Phorbasides A-E, cytotoxic chlorocyclopropane macrolide glycosides from the marine sponge Phorbas sp. CD determination of C-methyl sugar configurations

Article abstract of DOI:10.1021/jo702307t

(Chemical Equation Presented) Five new cytotoxic macrolide glycosides phorbasides A-E (3-7), each possessing a macrolide ring appended to a rare ene-yne-trans-2-chlorocyclopropane, were isolated from the same Western Australian sponge (Phorbas sp.) that provided phorboxazoles A and B. The structures of 3-7 were solved by analysis of spectroscopic data including NMR, MS, and CD. A synthesis of methyl 2-O-methyl-α-L-evalose from L-rhamnose was completed and used for configurational assignment of the sugar residue in 3. Acid-catalyzed methanolysis of 3 followed by two-step derivatization of the liberated O-methyl glycoside gave a vicinal 4-O-naphthoyl/tertiary 3-N-(2-aminonaphthyl)carbamate derivative that exhibited exciton coupled CD identical with that of the derivative prepared from synthetic 1,2-O-dimethyl-α-L-evalose.

Full text of DOI:10.1021/jo702307t

Phorbasides A-E, Cytotoxic Chlorocyclopropane Macrolide
Glycosides from the Marine Sponge Phorbas sp. CD Determination
of C-Methyl Sugar Configurations
‡
,†
†
§
John B. MacMillan, Guang Xiong-Zhou, Colin K. Skepper, and
§
,‡,
Tadeusz F. Molinski
*
Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences,
MC0358, 9500 Gilman DriVe, La Jolla, California 92093-0358, and Department of Chemistry, UniVersity
of California, DaVis, One Shields AVenue, DaVis, California 95616
tmolinski@ucsd.edu
ReceiVed NoVember 8, 2007
Five new cytotoxic macrolide glycosides phorbasides A-E (3-7), each possessing a macrolide ring
appended to a rare ene-yne-trans-2-chlorocyclopropane, were isolated from the same Western Australian
sponge (Phorbas sp.) that provided phorboxazoles A and B. The structures of 3–7 were solved by analysis
of spectroscopic data including NMR, MS, and CD. A synthesis of methyl 2-O-methyl-R-L-evalose from
L-rhamnose was completed and used for configurational assignment of the sugar residue in 3. Acid-
catalyzed methanolysis of 3 followed by two-step derivatization of the liberated O-methyl glycoside
gave a vicinal 4-O-naphthoyl/tertiary 3-N-(2-aminonaphthyl)carbamate derivative that exhibited exciton
coupled CD identical with that of the derivative prepared from synthetic 1,2-O-dimethyl-R-L-evalose.
Introduction
macrolide ring, glycosylated at C5 by a 3-C-methyl sugar and
terminated by a rare ene-yne 2-chlorocyclopropane. Glycosy-
lated polyketides with similar macrolide rings have been found
in cyanobacteria, (e.g., acutiphycin from Oscillatoria acutis-
In 1995 we reported the structures of phorboxazoles A (1a)
and B (1b, Figure 1) from the marine sponge Phorbas sp.
1
collected off the Western Australian coastline. Recently, we
disclosed the structures of two unrelated macrolidessphorbasides
A (3) and B (4)sobtained from minor fractions of the sponge
3
4
sima, lyngbyaloside from Lyngbya sp., lyngbouilliside from
5
L. bouillani ); however, the unique ene-yne 2-chlorocyclopro-
2
pane group has been encountered only once before in callipel-
extract. The phorbasides contain a common 14-membered
tosides A (2), B, and C from the New Caledonian sponge
6
*
To whom correspondence should be addressed. Phone: (858) 534-7115.
Callipelta sp. We now convey a full account of structure
Fax: (858) 822-0386.
‡
Present address: University of Texas, Southwestern Medical Center, Dept.
of Biochemistry, 5323 Harry Hines Blvd., Dallas, TX 75390.
(3) Barchi, J. J.; Moore, R. E.; Patterson, G. M. L. J. Am. Chem. Soc. 1984,
106, 8193–8197.
†
University of California, Davis.
University of California, San Diego.
§
(4) Luesch, H.; Yoshida, W. Y.; Harrigan, G. G.; Doom, J. P.; Moore, R. E.;
Paul, V. J. J. Nat. Prod 2002, 65, 1945–1948.
(
1) (a) Searle, P. A.; Molinski, T. F. J. Am. Chem. Soc. 1995, 117, 8126. (b)
Searle, P. A.; Molinski, T. F.; Brzezinski, L. J.; Leahy, J. W. J. Am. Chem. Soc.
996, 118, 9422. (c) Molinski, T. F. Tetrahedron Lett. 1996, 37, 7879.
2) (a) Skepper, C. K.; MacMillan, J. B.; Zhou, G.-X.; Masuno, M. N.;
(5) Tan, L. T.; Márquez, B. L.; Gerwick, W. H. J. Nat. Prod. 2002, 65, 925–
928.
1
(
(6) (a) Zampella, A.; D’Auria, M. V.; Minale, L.; Debitus, C.; Roussakis,
C. J. Am. Chem. Soc. 1996, 118, 11085–11088. (b) Zampella, A.; D’Auria, M. V.;
Minale, L.; Debitus, C. Tetrahedron 1997, 53, 3243–3248.
Molinski, T. F. J. Am. Chem. Soc. 2007, 129 (14), 4150–4151. (b) MacMillan,
J. B. Ph.D. Thesis, University of California, Davis, 2004.
1
0.1021/jo702307t CCC: $40.75  2008 American Chemical Society
J. Org. Chem. 2008, 73, 3699–3706 3699
Published on Web 04/16/2008

MacMillan et al.
appears to be the most potent member of this family of cytotoxic
compounds (IC50 ) 2 µM, against HCT-116 cells) reported to
date, which suggests that the cytotoxicity of glycosidic ene-
yne chlorocyclopropane macrolides is largely modulated by the
structures of the sugars.
Results
Examination of minor fractions obtained from the same
specimen of Phorbas sp. that yielded 1a (0.04% dry weight,
w/w) and 1b (0.017% w/w) showed minor components that were
further purified by reversed phase C18 HPLC to provide
-
3
phorbaside A (3, 1.29 mg, 5.5 × 10 % w/w), phorbaside B
-
3
(
×
4, 1.40 mg, 6.0 × 10 % w/w), phorbaside C (5, 3.0 mg, 1.3
-2
-4
10 % w/w), phorbaside D (6, 0.78 mg, 3.4 × 10 % w/w),
-4
and phorbaside E (7, 0.84 mg, 3 × 10 % w/w). Structure
elucidation of these very small quantities of compounds was
greatly assisted by 2D NMR experiments carried out at 600
MHz on an NMR spectrometer equipped with a cryoprobe that
1
provides a 4–5-fold greater H signal-to-noise than conventional
probes.
