Total synthesis and properties of the crambescidin core zwitterionic acid and crambescidin 359

Article abstract of DOI:10.1021/ja042875+

The total synthesis of the crambescidin core acid 9, crambescidins 359 (8) and 431 (7), and the properties of the crambescidin core are described. A key step of the synthetic route to guanidinium carboxylate 9 is Pd(0) catalyzed cleavage of the ester side chain of pentacyclic cinnamyl ester 15. This ester is also employed to prepare a small library of crambescidin alkaloid analogues that differ in their C14 side chain. The zwitterionic guanidinium carboxylate 9 was shown to readily decarboxylate to form crambescidin 359 (8). Decarboxylation of crambescidin core acid 9 was fastest under basic conditions. In the presence of base, up to eight deuterium atoms can be incorporated into the pentacyclic crambescidin core. Both deuterium incorporation and decarboxylation of crambescidin core acid 9 are the result of facile ring opening of the spirocyclic ether rings of the pentacyclic guanidinium moiety.

Full text of DOI:10.1021/ja042875+

Published on Web 02/18/2005
Total Synthesis and Properties of the Crambescidin Core
Zwitterionic Acid and Crambescidin 359
Zachary D. Aron and Larry E. Overman*
Contribution from the Department of Chemistry, 516 Rowland Hall, UniVersity of California,
IrVine, California 92697-2025
Received November 25, 2004; E-mail: leoverma@uci.edu
Abstract: The total synthesis of the crambescidin core acid 9, crambescidins 359 (8) and 431 (7), and the
properties of the crambescidin core are described. A key step of the synthetic route to guanidinium
carboxylate 9 is Pd(0) catalyzed cleavage of the ester side chain of pentacyclic cinnamyl ester 15. This
ester is also employed to prepare a small library of crambescidin alkaloid analogues that differ in their C14
side chain. The zwitterionic guanidinium carboxylate 9 was shown to readily decarboxylate to form
crambescidin 359 (8). Decarboxylation of crambescidin core acid 9 was fastest under basic conditions. In
the presence of base, up to eight deuterium atoms can be incorporated into the pentacyclic crambescidin
core. Both deuterium incorporation and decarboxylation of crambescidin core acid 9 are the result of facile
ring opening of the spirocyclic ether rings of the pentacyclic guanidinium moiety.
Introduction
alkaloids. The potential for these compounds as therapeutic
agents has been recognized, as they are currently undergoing
The crambescidin family of guanidinium alkaloids, isolated
from a variety of marine sponges, exemplifies the valuable
compounds that have been discovered through recent investiga-
tions of marine natural products.1 The first member of the
crambescidin family discovered, ptilomycalin A (1), was initially
isolated by Kashman, Kakisawa and co-workers from the Red
Sea sponge Hemimycale sp. and a Caribbean sponge originally
identified as Ptilocaulis spiculifer.2,3 Ptilomycalin A (1) and
many cognate crambescidin alkaloids display nanomolar cyto-
toxicities against several tumor cell lines.4-6 Antifungal activity
against Candida albicans, antiviral activity against herpes
simplex virus (HSV),2,7,8 anti-HIV activity,9 inhibition of HIV-1
envelope mediated cell fusion,10 and inhibition of the binding
of various proteins to HIV-1 Nef11 are possibly useful biological
activities reported also for members of this family of guanidine
early phase development as anti-cancer agents.12
Members of the crambescidin alkaloid family are character-
ized structurally by a unique pentacyclic guanidinium core
(Figure 1). Crambescidins that vary in their substitution at C14
are exemplified by crambescidins 800(2a),4,13 359 (8),14 431
(7),14 657 (10a),5 neofolitispates 1-3 (11-13),15 crambescidin
core acid 9,16 and celeromycalin (3).9 Additional members of
this family contain a hydroxyl group at C13: crambescidins
816 (4), 830 (5), and 844 (6).4 A small group, the 13,14,15-
isocrambescidins (not shown), have a different configuration
of the pentacyclic guanidinium moiety.8,17
Despite the broad range of crambescidin alkaloid structures,
there is limited understanding of structure-activity relationships
of these natural products. The presence of a side chain is well-
established to be important for anticancer activity.12,18 However,
simple spermidine amides of ω-hydroxycarboxylic acids lacking
a guanidine core are inactive.7,9 There is little understanding of
how the nature of the C14 side chain impacts biological activity.
One reason for the lack of systematic studies in this area has
been the inability to remove the C14 side chain from the more
available of these marine alkaloids without destroying the
(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2004, 67, 1216-1238.
(2) Kashman, Y.; Hirsh, S.; McConnell, O. J.; Ohtani, I.; Kusumi, T.; Kakisawa,
H. J. Am. Chem. Soc. 1989, 111, 8925-8926.
(3) The Caribbean sponge was originally identified as Ptilocaulis aff. Spiculifer.
A reexamination of the voucher specimen has led to this sponge being
characterized as belonging to the genus Batzella Topsent, 1891, which is
closely related to the genera Crambe and Monanchora, see: Tavares, R.;
Daloze, D.; Braekman, J. C.; Hajdu, E.; Van Soest, R. W. M. J. Nat. Prod.
1995, 58, 1139-1142.
(4) Jares-Erijman, E. A.; Sakai, R.; Rinehart, K. L. J. Org. Chem. 1991, 56,
5712-5715.
(11) Olszewski, A.; Sata, K.; Aron, Z. D.; Cohen, F.; Harris, A.; McDougall,
B. R.; Robinson, W. E. Jr.; Overman, L. E.; Weiss, G. A. Proc. Natl. Acad.
Sci. USA 2004, 101, 14079-14084.
(5) Rinehart, K. L.; Jares-Erijman, E. A. U. S. Patent 5,756,734; 1998.
(6) Ptilomycalin A recently was shown to induce differentiation of K562 cells
with arrest of the cell cycle at the S-phase, see: Aoki, S.; Kong, D.; Matsui,
K.; Kobayashi, M. Anticancer Res. 2004, 24, 2325-2330.
(7) Ohtani, I.; Kusumi, T.; Kakisawa, H.; Kashman, Y.; Hirsh, S. J. Am. Chem.
Soc. 1992, 114, 8472-8479.
(12) Aron, Z. D.; Pietraszkiewicz, H.; Overman, L. E.; Valeriote, F.; Cuevas,
C. Bioorg. Med. Chem. Lett. 2004, 14, 3445-3449.
(13) Berlinck, R. G. S.; Braekman, J. C.; Daloze, D.; Bruno, I.; Riccio, R.;
Ferri, S.; Spampinato, S.; Speroni, E. J. Nat. Prod. 1993, 56, 1007-1015.
(14) Braekman, J. C.; Daloze, D.; Tavares, R.; Hajdu, E.; Van Soest, R. W. M.
J. Nat. Prod. 2000, 63, 193-195.
(8) Jareserijman, E. A.; Ingrum, A. L.; Carney, J. R.; Rinehart, K. L.; Sakai,
R. J. Org. Chem. 1993, 58, 4805-4808.
(15) Venkateswarlu, Y.; Reddy, M. V. R.; Ramesh, P.; Rao, J. V. Indian J.
Chem. Sect. B 1999, 38, 254-256.
(9) Palagiano, E.; Demarino, S.; Minale, L.; Riccio, R.; Zollo, F.; Iorizzi, M.;
Carre, J. B.; Debitus, C.; Lucarain, L.; Provost, J. Tetrahedron 1995, 51,
3675-3682.
(16) Meragelman, K. M.; McKee, T. C.; McMahon, J. B. J. Nat. Prod. 2004,
67, 1165-1167.
(10) Chang, L.-C.; Whittaker, N. F.; Bewley, C. A. J. Nat. Prod. 2003, 66,
1490-1494.
(17) Shi, J.-G.; Sun, F.; Rinehart, K. L. Crambescidin Compounds, WO Patent
98/46575; 1998.
