preliminary structure−activity
relationships (SARs) for compound 2. In this letter, we report
the synthesis and biological evaluation of 20 new semisynthetic
analogues of this fascinating class of cytotoxic agents.
W
Most of the withanolides are highly oxygenated members of the
Solanaceae family.1,2 Withaferin A (1; Figure 1) has a wide
Previous authors have asserted that a 2-ene-1-one, a 5β,6β-
epoxide, and a 17β-oriented δ-lactone are essential for
biological activity.2,14,15 Accordingly, we initially sought to
minimize modifications at these positions. Moreover, since the
di- and triacetate of compound 2 displayed significant
selectivity against melanoma cell line B16F10 with a potency
in nanomolar range, we first focused on examining the effects of
different derivatives of the free hydroxyl group of 2.
Figure 1. Chemical structures of withaferin A and its C-19 oxygenated
analogue, withalongolide A.
Accordingly, we prepared a series of aliphatic esters of 2
(Scheme 1). After slight modifications of the standard
acetylation procedures, it was possible to isolate mono- (3−
5) and diacetylated analogues 6 and 712 from the same reaction
along with some recovered 2 (acetic anhydride was added in
two portions; see the Supporting Information for details).
Withalongolide A 4,19,27-triacetate 812 and withalongolide A
range of biological activities, such as antitumor, antimicrobial,
antioxidant, anti-inflammatory, immunomodulatory, protective,
and antiangiogenic effects.1−6 The antitumor activity of
withaferin A results from targeting multiple signaling pathways
such as HSP90 and NF-κB that may circumvent the
development of resistance among cancer cells.7−11
Recently, Zhang et al. reported the isolation, structures, and
cytotoxic activities of withanolides featuring the presence of a
rare C-19 hydroxy group.12,13 Withalongolide A (2; Figure 1)
was found to be less potent than withaferin A against the
carcinoma and melanoma cell lines tested. However, the
Received: July 11, 2013
Accepted: September 5, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthesis of Acetylated and Propionylated
Analogues of Withalongolide A (2)
Scheme 2. Synthesis of Benzoylated and Carbamate
Analogues of Withalongolide A (2)
a
a
a
Reagents and conditions: (a) For 3−7, (CH3CO)2O, pyridine, rt,
73% combined yield; (b) for 8, (CH3CO)2O, pyridine, DMAP, rt,
99%; (c) for 9, (CH3CH2CO)2O, pyridine, DMAP, rt, 99%.
4,19,27-propionate 9 were obtained by treating 2 with
appropriate anhydrides in the presence of pyridine and a
catalytic amount of 4-(dimethylamino)pyridine (DMAP).
The second series of analogues entailed the synthesis of
acetates and aromatic esters in assorted combinations along
with carbamates (Scheme 2). The reaction of 2 with p-
chlorobenzoyl chloride under typical acylation conditions
afforded a mixture from which mono- (10), di- (11 and 12),
and tri-p-chlorobenzoate ester 13 were isolated.16 The free C-
19 hydroxyl group of 12 was further acetylated with acetic
anhydride to afford compound 16. Carbamoylation only
occurred at the C-4 hydroxyl, even after treating 2 with excess
dimethylcarbamoyl chloride, to afford monosubstituted dime-
thylcarbamate analogue 14. Subsequent acetylation of the
remaining two hydroxyl groups with acetic anhydride delivered
compound 15 in good yield. Efforts to make carbonate
analogues from phenylchloroformate led to a multitude of
products, which seemed to be unstable.
Jaborosalactones are withanolides isolated from Jaborosa
species. We synthetically prepared close derivatives of known
jaborosalactones by chemically manipulating 8 as shown in
Scheme 3. Thus, the palladium-catalyzed, formate-mediated
reduction of triacetate 8 afforded a mixture of jaborosalactone
derivatives 1717 and 1818,19 through C-4 deoxygenation of the
acetate group.20 The structure of the diacetate analogue of
jaborosalactone V 17 was confirmed by single-crystal X-ray
diffraction analysis (Figure 2).17 Compound 18, a C-19 acetate
analogue of jaborosalactone B 27-acetate was obtained via
similar C-4 deoxygenation followed by subsequent epoxide
ring-opening.18,19 Analogue 19, a C-19 acetate derivative of
jaborosalactone E 27-acetate, was obtained by epoxide ring-
opening of compound 17 using hydrogen chloride solution in
ether.21 Besides being expected from a mechanistic perspective,
antirelationship of the C-5 chlorine and C-6 hydroxyl groups
was assigned based on NMR data comparison with the
literature values for jaborosalactone E and similar com-
pounds.22,23 Acetylation of a small amount of compound 19
a
Reagents and conditions: (a) For 10−13, p-chlorobenzoyl chloride,
triethylamine, DMAP, CH2Cl2, rt, 76% combined yield; (b)
(CH3)2NCOCl, triethylamine, DMAP, CH2Cl2, rt, 73%; (c)
(CH3CO)2O, pyridine, DMAP, rt, 70%; (d) (CH3CO)2O, triethyl-
amine, DMAP, CH2Cl2, rt, 95%.
