Structure and stereochemistry of a novel bioactive sphingolipid from a Calyx sp.

Article abstract of DOI:10.1016/S0040-4020(01)00958-9

Bioassay-directed fractionation of a sponge of the genus Calyx using a yeast bioassay for DNA-damaging agents yielded the novel sphingolipid calyxoside (1) as the major bioactive constituent. The structure of 1 was assigned as 1,3,26-trihydroxy-2,27-diaminooctacosan-18-one-1-β-D-glucoside by ¹H- and ¹³C NMR, DEPT, DQCOSY, HMQC, and HMBC spectra. The carbonyl group was located at C-18 by analysis of the EI-MS fragmentation of the amino derivative of its aglycone pentaacetate. Its absolute configuration was determined as 2S,3R,26S,27S by analysis of the ¹H NMR and CD spectra of its aglycone pentabenzoate.

Full text of DOI:10.1016/S0040-4020(01)00958-9

TETRAHEDRON
Pergamon
Tetrahedron 57 ,2001) 9549±9554
Structure and stereochemistry of a novel bioactive sphingolipid
from a Calyx sp.
Bing-Nan Zhou,a Michael P. Mattern,b Randall K. Johnsonb and David G. I. Kingstona,p
aDepartment of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, Blacksburg, VA 24061-0212, USA
bGlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA
Received 10 August 2001; accepted 4 October 2001
AbstractÐBioassay-directed fractionation of a sponge of the genus Calyx using a yeast bioassay for DNA-damaging agents yielded the
novel sphingolipid calyxoside ,1) as the major bioactive constituent. The structure of 1 was assigned as 1,3,26-trihydroxy-2,27-diaminooc-
tacosan-18-one-1-b-d-glucoside by 1H- and 13C NMR, DEPT, DQCOSY, HMQC, and HMBC spectra. The carbonyl group was located at C-
18 by analysis of the EI-MS fragmentation of the amino derivative of its aglycone pentaacetate. Its absolute con®guration was determined as
2S,3R,26S,27S by analysis of the 1H NMR and CD spectra of its aglycone pentabenzoate. q 2001 Published by Elsevier Science Ltd.
1. Introduction
show any absorption above l 210nm in its UV spectrum,
indicating the presence of amino group,s) and the absence
of any conjugation. Its molecular formula was C34H69N2O9,
as deduced from HRFAB-MS, and this was con®rmed by
HRFAB-MS of its octaacetate derivative 2. Its 13C NMR
and DEPT spectra in combination with its molecular
formula indicated the presence of a terminal methyl group
,dH 1.29 ppm ,d) and dC 16.0ppm), a ketone group , dC
214.3 ppm), an acetal group ,dC 104.0 ppm and dH 4.35
,d, Jˆ7.63 Hz), two oxygenated methylenes, six oxy-
genated methines and two methines with amino groups.
The remaining carbons and hydrogens were assigned to
a long alkyl chain based on the overlapping signals for
methylene protons and the corresponding carbon signals.
In continuation of our search for novel natural products with
potential as anticancer agents,1±4 a methanol extract of a
Calyx sp. was examined and found to show reproducible
and selective bioactivity in our yeast assay for DNA-
damaging agents,1 and it was thus selected for bioassay-direc-
ted fractionation. Sponges of the genus Calyx, belonging to
the family Oceanapiidae, have been the source of a number
of interesting compounds. They have been reported to contain
novel steroids with acetylene or cyclopropene groups in their
side chains,5,6 diketopiperazines with antibiotic and phyto-
toxic properties,7 unique diphenyloxolanes,8 pyridinetetra-
decanamines9 and 11-methylpentadecanoic acid.10
Because of the overlap of the terminal methyl signal with
methylene signals at higher ®eld and the overlap of some
signals in the d 3±4.2 ppm region of the 1H NMR spectrum
of 1 ,Table 1), calyxoside was converted to its octaacetate 2,
and the NMR spectra of 2 were used for structural analysis.