The molecular formula of phorbaside A (3), C33H49ClO10,
+
was derived from HRFABMS (m/z [M + Na] , 663.2885) while
+
that of phorbaside B (4) was C41H63ClO14 (m/z [M + Na]
8
37.4049). The UV spectra of both compounds exhibited
chromophores (λmax 245 nm, ꢀ 12000) that were assigned to a
conjugated 1-chloro-2-(E-alk-3′-en-1′-ynyl)cyclopropane group
attached to a 14-membered-ring macrolide as described in our
2
preliminary communication. Extensive analysis of 2D NMR
spectra of 3 (COSY, ROESY, HSQC, HMBC, and HSQC-
TOCSY) established the constitution and relative configuration
of the macrolide ring and attachment of the sugar group at C5.
Quantitative CD analysis of the hyperconjugated ene-yne
chromophore secured the absolute configuration of both the
macrolide ring and the trans-2-chlorocyclopropyl ring in 3 and
2
4
, as depicted. Analysis of UV, IR, and NMR spectroscopic
data showed that each of the phorbasides A-E (3-7) had an
identical macrolide ring (aglycone), but differed only in the
nature of the sugar group.
The sugar unit in 3 was identified as 2-O-methylevalose (6-
deoxy-3-C-2-O-dimethyl-manno-pyranoside) by analysis of vici-
nal coupling constants, ROESY correlations, and comparison
1
13
of H and C NMR chemical shifts with the evalose residue in
the glycoside callipeltoside C, but this did not reveal the absolute
configuration. A number of interesting features were seen for
the C5-glycone including a C-methyl group at C3′ and an
O-methoxyl at C2′. The constitution was determined on the basis
of COSY correlations from the anomeric proton H1′ (δ 4.92,
s) to H2′ (δ 3.11, s) and from H4′ (δ 3.35, d, J ) 9.6 Hz) to
1
H5′ (δ 3.64, dq, J ) 9.6, 6.6 Hz). The H NMR coupled spin
1
FIGURE 1. Structures of phorboxazoles A and B (1a and b),
system of the pyranose residue was discontinuous at C3′ due
to the presence of the quaternary center; however, HMBC
correlations were observed from H2′, H3′, and H4′ to C3′-Me
(C7′, δ 17.8, q) and from H2′ to C3′ (δ 72.3, s, see the
Supporting Information in ref 2).
6
a
19
phorbasides A-E (3-7), callipeltoside A (2), and derivatives.
elucidation of phorbasides A and B (3 and 4, Figure 1), and
three previously unreported minor metabolites, phorbasides C-E
5–7).
Each of the five compounds differs only in the structure of
the 3-C-methyl sugars attached at C5. To define the configu-
ration of the glycone residues, we developed a method for
assignment of C-methyl sugars by circular dichroism (CD) that
exploits exciton coupling between vicinal tertiary carbamate and
(
Phorbaside B (4). The molecular formula for phorbaside B
(4), C41H63ClO14, differed from that of 3 by addition of the
elements of C8H14O4 suggesting the presence of an additional
2
1
O-methylevalose residue. Differences in the H NMR spectrum
of 3 and 4 (ref 2, ) were found only in the sugar portion of the
spectra (Table S2 in the Supporting Information in ref 2); the
1
2
-naphthoate ester groups. The latter was used to confirm the
H NMR spectrum of 4 contained two anomeric protons (δ 4.83,
L-evalose configuration in phorbaside A (1b). Phorbaside C (5)
s, H1′; δ 5.71, s, H1″) and an additional three methyl signals
3
700 J. Org. Chem. Vol. 73, No. 10, 2008

Phorbasides A-E
δ 3.48, s; 1.29, d, J ) 6.6 Hz; 1.35, s). gCOSY and HMBC
(
cross peaks located the second 2-O-methylevalose residue at
C4′ of the first evalose residue [HMBC; δ 3.37, d, J ) 9.7 Hz,
H4′ to δ 92.4, C1″]. Therefore, phorbaside B had a 1″ f 4′
disaccharide linkage.
Phorbaside C (5). The molecular formula for phorbaside C
+
(
5) was C40H61ClO14 (HRFABMS m/z [M + Na] 823.3615),
or one CH2 group less than the formula of phorbaside B (4).
H NMR spectrum of 5 (Supporting Information, Figure S1)
1
showed only two OMe singlets (δ 3.23 and 3.38 ppm) compared
1
to the three found in 4. The remainder of the H NMR chemical
shifts for the macrolide ring of 5 matched those of 4, including
the shifts for H9 (δ 3.75, dd, J ) 9.0, 1.8 Hz), implying the
1
MeO group at C9 was intact. The H NMR chemical signals of
the C5-O-evalose residue of 5 showed similarity with those of
4
, but the second sugar residue showed major changes. The
signal for the anomeric proton H1″ of 5 (δ 5.42, s) was shifted
.28 ppm upfield from that of 4 (δ 5.71, s), while H2″ (δ 3.58,
0
s) shifted 0.48 ppm downfield with respect to that of 4 (δ 3.10,
s). The absence of an HMBC cross peak from the singlet H2″,
1
together with the differences of the H NMR spectrum in the
FIGURE 2. Conformations of R- and ꢀ-anomers of L-2-O-methyl-
evalose glycosides (L-2-O-methyl-6-deoxy-3-C-methyl-manno-pyra-
noside) and predicted H NMR JHH values. A arrows show ROESY
vicinity of C2″ strongly suggested the O-2″-desmethyl structure
. The remaining HMBC correlations and H NMR shifts were
consistent with a 1″ f 4′ disaccharide linkage, as in 4.
Phorbaside D (6). The molecular formula for phorbaside D
1
3
1
5
correlations (600 MHz, t
glycone in 3.
m
) 400 mS) observed for the corresponding
1
(
6) is C34H48ClNO14. Since the H signals for the macrolide
was positioned on C2″ based on HMBC correlations from the
methyl singlet at δ 3.39 ppm to C2″ (δ 85.6).
rings in 5 and 6 were similar (Supporting Information, Figure
S2), the N atom was located within the sugar group, which
strongly suggested the presence of one residue of callipeltose
see Figure 1, 3-O-4-N-carbamoyl-[4-amino-4,6-dideoxy-2-O,-
C-dimethyl-R-talopyranose). The IR spectrum of 6 revealed
a second carbonyl stretch at ν 1710 cm , while the C NMR
spectrum exhibited an extra sp carbon (δ 158.0, s)sboth
associated with a cyclic carbamate between C3′ and C4′. This
was verified by an HMBC cross peak between signals of H4′
δ 3.35, s) and the C8′ CdO carbon (δ 158.0, s). The H NMR
signal for H4′ was now a singlet, as a consequence of flattening
of the pyranose ring by the imidazolone ring, inversion of
configuration at C4′, and a syn relationship between H4′ and
H5′ (δ 3.91, d, J ) 6.0 Hz). This is consistent with a boat
configuration of the pyranose ring as assigned for callipeltoside
Configuration of L-2-O-Methylevalose Residues. Evaluation
of both vicinal homonuclear J couplings and ROESY correla-
tions in the evalose ring system of 3 defined the sugar relative
configurations in 3 and 4 (Figure 2). Vicinal J couplings for
the sugar group were limited due to interruption of the vicinal
proton pairs by the C3′ quaternary carbon and a J ) 0 Hz
coupling for H1′-H2′. Two pyranose conformations consistent
with the latter data and a large trans-diaxial J coupling between
H4′ and H5′ (J ) 9.6 Hz) are the L-R-chair and the L-ꢀ-chair
(
3
6
-1
13
2
1
(
(
Figure 2).