9
3380
J. AM. CHEM. SOC. 2005, 127, 3380-3390
10.1021/ja042875+ CCC: $30.25 © 2005 American Chemical Society

Crambescidin Core Zwitterionic Acid
A R T I C L E S
Scheme 1. Tethered Bignelli Approach to Crambescidin Alkaloids
been reported.18a,22-27 Of these, only our efforts and those of
Snider and co-workers resulted in preparation of the full
crambescidin core having a pendant C14 carboxylic ester side
chain.18a,24-26
Our strategy for the total synthesis of crambescidin alkaloids
such as crambescidin 657 (10a) employs a tethered Biginelli
condensation28 to combine two fragments: one, 18, encoding
C1-C13 and the urea precursor of the guanidine, and the other,
19, comprising C14-C20 and the C14 side chain (Scheme
1).24-27 To access the crambescidin core acid 9, the elaborate
side chain would be replaced with one that could be cleaved
under mild conditions to reveal a carboxylic acid substituent at
C14. In our earliest investigations,27,29 we found that an allyl
ester could be cleaved efficiently in the presence of a fully
constituted pentacyclic guanidine moiety.30 This strategy was
employed in the present investigation. However, rather than
using a simple allyl group, we chose to employ a trans-cinnamyl
ester (20 f 17 f 15), because this substituent would both
decrease the polarity of late stage intermediates and provide a
convenient chromophore.
Figure 1. Crambescidin natural products.
pentacyclic guanidine moiety. For example, Kashman and co-
workers reported that subjection of ptilomycalin A (1) to acid
or base hydrolysis, metal hydride reduction, catalytic hydroge-
nation or oxidation, resulted in the formation of intractable
mixtures.7,19,20 The inability to access core acid 9 by degradation,
and its reported isolation in low yield from natural sources only
earlier this year,16 combine to make chemical synthesis the only
viable way to obtain unnatural C14 side chain congeners of the
crambescidin alkaloids.
This paper describes the total synthesis, isolation,21 structural
characterization, and derivitization of the crambescidin core acid
9. Besides providing the first complete description of the
physical and chemical properties of this important species, these
studies resulted in the synthesis of crambescidins 359 (8) and
431 (7), and libraries of crambescidin analogues that vary at
C14. Evidence concerning the likely biosynthetic origin of
crambescidin 359, and a proposal of one potential mode of
action of the crambescidin alkaloids is also provided.
B. Synthesis of the Crambescidin Core Cinnamyl Ester
15. The starting point for this synthesis was the known
enantiopure methyl ester 21, which is available in five steps
and 58% overall yield from commercially available poly-(3-
hydroxybutyric) acid.24,27,29 Initial attempts to convert methyl
â-ketooctanoate 21 to its cinnamyl congener 20 by reaction with
cinnamyl alcohol in refluxing toluene using 4-(dimethylamino)-
pyridine (DMAP) as catalyst31 were complicated by competitive
Results
A. Synthetic Strategy. Several syntheses of members and
congeners of the crambescidin family of natural products have
(22) Heys, L.; Moore, C. G.; Murphy, P. J. Chem. Soc. ReV. 2000, 29, 57-
67.
(18) (a) Snider, B. B.; Shi, Z. P. J. Am. Chem. Soc. 1994, 116, 549-557. (b)
Black, G. P.; Coles, S. J.; Hizi, A.; Howard-Jones, A. G.; Hursthouse, M.
B.; McGown, A. T.; Loya, S.; Moore, C. G.; Murphy, P. J.; Smith, N. K.;
Walshe, N. D. A. Tetrahedron Lett. 2001, 42, 3377-3381. (c) Moore, C.
G.; Murphy, P., J.; Williams, Harri L.; McGown, Alan T.; Smith, Nigel K.
Tetrahedron Lett. 2003, 44, 251-254. (d) Georgieva, A.; Hirai, M.;
Hashimoto, Y.; Nakata, T.; Ohizumi, Y.; Nagasawa, K. Synthesis-Stuttgart,
2003, 1427-1432.
(23) Nagasawa, K.; Georgieva, A.; Koshino, H.; Nakata, T.; Kita, T.; Hashimoto,
Y. Org. Lett. 2002, 4, 177-180.
(24) Coffey, D. S.; McDonald, A. I.; Overman, L. E.; Rabinowitz, M. H.;
Renhowe, P. A. J. Am. Chem. Soc. 2000, 122, 4893-4903.
(25) Coffey, D. S.; Overman, L. E.; Stappenbeck, F. J. Am. Chem. Soc. 2000,
122, 4904-4914.
(26) Coffey, D. S.; McDonald, A. I.; Overman, L. E.; Stappenbeck, F. J. Am.
Chem. Soc. 1999, 121, 6944-6945.
(19) A patent application claims that the core acid is obtained upon reacting
ptilomycalin A with an unspecified mixture of NaOH, MeOH and water at
65 °C.20 However, no characterization data to support this assertion was
provided.
(27) Overman, L. E.; Rabinowitz, M. H.; Renhowe, P. A. J. Am. Chem. Soc.
1995, 117, 2657-2658.
(28) For a recent review, see: Aron, Z. D.; Overman, L. E. Chem. Commun.
2004, 253-265.
(20) Mai, S. H.; Nagulapalli, V. K.; Patil, A. D.; Truneh, A.; Westly, J. W.
Marine Compounds as HIV Inhibitors, WO Patent 93/01193; 1993.
(21) An early version of the synthesis described herein was described in
Overman, L. E.; Stappenbeck, F.; McDonald, A. I. Hexahydropyrrolo[1,2-
c]pyrimidines as Antiviral and/or Antitumor Agents WO Patent 01/00626,
January, 2001.
(29) Renhowe, P. A. Ph. D, Dissertation, University of California, Irvine.
1995.
(30) (a) Jeffery, P. D.; McCombie, S., W. J. Org. Chem. 1982, 47, 587-590.
For a review of allylic protecting groups, see: (b) Guibe, F. Tetrahedron
1998, 54, 2967-3042.
9
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A R T I C L E S
Aron and Overman
Scheme 2. Preparation of Cinnamyl â-ketoester 20
Thus, resubjection of 28, followed by separation of the resulting
epimers on silica gel, when repeated two times, provided
isomerically pure 15 in 53% overall yield from tricyclic urea
26.
C. Synthesis, Isolation and Single-Crystal X-ray Analysis
of Crambescidin Core Acid 9. A variety of palladium catalysts
and reaction conditions30 were examined for the deprotection
of cinnamyl ester 15 to form core acid 9. (Tetrakistriph-
enylphosphine)palladium (0.3 equiv) emerged as the preferred
catalyst and triethylammonium formate as the reducing agent
of choice (eq 1). When deprotection of 15 was carried out using
these reagents at room temperature in THF, guanidinium
retro-Claisen fragmentation to form ultimately cinnamyl hex-
anoate 22 (Scheme 2). Lowering the reaction temperature to
85 °C, necessary to avoid the retro-Claisen reaction, resulted
in incomplete conversion of 21 to 20. However, the use of a
large excess of cinnamyl alcohol (7 equiv) and azeotropic
removal of methanol with acetonitrile resulted in complete
consumption of 21, providing cinnamyl â-ketoester 20 in 74%
yield.
1
carboxylate 9 was formed in high yield (>90% yield by H
NMR analysis using an internal standard).
Using experimental procedures that had been optimized
during our earlier syntheses of crambescidin 800 (2a) and
congeners,24,27 the cinnamyl ester derivative 15 of the cramb-
escidin core acid was prepared by the sequence depicted in
Scheme 3. Dienyl urea 23, available in 14 steps from 3-bu-
tynol,24,27 first was converted in two steps to aminal 18. Tethered
Biginelli condensation of this intermediate with â-ketoester 20
provided a 1:6 mixture of pyrrolopyrimidines 24 and 17 in 63%
overall yield from urea 23. Epimers 24 and 17 could be
separated by HPLC, but were generally carried forward as a
mixture. The configurational assignments of these intermediates
were based on nOe studies of later intermediates and 1H NMR
correlations of the H13 methine hydrogens (24: 4.40 ppm, 17:
4.26 ppm) with reference compounds.32 Cleavage of the siloxy
protecting groups of 17 with tetrabutylammonium flouride
(TBAF) in DMF generated the corresponding diol, which
underwent acid-promoted spirocyclization in the presence of 1
equiv of p-toluenesulfonic acid at room temperature to form
tricyclic urea 2533 in 67% overall yield. Limiting the reaction
temperature was key to suppressing unproductive side reactions
during the spirocyclization step.