The 13C NMR spectrum of 2 in CDCl3 showed signals for a
ketone group ,dC 211.7 ppm) and eight acetyl groups ,dC
169.4, 169.5, 169.5, 169.7, 170.1, 170.8, 170.6, and 171.1;
dH 1.96, 1.96, 1.98, 2.00, 1.02, 2.03, 2.06, and 2.07). Its
DQCOSY and HMBC spectral data ,Table 1) indicated
the presence of three spin systems. The signals at dC
100.6 ppm ,d) and dH 4.44 ppm ,d, Jˆ7.9 Hz), dC
71.3 ppm ,d) and dH 4.92 ,dd, Jˆ7.9, 9.6 Hz), dC 72.6 ppm
,d) and dH 5.17 ,dd, Jˆ9.6, 9.9 Hz), dC 68.2 ppm ,d) and dH
5.04 ,dd, Jˆ9.9, 9.5 Hz), dC 71.8 ppm ,d) and dH 3.68 ,dd,
m), dC 61.8 ppm ,t) and dH 4.22 ,dd, Jˆ12.1, 4.1 Hz), and
dH 4.13 ,dd, Jˆ12.1, 2.3 Hz) suggested the presence of a
glucose moiety. The groups CH3CH,NHCOCH3)CH
2. Results and discussion
Partition of a CH2Cl2/methanol extract of the sponge
between aqueous MeOH and n-hexane, followed by par-
tition of the aqueous MeOH fraction with CH2Cl2, yielded
a bioactive aqueous MeOH fraction with an IC12 of
148 mg mL21 in the rad52Y yeast assay. Puri®cation of
this fraction by repeated chromatography on MCI gel ,a
porous polystyrene gel) with elution with 50% aqueous
MeOH and monitoring of the fractions by bioassay and by
1H NMR spectroscopy yielded the novel bioactive
compound 1, designated calyxoside.
Calyxoside ,1) gave a positive ninhydrin test and did not
Keywords: sphingolipid; calyxoside; structure and stereochemistry;
bioactivity.
Corresponding author. Tel.: 11-540-231-6570; fax: 11-540-231-7702;
e-mail: dkingston@vt.edu
p
,OCOCH3)CH2±
,OCOCH3)CH2±, were assigned to two additional spin
and
ROCH2CH,NHCOCH3)CH
0040±4020/01/$ - see front matter q 2001 Published by Elsevier Science Ltd.
PII: S0040-4020,01)00 958-9

9550
B.-N. Zhou et al. / Tetrahedron 57 72001) 9549±9554
Table 1. 1H- and 13C NMR data for calyxoside 1, its octaacetyl derivative 2, and oceanapiside 6
Atom no.
Calyxoside 1 ,in CD3OD)
Calyxoside octaacetate 2 ,in CDCl3)
H ,mult. J Hz) DQCOSY
6
dC
d
H ,mult. J Hz)
dC
d
HMBC ,C to H)
H-10, H-2
dC
58.9
57.2
80.1
33.1
26.7
1
66.7
4.02 ,dd, 11.5, 8.7)
3.86 ,dd, 11.5, 3.7)
3.40,m)
3.84 ,m)
1.48 ,m)
67.1
3.88 ,dd, 10.3, 3.9)
3.59 ,dd, 10.3, 4.5)
4.19 ,m)
H-2
2
3
4
5
56.7
70.1
34.1
27.0
50.8
74.1
31.5a
H-1a, H-1b, H-3
H-2, H-4
H-3
H-1a, H-1b, H-3
H-1a, H-1b, H-2
H-2
4.83 ,m)
1.51 ,m)
6±9
10
11
12
13±15
16
17
18
19
2029.6
21±23
24
25
26
27
28
10
43.4
214.3
43.4
24.8
43.4
214.3
43.5
1.51 ,m)
29.6
42.7a
211.7
42.8a
1.51 ,m)
2.35 ,t, 7.5)
H-17
H-16
2.43 ,t, 7.2)
H-17, H-19
2.43 ,t, 7.2)
2.35 ,t, 7.5)
H-20
H-21
26.2
34.5
73.1
53.5
16.9
104.5
74.8
77.7
71.4
26.2
34.5
73.1
53.5
16.0
104.5
74.8
77.7
71.6
78.1
62.6
1.42 ,m)
3.46 ,m)
3.12 ,m)
1.26 ,d)
4.35 ,d, 7.6)
3.25 ,m)
3.32 ,m)
