Synthesis and initial structure--activity relationships of modified salicylihalamides.

Article abstract of DOI:10.1021/ol0068086

[reaction:see text] The first stereoselective total synthesis of the potent antitumor compound (-)-salicylihalamide A is presented. The practicality of our approach provides for high material throughput and is highlighted by the rapid construction of a variety of modified congeners. Initial structure-activity relationships are derived from growth inhibition experiments with a human melanoma cancer cell line.

Full text of DOI:10.1021/ol0068086

ORGANIC
LETTERS
2
000
Vol. 2, No. 26
241-4244
Synthesis and Initial Structure−Activity
Relationships of Modified
Salicylihalamides
4
Yusheng Wu, Olga R. Seguil, and Jef K. De Brabander*
Department of Biochemistry, The UniVersity of Texas Southwestern Medical Center at
Dallas, Dallas, Texas 75390-9038
jef.de brabander@utsouthwestern.edu
Received October 31, 2000
ABSTRACT
The first stereoselective total synthesis of the potent antitumor compound (−)-salicylihalamide A is presented. The practicality of our approach
provides for high material throughput and is highlighted by the rapid construction of a variety of modified congeners. Initial structure−activity
relationships are derived from growth inhibition experiments with a human melanoma cancer cell line.
2
Natural products continue to be the primary source for
compounds with unique biological functions. Because such
compounds could potentially interact with proteins of
unknown function, or interfere with cell-signaling pathways,
growth regulation, and differentiation in unique ways, they
constitute extremely valuable research tools in discovery
biology efforts. In this context, a unique opportunity is
provided by the discovery of the marine natural products
macrocyclic salicylate natural products. Notably, salicyli-
halamides displayed a unique signature in the National
Cancer Institute’s human tumor 60-cell line panel, indicating
a potentially novel mechanism of antineoplastic activity.1
Unfortunately, a limited supply from an unidentified species
of the marine sponge Haliclona prevents any serious efforts
to explore salicylihalamide’s molecular pharmacology and
develop these compounds as new chemotherapeutic leads for
the treatment of cancer.
1
salicylihalamides A and B (1 and 2, Figure 1), the first
examples of a growing number of structurally related
Our laboratory has offered an initial solution to this
problem by exploring reactivity patterns and chemical
(
1) Erickson, K. L.; Beutler, J. A.; Cardellina II, J. H.; Boyd, M. R. J.
Org. Chem. 1997, 62, 8188-8192.
2) Lobatamides: (a) McKee, T. C.; Galinis, D. L.; Pannell, L. K.;
(
Cardellina, J. H., II; Laakso, J.; Ireland, C. M.; Murray, L.; Capon, R. J.;
Boyd, M. R. J. Org. Chem. 1998, 63, 7805-7810. (b) Lobatamide A is
identical to the structure of YM-75518, see: Suzumura, K.-I.; Takahashi,
I.; Matsumoto, H.; Nagai, K.; Setiawan, B.; Rantiatmodjo, R. M.; Suzuki,
K.-I.; Nagano, N. Tetrahedron Lett. 1997, 38, 7573-7576. CJ-12,950 and
CJ-13,357: (d) Dekker: K. A.; Aiello, R. J.; Hirai, H.; Inagaki, T.;
Sakakibara, T.; Suzuki, Y.; Thompson, J. F.; Yamauchi, Y.; Kojima, N. J.
Antibiot. 1998, 51, 14-20. Apicularens: (e) Kunze, B.; Jansen, R.; Sasse,
F.; H o¨ fle, G.; Reichenbach, H. J. Antibiot. 1998, 51, 1075-1080. (f) Jansen,
R.; Kunze, B.; Reichenbach, H.; H o¨ fle, G. Eur. J. Org. Chem. 2000, 913-
919. Oximidines: (g) Kim, J. W.; Shin-ya, K.; Furihata, K.; Hayakawa,
Y.; Seto, H. J. Org. Chem. 1999, 64, 153-155.
Figure 1. Salicylihalamide A and side chain modified congeners.
1
0.1021/ol0068086 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/29/2000

