Chemical synthesis and biological evaluation of palmerolide A analogues

Article abstract of DOI:10.1021/ja802803e

Molecular design and chemical synthesis of several palmerolide A analogues allowed the first structure activity relationships (SARs) of this newly discovered marine antitumor agent. From several analogues synthesized and tested (ent-1, 5-14, 21-26, 50, 51), compounds 25 (with a phenyl substituent on the side chain) and 51 (lacking the C-7 hydroxyl group) were the most interesting, exhibiting approximately a 10-fold increase in potency and equipotency, respectively, to the natural product. These findings point the way to more focused structure activity relationship studies.

Full text of DOI:10.1021/ja802803e

Published on Web 07/04/2008
Chemical Synthesis and Biological Evaluation of
Palmerolide A Analogues
K.C. Nicolaou,*,† Gulice Y. C. Leung, Dattatraya H. Dethe, Ramakrishna Guduru,
Ya-Ping Sun, Chek Shik Lim, and David Y.-K. Chen*
Chemical Synthesis Laboratory@Biopolis, Institute of Chemical and Engineering Sciences
(ICES), Agency for Science, Technology and Research (A*STAR), 11 Biopolis Way,
The Helios Block, #03-08, Singapore 138667
Received April 16, 2008; E-mail: kcn@scripps.edu; david_chen@ices.a-star.edu.sg
Abstract: Molecular design and chemical synthesis of several palmerolide A analogues allowed the first
structure activity relationships (SARs) of this newly discovered marine antitumor agent. From several
analogues synthesized and tested (ent-1, 5-14, 21-26, 50, 51), compounds 25 (with a phenyl substituent
on the side chain) and 51 (lacking the C-7 hydroxyl group) were the most interesting, exhibiting approximately
a 10-fold increase in potency and equipotency, respectively, to the natural product. These findings point
the way to more focused structure activity relationship studies.
Introduction
Intelligence gathering on molecules from nature has amassed
an invaluable body of knowledge and established itself as a
leading and fertile avenue for drug discovery and development,
as amply demonstrated over the past century.1 The recently
reported antitumor properties [selective cytotoxicity against
melanoma cancer cells UACC-62 (LC50 ) 18 nM)] of
palmerolide A (1, Figure 1), a substance isolated from the
circumpolar tunicate Synoicum adareanum collected from the
waters around Anvers Island on the Antarctic Peninsula,2
promise a new chapter in cancer research, provided chemical
synthesis can deliver sufficient quantities of the compound and
its congeners for further biological investigations. Indeed,
synthetic studies directed toward palmerolide A culminated in
total syntheses of both ent-palmerolide A (ent-1, Figure 2)3,4
and naturally occurring palmerolide A,4 as well as several
diastereoisomers and analogues [5-13 (Figure 2), 14 (Table
1)]4 of the natural product. Most importantly, these studies
resulted in the revision of the structure of palmerolide A to that
represented by structure 1. In this article we report the
application of our developed synthetic technologies to the
construction of several new palmerolide A analogues [21-26
(Table 1) and 50, 51 (Scheme 2)] and the biological evaluation
of all synthesized palmerolides against an array of tumor cells.
Figure 1. Structure of palmerolide A (1) and retrosynthetic analysis of
the palmerolide structure (1a) leading to building blocks 2a, 3a, 3b, 4a,
and 4b. TBS ) tert-butyldimethylsilyl; MOM ) methoxymethyl.
inhibitors,5 a series of enamide analogues (21-26) were
prepared as illustrated in Table 1. Thus, under our carefully
optimized conditions, the application of the Buchwald copper-
catalyzed coupling (CuI, K2CO3 and N,N′-dimethylethylenedi-
amine)6 allowed the coupling of macrocyclic iodide 144 and
Results and Discussion
Recognizing the structural and biological similarity between
palmerolide A (1) and other enamide containing ATPase
† Additional affiliations at The Scripps Research Institute and University
of California, San Diego.