31.4a
76.4
47.8
18.4
100.6
71.3
72.6
68.2
3.68 ,m)
61.8
1.51 ,m)
4.83 ,m)
4.19 ,m)
1.07 ,d, 6.9)
4.44 ,d, 7.9)
4.92 ,dd, 7.9, 9.6)
5.17 ,dd, 9.6, 9.9)
5.04 ,dd, 9.9, 9.5)
H-4
H-26
H-27
H-28
H-25
H-25, H-27
H-26, H-28
H-27
H-20
H-10, H-30
H-20, H-4
H-30, H-50
0, H-6a0, H-6b0
H-50
H-26
H-1a, H-1b, H-30
H-10, H-30, H-4'
H-10, H-40
H-30, H-6a0, H-6b0
H-10, H-40, H-6a0, H-6b0
H-40
20
30
40
3.29 ,m)
50
78,.0m3).40
62.4
71.8
3.66 ,dd, 11.8, 5.7)
3.89 ,dd, 11.8, 3.7)
60
4.22 ,dd, 12.1, 4.7)
4.13 ,dd, 12.1, 2.3)
6.09 ,d, 8.9)
2-NH
27-NH
H-2
H-27
5.57 ,d, 9.3)
b
c
a
These values may be interchanged.
b
c
d
d
C 169.4, 169.5, 169.7, 170.1, 170.6, 170.8, and 171.1.
H 1.96 ,s), 1.96 ,s), 1.98 ,s), 2.00 ,s), 2.02 ,s), 2.03 ,s), 2.06 ,s), and 2.07 ,s).
0
0
systems, consistent with the two terminal sequences of a
long chain sphingolipid type of compound.
,J1 ,2ˆ7.6 Hz in 1 and J1 ,2ˆ7.9 Hz in 2) indicated that
calyxoside is a b-glucoside. The long range correlation
between H-10 ,dH 4.35 ppm, d, Jˆ7.6 Hz) and C-1 ,dC
66.7 ppm, t) in the HMBC spectrum of 1 and the long
range correlations between H-10 ,dH 4.44 ppm, d, Jˆ
7.9 Hz) and C-1 ,dC 67.1 ppm, t) and H-1 ,dH 3.88 ppm,
dd, Jˆ10.30, 3.89 Hz, and dH 3.59 ppm, dd, Jˆ10.30,
4.5 Hz) with C-10 ,dC 100.6 ppm, t) in the HMBC spectrum
of calyxoside octaacetate 2 indicated that glucosylation was
on C-1. The assignment of the d stereochemistry to the
glucose moiety was based on the hydrolysis of calyxoside
by almond b-glucosidase, speci®c for b-d-glucosides or
When 1 was heated with 1% HCl in 50% aqueous MeOH, it
was hydrolyzed to glucose and the aglycone calyxinin ,3).
The water-soluble fraction of the hydrolyzate was treated
with acetic anhydride and pyridine to convert it to its
pentaacetate, which was identi®ed as glucose pentaacetate
by GC±MS comparison with authentic material. The
chemical shifts and coupling constants of the sugar moiety
in 1 and 2 con®rmed the assignment of the sugar as glucose,
and the coupling constant of the anomeric proton

B.-N. Zhou et al. / Tetrahedron 57 72001) 9549±9554
9551
Table 2. Partial 1H NMR data of the a-termini of compounds 8, 13, and model compounds
a-Terminus
Chemical shifts ,ppm)
d3-H d2-H
4.87 4.61, 4.65
J-values ,Hz)
dC2±NH
d1-H
JNH±H-2
JH-2±H-3
Model compound 11 ,erythro)a
Oceanin perbenzoate 8 ,erythro)a
Model compound 12 ,threo)a
7.105.41
7.08
6.63
8.7
4.1
4.1
5.38
5.56
4.88
4.89
4.61, 4.64
4.48, 4.57
8.7
8.6
9.2
3.8
4.8
Calyxinin pentabenzoate 13 ,erythro)
7.105.38
4.88
4.62, 4.66
a
See Ref. 15.
b-d-galactosides,11 con®rming that 1 is a sphingolipid 1-b-
d-glucoside.
dC-1, dC-2 and dC-3, consistent with glucosylation at C-1 in 1
and at C-3 in 6.