The cinnamyl ester 15 of the crambescidin core was prepared
in three additional steps from tricyclic intermediate 25. First
the alcohol group of 25 was protected as an R-chloroacetate to
avoid methyl ether formation in the subsequent step. At this
point, the minor trans tricyclic isomer derived from 24 was easily
removed by silica gel chromatography to provide isomerically
pure 26 in 74% yield. Exposure of keto urea 26 to excess MeOTf
in the presence of 2,6-di-tert-butyl-4-methylpyridine (DTBMP)
yielded methyl pseudourea 27. Rigorous exclusion of water was
necessary to achieve reproducibly high yields in this step. Finally
exposure of crude pseudourea 27 to anhydrous ammonia (18
psi) at 60 °C in allyl alcohol buffered with NH4Cl for 1.5 days
provided a 1.1:1.0 mixture of pentacyclic esters 28 and 15 in
78% yield. This mixture could be separated on silica gel.34 Our
previous studies had shown that the C14 stereocenter would
have been equilibrated under these aminolysis conditions.24,27
Purification of core acid 9 was challenging as this zwitterion
is a polar, highly crystalline solid, which is sparingly soluble
in most solvents. When used as an impure oil, acid 9 is slightly
soluble (∼0.05-0.1 M) in solvents such as benzene, methanol
or dichloromethane. However, pure crystalline 9 is soluble only
in mixtures of alcoholic and halogenated solvents. In addition
to demonstrating poor solubility, acid 9 decarboxylates under a
variety of conditions (vide infra). The low solubility and high
polarity of 9 effectively prohibited its isolation in pure form by
either normal or reverse phase chromatography. However, some
purification could be obtained by filtration through Davisil silica
gel (20-40% methanol/chloroform) to provide a 60% recovery
of a colorless oil that was ∼80% pure. Fortunately, core acid 9
precipitates from deallylation reactions when run at high
concentrations. Isolation of this material by filtration provided
analytically pure 9, [R]26405 -12.4 (c 0.1, 1:1 MeOH-CHCl3),
in 53-68% yield.35 Examination of the remaining solution
revealed the presence of residual 9; however, attempts to recover
this material from the filtrate led to decomposition and the
formation of black solids.
Single crystals of 9 could be grown as its methanol solvate
from mixtures of methanol and dichloromethane. X-ray analysis
of this material resulted in the first crystal structure of a
crambescidin alkaloid (Figure 2).
D. Decarboxylation of the Crambescidin Core Acid 9;
Total Synthesis of Crambescidin 359 (8). Core acid 9
decarboxylates upon storage at 0 °C or room temperature for
several weeks, the product of decarboxylation being consistently
observed by electrospray mass spectrometry in samples of 9.
Purification of the decarboxylation product by HPLC provided
crambescidin 359 (8) (eq 2). The chloride salt of this material
(35) (a) The corresponding material isolated from the sponge Monanchora
ungiculata, [R]25 -19 (c 0.04, MeOH)16 was apparently an oil as the
D
(31) Taber, D. F.; Amedio, J. C., Jr.; Patel, Y. K. J. Org. Chem. 1985, 50, 3618-
3619.
crystalline core acid is not soluble at that concentration in MeOH. The 1H
and 13C NMR spectra reported for natural 9 vary somewhat from what we
observe with a crystalline sample. 1H NMR signals for several hydrogens
of zwitterion 9 and the corresponding cationic acid vary considerably. The
material isolated by McKee and co-workers might have been a mixture of
these materials. (b) The absolute configuration of 9 is depicted incorrectly
in reference 16.
(32) McDonald, A. I.; Overman, L. E. J. Org. Chem. 1999, 64, 1520-1528.
(33) This intermediate contained ∼15% of the C13 epimer formed during the
tethered Biginelli condensation.
(34) Small quantities of other diastereoisomers, possibly epimers at C10, were
removed at this point.
9
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Crambescidin Core Zwitterionic Acid
A R T I C L E S
Scheme 3. Synthesis of Pentacyclic Cinnamyl Ester 15
Table 1. Base Catalyzed Decarboxylation of Crambescidin Acid 9
(8, X ) Cl) exhibited spectroscopic properties identical to those
reported for natural crambescidin 359 (8).14
time
(h)
[buffer]
(mM)
buffer ratio
(OH:ONa)a
% conv.
43
43
43
53
53
53
25
50
75
50
50
50
1:1
1:1
1:1
2:1
1:1
1:2
43
56
59
44
63
73
a p-methoxyphenol buffers
To gain additional insight into the decarboxylation of core
acid 9, it was exposed to several buffers in a 1:1 mixture of
CDCl3-CD3OD (at ∼0.006 M); reaction progress was moni-
tored by the dissappearance of the C14 methine hydrogen of 9
using 1,3,5-trimethoxybenzene as an internal standard. At low
(formate buffer) or neutral (p-nitrophenol buffer) pH, decar-
boxylation of 9 occurs in a pH independent manner to give
∼20% of crambescidin 359 (8) after 2.5 days at room temper-
ature. However under basic conditions, acid 9 undergoes rapid,
pH dependent, decarboxylation at room temperature. As sum-
marized in Figure 3, decarboxylation of 9 occurs substantially
faster in the presence of a p-methoxyphenol buffer (2.5 equiv
of both p-methoxyphenol and sodium p-methoxyphenoxide, pKa
of p-methoxyphenol ) 10.20 in H2O) than in a comparable
p-bromophenol buffer (p-bromophenol pKa ) 9.00 in H2O).
The mechanism of base catalysis of the decarboxylation of 9
was studied briefly, however the low solubility of 9 and its
limited availability prevented a detailed examination. Increasing
the concentration of the 1:1 p-methoxyphenol/sodium p-meth-
oxyphenolate buffer by a factor of 3, increased the rate of
decarboxylation slightly (∼35%, see Table 1). Changing the
ratio of phenoxide to phenol from 1:2 to 2:1 (equivalent to a
change in pH of 0.6 units) increased the rate by 65% (Table 1).
These results are consistent with decarboxylation being both
specific base and general base (or specific base/general acid)
catalyzed.
E. Incorporation of Deuterium into the Crambescidin
Core. While studying the decarboxylation of core acid 9 in
deuterated solvents, incorporation of deuterium into the product,
crambescidin 359 (8), was observed. In deuterated solvents at
high pH, crambescidin 359 (8) incorporated up to eight
Figure 2. X-ray model of crambescidin core acid 9 methanol solvate.
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A R T I C L E S
Aron and Overman
Table 2. Deuterium Incorporation into Crambescidin 359 (8) at
Various PD and Buffer Concentrations
time
(h)
[buffer]
(mM)
average no.
pD
of D incorporated
62
62
62
62
62
12.0
12.5
13.0
13.0
13.0
40
40
40
20
10
1.0
2.8
3.8
2.2
1.1
Figure 3. Decarboxylation of core acid 9 in a variety of buffers.
Figure 5. Deuterium incorporation into crambescidin 359 (8) as a function
of time at 63 °C and pD ) 13.
Deuterium incorporation into the crambescidin core was
studied also in D2O.38 The low solubility of cinnamyl ester 15
in D2O prohibited the collection of meaningful data for this
substrate. Accordingly, only core acid 9 and crambescidin 359
(8) were examined. Studies were carried out over the pD range
4-12.5; however, deuterium incorporation at room temperature
was only seen at high pH. As had been observed in organic
solvents, core acid 9 underwent extensive decarboxylation at
pD > 12.39 Although core acid 9 did not incorporate deuterium
in organic solvents, deuterium incorporation into both residual
core acid 9 and crambescidin 359 (8) occurred with similar rates
in D2O in the pD range 9-12.5.40
Crambescidin 359 (8) was chosen for detailed study in
aqueous medium, with deuterium incorporation being examined
in various phosphate buffers. The rate of decarboxylation
increased with both increasing pD and buffer concentration
(Table 2). These variations in rate indicate that deuterium
incorporation into crambescidin 359 (8), similar to decarboxy-
lation of core acid 9, is both specific base and general base (or
specific base/general acid) catalyzed.