Configuration (b) was proposed for the ꢀ-2-O-methylevalose
residue in callipeltoside C on the basis of interpretation of
6b
NOE’s; however, our measurements of 3 support configuration
(a) with an axial R-glycoside linkage (L-R-chair) at C5 as
follows. ROESY (600 MHz, tm ) 400 mS) correlations were
observed in 3 from the anomeric H1′ proton to H5 on the
macrolide ring and from H1′ to the syn-facial C2′-OMe group
6
A by Zampella and co-workers. The latter relative configuration
was verified by the appearance of a ROESY cross peak between
H4′ and H5′ signals. Formally, callipeltose derives from
intramolecular SN2 displacement of the C4′-OH group by the
pendant carbamoyl NH2 group with inversion at C4′. A putative
precursor for the oxazolidinone ring of callipeltose, 2-O-methyl-
-O-carbamoylevalose, has been reported in the structures of
glycosylated macrolides auriside B and callipeltoside B.
Phorbaside E (7). Phorbaside E (7), C40H61ClO14 (HR-
FABMS m/z [M + Na] 823.3750) is isomeric with phorbaside
C (5). Analysis of the H NMR, COSY, and HMBC spectral
data (Supporting Information, Figure S3) revealed differences
in the sugar moieties between 7 and 5 that suggested the
glycoside linkage between the two evalose sugar residues was
(
δ 3.45, s), but not from H1′ to H5′ or H1′ to H3-7′. These
8
results are consistent only with an axial L-R-glycoside linkage.
Similar ROESY correlations were seen in the 2-O-methylevalose
residue at C5 in 4 (see the Supporting Inforamtion in ref 2a).
Additional ROESY cross peaks were observed in 3 from C7′
to H2′ (δ 3.11, s)sconfirming the syn relationship between the
oxygenated substituents at C2′ and C3′sand from the C2′ MeO
proton signal to the H23 methyl group (δ 0.89, d, J ) 6.6 Hz).
Since the absolute configuration of the macrolide ring in 3 had
3
7
6
+
1
2
a
been assigned, the latter correlation was suggestive of an
L-sugar, although the relatively weak magnitude of the cross
peak did not lend itself to an unequivocal assignment.
1
″f2′ rather than 1″f4′ found in phorbasides B and C.
Phorbaside E (7) also differs in location of the MeO group (C2″
in 7, C2′ in 5).The key differences were the downfield shift of
H2′ in 7 to δ 3.57 ppm from 3.15 ppm in 5, and HMBC
correlations revealed cross-peaks between H2′ (δ 3.57, s) and
the C1″ anomeric carbon (δ 95.6 ppm). The sugar MeO group
To resolve this matter, we assigned the sugar configuration
in 3 independently by synthesis of the methyl glycoside of R-L-
2
-O-methylevalose and a novel application of exciton coupled
(
8) The R-configuration of the glycoside linkages in 3 and 4 contrasts with
the ꢀ-configuration assigned to callipeltoside C (ref 6b; note, 2-O-methylevalose
is depicted in the D-configuration in this paper).
(
7) Sone, H.; Kigoshi, H.; Yamada, K. J. Org. Chem. 1996, 61, 8956–8960.
J. Org. Chem. Vol. 73, No. 10, 2008 3701

MacMillan et al.
SCHEME 1. Synthesis of Methyl r-L-Evalopyranoside (17)
TABLE 1. Reduction of 13
entry
no.
reagent
NaBH4
L-selectride THF
SmI2
solvent
EtOH
T (°C)
t (h) % yield 14:15
1
2
3
0
0
0.16
0.5
97
0:100
0:100
86
b
THF/
i-PrOH
EtOH
i-PrOH
i-PrOH
rt f reflux 16
b
b
4
5
6
7
Na°
Na°
Li°
0
1
2
-10
-10
a
1.7
0.7
3
8
100:0
100:0
a
Na°
t-BuOH/ -30
NH3(l)
a
b
Based on NMR integration of the anomeric H1 signal. Decomposition.
SCHEME 2. Synthesis of Dichromophoric Derivative 9
CD. Pair-wise exciton coupling CD (ECCD) of dibenzoate
derivatives has been used to assign absolute configuration in
pyranoses; however, these methods are applicable only to
from Phorbaside A (3) and Methyl 2-O-methyl-r-L-evalose
(
17)
9
secondary and primary OH groups. The evalose sugar residue
poses two problems for analysis by ECCD; evalose is not
commercially available and the C3 tertiary alcohol is resistant
to benzoylation. These difficulties were overcome by a multistep
synthesis of a bis-chromophoric derivative of L-evalose, 9, from
L-rhamnose (8). Compound 9 embodies an N-aryl carbamate
derivative of the sterically hindered C3 tertiary alcohol and
provides an opportunity to interrogate absolute configuration
through exciton coupling with the adjacent 2-napthoate ester.
L-Rhamnose (8) was converted to the O-methyl glycoside (10,
Scheme 1) that was further protected as the acetonide 11
1
0
(
acetone, CuSO4, reflux). Oxidation of 11 (CrO3, pyridine,
11
CH2Cl2, CH3CO2H) gave the ketone 12 in 85% yield. The
C3-methyl branch was introduced according to Klemer by
kinetic deprotonation of 12 (1 equiv, LDA, -78 °C) followed
by the addition of MeI to give 13 (71% yield). Reduction of
the carbonyl group in 13 by hydride under thermodynamic
conditions was expected to give the desired C4 equatorial
1
2
1
2
alcohol 14; however, literature precedence suggested that
NaBH4 reduction proceeds under kinetic control by axial attack
of hydride from the less hindered ꢀ-face to give the undesired
equatorial alcohol 15. Attempted reduction of ketone 13 under
a variety of kinetic and thermodynamic conditions (Table 1)
produced only 15 or poor conversion to 14 (entries 6 and 7,
<
8%). Successful conversion of 13 to the desired diastereomer
4 was achieved by using a variant of Lipták’s procedure that
allowed hydride reduction to proceed under the direction of the
1
13
C3 hydroxyl. Removal of the acetonide in 13 (acetone, PPTS,
reflux) gave keto-diol 16, which was reduced (NaBH(OAc)3,
AcOH) to alcohol 17 exclusively. Reprotection of 17 as a C2/
C3 acetonide (acetone, CuSO4, reflux) gave 14 (Scheme 2),
which was treated with 2-naphthoyl chloride (pyridine, CH2Cl2)
13
(
9) Chang, M.; Meyers, H. V.; Nakanishi, K.; Ojika, M.; Park, J. H.; Park,
M. H.; Takeda, R.; Vázquez, J. T.; Wiesler, W. T. Pure Appl. Chem. 1989, 61,
1
193–1200.
(
10) Jary, J.; Capek, K.; Kovar, J. Collect. Czech. Chem. Commun. 1963,
2
8, 2171–2176.