Treatment of the aglycone calyxinin ,3) with acetic
anhydride in pyridine converted it to calyxinin pentaacetate
,4), which had the molecular composition C38H68N2O9
,HRFAB-MS), indicating that the aglycone 3 contains 28
In agreement with previous studies on oceanapiside, direct
attempts to determine the position of the carbonyl group in
1 or 2 by mass spectrometry were unsuccessful, since
fragmentation was dominated by cleavages a to the amino
groups and no ions could be assigned with con®dence to
cleavages a to the carbonyl group. The position of the
carbonyl group in rhizochalin was assigned by conversion
of its acetate to a mixture of esters by Baeyer±Villiger
oxidation followed by EI-MS, while its location in
oceanapiside was assigned by MALDI MS/MS on a deuter-
ated sample which took more than two months to prepare.
Neither of these approaches seemed attractive, and we
elected to convert the carbonyl group of calyxinin penta-
acetate ,4) to an amino group to facilitate cleavage a to the
erstwhile carbonyl group. Compound 4 was thus converted
to 18-aminocalyxinin pentaacetate ,7) by reductive
amination with lithium cyanoborohydride in the presence
1
carbons. The H- and 13C NMR spectra of 4 con®rmed the
presence of a carbonyl group and the terminal functional
groups. This evidence thus established the structure of
calyxoside as an unusual glucosylated ketosphingolipid,
but did not serve to identify the location of the carbonyl
group or the stereochemistry of the two aminoalcohol end
units.
Two ketosphingolipids have been reported previously,
rhizochalin ,5),12,13 a sphingolipid 3-galactoside, and
oceanapiside ,6),14 a sphingolipid 3-b-d-glucoside. The
13C NMR spectrum of calyxoside was very similar to the
literature spectrum of oceanapiside except for the values of

9552
B.-N. Zhou et al. / Tetrahedron 57 72001) 9549±9554
Table 3. Partial 1H NMR data of the v-termini of compounds 8, 13, and model compounds
v-Terminus
Chemical shifts ,ppm)
J-values ,Hz)
dC27-NH
d26-H
d27-H
d28-H
JNH±H-27
JH-26±H-27
Model compound 9 ,erythro)a
Model compound 10 ,threo)a
6.97
6.39
6.38
6.38
5.24
5.24
5.21
5.21
4.46
4.53
4.53
4.53
0.97
0.94
1.28
1.29
7.8
9.0
8.9
9.05.3
2.6
5.2
5.2
Oceanin perbenzoate 8 ,ent-threo)a
Calyxinin pentabenzoate 13 ,threo)
a
See Ref. 15.
of ammonium acetate and molecular sieves. The predomi-
nant fragment ions of 7 at m/z 441 ,C24H45N2O5) and 285
,C15H28N2O3), corresponding to cleavage of the C-18/C-19
and C-17/C-18 bonds, respectively, indicated that the amino
group was located on C-18. The carbonyl group in 1 could
thus be assigned to the C-18 position.
We elected to use this approach to assign the stereo-
chemistry of calyxoside. Calyxinin pentabenzoate 13 was
thus prepared, and its 1H NMR spectrum was compared with
those of the model compounds 9±12 as well as that of
oceanin octabenzoate 8, as shown in Tables 2 and 3. The
threo and erythro con®gurations at the a- and v-termini
could be differentiated by comparison of their chemical
shifts and coupling constants with those of the model
compounds 9±12. The similarities of dC-2,NH, JNH±H-2, and
JH-2±H-3 as well as of dH-27, JNH±H-27, and JH-26±H-27 of 13 with
the corresponding values of 8 and of the appropriate model
compounds indicated that calyxinin and calyxoside have the
same relative con®guration as oceanin, with the erythro
con®guration at the a-terminus and the threo con®guration
at the v-terminus.
The stereochemistry of calyxoside remained to be assigned.