1
Figure 4. (a) 800 MHz H NMR spectrum of crambescidin 359 (8) after
incorporation of eight nonexchangeable deuterium atoms; the signals marked
with an X are from an unknown impurity having an isolated spin system.
(b) 800 MHz H NMR of unlabeled crambescidin 359 (8).
1
nonexchangeable deuterium atoms. For example, incubating a
sample of crambescidin 359 (8) with a 1:1 mixture of D2O and
methanol containing a 0.1 M phosphate buffer (pD ) 13 in
H2O) at 65 °C for 25 h resulted in the incorporation of 7.9
deuterium atoms. Deuterium incorporation was identified by 1H
NMR spectroscopy to have occurred at C7, C9, C14, and C16
(Figure 4).
To gain a preliminary understanding of deuterium incorpora-
tion into the crambescidin pentacyclic guanidinium moiety,
samples of crambescidin 359 (8), core acid 9 and cinnamyl ester
15 were dissolved in a 1:1 mixture of CDCl3-CD3OD in the
presence of a 0.015 M p-methoxyphenol/sodium p-methoxy-
phenolate (1:1) buffer. These reactions then were monitored as
a function of time by quenching aliquots into acidic methanol,
which also washed out any exchangeable deuterium atoms. The
average amount of deuterium incorporated at a given time was
calculated from isotope patterns.36,37 After 55 h at room
temperature, crambescidin 359 (8) had incorporated an average
of 2.8 deuterium atoms, whereas cinnamyl ester 15 had
incorporated an average of 1.2 deuterium atoms. As was
expected, core acid 9 underwent extensive decarboxylation under
these reaction conditions to form crambescidin 359 (8). To our
surprise, 9 did not incorporate deuterium, despite the concurrent
incorporation of deuterium into crambescidin 359 (8).
The amount of deuterium incorporated into crambescidin 359
(8) was studied also as a function of time at 63 °C and pD )
13 (Figure 5). Two features are apparent in the data obtained
from the experiment summarized in Figure 5: deuterium
incorporation exhibits a small induction period40 and a sudden
decrease in rate after the incorporation of approximately four
deuterium atoms. Data collected between 10 min and 8.5 h is
consistent with the presence of two parallel pseudo-first-order
processes, with approximate values for kobs of 1.7 and 0.17 h-1 41
.
(38) In experiments performed in water, the ionic strength of buffer solutions
was maintained constant by the addition of cesium iodide.
(39) Accurate quantification of the rate of decarboxylation of 9 in aqueous media
was prohibited by both the low solubility of 9 in D2O and substantially
different ionization potentials of core acid 9 and crambescidin 359 (8).
(40) This is best seen in the tabulated data, see Supporting Information.
(36) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1993, 115, 6317-6321.
(37) Seto, H.; Mizukai, K.; Fujioka, S.; Koshino, H.; Yoshida, S. J. Chem. Soc.
Perkin 1. 2002, 21, 2395-2399.
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Crambescidin Core Zwitterionic Acid
A R T I C L E S
Scheme 4. Synthesis of Iodides 30a-g
To identify the site of the initial, rapid deuterium incorpora-
tion, a sample of crambescidin 359 (8) was exposed to a 1:1
p-methoxyphenol/sodium p-methoxyphenolate buffer in a 1:1
mixture of CDCl3 and CD3OD at room temperature. This
reaction was halted after 7 days, at which time approximately
1
4 nonexchangeable deuterium atoms had been introduced. H
NMR analysis revealed that deuterium incorporation had
occurred predominantly at C7 and C9.
F. Reaction of Crambescidin 359 (8) with Basic Ethaneth-
iol. The behavior of crambescidin 359 (8) in basic ethanethiol
was examined to determine whether the unveiling of an
N-amidinyl iminium ion (vide infra) under basic conditions
would lead to reaction with incipient nucleophiles. Incubation
of crambescidin 359 (8) with ethanethiol in the presence of a
1:1 p-methoxyphenol/sodium p-methoxyphenolate buffer for 3
days at room-temperature resulted in partial conversion of 8 to
an adduct of 8 and ethanethiol. This adduct was observed by
both high-resolution electrospray mass spectrometry (HRESMS)
and HPLC-MS analysis.42 Unfortunately, all attempts to isolate
this species were unsuccessful as it reverted to the starting
materials upon removal of the solvent.
ethyl iodide, carried out in the presence, or absence, of added
CsCO3 provided the corresponding ester 7 in trace amounts only.
Larock has reported that silver salts promote the esterification
of carboxylic acids with alkyl halides.46 Accordingly, the
coupling of carboxylate 9 with allyl 6-iodohexanoate (30b) was
examined in the presence of a range of bases and silver additives.
Carrying out this reaction in CH2Cl2 at room temperature with
7 equiv of iodide 30b, 1.5 equiv of CsCO3 and 3 equiv of
AgNO3 proved optimal, providing ester 14b in good yield from
cinnamyl ester 15 (eq 4).47 In a similar fashion, crambescidin
431 (7) was isolated as its trifluoroacetate salt in 21% overall
yield from cinnamyl ester 15.
G. Esterification of Core Acid 9. Total Synthesis of
Crambescidin 431 (7) and Preparation of a Small Library
of Crambescidin Analogues. Early scouting studies indicated
that esters of core acid 9 could not be prepared by acyl transfer,43
undoubtedly a result of a high degree of steric congestion around
the C14 carboxylate carbon (see Figure 2). Accordingly, the
conversion of core acid 9 to ester derivatives was pursued using
the carboxylate as a nucleophile. We initially examined Mit-
sunobu coupling. However, coupling of allyl 16-hydroxyhexa-
decanoic acid (29a) with a sample of partially purified core acid
944 in the presence of triethylammonium camphorsulfonate45
provided ester 14a in low yield (eq 3).
Crambescidin 431 (7) was isolated initially as the trifluoro-
acetate salt, the 13C NMR spectra of which correlated well with
data reported for the natural product.14 However, the 1H NMR
spectrum of the trifluoroacetate salt showed significant discrep-
1
ancies with the H NMR data reported for crambescidin 431.
NMR spectra of crambescidin alkaloids is known to vary
somewhat depending upon the counterion.24 Thus, the trifluo-
roacetate salt of synthetic crambescidin 431 (7) was washed
with saturated ammonium chloride to form the chloride salt.
1
The H NMR spectrum of this material matched closely with
We turned to the coupling of the crude zwitterionic core acid
9 with alkyl iodides. The direct reaction of carboxylate 9 with
that reported for natural 7.14,40
To access side chain analogues of crambescidin 657 (10a)
and 800 (2a) that varied in the length of the ω-hydroxyalkanoic
acid unit, a series of straight chain allyl ω-iodocarboxylates was
required. These compounds, 30a-g, were obtained by the
straightforward two step sequence summarized in Scheme 4.
(41) For a description of the interpretation of data of this type, see Frost, A. A.;
Pearson, R. G. Kinetics and Mechanism; A Study of Homogeneous Chemical
Reactions; John Wiley & Sons: New York, 1961, p 160-164.
(42) That an adduct between crambescidin 359 (8) and ethanethiol was formed
was further confirmed by the repetition of this experiment using partially
deuterated crambescidin 359; deuterium was observed in the adduct.
(43) McDonald, A. I. Ph.D. Dissertation, University of California, Irvine, 2000.
(44) For these reactions, core acid 9 was purified on Davisil resulting in a 60%
yield of material that was ∼80% pure.
(46) Larock, R. C. J. Org. Chem. 1974, 39, 3721-3727.
(47) Unfortunately, ester 14a could not be separated from triphenylphosphine
oxide. Conversions were obtained by isolating material contaminated with
triphenylphosphine oxide and calculating the yield by 1H NMR calibrated
with an internal standard.