(
(
11) Garegg, P. J.; Samuelsson, B. Carbohydr. Res. 1978, 67, 267–270.
12) Klemer, A.; Stegt, H. J. Carbohydr. Chem. 1985, 4, 205–213.
(13) Gyergyói, K.; Tóth, A.; Bajza, I.; Lipták, A. Synlett 1998, 127–128.
3
702 J. Org. Chem. Vol. 73, No. 10, 2008

Phorbasides A-E
to provide the 4-O-naphthoate ester 18 in 82% yield. Removal
of the acetonide (MeOH, p-TsOH, reflux) followed by O-
methylation of the secondary OH group (Ag2O, CH3I, CH3CN)
provided 1,2-O-dimethyl-4-O-naphthoyl-L-evalose (19) in 55%
yield.
To introduce a second chromophore into the sugar by
acylation of the sterically hindered tertiary alcohol 19, we chose
to use an N-aryl isocyanate as a more reactive electrophile for
conversion of the tertiary OH group to a carbamate. 6-Meth-
1
4
oxynaphthylisocyanate (20), prepared from commercially
available 6-methoxy-2-naphthoic acid by Curtius rearrangement
of the corresponding acyl azide (diphosphoryl azide, reflux,
benzene), added smoothly to 19 (pyridine, 60 °C) to give
carbamate 9.
Phorbaside A (3) was degraded to 9 in three steps (Scheme
2
): microscale methanolysis of 3 (50 µg, MeOH, HCl, 80 °C)
followed by removal of the volatiles, treatment of the crude
product with 2-naphthoyl chloride (pyridine, CH2Cl2), and
heating the residue with 20 (pyridine, 60 °C). Purification of
the crude product by normal phase HPLC (4% i-PrOH:n-hexane)
gave 9 with a retention time identical with the synthetic standard
prepared from L-rhamnose. The collected HPLC fraction
containing pure 9 was used directly for CD measurements, as
follows (Figure 3).
The CD spectrum of 9 (Figure 3a), derived from L-rhamnose
(
4% i-PrOH:n-hexane), gave a weak positive bisignate Cotton
effect [λmax 241 nm (∆ꢀ + 2.6), 226, (-1.5); A ) 4.1]. This is
consistent with a conformation of 9 (Figure 3b) that subtends a
positive helicity between the C-O bonds at C3 and C4. The
CD spectrum of the O-methyl glycoside 9 prepared from
phorbaside A was identical in sign and magnitude with that of
authentic 9 prepared from L-rhamnose.
FIGURE 3. CD spectra (4% i-PrOH:n-hexane): (a) synthetic 9 (blue,
solid line, see Schemes 1 and 2) and (b) 9 derived from phorbaside A
Thus, the C3′-methyl sugar in 3 was verified as L-2-O-
methylevalose. By association, the evalose units in the co-
occurring natural products 4, 5, and 7 are also likely to have
the L-configuration. Since the absolute configurations at C13,
C18, and C19 in 3 and 4 had been assigned earlier from analysis
(
red line). The electronic transition dipole moments of the two
chromophores in 9 constitute a positive helicity.
Evalose and callipeltose have manno-pyranoside and talo-
2
of the CD spectrum of the native ene-yne chromophore, and
pyranoside relative configurations, respectively; however, evalose
the relative configuration of the macrolide ring established
from ROESY correlations, the complete configuration of 3–4
can be stated as 2S,3S,5S,6R,7R,8R,9R,13R,18R,19S,1′R,2′R,3′R,-
16
occurs in nature in both L- and D-configurations. We chose to
evaluate the sugar configuration of 3 using an independent
approach. The method described here is a variant on the well-
established sugar dibenzoate method developed by Nakanishi
1
5
4
′S,5′S.
9
and co-workers. Use of the dibenzoate method is precluded
for assignment of C-methyl sugars due to the lack of reactivity
at tertiary OH groups, but the present variation circumvents this
limitation by exploiting sequential O-naphthoylation of a
secondary OH group followed by smooth reaction of the tertiary
Discussion
Determination of configuration in C-methyl sugars adds a
level of difficulty to carbohydrate analysis for a number of
reasons. C-Branched sugars exhibit more conformational het-
erogeneity due to lower barriers of inversion between chair
conformers or the presence of other conformers (e.g., twist-
chair and boat). Earlier, we had assigned the absolute config-
uration of the aglycon in 3 and 4 by analysis of macrolide
dipolar couplings from ROESY spectra and independent CD
analysis of the ene-yne chlorocyclcopropane group, which
OH group with an aryl isocyanate.
The 1H and 13C NMR values of the R-L-2-O-methylevalose
residue in 3 and 4 were entirely consistent and similar to those
of methyl R-L-evalopyranoside (cf. Figure 2a, R ) Me), the
anomeric configuration of which was established beyond doubt
17
by Rheingold and co-workers by use of X-ray crystallography.
Coincidently, methanolysis of L-evalose glycosides is known
to give the methyl R-L-evalose, not methyl ꢀ-L-evalose. This is
also readily apparent from the present work where microscale
methanolysis of 3 gave methyl R-L-2-O-methylevalose identical
2
relayed the configurations of C18 to C13. Although the
configuration of callipeltose in 6 was most likely to be the same
as that in callipeltoside A, the sugar configuration of evalose
residues in 3, 4, 5, and 7 was not so obvious for two reasons.
(
16) For example, in the orthosomycin antibiotics, everninomicin: (a)
(
14) Asis, S. E.; Bruno, A. M.; Martinez, A. R.; Sevilla, M. V.; Gaozza,
C. H.; Romano, A. M.; Coussio, J. D.; Ciccia, G. Farmaco 1999, 54 (8), 517–
23.
15) The sugar units in phorbasides C-E 5–7 have analogous configurations,
the only difference being 4′R for the callipeltose unit of 6.
Ganguly, A. K.; Saksena, A. K. J. Chem. Soc., Chem. Commun. 1973, 15, 531–
532. (b) Ollis, W. D.; Smith, C.; Wright, D. E. J. Chem. Soc., Chem. Commun.
1974, 881–882.
5
(
(17) Giuliano, R. M.; Kasperowicz, S.; Boyko, W. J.; Rheingold, A. L.
Carbohydr. Res. 1989, 185, 61–67.
J. Org. Chem. Vol. 73, No. 10, 2008 3703

MacMillan et al.
1
9
TABLE 2. Cytotoxicity of Phorbasides (3–7), Callipeltoside A (2),
and Analogues
(IC50 > 100 µM). Interestingly, a synthetic diastereomer 23
of callipeltoside A with opposite configurations at the two
chlorocyclopropane stereocenters C20, C21 (i.e., the same as
the C18,C19 stereocenters of phorbasides A-E) was about twice
compd
cell line
IC50 (µM)
ref
a
a
a
a
a
b
b
c
c
c
c
3
4
5
6
7
2
2
2
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
NSCLCN6
P388
A2780
A2780
A2780
A2780
30.0
1
9
as potent (IC50 7.0 µM) under the same conditions.