3
compatibility issues related to salicylihalamides and other
efficient chemistry we had developed for the synthesis of 4.
Therefore, a series of optimization experiments were per-
formed that quickly led to the use of allyl diethylphospho-
noacetate in the Horner-Wadsworth-Emmons homologa-
tion, delivering allyl ester 6 as a single E isomer. Subsequent
macrocyclic salicylate natural products.4 These studies
culminated in a revision of the absolute configuration of (-)-
salicylihalamide A (1) through the first total synthesis of its
,5
3
enantiomer. To facilitate the search for a cellular target, we
required structural variants that would allow for the introduc-
tion of a suitable reporter without compromising biological
activity. Herein, we report a practical synthesis of naturally
occurring (-)-salicylihalamide A and a series of modified
congeners (generalized structure 3, Figure 1) to establish the
first structure-activity relationships (SAR).
treatment with BBr
3
was followed by bis-silylation of 7.
Isocyanate 10 was now in reach by Pd-catalyzed deprotection
8
9
of allyl ester 8, acyl azide formation, and Curtius rear-
rangement. Importantly, this sequence gave us ∼0.5 g of
isocyanate 10 with a dramatically improved overall yield
(75% from 4).
Our first objective was to deliver naturally occurring (-)-
Addition of a 1:1 mixture of (1Z,3Z)- and (1Z,3E)-1-lithio-
1,3-hexadiene to isocyanate 10 and final deprotection af-
forded a 1:1 mixture of salicylihalamide A (1) and C22-E
3
salicylihalamide A. Following our initial route, homologa-
tion of the aldehyde6 derived from 47 with trimethyl
phosphonoacetate yielded 5 as a 4:1 mixture of E/Z isomers
3,5d-g,10
isomer 11.
This mixture was indistinguishable, within
(
Scheme 1). Subsequent routine transformations then deliv-
the limits of experimental error, from natural salicylihalamide
A on the basis of comparative testing in the National Cancer
11
Institute 60-cell screen. Careful examination of the product
mixture identified the presence of two additional compounds,
which were purified and characterized as salicylihalamide
Scheme 1a
1
2
dimers 12 and 13.
We next turned our attention to a series of side chain
modified analogues. The best starting point to initiate these
efforts would take advantage of the extremely efficient and
high-yielding construction of isocyanate 10. Carbamate 16
was prepared by heating 10 in the presence of n-pentanol
followed by deprotection (50%, 2 steps). Tetrahydrosalicyli-
halamide 14 and the corresponding dimer 15 were obtained
in a manner identical to that described for 1. While lacking
the enoyl functionality (potential Michael acceptor), ana-
logues 14 and 16 displayed significant growth inhibitory
activity against the human melanoma cell line SK-MEL-5
(3) Wu, Y.; Esser, L.; De Brabander, J. K. Angew. Chem., Int. Ed. 2000,
in press.