(1) Nicolaou, K. C.; Montagnon, T. Molecules That Changed The World;
Wiley-VCH: Weinheim, 2008; p 366.
(4) (a) Nicolaou, K. C.; Guduru, R.; Sun, Y.-P.; Banerji, B.; Chen, D. Y.-
K. Angew. Chem., Int. Ed. 2007, 46, 5896–5900. (b) Nicolaou, K. C.;
Sun, Y.-P.; Guduru, R.; Banerji, B.; Chen, D. Y.-K. J. Am. Chem.
Soc. 2008, 130, 3633–3644.
(2) Diyabalanage, T.; Amsler, C. D.; McClintock, J. B.; Baker, B. J. J. Am.
Chem. Soc. 2006, 128, 5630–5631.
(5) Xie, X.-S.; Padron, D.; Liao, X.; Wang, J.; Roth, M. G.; De Brabander,
J. K. J. Biol. Chem. 2004, 279, 19755–19763.
(3) Jiang, X.; Liu, B.; Lebreton, S.; De Brabander, J. K. J. Am. Chem.
Soc. 2007, 129, 6386–6387.
(6) Jiang, L.; Job, G. E.; Klapars, A.; Buchwald, S. L. Org. Lett. 2003, 5,
3667–3669.
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A R T I C L E S
Nicolaou et al.
Table 1. Preparation of Palmerolide A Analogues 21-26 through
Copper-Mediated Coupling Reactionsa
a Reagents and conditions: 15-20 (2.0 equiv), CuI (1.5 equiv),
K2CO3 (5.0 equiv), N,N′-dimethylethylenediamine (3.0 equiv), DMF, 23
°C, 1 h. DMF ) N,N′-dimethylformamide.
stannane 3b and carboxylic acid 4b were required. Their
constructions are shown in Scheme 1. Thus, asymmetric Keck
allylation8 of aldehyde 274 under the standard conditions [(R)-
BINOL, Ti(Oi-Pr)4; then n-Bu3Sn(allyl)], followed by removal
of the TMS group (K2CO3, MeOH) from the resulting product,
furnished allylic alcohol 28 in 97% overall yield and >90% ee
(by Mosher ester analysis).9 Installation of the carbamate group
[Cl3C(CO)NCO, K2CO3, 98% yield],10 followed by manipula-
tion of the acetylenic moiety [(i) AgNO3, NBS; (ii) Pd(dba)2
cat., n-Bu3SnH],11 then led to the desired vinyl stannane 3b
(72% overall yield). The carboxylic acid fragment 4b was
prepared from 6-heptene-1-ol (30) by a three-step sequence
involving oxidation (NMO, TPAP cat., 80% yield),12 Wittig
olefination (Ph3PdCHCO2Me, 72% yield), and saponification
(aq. KOH, 90% yield) as shown in Scheme 1.
Figure 2. Synthesized palmerolide A diastereoisomers and analogues (ent-
1, 5-13).4
primary amides 15-207 to afford palmerolide A analogues
21-26 in moderate to good yields (31-54%), as summarized
in Table 1.
In addition to the side-chain enamide analogues, a number
of macrolide analogues of palmerolide A were designed and
pursued by chemical synthesis as shown in Schemes 1 and 2.
In these designs we sought single deletions of the hydroxyl
groups situated on the 20-membered macrocycle (i.e., 50 and
51, Scheme 2) as well as the deletion of both hydroxyl groups
(i.e., 52, Scheme 2). For the synthesis of these analogues, and
according to our general strategy (Figure 1), fragments vinyl
With all building blocks (2a,4 3a,4 3b, 4a,4 4b) in hand the
next task became their union and further elaboration as
summarized in Scheme 2. In accordance with our previously
established procedures,4 hydroxy vinyl iodide 2a was coupled
(8) (a) Keck, G. E.; Tarbet, K. H.; Geraci, L. S. J. Am. Chem. Soc. 1993,
115, 8467–8468. (b) Codesido, E. M.; Cid, M. M.; Castedo, L.;
Mourino, A.; Granja, J. R. Tetrahedron Lett. 2000, 41, 5861–5864.