2.0
61.9
10.2
16.6
22.4
Callipeltoside A showed modest activity against NSCLCN6
human bronchopulmonary nonsmall cell lung carcinoma cells
(IC50 ) 11.26 µg/mL) and the murine cell line P388 (15.16
µg/mL), but appeared to induce a blockade of cell proliferation
d
d
6a
20.0
17.0
at the G1 phase in the cell cycle. These results and the higher
2
2
2
1
2
3
potency of the disaccharide phorbaside C (5) suggest the latter
may be an interesting synthetic target for the preparation of
additional quantities for advanced biological evaluation.
None of the phorbasides showed antifungal activity in a
standard paper disk assay at 10 µg per disk against Candida
albicans ATCC 14503, C. glabrata, C. krusei, and C. albicans
UCD-FR1.
>100
7.0
a
This work. Inhibition of cell growth and IC50’s determined from
b
c
dose responses of MTS end points. Zampella et al. (ref 6a), Trost et
al. (ref 19). Values converted from literature values of IC50 in µg/mL.
d
with that synthesized from methyl R-L-rhamnoside (10). The
favored axial R-anomeric configuration is also seen in each
glyoside linkage of 3–7.
Conclusion
The L-absolute configuration of the 2-O-methylevalose residue
in 3 was unambiguously defined by CD correlation with our
synthetic standard. The split-Cotton effect of the vicinal car-
bamate-naphthoate pair in 9 is relatively weak (A ≈ 4.5)
The complete stereostructures of phorbasides A-E (1–5)
are defined. The structures of phorbasides contain the same
macrolide ring but differ in the sugar units. A novel
application of exciton coupling CD, which employs pairwise
interactions between the vicinal tertiary N-carbamoyl-2-
aminonaphthyl group and a secondary O-naphthoate ester,
was used to define the L-configuration of evalose sugar units
in phorbasides A and B. The compounds exhibit variable
cytotoxicity that appears to depend upon the presence of a
free 2′- or 2″-hydroxyl group.
compared to typical values for vicinal dibenzoyl pyranoses (A
8
≈
62) for a number of reasons. Greater rotational freedom of
the O-(CO)-N bond allows reorientation through several
conformers that are separated by relatively low energy barriers,
with consequent averaging of the effective electronic dipole
moment vector. A weaker transition dipole moment of the
N-arylamine group in 9 compared to O-benzoate esters may
further reduce the A value. The latter limitation may be
overcome by the use of N-acylisocyanates to prepare tertiary
N-aryl carbamates of tertiary alcohols with larger electronic
transition dipole moments, but the limitation of conformational
averaging remains. Nevertheless, vicinal N-arylcarbamate-O-
naphthoate pairs give CD spectra with sufficient signal at
submicromole amounts and an unequivocal sign for the split
Cotton effect allows unambiguous configurational assignment
of C-3-methyl sugars. The method should be applicable to other
bioactive glycosidic macrolides containing C-methyl pyranosides
with tertiary OH groups and possibly extendable to C-methyl
Experimental Section
General Procedures. 1D and 2D NMR spectra for phorbasides
A-E were measured on an NMR spectometer equipped with a
1
3
1
15
14 T cryomagnet and a { C}- H/ N cold probe with a
preamplifier cooled to T ) ∼20 K. FTIR spectra were acquired
on thin films dispersed on NaCl or ZnSe plates. Circular
dichroism spectra were measured on a grating spectropolarimeter
equipped with a photoelastic modulator. NMR spectra of
synthetic compounds were acquired on an NMR spectrometer
1
3
1
equipped with a 9.3 T cryomagnet and an inverse detect { C} H
1
13
cryoprobe or 7T with a H, C dual-tuned probe. High-resolution
mass spectra were measured by R. New (University of California,
Riverside, Mass Spectrometry Facility). All solvents were HPLC
grade or distilled from glass.
1
8
furanosides (e.g., trachycladine).
Biological Activity. Phorbasides exhibited moderate to potent
cytotoxicity against cultured HCT 116 cells (human colon
cancer, Table 2). Phorbaside C (5) appears to be the most potent
member among the family of phorbasides and callipeltosides
with an IC50 of 2.0 µM, while phorbaside E (7) was somewhat
less active (IC50 ) 10.2 µM). Both phorbasides A (3) and D
Isolation of Phorbasides A-E. The sponge Phorbas sp. was
collected in Muiron Island, Western Australia in 1993. The sample
was immediately frozen and stored at -20 °C until extraction (∼2
3
months). Two batches of the CHCl -soluble fraction (93-054-C3
1
and 93-054-C2) of the MeOH extract of Phorbas sp. (93–054)
(
5) were substantially less cytotoxic (IC50 ) 30 and 61.9 µM,
were separated under HPLC conditions A (C18, 5 µ, 100 Å, 78:22
respectively). On the basis of these results it appears that a free
hydroxyl at C2′ or C2″ is required for enhanced cytotoxic
activity. Trost and co-workers had earlier shown that the sugar
2
MeOH/H
2
O, 3 mL/min) as described previously. The second-
eluting peaks (94-054-C3-1-2 and 94-054-C2-2-2) were pooled and
further fractionated under HPLC conditions B (C18, 3 µ, 60:40
3 2
CH CN/H O, 1.0 mL/min, UV detect λ 254 nm) to give 93-054-
1
9
residue of callipeltoside A (2) was essential for activity; the
removal of the chlorine atom from the cyclopropane ring
did not affect activity, but the configuration of the trans-2-
chlorocyclopropane did. Incubation of 2 with the A2780 human
ovarian carcinoma cell line showed modest inhibitory activity
C3-1-2a, phorbaside D (6, 0.78 mg). The third-eluting peaks and
fourth-eluting peaks were similarly separated under conditions B
to give phorbasides A (3,1.29 mg) and phorbaside B (4, 1.30 mg),
2
as reported earlier. Peaks (93-054-C3-3, 11 mg and 93-054-C2-4,
9
.0 mg) were separated in parallel by HPLC under conditions A.
(
)
IC50 ) 20.0 µM), similar to the des-chloro derivative 21 (IC50
The pooled third-eluting peaks (1.79 mg) were further purified under
17.4 µM); however, the algycon 22 was essentially inactive
conditions B to give 93-054-C2-C4-2a, phorbaside E (7, 0.84 mg).
1
The CCl
4
-soluble fraction of the MeOH extract of the sponge was
(
(
18) Searle, P. A.; Molinski, T. F J. Org. Chem. 1995, 60 (13), 4296–4298.
19) Trost, B. M.; Gunzner, J. L.; Dirat, O.; Rhee, Y. H. J. Am. Chem. Soc.
purified under condition A to give five peaks. Peaks 2 and 3 were
2
002, 124 (35), 10396–10415.
found to contain additional phorboxazoles A (1a, 1.44 mg) and B
3
704 J. Org. Chem. Vol. 73, No. 10, 2008

Phorbasides A-E
1
(
1b, 1.00 mg), respectively, and the fifth-eluting peak (93-054-
min. The temperature was lowered to -78 °C and ketone 12
(650 mg, 3.09 mmol) was added dropwise via cannula to give
a deep yellow solution. After 30 min, dry HMPA (4.7 mL, 26
mmol) and methyl iodide (1.6 mL, 26 mmol) were added by
syringe and the mixture was stirred for 3 h at -78 °C. The
B-2A-3-1-3, 3.52 mg) was resolved under conditions B to give 93-
54-B-2A-3-7A, phorbaside C (5, 1.40 mg).