(4) For our synthetic efforts toward apicularen A, see: Bhattacharjee,
A.; De Brabander, J. K. Tetrahedron Lett. 2000, 41, 8069-8073.
(5) For other synthetic efforts, see: (a) Georg, G. I.; Blackman, B.;
Mossman, C. J.; Yang, K.; Flaherty, P. T. Abstracts of Papers, 219th
National Meeting of the American Chemical Society, San Francisco, March
2
000; American Chemical Society: Washington, DC, 2000; ORGN 807.
(
b) F u¨ rstner, A.; Thiel, O. R.; Blanda, G. Org. Lett. 2000, 2, 3731-3734.
(
c) Feutrill, J. T.; Holloway, G. A.; Hilli, F.; H u¨ gel, H. M.; Rizzacasa, M.
A. Tetrahedron Lett. 2000, 41, 8569-8572. For studies related to the
enamide side chain, see: (d) Snider, B. B.; Song, F. Org. Lett. 2000, 2,
4
3
07-408. (e) Kuramochi, K.; Watanabe, H.; Kitahara, T. Synlett 2000, 397-
99. (f) Shen, R.; Porco, J. A., Jr. Org. Lett. 2000, 2, 1333-1336. (g)
Stefanuti, I.; Smith, S. A.; Taylor, R. J. K. Tetrahedron Lett. 2000, 41,
3
735-3738.
(
6) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-7287.
(7) In our previously reported synthesis of (+)-salicylihalamide A, all
3
three stereocenters in ent-4 were introduced by reagent-controlled reactions.
The same chemistry was exploited for the preparation of 4, except for the
use of antipodal chiral reagents.
a
2 2
Reagents and conditions: (a) Dess-Martin periodinane, CH Cl ;
(
b) allyl diethylphosphonoacetate, NaH, THF, 0 °C, 97% (two
steps); (c) BBr , CH Cl , -78 °C, 92%; (d) TBSCl, imidazole,
DMF, 95%; (e) cat. Pd(PPh , morpholine, THF, 97%; (f)
PhO) P(O)N , Et N, PhH; (g) PhH, 80 °C, 93% (two steps); (h)
-bromo-1,3-hexadiene (1:1 mixture of 1Z,3Z and 1Z,3E isomers)
or 1-bromohexane, t-BuLi, Et O, -78 °C, then add 10, -78 °C;
(
8) Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. Engl. 1984, 23,
3
2
2
7
1-72.
3 4
)
(
9) Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974, 30, 2151-
(
1
2
3
3
2
157.
(10) The antipodal geometrical isomers were previously separated and
3
2
fully characterized individually. The spectroscopic data of the mixture (1
and 11) were in full accord with those previously obtained.
(
in the same screen, indicating that biological function is at least partly
dependent on a correctly configured macrolactone. We thank Dr. Michael
R. Boyd (National Cancer Institute) for testing our compounds in the 60-
cell line panel.
(
i) HF‚pyr., pyr./THF, 20% for 1/11, 10% for 12, 10% for 13, 22%
11) The corresponding enantiomers were completely devoid of activity
for 14, 14% for 15 (two steps); (j) 1-pentanol, PhH, 75 °C, then
step i, 50% (two steps).
(
12) These dimers are presumably formed through reaction of the
3
ered isocyanate 10 in 20-30% overall yield from 4. The
intermediate lithiated amide with a second molecule of isocyanate 10. For
moderate yields and stereoselectivity detracted from the
a similar observation, see ref 5e.
4242
Org. Lett., Vol. 2, No. 26, 2000