showed a downfield shift of H-6 from δ 3.99 to 5.12 ppm
suggesting the presence of a hydroxyl group at C-6 (data not
shown).21,24 The C-6 free hydroxyl group of compound 18 was
further acetylated to provide triacetylated analogue 20.
The α,β-unsaturated ketone of withaferin A is a Michael
acceptor.10 Therefore, we sought to modify the α,β-unsaturated
ketone in order to modulate its reactivity. Initial attempts to
perform Diels−Alder reactions on the Δ2,3-olefin were
unsuccessful. Subsequent efforts were directed toward the
synthesis of α-substituted aryl analogues of the ring A enone of
2. Compound 2 and its triacetate analogue 8 were subjected to
α-iodination conditions using iodine and DMAP to afford their
corresponding α-iodoenone analogues 21 and 22 (Scheme
4).25 However, attempts to modify these α-iodoenones through
Suzuki cross-coupling with boronic acids26,27 to obtain α-aryl
analogues met with no success; only decomposition of the α-
iodoenones was observed.
The final analogue was prepared via ring-closing macro-
cyclization28,29 to afford a rare steroidal macrocycle (Scheme
5).30,31 Thus, bis-acylation of withalongolide monoacetate 3
with 4-pentenoic anhydride afforded 23. Ring-closing meta-
thesis with Grubb’s II catalyst exclusively afforded the 14-
membered macrocycle 24 with E-configuration.32
All synthesized analogues of 2, along with withaferin A as a
positive control, were tested for their cytotoxic activity against
four cancer cell lines: head and neck squamous cell carcinoma
(HNSCC, JMAR), breast cancer cells (MDA-MB-231),
B
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Scheme 3. Synthesis of Jaborosalactone Derivatives from
Triacetate 8
Scheme 5. Synthesis of Steroidal Macrocycle 24 from
a
a
Withalongolide Monoacetate 3
a
Reagents and conditions: (a) (H2CCHCH2CH2CO)2O, pyridine,
DMAP, rt, 92%; (b) Grubb’s II catalyst, CH2Cl2, 37 °C, 87%.
Table 1. Cytotoxicity Activity (IC50 Values in μM) of
a
Withalongolide A Analogues against Five Cell Lines
compd
JMAR
MDA-MB-231
SKMEL-28
DRO81-1 MRC-5
1
1.00
3.10
1.22
3.06
2.48
0.515
0.600
0.655
1.00
1.72
4.64
>10
0.540
1.50
1.00
0.780
1.70
2.70
5.30
2.30
1.80
1.20
0.490
0.295
0.310
0.615
2.55
2.35
>10
a
Reagents and conditions: (a) Pd(OAc)2, PPh3, HCO2NH4, dioxane,
2
1.60
reflux, 50% combined yield; (b) HCl solution in ether, CH2Cl2, rt,
70%; (c) (CH3CO)2O, pyridine, DMAP, rt, 87%.
3
0.415
1.15
0.705
1.23
1.05
4
0.780
0.570
0.155
0.0580
0.110
0.130
1.85
5
0.890
0.205
0.635
0.655
0.365
1.40
1.22
6
0.230
0.170
0.110
0.285
2.25
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2.62
1.11
1.90
>10
>10
>10
Figure 2. Single-crystal X-ray structure of analogue 17.