(9) For asymmetric Brown allylation of 4-pentynal to give ent-28, see:
(a) White, J. D.; Kim, T.-S.; Nambu, M. J. Am. Chem. Soc. 1995,
117, 5612–5613.
(10) Kocovsky, P. Tetrahedron Lett. 1986, 27, 5521–5524.
(11) Boden, C. D. J.; Pattenden, G.; Ye, T. J. Chem. Soc., Perkin Trans.
1 1996, 20, 2417–2419.
(7) The primary amides were prepared from the corresponding carboxylic
(12) Griffith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A. D. Chem.
Commun. 1987, 21, 1625–1627.
acids [DCC, N-hydroxysuccinimide, NH4OH].
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Synthesis and Evaluation of Palmerolide A Analogues
A R T I C L E S
Scheme 2. Synthesis of Palmerolide A Analogues 50 and 51a
Scheme 1. Synthesis of Vinyl Stannane 3b and Carboxylic Acid
4ba
a Reagents and conditions: (a) (R)-BINOL (0.4 equiv), Ti(Oi-Pr)4 (0.8
equiv), 4 Å molecular sieves, CH2Cl2, reflux, 1 h; then 27, n-Bu3Sn(allyl)
(1.1 equiv), CH2Cl2, -78 f -20 °C, 24 h; (b) K2CO3 (3.0 equiv), MeOH,
23 °C, 7 h, 97% (> 90% ee), two steps; (c) trichloroacetyl isocyanate (2.0
equiv), CH2Cl2, 23 °C, 1 h; then K2CO3 (3.0 equiv), MeOH, 23 °C, 1 h,
98%; (d) NBS (1.2 equiv), AgNO3 (0.1 equiv), acetone, 23 °C, 1 h, 90%;
(e) Pd(dba)2 (0.05 equiv), PPh3, (0.2 equiv), n-Bu3SnH (2.2 equiv), THF,
30 min, 23 °C, 80% (> 95:5 E/Z stereoselectivity); (f) NMO (4.5 equiv),
TPAP (0.03 equiv), CH2Cl2, 23 °C, 1 h, 80%; (g) Ph3PdCHCO2Me (1.2
equiv), CH2Cl2, 23 °C, 8 h, 72%; h) KOH (5.0 equiv), dioxane/H2O (4:1),
23 °C, 24 h, 90%. TMS ) trimethylsilyl; DIP ) diisopinocampheyl; NBS
) N-bromosuccinimide; dba ) dibenzylideneacetone; TPAP ) tetra-n-
propylammonium perruthenate; NMO ) N-methylmorpholine-N-oxide.
with carboxylic acids 4a and 4b under Yamaguchi conditions13
(2,4,6-trichlorobenzoyl chloride, Et3N, 4-DMAP) to afford esters
31 (90% yield) and 32 (90% yield), respectively. Attachment
of the vinyl stannane 3b to ester vinyl iodides 31 and 32, and
vinyl stannane 3a to 32 through Stille coupling14 reactions
[Pd(dba)2 cat., AsPh3, LiCl] led to hexaenes 33 (56% yield)
and 43 (63% yield), and 38 (65% yield), respectively. These
products were then converted to the required ring closing
metathesis15 substrates 37 (four steps, 43% overall yield), 42
(four steps, 30% overall yield), and 46 (three steps, 51% overall
yield) by standard procedures involving TBAF-induced desi-
lylation (34, 39, 44), DMP oxidation (35, 40, 45), Takai
olefination16 (CrCl2, CHI3; 36, 41, 46), and MOM removal
(TMSCl, MeOH; 37, 42). Interestingly, while 37 and 42
underwent smooth ring closing metathesis with Grubbs II
catalyst [CH2Cl2, 0.005 M, 25 °C] to afford the desired
macrocycles 47 (78% yield) and 48 (81% yield), respectively,