[
GI50 0.38 µM (14); 0.5 µM (16)].13 Interestingly, salicyli-
a putative binding protein through a protonation/nucleophilic
addition mechanism (RC(O)NHCHdCHR f RC(O)NH d
+
halamide dimers 12, 13, and 15 were found to be effective
at a concentration range similar to that of the parent
salicylihalamides [GI50 0.04 µM (12); 0.1 µM (13); 0.6 µM
CH-CH
2
R f RC(O)NHCHXR′-CH R, XR′ ) nucleophilic
2
1
6
amino acid residue). The most straightforward and least
intrusive way to knock out the chemical reactivity associated
with the acylated enamine would constitute a hydrogenation
of salicylihalamide’s enamine double bond. It was antici-
pated, however, that the presence of the endocyclic double
bond would pose a serious chemoselectivity problem. For
example, direct hydrogenation of biologically active salicyli-
halamide carbamate 16 is likely to deliver the doubly
saturated analogue 22 (Scheme 2). To separate the effect of
endocyclic double bond saturation from enamine saturation
on biological activity, it was imperative to prepare the
saturated macrolactone mutant 21. Our point of departure
13
(15)]. This observation indicates that substantial sterically
demanding modifications can be made without abrogating
biological activity and can be exploited for the development
of analogues and probe reagents.
Side chain modified analogues that lack salicylihalamide’s
characteristic N-acyl enamine functionality are attractive
candidates for the following reasons: (1) they are expected
to confer increased acid stability, (2) they can potentially be
prepared via shorter sequences, and (3) they would answer
an important question related to the functional role of the
N-acyl enamine moiety. Octanoate 18, a compound with
identical chain length and similar hydrophobicity to that of
salicylihalamide, is representative of this class of compounds
and was prepared from alcohol 4 via a Mitsunobu esterifi-
17
entailed a transfer hydrogenation of (Z)-cycloalkene 19 with
concomitant removal of the p-methoxybenzyl (PMB) ether.18
Subsequent conversion of 20 to 21 took full advantage of
the chemistry outlined for the preparation of 16 (Scheme 1)
without complication. Hydrogenation of this material then
yielded the fully saturated salicylihalamide 22.
14
cation followed by deprotection (Scheme 2). An additional
Scheme 2a
While lacking the endocyclic double bond of carbamate
6 (GI50 0.5 µM), analogue 21 retained a significant, although
1
attenuated, level of growth inhibitory activity (GI50 8 µM).13
This is of significance because we now have a calibration
point for comparing the effect of the enamine to amine
permutation (21 f 22), which completely abolished the
antiproliferative potential of 21. The question remains,
however, if an increased conformational flexibility of the
side chain or the inability to form a covalent bond is at the
origin of this deleterious (with respect to biological activity)
mutation. The final answer will have to come from labeling
studies with a yet to be identified cellular target.
In summary, we have synthesized for the first time (-)-
salicylihalamide A, a compound that is no longer available
from natural sources. Optimization of the chemistry involved
has put us in the comfortable position of high material
throughput, which was exploited for the synthesis of a variety
of synthetic salicylihalamides. Initial SAR studies have
revealed an important role for both the side chain and the
macrolactone and have indicated possibilities for the devel-
opment of salicylihalamide-based molecular probes.
Acknowledgment. Financial support provided by the
Robert A. Welch Foundation and junior faculty awards
administered through the Howard Hughes Medical Institute
a
Reagents and conditions: (a) octanoic acid, DEAD, PPh
, CH Cl , -78 °C, 80% (two steps); (c) Pd/C, NH
MeOH 70%; (d) Pd/C, H , MeOH, 30%.
3
, Et
2
O;
(
b) BBr
3
2
2
4
O
2
CH,
(13) Growth inhibition was determined 2 days after the addition of the
compounds by the MTT assay (Mosmann, T. J. Immunol. Methods 1983,
2
6
5, 55-63). The GI50 values were calculated on the basis of triplicate assays
at four different concentrations of the drug. Synthetic salicylihalamide A
(
1/11) was used as a positive control (GI50 0.07 µM).
member is represented by allyl ester 7, an intermediate for
the synthesis of salicylihalamides (Scheme 1). The inability
of analogues 7 and 18 to arrest cell growth13 indicates a
specific role for the side chain, perhaps expressed in the
N-acyl enamine functionality.15
At this point, a more in depth evaluation of the functional
role for the acylated enamine was conducted. Indeed, it is
possible that salicylihalamides form a covalent complex with
(14) Mitsunobu, O. Synthesis 1981, 1-28.
(15) However, an intact side chain is not sufficient for biological activity;
see footnote 11.
(16) For an example of an enamide-containing natural product, and
proposed modification of an N-acyliminium ion by an enzyme active site
nucleophile, see: Gentle, C. A.; Bugg, T. D. H. J. Chem. Soc., Perkin Trans.
1
1999, 1279-1285.
(17) Bieg, T.; Szeja, W. Synthesis 1985, 76-77.
(18) While compound 20 is in principle accessible from 4, we opted to
make a productive use of 19, a minor isomeric byproduct obtained en route
to the synthesis of 4.3
Org. Lett., Vol. 2, No. 26, 2000
4243

1
and the University of Texas Southwestern Medical Center
are gratefully acknowledged. We also thank Dr. Michael G.
Roth and Maria G. Kosfiszer (Department of Biochemistry)
for invaluable assistance with the cell-based assays and Dr.
Michael R. Boyd (National Cancer Institute) for providing
a sample and NMR spectra of natural salicylihalamide A.
Supporting Information Available: H NMR spectra of
1/11, ent-1, and ent-11, the procedure for the preparation of
1/11 and 12-16, and characterization data and H NMR
spectra for compounds 6-9, 12-18, and 20-22.
1
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