0
Phorbaside A (3). UV (MeOH) λmax 236 nm (ꢀ 12000); IR
ZnSe) ν 3455, 2923, 1740 cm- ; for H and C NMR see the
1
1
13
(
+
Supporting Information in ref 2. HRFABMS m/z [M + Na]
mixture was quenched by addition of saturated NH
mL) and extracted with Et
O (2 × 40 mL). The combined ether
layers were washed with brine, dried over MgSO , and concen-
4
Cl (aq, 30
6
63.2885 (calcd for C33
H
49ClO10Na, 663.2912).
2
Phorbaside B (4). UV (MeOH) λmax 235 nm (ꢀ 12000); IR
4
ZnSe) ν 3457, 2920, 1741 cm- ; for H and C NMR see the
1
1
13
trated to give a yellow oil that was purified by flash chroma-
(
+
Supporting Information in ref 2. HRFABMS m/z [M + Na]
tography (silica, 10:90 ethyl acetate:n-hexane) to give 13 as a
1
2
8
37.4049 (calcd for C41
H
63ClO14Na, 837.3804).
clear oil (0.7 g, 71% yield). [R]
D
-108.2 (c 3, CHCl
3
) {lit.
1
Phorbaside C (5). UV (MeOH) λmax 235 nm (ꢀ 12000); IR
ZnSe) ν 3450, 2925, 1740 cm ; for H and C NMR and HMBC,
[R]
D
-114.6 (c 1.5, CHCl
3
)}; H NMR (CDCl ) δ 4.88 (s, 1H),
3
-1
1
13
(
4.30 (q, 1H, J ) 6.4 Hz), 4.04 (s, 1H), 1.45 (s, 3H), 1.38 (s,
+
13
see Table S1, Supporting Information. HRFABMS m/z [M + Na]
23.3615 (calcd for C40 61ClO14Na, 823.3648).
Phorbaside D (6). UV (MeOH) λmax 236 nm (ꢀ 12300); IR
3
3H), 1.37 (s, 3H), 1.35 (d, 3H, J ) 6.4 Hz); C NMR (CDCl )
8
H
204.9, 110.7, 98.3, 83.2, 68.9, 56.2, 27.6, 27.4, 21.7, 15.4.
Methyl 6-Deoxy-2,3-O-isopropylidene-r-L-talo-pyranoside (15).
A stirred solution of ketone 13 (200 mg, 0.87 mmol) in EtOH (10
ZnSe) ν 3450, 2925, 1740, 1701 cm- ; for H, C NMR, and
1
1
13
(
HMBC, see Table S2, Supporting Information. HRFABMS m/z
mL) was treated portionwise with NaBH
5 min After an additional 15 min, the mixture was quenched by
the addition of solid NH Cl (60 mg), and the mixture was extracted
with Et
O (2 × 15 mL). The combined ether layers were dried
over MgSO and concentrated to give 15 as a clear oil (194 mg,
96% yield). [R]
4
(60 mg,1.6 mmol) over
+
[
(
M + Na] 688.2885 (calcd for C34H48ClNO10Na, 688.2864).
Phorbaside E (7). UV (MeOH) λmax 235 nm (ꢀ 12000); IR
4
ZnSe) ν 3448, 2922, 1742 cm- ; for H, C NMR and HMBC,
1
1
13
2
+
see Table S3, Supporting Information. HRFABMS m/z [M + Na]
4
13
8
23.3750 (calcd for C40
O-Methyl r-L-Rhamnose (10). Trimethysilyl chloride (15 mL)
was added to anhydrous MeOH (100 mL) followed by L-rhamnose
25 g, 0.15 mol). The mixture was heated at reflux for 19 h with
stirring, cooled, and neutralized by addition of PbCO . Lead salts
H
61ClO14Na, 823.3648).
D
-54 (c 2, CHCl
) 4.91 (s, 1H), 3.88 (q, 1H, J ) 6.4 Hz), 3.39 (s,
3H), 3.15 (s, 1H), 2.42 (br s, 1H), 1.55 (s, 3H), 1.40 (s, 3H). 1.36
3
) {lit. [R]
D
3
-55.0 (c 1, CHCl )};
1
H NMR (CDCl
3
1
3
(
3
(s, 3H), 1.34 (d, 3H, J ) 6.4 Hz); C (CDCl ) δ 109.4, 98.5, 79.7,
75.2, 67.6, 55.2, 27.8, 26.3, 21.9, 15.4.
3
were removed by filtration and the filtrate was concentrated under
reduced pressure to give a syrupy residue. The residual water was
removed by azeotropic distillation under reduced pressure with
toluene (2 × 200 mL) to give the crude methyl glycoside as a white
4,5-Dihydroxy-6-methoxy-2,4-dimethyldihydropyran-3-one (16).
A solution of ketone 13 (700 mg, 3.24 mmol) in methanol (25 mL)
containing p-toluenesulfonic acid hydrate (60 mg) was heated at
reflux for 24 h. The volatiles were removed under reduced pressure
to give a yellow oil that was taken up in 1:1 EtOAc/n-hexane and
eluted through a plug of silica gel with the same solvent to give 16
2 2
foam. The product was taken up in 5% MeOH:CH Cl and eluted
through a plug of silica gel with the same solvent to give 10 as a
3
2
4
0:1 mixture of R-L:ꢀ-L anomers (20 g, 78% yield). [R]
D
-61.9 (c
3
as a clear foam (612 mg, 97% yield). [R]
D
-102.2 (c 2.5, CHCl
3
)
9
1
20
1
.0, H
2
O) {lit. [R]
D
-62.4 (c 9.87, H
2
O)}; H NMR (CD OD) δ
{lit. [R]
D
-105 (c 2.0, CHCl
3
)}; H NMR (CDCl ) δ 4.87 (s,
3
.55 (s, 1H), 3.78 (d, 1H, J ) 2.6 Hz), 3.58 (dd, 1H, J ) 7.2, 2.6
1H), 4.51 (d, 1H, J ) 6.4 Hz), 3.52 (s, 3H), 3.43 (s, 1H), 1.54 (s,
13
Hz), 3.55 (m, 1H), 3.38 (m, 1H), 3.37 (s, 3H), 1.3 (d, 3H, J ) 6.4
Hz).
3
3H), 1.34 (d, 3H, J ) 6.4 Hz); C NMR (CDCl ) δ 203.9, 100.3,
86.6, 78.3, 66.8, 56.0, 23.9, 13.8.
Methyl 6-Deoxy-2,3-isopropylidene-r-L-talo-pyranoside (11).