Scheme 4. Synthesis of α-Iodoenone Derivatives
>10
>10
>10
>10
>10
2.09
0.970
>10
0.610
0.175
>10
0.515
0.205
>10
0.585
0.0865
>10
1.75
0.510
>10
1.22
>10
0.790
>10
0.710
>10
1.10
2.05
>10
>10
2.16
>10
0.800
>10
1.17
1.35
3.15
>10
>10
>10
>10
>10
9.11
5.25
9.10
0.475
0.865
0.360
0.830
1.44
0.965
0.210
0.625
0.245
0.275
0.470
0.205
0.115
0.580
0.225
a
Analogues 12, 13, 16, 18, and 20 were inactive with IC50 ≥ 10 μM for
all cell lines tested.
melanoma (SKMEL-28), and colon cancer cells (DRO81-1), in
addition to normal fetal lung fibroblast (MRC-5) cells (Table
1). Compound 2 was less potent than withaferin A, as
previously reported.12 Acetylation of compound 2 resulted in
more potent analogues with diacetates (6 and 7) and triacetate
8 exhibiting enhanced cytotoxic activity than the monoacety-
lated analogues (3, 4, and 5). Notably, compound 7 was found
to be selectively cytotoxic toward DRO81-1 with an IC50 value
of 0.0580 μM.12 The tripropionylated analogue 9 was also
active with IC50 values in the range of 0.130−1.00 μM. The
increased cytotoxic potency of the di- and triacetate analogues
of 2 could be due to increased lipophilicity leading to enhanced
cell permeability.16,33,34
Although the mono-p-chlorobenzoylated derivative 10 and
C-19,27-dibenzoylated analogue 11 were active, the C-4,27-
dibenzoylated analogue 12 and tribenzoylated compound 13
were not. Compounds 14 and 15 exhibited higher cytotoxic
activity than 2, with compound 15 displaying 3−5 times
C
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increased cytotoxicity against breast cancer and melanoma cells
compared to 1. Compound 15 also demonstrated modest
selectivity toward DRO81-1 cells with an IC50 value of 0.0865
μM, similar to compound 7.
to purchase the X-ray diffractometer. We thank Sarah
Neuenswander and Dr. Justin Douglas for assistance with
NMR, Patrick Porubsky for HRMS and HPLC data, and
Robert Gallagher and Rao Gollapudi for assistance in the
isolation of withalongolide A. M.S.C and J.B. thank Dr. G.
Juillard, University of California, Los Angeles, CA, for a gift of
the DRO81-1 cell line as well as Dr. Lisa Shnayder (KUMC)
and Dr. Jeffrey Myers, University of Texas, Houston, TX, for
the gift of the HNSCC JMAR cell line.
Jaborosalactone V diacetate 17 was found to be equipotent
to withaferin A 1, suggesting that C-4 hydroxyl group is not
crucial for activity.2,15 Compound 19, containing a 5α,6β-
chlorohydrin, was slightly less potent than the 5β,6β-epoxy
analogue 17, with IC50 values in the range of 0.800−2.16 μM
against melanoma and carcinoma cells. The cytotoxic activity
was abolished in epoxide-lacking analogues 18 and 20.2,15
Even though the 2-iodoenone analogue 22 displayed
comparable cytotoxicity to its parent compound 8, compound
21 showed very weak activity compared to 2. Interestingly,
macrocycle 24 exhibited increased potency compared to its
acyclic analogue 23 across all cell lines tested with IC50 values
in the range of 0.205−0.965 μM.28,29 Most of the active
analogues were moderately selective toward cancer cells
compared to normal fibroblast cells.
In our efforts to contribute toward the development of
anticancer-related therapeutics, we have identified additional
cytotoxic agents based on the natural product withalongolide A
2. Many of these analogues were more potent than the parent
compound, and the SAR profile was in good agreement with
those previously reported for withanolides having similar
structures.1,2,15,16 The selectivity of analogues 7 and 15 toward
colon cancer cells (DRO81-1) is intriguing, and further efforts
are underway to study these analogues as potential anticancer
agents.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, cytotoxicity assay, details of the
crystal structure of compound 17, characterization data, and 1H
and 13C NMR spectra of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(J.A.) E-mail: jaube@ku.edu.
■
Funding
H.F.M. acknowledges financial support from the Office of
Research and Graduate Studies and the receipt of a Mitscher
Fellowship, both from University of Kansas. This study was
supported, in part, by Grant IND 0061464 (awarded to B.N.T.)
from the Kansas Bioscience Authority (KBA) and Center for
Heartland Plant Innovations (HPI). The authors also acknowl-
edge partial financial assistance from Grant NFP0066367 from
the Institute for Advancing Medical Innovation (IAMI)
(awarded to M.S.C. and B.N.T.) as well as from resources
from the Department of Surgery at the University of Kansas
Medical Center as well as the Department of Surgery at the
University of Michigan (to M.S.C). Partial support of the in
vitro experiments was provided by the University of Kansas
Center for Cancer Experimental Therapeutics NIH-COBRE
P20 RR015563 (PI, B.N.T.; project award PI, M.S.C.).
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We acknowledge Dr. Victor Day for the X-ray crystal structure
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