(45) Hughes, D. L.; Reamer, R. A.; Bergan, J. J.; Grabowski, E. J. J. J. Am.
Chem. Soc. 1988, 110, 6487-6491.
9
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A R T I C L E S
Aron and Overman
Scheme 5. Synthesis of Crambescidin 657 and 800 Analogues
2b-g and 10a-g
Figure 6. X-ray model of crambescidin core acid 9. The plane is drawn
orthogonal to the guanidinium π-system and passing through the two
disubstituted guanidinium nitrogen atoms.
Scheme 6. Synthesis of Crambescidin Analogs Having Short
Hydrocarbon Side Chains
Discussion
A. Conformation of Crambescidin Core Acid 9. Cramb-
escidin alkaloids are known to bind anions through hydrogen
bonding interactions with their guanidinium functional group.7,50
The selectivity of these interactions undoubtedly is defined by
the absolute and relative configuration of the pentacyclic ring
system and the conformations of the spiroaminal rings that frame
the guanidinium hydrogens. A second perspective of the X-ray
model of crambescidin core acid 9 is shown in Figure 6.
Whereas both oxygens of the spiroaminal rings are disposed
axially, an observation consistent with earlier 1H NMR studies,50
the orientation of these two rings with respect to the guanidinium
nitrogens is notably different.The chair tetrahydropyran ring of
9 projects 1.2 Å past a plane passing through the disubstituted
guanidinium nitrogen atoms (Figure 6), whereas the 2,3,4,5-
tetrahydrooxepine ring exists in a flatter conformation in which
few atoms of this ring project past this perpindicular plane. Thus,
nonbonded steric interactions between anions hydrogen-bonding
to the guanidinium hydrogens and the pentacyclic crambescidin
core would be most pronounced with the hydropyran ring of
these alkaloids.
With iodides 30b-g in hand, we turned to the synthesis of
analogues of crambescidin 657 (10a) and crambescidin 800 (2a)
(Scheme 5). Using the optimized carboxylate alkylation condi-
tions developed for preparing 14b, crambescidin 657 analogues
10b-g were generated from pentacyclic cinnamyl ester 15 in
3 steps and 43-58% overall yield. Using procedures developed
during our total synthesis of crambescidin 800 (2a),24 these
analogues were coupled with protected hydroxyspermidine 3148
and the resulting amide was deprotected by reaction with
anhydrous HCl in ethyl acetate to provide crambescidin 800
analogues 2b-g.
Purification of analogues 2b-g was typically difficult.
Although analogues 2d and 2f could be obtained in greater than
98% purity by C18 reverse phase preparative HPLC, analogues
2b, 2c, 2e, and 2g were not completely pure after preparative
reverse phase HPLC. Fortunately, it was found that material
that had been purified by reverse phase HPLC could be further
purified by silica gel chromatography. In this way, analogues
2b, 2c, 2e, and 2g were obtained in greater than 98% purity.49
A series of crambescidin analogues containing simple hy-
drocarbon ester side chains was prepared also (Scheme 6). The
low yields realized in the synthesis of 33-36 reflect losses
during reverse phase preparative HPLC purification of the
chloride salts, rather than poor yields in the alkylation reactions.
B. Deuterium Incorporation Into the Crambescidin Pen-
tacyclic Core. In the presence of base, crambescidin 359 (8)
incorporates deuterium at C7, C9, C14, and C16, positions
adjacent to the two spiroaminal carbons, C8 and C15. A
plausible mechanism for this process is depicted in Scheme 7.
Reversible opening of the spiroaminal rings of the crambescidin
core generates N-amidinyl iminium ion intermediates 39 and
42,51 which incorporate deuterium by virtue of equilibrium with
their respective enamonium ion congeners 40/41 and 43/44.
(50) Anion binding has also been studied with simpler crambescidin analogues,
see: Murphy, P. J.; Williams, H. L.; Hibbs, D. E.; Hursthouse, M. B.;
Malik, K. M. A. Chem. Commun. 1996, 445-447.
(51) For simplicity, the conjugate acids of 39 and 40, which would be present
also, are not shown.
(48) Coffey, D. S.; McDonald, A. I.; Overman, L. E. J. Org. Chem. 1999, 74,
8741-8742.
(49) The yields obtained reflect largely the ease or difficulty of purification.
9
3386 J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005

Crambescidin Core Zwitterionic Acid
A R T I C L E S
Scheme 7. Specific Base Catalyzed Mechanism for Incorporation of Deuterium at C7, C9, C14, and C16 of Crambescidin 359 (8)
That deuterium is incorporated fastest at C7 and C9 demon-
strates that equilibration of pentacyclic guanidine 37 with
tetracyclic guanidinium ions 39, 40, and 41 is faster than the
related equilibration of pentacyclic guanidine 38 with tetracyclic
guanidinium ions 42, 43, and 44.52 Most likely this rate
difference resides in the ring-opening step, which would be
expected to be faster for the spiro 2,3,4,5-tetrahydrooxepine
ring.53
Deuterium incorporation into crambescidin 359 (8) occurs
at room temperature at an appreciable rate only at pD g 12. If
the pKa of the guanidinium functional group of the pentacyclic
crambescidin core is approximated by that of tetramethylguani-
dinium (pKa ) 13.6 in water),54 ∼3% would be deprotonated
in D2O at pD ) 12 (phosphate buffer). In nonaqueous solvents,
the extent of deprotonation could be greater.54 Base catalysis
of deuterium incorporation would be expected if spiroaminal
ring-opening were rate-determining. Deprotonation of the guani-
dinium ion generates a more electron-rich guanidine functional
group, which by virture of donation of electron density into the
σ* orbital of the spirocyclic carbon-oxygen bond, would
facilitate ring-opening.55 Specific base catalysis is depicted
in Scheme 5, however, the observed small increase in rate
with increasing buffer concentration implies the existence of a
related general base (or specific base-general acid) catalyzed
process.
C. A Possible Role for Spiroaminal Ring Opening in the
Biological Activity of Crambescidin Alkaloids. The proposal
that deprotonation of the guanidinium cation of a crambescidin
alkaloid initiates spirocyclic ring opening suggests a potential
mechanism for at least some of the biological activity of these
alkaloids. Base-promoted opening of a spiroaminal ring upon
binding of a crambescidin alkaloid to a receptor56 would reveal
an N-amidinyl iminium ion electrophile,57 which could lead to
covalent binding to form an adduct such as 45 (eq 5). The
observation that an adduct is formed when crambescidin 359
(8) is exposed to ethanethiol in a p-methoxyphenol/p-methoxy-
phenolate buffer is consistent with this proposal.
D. Mechanism of Decarboxylation of Crambescidin Core
Acid 9. Base-catalyzed decarboxylation of crambescidin core
acid 9 likely occurs by a sequence of steps closely related to
those involved in deuterium incorporation. Catalyzed (specific
and/or general base) opening of the hydropyran ring of zwit-
terionic guanidinium carboxylate 9 generates N-amidinyl imi-
nium ion 46 (Scheme 8). The axial disposition of the carboxylate
functionality in this intermediate provides ideal orbital overlap
for decarboxylation with concomitant formation of neutral
N-amidinyl enamine 47. Protonation of this intermediate at
C14, followed by spirocyclization would yield crambescidin
359 (8).
(52) Another manifestation of this rate difference is the slight epimerization
that is observed at C14 of cinnamyl ester 15 after the incorporation of four
deuterium atoms.
(53) Typically, seven-membered rings possess greater ring strain (3-6 kcal/
mol) than their six-membered counterparts, e.g., see: Dudev, T.; Lim, C.
J. Am. Chem. Soc. 1998, 120, 4450-4458.
(54) Under the nonaqueous conditions used to examine deuterium incorporation
into crambescidin core acid 9 and crambescidin 359 (p-methoxyphenol
buffers in 1:1 CD3OD-CHCl3), the guanidinium acid might be more
extensively ionized. The pKa of tetramethylguanidine increases by 10 going
from water to acetonitrile (pKa ) 13.6 in water; 23.3 in acetonitrile), whereas
the pKa of phenol increases by 17 with a similar solvent transition (pKa )
10.0 in water; 27.2 in acetonitrile), see: Rodima, T.; Kaljurand, I.; Pihl,
A.; Ma¨emets, V.; Leito, I.; Koppel, I. A. J. Org. Chem. 2002, 67, 1873-
1881.