Fortunately the absolute con®guration of oceanapiside ,6)
1
was recently determined by a careful analysis of the H
NMR and CD spectra of its perbenzoyl aglycone ,8) in
comparison with the corresponding spectra of the synthetic
model compounds 9±12.15 In this work it was observed that
the CD spectrum of the aglycone perbenzoate 8 was the sum
of the expected local CD spectra at each terminus, since
the 22 carbons separating the termini greatly diminished
intramolecular exciton coupling and intramolecular effects
were negligible due to the high dilution of sample
,1024±1025 M) used in the determination.
The CD spectrum of calyxinin pentabenzoate 13 showed
D1 values of 25.06 at 237 nm and 10.40 at 220 nm. Com-
parison with the data in Table 4 indicated that the absolute
con®guration of calyxinin pentabenzoate is ,2S,3R) at the
a-terminus and ,26S,27S) at the v-terminus. Calyxoside
thus differs in absolute con®guration from oceanapiside,
which is ,2S,3R) at the a-terminus but ,26R,27R) at the
v-terminus.
It could thus be concluded that calyxoside 1 is a new natural
product with the structure ,2S,3R,26S,27S)-2,27-diamino-
1,3,26-trihydroxynonacosan-18-one-1-b-d-glucoside. Its
structure is unusual in being only the sixth member of a
small class of `two-headed' sphingolipids. In addition to
the keto compounds rhizochalin12 and oceanapiside14,15
previously mentioned, the other members include the poly-
enes leucettamols A and B16 and BRS1.17
Calyxoside ,1) was tested against the RS322 ,rad52), the
RS188N ,RAD1) and RS321,rad52.top1) yeast strains.
Table 4. CD spectral values ,MeOH) for compounds 13, 8, and model compounds
Compound
a-Terminus stereochemistry
v-Terminus stereochemistry
D11,237 nm)
D12,220nm)
A value ,D112D12)
Calyxinin pentabenzoate 13
Oceanin octabenzoate 8a
12110a
erythro
erythro
threo
erythro
threo
erythro
threo
erythro
threo
erythro
threo
25.06
29.61
20.57
25.13
29.19
213.75
26.57
211.13
12.06
25.63
10.40
17.12
23.19
10.73
11.92
15.85
13.86
17.86
21.25
12.67
25.46
216.73
12.62
ent-threo
threo
threo
erythro
erythro
ent-threo
ent-threo
ent-erythro
ent-erythro
11110a
25.86
1219a
211.11
219.60
210.43
218.99
13.31
1119a
121ent-10a
111ent-10a
101ent-9a
111ent-9a
28.30
a
See Ref. 15.

B.-N. Zhou et al. / Tetrahedron 57 72001) 9549±9554
9553
Compound 1 had IC12 values of 62 and 36 mg mL21 in
RS321 and RS322, respectively, but was inactive
aqueous MeOH fraction ,1.1682 g) was subjected to cc on
MCI gel ,30g) with 50% aqueous MeOH; 33 fractions of
10mL each were collected. Fractions 13±17 were bioactive
and gave typical lipid signals in their 1H NMR spectra. After
repeated cc on MCI gel with 50% aqueous MeOH, calyxo-
side ,1, 98 mg) was obtained from fractions 13±15. It had
IC12 values of 36.2 mg mL21 against RS322 and
62.5 mg mL21 against RS321. None of the fractions nor
calyxoside ,1) showed any activity against the wild-type
RS188 yeast strain.
,IC12.1000 mg mL21
)
in the repair-pro®cient strain
RS188N. These data are characteristic of a selective
DNA-damaging agent which does not act as a topo-
isomerase I or topoisomerase II inhibitor. Compound 1
was also tested for cytotoxicity in several mammalian cell
lines, with the following results ,cell line: IC50 value,
mg mL21): HFF, 20; MRC-5, 20, SW480, 5.0, HT-29, 10,
Saos-2, 5.0, DLD-1, 5.0, H460, 3.0. These data indicate that
1 has a relatively weak cytoxicity, without any strong
selectivity for any cell line; it is thus unlikely to be an
attractive candidate for further development based on this
activity pro®le.