substrate 46 (lacking both allylic hydroxyl groups) failed to
afford any macrocyclic product (i.e., 49) under the same or more
forcing conditions, leading instead to decomposition and/or
polymerization. These observations suggest further mechanistic
investigations in order to clarify the reasons behind the
requirement for at least one allylic hydroxyl group for ring
a Reagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride (1.1 equiv),
Et3N (2.0 equiv), 4a or 4b (1.2 equiv), 4-DMAP (1.0 equiv), toluene, 23 °C,
12 h, 31: 90%; 32: 90%; (b) 3a or 3b (1.2 equiv), Pd(dba)2 (0.25 equiv), AsPh3
(3.0 equiv), LiCl (3.0 equiv), NMP, 23 °C, 12 h, 33: 56%; 38: 65%; 43: 63%;
(c) TBAF (1.0 M in THF, 1.2 equiv), THF, 23 °C, 1 h, 34: 80%; 39: 80%; 44:
80%; (d) Dess-Martin periodinane (1.1 equiv), NaHCO3 (5.0 equiv), CH2Cl2,
23 °C, 20 min, 35: 78%; 40: 75%; 45: 80%; (e) CrCl2 (10.0 equiv) CHI3 (3.0
equiv), THF/dioxane (1:6), 23 °C, 2 h, 36: 80% (>95:5 E/Z); 41: 81% (>95:5
E/Z); 46: 80% (>95:5 E/Z); (f) TMSCl (5.0 equiv), MeOH, 40 °C, 1 h, 37:
87%; 42: 62%; (g) Grubbs II cat. (0.05 equiv), CH2Cl2, 23 °C, 1 h, 47: 78%,
48: 81%; (h) 3-methyl-2-butenamide (2.0 equiv), CuI (1.5 equiv), K2CO3 (6.0
equiv), N,N′-dimethylethylenediamine (3.0 equiv), DMF, 23 °C, 1 h, 50: 45%,
51: 40%. 4-DMAP ) 4-dimethylamminopyridine; TBAF ) tetra-n-butylam-
monium fluoride; NMP ) N-methylpyrrolidone.
(13) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
(14) Yin, L.; Liebscher, J. Chem. ReV. 2007, 107, 133–173.
(15) (a) Grubbs, R. H. Tetrahedron 2004, 60, 7117–7140. (b) Nicolaou,
K. C.; Bulger, P. G.; Sarlah, D. Angew. Chem., Int. Ed. 2005, 44,
4490–4527. (c) Gradillas, A.; Perez-Castells, J. Angew. Chem., Int.
Ed. 2006, 45, 6086–6101.
closure in these systems. Finally, installation of the enamide
moiety onto the growing molecules (47 and 48) through the
(16) Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408–
7410.
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A R T I C L E S
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Table 2. Cytotoxicity of Natural and Synthetic Palmerolides against Selected Cancer Cell Lines (GI50 Values in µM)a
cell line/
compound
entry
UACC-62c
MCF-7c
SF268c
NCI-H460c
IA9d
PTX22d
A8d
1
2
3
4
5
6
7
8
doxorubicin
0.294 ( 0.141
0.022 ( 0.016
0.057 ( 0.007
0.062 ( 0.001
8.077 ( 0.194
>10
0.056 ( 0.005
0.006 ( 0.001
0.040 ( 0.007
0.065 ( 0.011
6.260 ( 0.174
>10
5.415 ( 0.247
5.567 ( 0.255
7.299 ( 0.430
0.200 ( 0.026
>10
8.257 ( 0.047
>10
8.786 ( 0.152
7.025 ( 0.362
>10
0.755 ( 0.004
0.796 ( 0.166
7.397 ( 0.262
0.007 ( 0.000
0.071 ( 0.008
7.585 ( 0.252
0.074 ( 0.000
0.129 ( 0.048
0.026 ( 0.011
0.030 ( 0.012