The mechanism of the slower pH independent decarboxyl-
ation reaction is undoubtedly similar. Because of the excellent
(56) Zhou, G.; Somasundaram, T.; Blanc, E.; Parthasarathy, G.; Ellington, W.
R.; Chapman, M. S. Proc. Nat. Acad. Sci. U.S.A. 1998, 95, 8449-8454.
(57) Overman, L. E.; Wolfe, J. P. J. Org. Chem. 2001, 66, 3167-3175.
(55) Kirby, A. J. Stereoelectronic Effects; Oxford University Press: Oxford;
New York, 1996.
9
J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005 3387

A R T I C L E S
Aron and Overman
Scheme 8. Mechanism of Base Catalyzed Decarboxylation
Conclusion
The first practical method to obtain the crambescidin core
acid 9 has been developed. This enantioselective total synthesis
was accomplished in 29 total steps and 2.8% overall yield from
commercially available 3-butyn-1-ol by way of 15 isolated
intermediates. A key step in this synthesis was palladium
mediated deprotection of cinnamyl ester 15, an intermediate that
should be useful for the synthesis of various crambescidin
alkaloid analogues. In the present study, this ester was employed
to prepare crambescidins 431 (7) and 359 (8), and a library of
crambescidin analogues.61
Crambescidin core acid 9 provided single crystals, allowing
the first X-ray structure of the fully constituted crambescidin
core to be accomplished. Zwitterionic guanidinium carboxylate
9 was shown to readily decarboxylate to form crambescidin 359
(8), which was fastest under basic conditions. In the presence
of base, up to eight deuterium atoms can be incorporated into
the crambescidin core. Deuterium incorporation and decarboxy-
lation of crambescidin core acid 9 are the result of ring opening
of the spirocyclic ether rings of the pentacyclic crambescidin
moiety. This facile ring opening generates electrophilic N-
amidinyl iminium ion intermediates, which in vivo could result
in covalent binding.
overlap of the C14-C22 and C-O σ-bonds of guanidinium
carboxylate 9, concerted decarboxylation with concomitant
opening of the C14 spiroaminal is plausible (eq 6).
Experimental Section.
Preparation of Pentacyclic Guanidine Esters 28 and 15. A
solution of tricyclic urea 26 (0.29 g, 0.46 mmol), methyl triflate (1.4
mL, 9.1 mmol), 2,6-di-tert-butyl-4-methylpyridine (0.50 mL, 2.3 mmol)
and CH2Cl2 (23 mL) was maintained at room temperature for 9 h. This
solution was diluted with Et2O (150 mL) and washed with 1 N NaOH
(2 × 15 mL) and brine (15 mL). The organic layer was dried (MgSO4),
filtered, and concentrated to give pseudourea 27. This intermediate was
used without further purification.
Ammonia was bubbled through a solution of this sample of crude
27, NH4Cl (0.055 g, 1.0 mmol), and allyl alcohol (5 mL) for 20 min at
room temperature. The reaction vessel then was sealed and heated to
60 °C. After 20 min, the pressure typically varied, depending on reaction
scale, from 18 to 25 psi. If the pressure was higher than 18 psi, the
reaction vessel was vented at this stage to adjust the pressure to 18
psi. The sealed reaction vessel was maintained at 60 °C and 18 psi for
36 h. The reaction then was cooled to room temperature, diatomaceous
earth (1 g) was added and the mixture was concentrated to give a fine
powder. Purification of this powder by silica gel MPLC (100:0.3:0.1-
100:0.6:0.1 CHCl3:i-PrOH:CF3CO2H) provided 95 mg (0.15 mmol) of
15, 110 mg (0.17 mmol) of 28 and 21 mg (0.033 mmol) of a 1.5:1
mixture of 28:15 (78% overall yield). Resubjection of 28 and the 1.5:1
mixture of 28:15 to these reaction conditions (twice) provided, after
MPLC purification, an additional 60 mg (53% combined yield) of pure
15 trifluoroacetate salt: 1H NMR (500 MHz, CDCl3) δ 10.02 (br s,
1H), 9.82 (br s, 1H), 7.38-7.42 (m, 2H), 7.32-7.36 (m, 2H), 7.23-
7.30 (m, 1H), 6.68 (d, J ) 15.9 Hz, 1H), 6.26 (dt, J ) 15.9, 6.6 Hz,
1H), 5.63-5.67 (m, 1H), 5.47 (d, J ) 10.8 Hz, 1H), 4.73 (d, J ) 6.6
Hz, 2H), 4.50-4.52 (m, 1H), 4.31 (quint, J ) 4.9 Hz, 1H), 3.96-4.10
(m, 2H), 2.99 (d, J ) 4.7 Hz, 1H), 2.42-2.51 (m, 2H), 2.22-2.37 (m,
As noted earlier, under basic conditions deuterium was not
incorporated into core acid 9 in organic solvents. In aqueous
media in contrast, 9 incorporated deuterium at a rate similar to
that of decarboxylation product crambescidin 359 (8). These
observations are readily accounted for by the closely related
mechanisms we have proposed for base catalyzed decarboxy-
lation and deuterium incorporation. Apparently in less polar
solvents, intermediate 46 decarboxylates more rapidly than it
tautomerizes. This scenario is reasonable as decarboxylation
transforms zwitterionic 46 to a neutral product, a process that
would be dramatically faster in less polar solvents.58 Tautomeric
equilibration of 46 with enamine isomers would not involve
similar charge dissipation.
Decarboxylation of crambescidin acid 9 to form crambescidin
359 (8) is a probable step in the biosynthesis of 8. We speculate
that guanidinium carboxylate 9 arises from esterase cleavage
of more abundant crambescidin alkaloids, such as crambescidin
800 (2a). There is evidence that the spiroaminal rings of
ptilomycalin A (1) protect the carboxylic ester from cleavage
by the pendant spermidine moiety.59 Perhaps cleavage of the
ester side chain is facilitated by prior opening of the tetrahy-
dropyran ring.60
(60) Incubating ptilomycalin A (1) at room temperature in a pH 13.0 phosphate
buffer and monitoring of the reaction by LCMS identified crambescidin
359 (8) as a major product. Trace quantities of the crambescidin core acid
(9) were observed by ESMS.
(61) Using this library, in vitro cytotoxicity against several cancer cell lines
was shown to increase with increasing length of the ω-hydroxyalkanoic
acid side chain fragment.12 Moreover, tumor selectivity was shown to vary
widely with side chain structure, with analogues having short, nonpolar
side chains being both potent and showing good selectivity for some solid
tumors.12 Members of this library were also used in recent studies that
identified the first small molecular inhibitors of the HIV-1 protein Nef.11
(58) (a) Kemp, D. S.; Paul, K. G. J. Am. Chem. Soc. 1975, 97, 7305-7312. (b)
For a recent study, see: Catala´n, J. D´ıas, C.; Garcia-Blanco, F. J. Org.
Chem. 2000, 65, 3409-3415.
(59) Grillot, A. L.; Hart, D. J. Tetrahedron 1995, 51, 11377-11392.