3.4.1. Calyxoside 1. Colorless gel-like substance; [a]25
ˆ
D
215.8 ,c 0.321, MeOH); IR ,KBr, ®lm) nmax 3600±2900
,OH) and 1712 cm21 ,CO); 1H NMR and 13C NMR data, see
Table 1. HRFAB-MS ,M1H)1 m/z 649.5024 ,M1H)1
,C34H69N2O9 calcd m/z 649.5037).
3. Experimental
3.4.2. Calyxoside octaacetate 2. Calyxoside 1 ,4 mg) was
dissolved in acetic anhydride ,0.2 mL) and pyridine ,20 mL)
and the mixture stirred for 12 h at room temperature. The
product was worked up in the usual way to give calyxoside
octaacetate ,3 mg) as a colorless gel-like substance:
3.1. General experimental procedures
The CD spectrum was recorded on a JASCO J720Spectro-
polarimeter. NMR spectra were recorded on a Varian Unity
400 NMR instrument at 399.951 MHz for 1H and
100.578 MHz for 13C, using standard Varian pulse sequence
programs. Low resolution mass spectra were taken on a VG
7070 E-HF at VPI&SU. High resolution mass spectra were
obtained on a Kratos MS50instrument at the Nebraska
Center for Mass Spectrometry. MCI gel ,75±150 mm par-
ticle size) was obtained from Mitsubishi Chemicals. Other
conditions were as previously described.1
[a]25 ˆ25.23 ,c 0.38, CHCl3), 1H-NMR 13C-NMR,
D
DQCOSY, and HMBC data, see Table 1; HRFAB-MS m/z
985.5820,M 1H)1 ,calcd for C50H85N2O17, m/z 985.5848).
3.4.3. Hydrolysis of calyxoside. A solution of calyxoside
,1, 10.2 mg) was mixed with 1% HCl ,2 mL) in 50% aq.
MeOH solution. The reaction mixture was stirred at room
temperature overnight. The MeOH was removed by
evaporation and the reaction mixture diluted with H2O
,2 mL), neutralized with NH4OH, and extracted with
CH2Cl2. The aqueous solution was evaporated to dryness,
and the residue was stirred in acetic anhydride ,0.5 mL) and
pyridine ,3 drops) at room temperature overnight. After
removing the reagents with N2, the product was analyzed
by GC±MS. The retention time of the major peak and its
EI-MS spectrum were the same as those of an authentic
standard of d-glucose pentaacetate.
3.2. Yeast bioassay
The experimental methods utilized in the Saccharomyces
cerevisieae bioassay using DNA repair de®cient mutants
were performed as previously described.18 The IC12 values
refer to the concentration in mg mL21 required to produce a
zone of inhibition 12 mm diameter around a 100 mL well
during a 48 h incubation period at 378C.
3.3. Materials
Evaporation of the CH2Cl2 extract yielded the aglycone
calyxinin ,3) as a colorless gum-like substance: [a]25
ˆ
D
1
Calyx sp. 2 ,Oceanapiidae) ,SCLID 41689/88) was
collected by Dr Patrick Colin, Coral Reef Research Foun-
dation, Palau, in Sulawesi in May 1993. A voucher speci-
men, number 0CDN1359, was deposited at the Smithsonian
Institution. The taxonomic assignment was made by Dr
Michelle Kelly, NIWA, NZ. The material was extracted
with CH2Cl2/MeOH at the NCI Frederick Research and
Development Center, Frederick, MD to yield extract
C011149, also known as SCLID 41688.
22.17 ,c 0.23, MeOH); H NMR d ppm ,Hz) in CD3OD/
CDCl3: 1.06 ,3H, t, Jˆ6.6 Hz), 1.2±1.6, 2.44 ,4H, t, Jˆ
7.21 Hz), 2.72 ,2H, m), 3.21 ,1H, m), 3.49 ,4H, m), 3.72
,1H, m), and 13C NMR d ppm in CD3OD/CDCl3: 18.9, 24.6,
24.7, 26.4, 26.8, 30.0,0), 30.0,4), 30.3±30.5, 34.2, 34.4,
43.4, 52.2, 57.6, 63.8, 76.4.