0.048 ( 0.006
9.475 ( 0.593
>10
0.008 ( 0.001
0.007 ( 0.001
0.010 ( 0.001
0.017 ( 0.004
6.589 ( 0.054
>10
0.033 ( 0.007
0.006 ( 0.001
0.038 ( 0.003
0.059 ( 0.001
>10
0.201 ( 0.049
0.079 ( 0.001
0.066 ( 0.007
0.073 ( 0.005
>10
0.051 ( 0.017
0.021 ( 0.015
0.018 ( 0.003
0.049 ( 0.004
8.844 ( 1.301
>10
8.634 ( 1.860
6.145 ( 0.922
8.477
Taxol
natural 1b
synthetic 1
ent-1
5
6
7
8
9
10
11
12
13
14
21
22
23
24
25
26
50
51
>10
>10
5.398 ( 0.362
8.129 ( 1.187
8.768 ( 0.698
0.322 ( 0.088
>10
6.830 ( 0.077
7.961 ( 0.584
9.638 ( 0.362
0.281 ( 0.118
>10
6.108 ( 0.134
7.028 ( 0.192
8.664 ( 0.494
0.075 ( 0.003
>10
>10
7.131 ( 1.143
>10
7.052 ( 0.474
5.865 ( 0.590
>10
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.288 ( 0.017
>10
0.627 ( 0.016
>10
0.083 ( 0.006
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6.837 ( 0.223
7.291 ( 0.137
0.430 ( 0.047
0.078 ( 0.001
3.796 ( 0.306
0.007 ( 0.000
0.061 ( 0.000
6.396 ( 0.106
0.055 ( 0.002
>10
>10
8.851
>10
>10
7.774 ( 1.094
0.618 ( 0.051
0.378 ( 0.141
7.944 ( 0.430
0.009 ( 0.001
0.067 ( 0.002
7.135 ( 0.667
0.072 ( 0.001
>10
6.700 ( 0.411
0.460 ( 0.042
0.072 ( 0.004
3.514 ( 1.379
0.006 ( 0.000
0.057 ( 0.001
6.691 ( 0.439
0.061 ( 0.013
0.641 ( 0.000
0.735 ( 0.084
8.822 ( 0.083
0.009 ( 0.001
0.067 ( 0.000
6.979 ( 0.531
0.063 ( 0.001
0.592 ( 0.007
0.491 ( 0.132
>10
0.741 ( 0.003
0.889 ( 0.029
>10
0.007 ( 0.001
0.054 ( 0.000
8.764 ( 0.315
0.060 ( 0.004
0.039 ( 0.002
0.081 ( 0.006
8.062 ( 0.037
0.076 ( 0.000
a Antiproliferative effects of tested compounds against human tumor cell lines and drug-resistant cell lines in a 48 h growth inhibition assay using the
sulforhodamine B staining methods. Human cancer cell lines: breast (MCF-7), lung (NCI-H460), CNS (SF268), melanoma (UACC62), ovarian (IA9),
and its drug-resistant mutants PTX22 (Taxol-resistant) and A8 (epothilone-resistant). Growth inhibition of 50% (GI50) is calculated as the drug
concentration which caused a 50% reduction in the net protein increase in control cells during drug incubation. GI50 values for each compound are given
in µM and represent the mean of 2-5 independent experiments ( standard error of the mean. b A natural sample of 1 was kindly provided by Professor
B. J. Baker, University of South Florida, Tampa. c These cell lines were provided by the National Cancer Institute (NCI), Division of Cancer Treatment
and Diagnosis (DCTD). d These cell lines were provided by Professor Paraskevi Giannakakou, Weill Medical College of Cornell University.
developed copper-catalyzed protocol allowed access to pal-
merolide A analogues 50 (45% yield) and 51 (40% yield),
respectively, while analogue 52 remained elusive through this
particular strategy.