9
3388 J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005

Crambescidin Core Zwitterionic Acid
A R T I C L E S
3H), 2.07-2.21 (m, 2H), 1.50-1.89 (m, 6H), 1.35-1.48 (m, 2H), 1.15-
1.35 (m, 3H), 1.05 (d, J ) 6.10 Hz, 3H), 0.83 (t, J ) 7.16 Hz, 3H);
13C (125 MHz, CDCl3)62 168.0, 149.0, 135.9, 135.7, 133.7, 129.8, 128.7,
128.5, 126.7, 121.9, 83.6, 80.7, 71.0, 67.3, 65.9, 53.9, 51.9, 50.1, 36.9,
32.1, 32.0, 30.6, 29.7, 29.1, 26.8, 23.5, 21.5, 18.4, 10.0 ppm; IR (film)
3212, 3088, 2941, 2864, 1679, 1625 cm-1; [R]26 -17.5, [R]26
4.8 Hz, 1H), 2.40-2.49 (m, 1H), 2.28-2.40 (m, 2H), 2.12-2.22 (m,
1H), 1.90-2.04 (m, 2H), 1.85-1.88 (m, 1H), 1.76-1.82 (m, 3H), 1.63-
1.74 (m, 2H), 1.51-1.62 (m, 1H), 1.38-1.50 (m, 2H), 1.25-1.35 (m,
2H), 1.11 (d, J ) 6.2 Hz, 3H), 0.85 (t, J ) 7.2 Hz, 3H); [R]24405 -45,
[R]24435 -6, [R]24546 -88, [R]24577 -109, [R]24589 -81 (c 0.006, MeOH).
A sample of 9 (6 mg) was dissolved in a 1:1 mixture of CH2Cl2 and
MeOH (5 mL); slow evaporation of this solution at room temperature
over 7 d resulted in colorless needles that were suitable for X-ray
analysis (CCDC 253224).
405
435
-12.2, [R]26 -4.75, [R]26 -4.73, [R]26 -3.74 (c 0.3, CH2Cl2);
546
577
589
HRMS (ES) calculated for C33H42N3O6 (M+): 520.3175, found:
520.3154.
28 trifluoroacetate salt: 1H NMR (500 MHz, CDCl3) δ 10.48 (s,
1H), 10.17 (s, 1H), 7.38-7.39 (m, 2H), 7.32-7.36 (m, 2H), 7.26-
7.30 (m, 1H), 6.68 (d, J ) 16.0 Hz, 1H), 6.27 (dt, J ) 15.9, 6.5 Hz,
1H), 5.62-5.69 (m, 1H), 5.48 (d, J ) 10.6 Hz, 1H), 4.79-4.88 (m,
2H), 4.42-4.50 (m, 1H), 4.35 (dt, J ) 11.6, 7.7 Hz, 1H), 4.08-4.17
(m, 1H), 3.77-3.82 (m, 1H), 2.55-2.64 (m, 2H), 2.45 (d, J ) 11.5
Hz, 1H), 2.29-2.34 (m, 3H), 2.13-2.16 (m, 2H), 1.94-1.97 (m, 1H),
1.80-1.90 (m, 1H), 1.59-1.77 (m, 4H), 1.50-1.59 (m, 1H), 1.39-
1.49 (m, 1H), 1.23-1.34 (m, 2H), 1.01-1.11 (m, 4H), 0.82 (t, J ) 7.2
Hz, 3H); 13C (125 MHz, CDCl3)62 167.4, 147.7, 135.7, 134.9, 133.4,
129.6, 128.5, 128.2, 126.4, 122.0, 83.7, 81.6, 70.8, 67.6, 66.0, 53.7,
53.5, 53.3, 37.1, 36.2, 32.1, 30.9, 30.0, 29.7, 29.3, 23.9, 21.5, 18.2,
10.5 ppm; IR (film) 3231, 3111, 3026, 2968, 2937, 1737, 1675, 1613
Preparation of Crambescidin 359 Chloride Salt (8). A mixture
of cinnamyl ester 15 formate salt (15 mg, 0.026 mmol), triethylam-
monium formate (0.13 mL, 1 M in THF), palladium tetrakis(triph-
enylphosphine) (8.6 mg, 0.0079 mmol) and THF (2.6 mL) was
maintained at room temperature for 12 h. The reaction then was
concentrated and the residue was dried at 0.5 Torr for 12 h. The crude
core acid 9 was used without further purification.
A solution of this sample of acid 9, p-methoxyphenol (32 mg, 0.26
mmol), sodium methoxide (0.13 mL, 1 M in MeOH), MeOH (2.1 mL)
and chloroform (2.1 mL) was maintained at room temperature. After 4
days, the reaction solution was concentrated to dryness and the residue
was dissolved in CH2Cl2 (2 mL) and MeOH (2 mL). This solution was
heated to 75 °C and allowed to concentrate to near dryness over 1 day.
The residue was dissolved in MeOH and purified by C18 reverse phase
preparative HPLC (1:9-4:6 MeOH:0.1% trifluoroacetic acid in water)
to give 7.0 mg (0.015 mmol, 58%) of the triflouroacetate salt of
crambescidin 359 (8) as a pale yellow oil:1H NMR (500 MHz, CDCl3)
δ 10.43 (br s, 1H), 10.04 (br s, 1H), 5.65 (br t, J ) 10.4 Hz, 1H), 5.48
(br d, J ) 10.8 Hz, 1H), 4.47 (d, J ) 10.3 Hz, 1H), 3.98-4.08 (m,
2H), 3.76-3.85 (m, 1H), 2.63 (t, J ) 13.4, 1H), 2.56 (dd, J ) 12.7,
4.6 Hz, 1H), 2.18-2.38 (m, 6H), 1.78-1.82 (m, 2H), 1.61-1.75 (m,
12H),63 1.38-1.59 (m, 5H), 1.12-1.34 (m, 14H), 1.20 (d, J ) 6.2 Hz,
3H), 0.81 (t, J ) 1.2 Hz, 3H); 13C (125 MHz, CDCl3)62 148.5, 133.7,
129.8, 83.7, 80.2, 70.7, 66.8, 53.1, 40.0, 37.1, 36.1, 33.6, 32.3, 30.1,
30.0, 29.7, 29.1, 23.7, 21.6, 18.1, 10.2 ppm; IR (film) 3231, 3115, 2972,
2880, 1675, 1652, 1606 cm-1; HRMS (ES) calculated for C21H34N3O2
(M+): 360.2651; found: 360.2643.
cm-1; [R]26405 +6.5, [R]26435 +16.8, [R]26546 +6.7, [R]26577 +2.2, [R]26
589
+1.7 (c 0.3, CH2Cl2); HRMS (ES) calculated for C33H42N3O6 (M+):
520.3175, found: 520.3154.
Alternatively, 15 and 28 could be isolated as their formate salts by
MPLC (100:0.3:0.2-100:0.6:0.2 CHCl3:i-PrOH:HCO2H) purification.
Formate salts show a diagnostic 1H NMR signal at 8.67 ppm.
Subsequent transformations of cinnamyl ester 15 were little effected
by the nature of its counterion.
Preparation of Crambescidin Core Acid 9. A vial containing the
formate salt of ester 15 (23 mg, 0.041 mmol), palladium tetrakis-
(triphenylphosphine) (4.5 mg, 0.0041 mmol) and a solution of triethyl-
ammonium formate in THF (0.21 mL, 1 M) was maintained at room
temperature for 14 h, diluted with freshly distilled acetone (3 mL) and
filtered through a medium porosity sintered glass frit. The filtrate was
washed with 10 mL of freshly distilled acetone to give 7.9 mg of
zwiterionic 9 as a colorless crystalline solid (0.020 mmol, 47%): 1H
NMR (500 MHz, 1:1 CDCl3:CD3OD - calibrated to CDCl3): δ 5.38-
5.42 (m, 1H), 5.19 (d, J ) 10.9 Hz, 1H), 3.97-4.02 (m, 1H), 3.88 (dt,
J ) 8.6, 4.8 Hz, 1H), 3.62-3.70 (m, 1H), 3.42-3.53 (m, 1H), 2.38 (d,
J ) 4.7 Hz, 1H), 2.16 (dd, J ) 12.7, 4.6 Hz, 1H), 2.08 (br t, J ) 15.2
Hz, 1H), 1.73-1.96 (m, 5H), 1.63 (dd, J ) 13.9, 5.8 Hz, 1H), 1.30-
1.53 (m, 6H), 1.13-1.28 (m, 3H), 0.92 (q, J ) 12.0 Hz, 1H), 0.76 (d
J ) 6.2 Hz, 3H) 0.52 (t, J ) 7.2 Hz, 3H); 13C (125 MHz, 1:1 CDCl3:
CD3OD - calibrated to CDCl3) 173.1, 148.8, 132.7, 129.8, 83.1, 80.8,
70.2, 65.9, 53.6, 53.1, 52.1, 36.8, 36.0, 31.6, 30.8, 29.6, 28.5, 26.3,
The chloride salt of crambescidin 359 (19) was generated by
dissolving a sample of its triflouroacetate salt (7 mg, 0.015 mmol) in
a mixture of CH2Cl2 (3 mL) and saturated aqueous NH4Cl (3 mL).