Anhydrous CuSO (1.2 g) was added to a solution of 10 (600
4
Methyl 6-Deoxy-3-C-methyl-r-L-manno-pyranoside (17). Ke-
tone 16 (550 mg, 2.86 mmol) was dissolved in glacial acetic
mg, 3.4 mmol) in acetone (100 mL) and the mixture was heated
to reflux for 24 h. CuSO was removed via filtration and the
filtrate concentrated under reduced pressure to give 11 as a clear
acid (15 mL) and the solution was treated with NaBH(OAc)
(1.8 g, 8.6 mmol) in three portions. After 5 h, H O (15 mL)
was added, followed by 6 M (NaOH) until the mixture was
neutral. The mixture was extracted with Et
O (2 × 40 mL) and
the combined organic phases dried over Na SO and concentrated
3
4
2
9
oil (660 mg, 94% yield). [R]
D
-10.2 (c 2.0, H
2
O) {lit. [R]
D
2
1
-
10.8 (c 1.65, H
2
O)}; H NMR (CDCl ) δ 4.82 (s, 1H), 4.1 (d,
3
2
4
1
3
3
5
H, J ) 6.0 Hz), 4.04 (dd, 1H, J ) 6.4, 6.0 Hz), 3.6 (m, 1H),
under reduced pressure to give a white foam. Purification of the
.37 (m, 1H), 3.35 (s, 3H), 1.51 (s, 3H), 1.31 (s, 3H), 1.27 (d,
residue by flash chromatography afforded 17 as a white solid
1
3
13
H, J ) 6.4 Hz); C (CDCl
3
) 109.5, 98.1, 78.3, 75.7, 74.4, 65.7
(493 mg, 87%). [R]
D
-81.5 (c 1.2, MeOH) {lit. [R] -83.3
D
1
4.9, 27.9, 26.1, 17.5.
3
(c 0.85, MeOH)}; H NMR (CD OD) δ 4.56 (s, 1H), 3.58 (dd,
Methyl 6-Deoxy-2,3-isopropylidene-r-L-lyxo-pyranosid-4-
ulose(12). Chromium(VI) oxide (1.4 g, 14 mmol) was added to a
mixture of CH Cl (80 mL) and pyridine (8 mL). After 10 min,
1H, J ) 9.8, 6.4 Hz), 3.47 (s,1H), 3.39 (d, 1H, J ) 9.8 Hz),
13
3.34 (s, 3H), 1.26 (d, 3H, J ) 6.4 Hz), 1.24 (s, 3H); C δ 103.2,
76.4, 76.1, 73.6, 68.6, 55.3, 19.2, 18.5.
2
2
when the mixture had turned maroon, alcohol 11 (4 g, 18 mmol)
was added to form a thick slurry. Acetic anhydride (8 mL) was
added and the mixture was vigorously stirred for 30 min. The crude
mixture was filtered through a plug of silica gel in ethyl acetate
and the filtrate concentrated under reduced pressure to give a pale
yellow oil. Separation of the crude product by flash chromatography
Methyl 6-Deoxy-2,3-O-isopropylidene-3-C-methyl-r-L-man
no-pyranoside (14). Alcohol 17 (450 mg, 2.34 mmol) was
dissolved in acetone (15 mL) and CuSO
was heated at reflux for 16 h. CuSO
and the volatiles were removed under reduced pressure to give 14
4
(300 mg) and the mixture
4
was removed by filtration
1
13
as a clear oil (490 mg, 93% yield). H NMR and C NMR data
1
2
13
(
silica, 10:90 ethyl acetate:n-hexane) gave ketone 12 as a clear
were consistent with literature values.
1
oil (3.6 g, 85% yield). H NMR (CDCl
3
) δ 4.80 (s,1H), 4.42 (s,1H),
.22 (q, 1H, J ) 6.4 Hz), 3.46 (s, 3H), 3.38 (s, 1H), 1.48 (s, 3H),
.40 (d, 3H, J ) 6.4 Hz), 1.36 (s, 3H); 13C NMR (CDCl
) δ 204.2,
11., 98.2, 78.7, 75.9, 69.8, 55.8, 26.7, 25.5, 16.0. The product
Methyl 6-Deoxy-2,3-O-isopropylidene-3-C-methyl-4-O-na-
phthoyl-r-L-manno-pyranoside (18). To a solution of 14 (490 mg,
2.11 mmol) in CH Cl (25 mL) was added 2-naphthoyl chloride
4
1
1
3
2
2
(521 mg, 2.7 mmol) in CH Cl over 5 min. Pyridine (250 µL) was
2
2
was used immediately in the next step.
added and the reaction mixture was stirred for 8 h at room
temperature then quenched with NH Cl (aq). The mixture was
extracted with CH Cl
(2 × 20 mL), and the combined organic
Methyl 6-Deoxy-2,3-isopropylidene-3-C-methyl-r-L-lyxo-py-
4
1
2
ranosid-4-ulose (13). Freshly distilled dry diisopropylamine
2
2
(
0.46 mL, 3.3 mmol) was added to a 3-necked flask containing
THF (40 mL). n-Butyllithium (2.4 M in hexanes, 1.4 mL, 3.3
mmol) was added dropwise at 0 °C and allowed to stir for 30
(
20) Chatterjee, D.; Bozic, C.; Aspinalli, G. O.; Brennan, P. J. J. Biol. Chem.
1988, 263, 4092–4094.
J. Org. Chem. Vol. 73, No. 10, 2008 3705

MacMillan et al.
concentrated to a yellow film, then dissolved in CH Cl (200 µL)
before addition of pyridine (20 µL) and 2-naphthoyl chloride (100
µg). The mixture was allowed to stir for 8 h and monitored by
TLC for the formation of 4-O-naphthoate ester. The solvent was
layers were dried over MgSO
oil that was purified by chromatography (silica, 10:90 EtOAc:n-
hexane) to afford 18 as a yellow oil (682 mg, 82%). [R] -94.3 (c
); IR ν 2820, 1738, 1605 cm- ; H NMR (CDCl
) δ
4
and concentrated to give a yellow
2
2
D
1
1
1
7
3
3
1
8
.4, CHCl
3
3
.29–7.46 (m, 7H), 5.1 (d, J ) 9.4 Hz), 4.88 (s, 1H), 3.81 (s, 1H),
2
removed under a stream of N and the residue dried under high
.64 (dd, 1H, J ) 9.4, 6.4 Hz), 3.34 (s, 3H), 1.54 (s, 3H), 1.46 (d,
vacuum. The sample was redissolved in pyridine (300 µL) and
treated with 2-isocyanato-6-methoxynaphthalene (20, 100 µg) before
being heated to 50 °C for 12 h. After removal of pyridine under
reduced pressure, the sample was resuspended in EtOAc (100 µL)
and the mixture centrifuged to remove solid material. The super-
natant was concentrated and redissolved in EtOAc to give “sample
A” (20 µL).
1
3
3
H, J ) 6.4 Hz); C NMR (CDCl ) δ 178.2, 135.6, 133.2, 132.5,
31.4, 131.1, 130.6, 129.7, 128.2, 126.5, 124.4, 109.3, 98.2, 85.9,
3.1, 67.3, 61.2, 55.2, 27.09, 26.9, 18.1, 17.2; HR FABMS m/z [M
+
+
H] 387.1801 (calcd for C22
H
27
O
6
, 387.1808).