Some interesting trends were observed upon changing the
enamide appendage. Thus, substituting the isopropene moiety
of palmerolide A with a methyl group (compound 21) resulted
in more than a 100-fold loss of activity, whereas polar groups
such as those embedded in pyridine enamide analogues 22 and
23 and thiazole analogue 24 retained some activity. A rather
dramatic reversal of this trend, however, occurred when the
enamide side chain was restored to a nonpolar aromatic system
as in compound 25 which exhibited almost a 10-fold increase
in potency from that of palmerolide A (1) against several of
the cells tested. Interestingly, when the isopropene substituent
on the enamide moiety of palmerolide A was changed to its
saturated counterpart (compound 26), its potency remained more
or less intact. Finally, it was intriguing to observe that while
analogue 50 (lacking the C-10 hydroxyl group) has lost
significant activity across the entire panel of cell lines tested,
analogue 51 (missing the C-7 hydroxyl group) exhibited
equipotent activity to palmerolide A. Considering the easier
access through chemical synthesis to such deoxygenated ana-
logues, than the natural substance, this finding is valuable and
path pointing for future explorations within the field.
The synthesized compounds were tested against a panel of
cancer cells, including breast (MCF-7), melanoma (UACC-62),
CNS (SF268), lung (NCI-H460), ovarian (1A9), Taxol-resistant
ovarian (PTX22),17 and epothilone-resistant ovarian (A8)18 cells
using doxorubicin, Taxol, and natural palmerolide A (1) as
standards; the results are summarized in Table 2. Both natural
and synthetic palmerolide A (1) exhibited the same potent
activity against all cell lines tested, whereas ent-1 was at least
100-fold less active than 1. Simultaneously inverting the
stereochemistry at C-7/C-10/C-11 (compound 5), C-7/C-10/C-
11/C-19 (compound 6), and C-7/C-10/C-11/C-20 (compound
7) resulted in significant loss of activity. However, removing
the carbamate group from the C-11 oxygen of palmerolide A
(1) (compound 9) resulted in a ca. 5-fold decrease of activity
across most of the cell lines, whereas the same change in ent-1
(compound 8) did not have much effect on its potency.
Removing the carbamate group from diastereoisomers 6 and 7
(compounds 10 and 11) did not have a significant effect on the
potency of these analogues. The carbonate derivative (compound
12) of decarbamated ent-1 was also devoid of significant activity
and so were the vinyl iodide precursors of ent-1 and 1
(compounds 13 and 14). These findings point to the importance
of the enamide functionality for the biological activities of
palmerolide A.
Conclusion
The described chemistry rendered several palmerolide A
analogues for biological evaluation, allowing the first structure
activity relationships (SARs) of this newly discovered and
promising antitumor agent to be elucidated. These early conclu-
sions point the way for further, more focused studies aiming at
the design and synthesis of even more potent and selective
palmerolide A analogues as biological tools and potential drug
candidates.
(17) Giannakakou, P.; Sackett, D. L.; Kang, Y.-K.; Zhan, Z.; Buters,
J. T. M.; Fojo, T.; Poruchynsky, M. S. J. Biol. Chem. 1997, 272,
17118–17125.
(18) Giannakakou, P.; Gussio, R.; Nogales, E.; Downing, K. H.; Zaharevitz,
D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K. C.; Fojo, T. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 2904–2909.
Acknowledgment. This article is dedicated to Professor Chi-
Huey Wong on the occasion of his 60th birthday. Professor K.C.N.
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10022 J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008

Synthesis and Evaluation of Palmerolide A Analogues
A R T I C L E S
is also at the Department of Chemistry and The Skaggs Institute
for Chemical Biology, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, California 92037 (USA) and the
Department of Chemistry and Biochemistry, University of Cali-
fornia, San Diego, 9500 Gilman Drive, La Jolla, California 92093
(USA). We thank Ms. Doris Tan (ICES) for high resolution mass
spectrometric (HRMS) assistance and Dr. Han Weiping (Singapore
Bioimaging Consortium (SBIC), A*STAR) for the biological
screening facility. Financial support for this work was provided by
A*STAR, Singapore.
Supporting Information Available: Experimental procedures
and compound characterization (PDF, CIF). This material
is available free of charge via Internet at http://pubs.acs.
org.
JA802803E
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