3.4.4. Calyxinin pentaacetate 5. Calyxinin ,5.67 mg) was
mixed with acetic anhydride 0.2 mL and anhydrous pyridine
,3 drops) and stirred overnight at room temperature. The
reaction mixture was worked up in the usual way to give
3.4. Isolation of calyxoside
1
calyxinin pentaacetate ,5 mg); H NMR d ppm ,Hz) in
CDCl3 1.10,3H, d, Jˆ6.80Hz, 3 £H-28), 1.2±1.7, 1.99
,3H, s, CH3CO±), 2.00 ,3H, s, CH3CO±), 2.06 ,3H, s,
CH3CO±), 2.07 ,3H, s, CH3CO±), 2.09 ,3H, s, CH3CO±),
2.37 ,4H, t, Jˆ7.41 Hz, 2£H-17 and 2£H-19), 4.06 ,1H, dd,
Jˆ11.59, 3.92 Hz, H-1b), 4.20,1H, m, H-27), 4.25 ,1H, dd,
Jˆ11.59, 6.08 Hz, H-1a), 4.39 ,1H, m, H-2), 4.85 ,1H, m,
H-26), 4.90,1H, m, H-3), 5.53 ,1H, d, Jˆ8.86 Hz,
±NHCOCH3), 5.89 ,1H, d, Jˆ9.27 Hz, ±NHCOCH3); 13C
NMR d ppm in CDCl3 18.5 ,C-28), 28.8±31.5 ,±CH2± and
The MeOH extract SCLID 41688 ,3.36 g, IC12
625 mg mL21 in the RS322 ,Rad52Y) assay and IC12
1250 mg mL21 in the RS321 assay) was partitioned between
n-hexane and MeOH/H2O ,60:40) and this was then diluted
to MeOH/H2O ,50:50) and extracted with CH2Cl2. The
resulting aqueous MeOH fraction ,1.17 g) was bioactive
with IC12 148 mg mL21 in the RS322 assay and IC12
125 mg mL21 in the RS321 assay, and the n-hexane and
CH2Cl2 fractions were inactive in this assay. The bioactive

9554
B.-N. Zhou et al. / Tetrahedron 57 72001) 9549±9554
CH3CO± signals), 42.7 ,C-17 or C-19), 42.8 ,C-17 or C-19),
47.1 ,C-27), 50.5 ,C-2), 62.6 ,C-1), 74.0 ,C-3), 76.4 ,C-26),
169.4 ,±CvO), 169.7 ,±CvO), 170.9 ,±CvO), 170.9
,±CvO), 171.0,±C vO), 211.7 ,C-18); HRFAB-MS m/z
697.4991 ,M1H)1 ,calcd for C38H69N2O9, m/z 697.5003).
for making the extract of Calyx sp. available through the
NCI Natural Products Repository. We also thank Mr Kim
Harich and Mr William R. Bebout, Sr., ,Virginia Poly-
technic Institute and State University) and the Nebraska
Center for Mass Spectrometry for low and high resolution
mass spectra, respectively.
3.4.5. Reductive amination of calyxinin pentaacetate 5 by
lithium cyanoborohydride. Calyxinin pentaacetate ,5,
4.13 mg, 0.0059 mmol) was mixed with molecular sieves
,3.3 mg), NH4OAc ,12.2 mg, 0.052 mmol) in 1 mL absolute
MeOH. A solution of LiBH3CN ,0.33 mg, 0.0052 mmol) in
absolute MeOH was added dropwise. The reaction mixture
was stirred for 48 h at room temperature. After removal of
most of the MeOH, the residue was taken up in H2O ,5 mL)
and extracted with CH2Cl2 ,4£5 mL). After washing
with saturated NaCl solution and water and drying with
anhydrous Na2SO4, the CH2Cl2 fraction was evaporated in
vacuo to give 2.1 mg 18-aminocalyxinin pentaacetate ,7):
EI-MS m/z 696 ,M21)1, 441 ,C24H45N2O5)1, 381, 285
,C15H28N2O3)1, 225.
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Acknowledgements
This work was supported by a National Cooperative Drug
Discovery Group award to the University of Virginia ,U19
CA 50771, Dr S. M. Hecht, Principal Investigator), and this
support is gratefully acknowledged. We thank Dr Gordon
Cragg, Dr David Newman, and the National Cancer Institute