This mixture was vigorously stirred for 6 h, partitioned and the aqueous
layer was extracted with CH2Cl2 (5 × 3 mL). The combined organic
layers were dried (MgSO4), filtered, concentrated and the residue was
filtered through a plug of silica gel (1:20 MeOH: CH2Cl2) to give 5.9
mg (0.015 mmol, 99%) of crambescidin 359 chloride salt (8) as a
colorless oil: HRMS (ES) calculated for C21H34N3O2 (M+): 360.2651,
found: 360.2638; 1H and 13C NMR spectra for this sample agreed well
with data reported for the natural product.14
1
22.9, 20.7 17.9, 9.3 ppm; IR (film) 2961, 2926, 1637 cm-1; H NMR
Representative Procedure for the Synthesis of Crambescidin 657
Analogues. Preparation Crambescidin 657 Analogue 10b. A mixture
of the formate salt of ester 15 (22 mg, 0.039 mmol), palladium tetrakis-
(triphenylphosphine) (13 mg, 0.012 mmol), a solution of triethylam-
monium formate (1 M in THF, 0.20 mL) and THF (3.9 mL) was
maintained at room temperature for 16 h, concentrated and the residue
was dried (0.05 Torr, 6 h) to give the crude core acid 9 as a yellow oil.
This material was used without further purification.
(500 MHz, CD3OD) δ 5.70-5.75 (m, 1H), 5.52 (br d, J ) 11.1 Hz,
1H), 4.31-4.38 (m, 1H), 4.22 (dt, J ) 8.7, 5.5 Hz, 1H), 3.96-4.04
(m, 1H), 3.78-3.86 (m, 1H), 2.69 (d, J ) 5.0 Hz, 1H), 2.53 (dd, J )
12.8, 4.8 Hz, 1H), 2.43 (br t, J ) 15.5 Hz, 1H), 2.11-2.30 (m, 3H),
1.99-2.10 (m, 2H), 1.80-1.90 (m, 2H), 1.70-1.79 (m, 3H), 1.67 (br
d, J ) 13.9 Hz, 1H), 1.42-1.58 (m, 3H), 1.22-1.38 (m, 2H), 1.08 (d,
J ) 6.2 Hz, 3H), 0.89 (t, J ) 7.3 Hz, 3H); [R]26 -12.4, [R]26
405
435
-2.8, [R]26 -5.4, [R]26 -5.1, [R]26 -2.9 (c 0.1, 1:1 CH2Cl2:
546
577
589
MeOH); [R]24 -17, [R]24 -54, [R]24 -120, [R]24 -102,
405
435
546
577
Cesium carbonate (19 mg, 0.06 mmol) was added to a solution of
this sample of core acid 9, allyl 6-iodohexanoate (30b, 77 mg, 0.27
mmol) and CH2Cl2 (0.39 mL). After 5 min, AgNO3 (20 mg, 0.12 mmol)
was added and the reaction was stirred at room temperature. After 16
h, this mixture was filtered through diatomaceous earth (∼300 mg),
which was washed with CH2Cl2 (∼8 mL). The combined filtrates were
concentrated and the residue was purified on silica gel (10:1:1000
HCO2H:i-Pr:CHCl3; 1:9 MeOH:CHCl3) to give a 1:0.5 mixture of ester
[R]24589 -88 (c 0.006, MeOH); HRMS (ES): calculated for C22H33N3O4
(M + H+): 404.2549, found: 404.2555.
Data for the trifluoroacetate salt formed from 9 and excess
trifluoroacetic acid: 1H NMR (500 MHz, CD3OD) δ 5.70-5.75 (m,
1H), 5.52 (br d, J ) 11.0 Hz, 1H), 4.34-4.42 (m, 2H), 4.02-4.10 (m,
1H), 3.80-3.88 (m, 1H), 3.01 (d, J ) 5.0 Hz, 1H), 2.64 (dd, J ) 13.0,
(62) The carbon signals of the trifluoroacetate counterion were typically not
observed because of coupling with the fluorine atoms and line broadening
resulting from exchange.
(63) Excess signals are attributed to water.
9
J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005 3389

A R T I C L E S
Aron and Overman
14b and triphenylphosphine oxide (as determined by 1H NMR analysis).
This sample was carried forward without further purification.
Acknowledgment. This work was supported by the Heart,
Lung & Blood Institute of the NIH (HL-25854) with additional
financial support being provided by Pharma Mar. We thank Dr.
J. Ziller for the X-ray diffraction study of 9, Dr. B. Nguyen for
800 MHz 1H NMR data, Dr. J. Greaves for assistance with mass
spectrometric measurements of deuterium incorporation, Dr. A.
McDonald for early studies in this area, Professor J. C.
Braekman for copies of NMR spectra of natural crambesidins
359 and 431, and Professor K. Nagasawa for copies of NMR
spectra of synthetic crambesidin 359. Z.A. was supported in
part by a Lilly Graduate Fellowship.
A solution of triethylammonium formate (1 M in THF, 0.080 mL,
0.080 mmol) was added to a solution of this sample of ester 14b,
palladium tetrakis(triphenylphosphine) (9.0 mg, 0.008 mmol) and THF
(0.80 mL). This solution was maintained at room temperature for 10
h. The reaction then was concentrated onto diatomaceous earth and
the residue was purified on silica gel (1000:5:10 CHCl3:i-PrOH:HCO2H;
1:9 MeOH:CHCl3) to give 11 mg (0.021 mmol, 54%) of crambescidin
657 derivative 10b as a yellow oil: 1H NMR (500 MHz, CDCl3) δ
10.35 (br s, 1H), 10.02 (br s, 1H), 5.64-5.70 (m, 1H), 5.48 (d, J )
10.2 Hz, 1H), 4.51 (d, J ) 8.7 Hz, 1H), 4.29 (dt, J ) 9.2, 5.13 Hz,
1H), 4.14-4.21 (m, 1H), 4.06-4.13 (m, 1H), 3.96-4.04 (m, 1H), 3.90-
3.96 (m, 1H), 2.95 (d, J ) 5.0 Hz, 1H), 2.54-2.64 (m, 2H), 2.14-
2.39 (m, 7H), 1.50-1.92 (m, 13H),63 1.30-1.47 (m, 5H), 1.14-1.27
(m, 2H), 1.04 (d, J ) 6.2 Hz, 3H), 0.83 (t, J ) 7.2 Hz, 3H); 13C (125
MHz, CDCl3) 177.0, 168.2, 148.8, 133.6, 129.9, 83.7, 80.7, 70.9, 67.1,
65.0, 53.9, 53.4, 51.9, 50.5, 37.0, 36.3, 35.2, 32.0, 29.7, 29.1, 28.2,
26.7, 25.2, 24.8, 23.6, 21.5, 18.2, 14.1, 10.1 ppm; IR (film) 2926, 2856,
2362, 2339, 1733, 1656, 1613 cm-1; HRMS (ES) calculated for
C28H43N3O6 (M + H+): 518.3230, found: 518.3224.
Supporting Information Available: A. General experimental
details; experimental procedures and characterization data for
all new compounds not reported in Experimental Section of the
paper; Tabular NMR data for crambescidin 359 (9); descriptions
of the deuterium incorporation and decarboxylation experiments
as well as tables of raw data; CIF file for X-ray structure of 9.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Other crambescidin 657 derivatives described in Scheme 5 were
prepared similarly. Samples for biological testing were purified by both
silica gel chromatography (as described) and C18 reverse phase
preparative HPLC (30:70-95:5 MeCN:0.1% CF3CO2H (v/v) in H2O).
JA042875+
9
3390 J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005