Methyl 4-O-(2′-Naphthoyl)-r-L-evalose (19). A solution of
methyl 2,3-O-isopropylidene-4-O-(2′-naphthoyl)-R-L-evalose (18)
600 mg, 1.55 mmol) and p-TsOH (40 mg) in methanol (15 mL)
(
HPLC and CD Analysis of L-Evalose Derivative 9 Ob-
tained from L-Rhamnose and Phorbaside A. HPLC analysis of
synthetic 9 (silica, ISCO 5 µ, 4.6 × 100 mm, 4% i-PrOH:n-hexane,
was heated at reflux for 14 h. The volatiles were removed under
reduced pressure to give the corresponding diol as a white solid
(
502 mg). A portion of the crude diol (350 mg, 1.0 mmol) in
CH CN (15 mL) was treated with freshly prepared Ag O (916 mg,
.0 mmol), followed by slow addition of iodomethane (187 mg,
.3 mmol), and the mixture was then heated at reflux for 5 h. Ag
UV detection: λ )254 nm) gave a single peak t ) 6.7 min.
R
Analytical HPLC of sample “A” (above) gave an identical peak
with the same retention time. The remainder of sample “A” (see
above) was purified by semipreparative HPLC under the same
3
2
4
1
2
O
was removed by filtration to give a yellow oil that was purified by
SiO chromatography (2:8 EtOAc:n-hexane) to provide 19 as a
white solid (200 mg, 55% yield). [R]
NMR (CDCl ) δ 7.28–7.46 (m, 7H), 5.14 (d, 1H, J ) 9.2 Hz),
.59 (dd, 1H, J ) 9.2, 6.4 Hz), 3.41 (s, 3H), 3.32 (s, 3H), 3.14 (s,
R
conditions. The fractions eluting at t ) 6.7 min from repetitive
injections of the remainder of sample “A” were collected and
measured directly by CD in the solvent used for elution (4% i-PrOH/
n-hexane). The CD spectrum of synthetic 9 (ꢀ 52000, c ) 7.9 ×
2
1
D
3
-86.4 (c 1.5, CHCl ); H
3
-6
3
1
1
1
10 M) was measured under the same conditions and the
concentration of sample “A” was normalized from the UV
extinction coefficient (see Figure 3).
1
3
3
H), 1.51 (s, 3H), 1.42 (d, 3H, J ) 6.4 Hz); C NMR (CDCl )
78.4, 135.4, 133.5, 132.1, 131.3, 131.1, 129.8, 129.2, 128.2, 126.5,
24.1, 97.9, 85.6, 82.0, 67.4, 61.3, 55.2, 53.1, 18.1, 16.9; HR
Cytotoxicity Measurements: HCT116. Compounds were as-
sayed with compounds in DMSO (final concentration, 1% v/v) and
run against etoposide as positive control. Human colon tumor cells
(HCT-116) were incubated in 96-well plates for 72 h before addition
of MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, Promega CellTiter
96 Aqueous cell proliferation assay, Technical Bulletin No. 169).
Dose responses were obtained from well absorbances (λ 490 nm),
after correction for background, and expressed as a percentage of
the negative control (DMSO, only).
+
FABMS m/z 361.1672 [M + H] (calcd for C20
25 6
H O , 361.1651).
2
-Isocyanato-6-methoxynaphthalene (20). A stirred solution
of 6-methoxy-2-naphthoic acid (201 mg, 1.03 mmol) in benzene
10 mL) was treated dropwise with diphenylphosphoryl azide (343
(
mg, 1.25 mmol). The mixture was stirred at 50 °C overnight, then
heated to reflux (16 h total) to complete the reaction, before cooling
and concentration. Filtration of the mixture through a plug of silica
1:1 EtOAc:hexane) gave pure 20. IR ν 2250 cm-1
14
(
.
N-(6″-Methoxy-2″-naphthyl)-[1,2-O-dimethyl-r-L-3-C-methyl-
-O-(2′-naphthoyl)-6-deoxy-manno-3-O-pyranosyl] Carbamate
4
(
Acknowledgement. We thank S. Lievens (UC Davis) for
cytotoxicity measurements of 3–7, Sara Kelly and W. Fenical
9). Evalose derivative 19 (25 mg, 0.07 mmol) was dissolved in
14
pyridine (5 mL). Freshly prepared 20 (19.9 mg, 1.4 mmol) was
added as a solution in pyridine (1 mL) and the mixture was heated
to 50 °C for 5 h. The mixture was quenched by the addition of 1
(Scripps Institution of Oceanography) for preliminary cytotox-
icity measurements, J. DeRopp (UC Davis) for assistance with
measurements of 600 MHz NMR spectra, and R. New (UC
Riverside) for MS data. This research was supported by funding
from NIH (AI39987 and CA122256).
mL of saturated aq NaHCO
MgSO , and concentrated to give a yellow solid that was purified
by SiO chromatography (1:9 EtOAc:n-hexane) to afford 9 as a
yellow solid (32 mg, 79% yield). UV (MeOH) λmax 238 nm (ꢀ
3 2 2
, extracted with CH Cl , dried over
4
2
1
Note Added in Proof. An independent confirmation of the
L-2-O-methylevalose in callipeltoside C has been revealed by
total synthesis. Carpenter, J.; Northrup, A. B.; Chung, deM.;
Wiener, J. J. M.; Kim, S-G.; MacMillan, D. W. C. Angew.
Chem. Int. Ed. 2008, early View, DOI: 10.1002/anie.200800086
5
7
(
1
1
1
8
2000), 270 (13000); [R]
D
-67.2 (c 4, CHCl
3 3
); H NMR (CDCl )
.2–7.45 (m, 13H), 5.32 (d, 1H, J ) 9.2 Hz), 4.84 (s, 1H), 3.71
dd, 1H, J ) 9.2, 6.4 Hz), 3.31 (s, 3H), 3.24 (s, 3H), 3.12 (s, 1H),
.52 (s, 3H), 1.45 (d, 3H, J ) 6.4 Hz); 13C δ 173.1, 157.6, 139.2,
37.6, 136.2, 136.1, 135.5, 132.7, 132.4, 131.6, 131.5, 130.9, 130.6,
30.2, 129.5, 128.2, 128.1, 126.3, 125.4, 124.2, 123.2, 97.2, 85.2,
4.9, 68.2, 59.2, 56.2, 53.1, 19.1, 18.4; HR FABMS m/z [M +
Supporting Information Available: 1H NMR, HSQC,
HMBC, and HSQC TOCSY of 3-7. This material is available
free of charge via the Internet at http://pubs.acs.org.
+
H] 560.2282 (calcd for C32
H34NO
8
, 560.2284).
Methanolysis of Phorbaside A and Derivatization. Phorbaside
A (3, 50 µg) was dissolved in dry MeOH (1 mL) that had been
purged with HCl (g) and heated to 100 °C for 6 h. The sample was
JO702307T
3
706 J. Org. Chem. Vol. 73, No. 10, 2008