Notes
J ournal of Natural Products, 2000, Vol. 63, No. 4 539
scale referenced to the solvent peak CHCl3 at 7.25 and CDCl3
at 77.0. For the P-388 assay a two-fold dilution series of the
sample was incubated for 72 h with murine leukemia cells
(ATCC CCL 46 P-388D1). The concentration of the sample
required to inhibit cell growth to 50% of the growth of a solvent
control was determined using the absorbance obtained upon
staining with MTT tetrazolium. As a positive control for this
assay, mitomycin C, at a concentration of 0.06 µg/mL, inhibited
the growth of P-388 cells by 43-75%.
Collection a n d Extr a ction . B. novae-zelandiae was col-
lected from Port Adventure, Stewart Island, New Zealand, in
J anuary 1994 [PERU code 940127-04, University of Otago
Herbarium (OTA) specimen 046765] and from the Charleston
area, West Coast, South Island, in J anuary 1997 (970129-11).
Initial screening was carried out using extracts produced by
shaking air-dried (30 °C), ground material (5.0 g) overnight
in EtOH (50 mL).
Isola tion of Na vicu lyl Ca ffea te (1). A bulk extract of
collection 970129-11 (67 g) was prepared by blending with
EtOH (1 × 350 mL, 3 × 150 mL) then CHCl3 (2 × 150 mL).
The solvent was removed from the combined, filtered extracts
to give a green gum (1.92 g, P-388 GI50 5.4 µg/mL). The crude
extract was subjected to reversed-phase flash chromatography
(1.92 g precoated on 2 g C18, loaded on a 20-g C18 column),
developed in 15-mL steps from H2O through MeCN to CHCl3.
Fractions eluted with H2O-MeCN (1:9), MeCN, and CHCl3
had GI50s < 10 µg/mL).
the dark for 18 h. The solution was concentrated to give an
orange oil. Column chromatography over Si gel eluting with
0-100% EtOAc in CH2Cl2 gave the crude ester (71 mg). A
CH2Cl2 solution of this was stirred with polyvinylpyrrolidine
(PVP) for 15 min, then column chromatography over PVP
eluting with CH2Cl2-EtOAc-MeOH gave the pure ester as a
white crystalline solid (33 mg, 41%): registry number 117614-
99-0; mp (EtOAc-Hexane) 121 °C.
Syn th esis of Na vicu lyl Ca ffea te (1). A solution of caffeic
acid (20 mg, 0.11 mmol), naviculol (24 mg, 0.11 mmol), and
DCC (25 mg, 0.12 mmol) in dry THF (2 mL) was held at room
temperature in the dark for 18 h. The solution was concen-
trated to give an orange oil. Column chromatography over Si
gel, eluting with 0-100% EtOAc in CH2Cl2, gave recovered
naviculol (9 mg, 37%), followed by the pure ester 1 (11 mg,
28%): colorless gum; [R]28 +29°, [R]28
+16°, [R]28
D
577 nm
546 nm
+11°, [R]28
-147°, [R]28
-180° (c 0.5, MeOH); 1H and
435 nm
405 nm
13C NMR matched the natural product, see Table 1; P-388 GI50
see Table 2.
,
Ack n ow led gm en t. We thank the New Zealand Depart-
ment of Conservation and Timberlands for permission to
collect; R. Tangney for taxonomic expertise; S. Lorimer and
N. Brennan for organizing collections; G. Ellis, D. Newman,
and the NCI for biological assays; M. Thomas and W. Redmond
for NMR; B. Clark for MS; and Y. Asakawa, T. Hashimoto,
and M. Toyota for a sample of bazzanenyl caffeate. This
research was supported by the New Zealand Foundation for
Research, Science and Technology, and by a New Zealand
Lottery Health Research Committee grant to R. Smith for an
indirect-detection NMR probe.
The least polar reversed-phase fraction (1.01 g, eluted with
1:1 CHCl3-MeCN, P-388 GI50 8.2 µg/mL) was subjected to
flash chromatography on Si gel (12 g), developed in steps from
cyclohexane to EtOAc. Fractions eluted with 6:1, 5:1, and 4:1
cyclohexane-EtOAc, which showed a UV-active spot on TLC
(2:1 cyclohexane-EtOAc) at Rf 0.3, were combined (88 mg).
The main component in this sample was purified by
reversed-phase HPLC (Merck Lichrospher 100 RP18, 250 × 10
mm, mobile phase 5 mL/min 15% H2O-85% MeOH, detection
at 206 nm). Compound 1 (3 mg), with retention time 25.5 min,
was recovered by adding H2O (35 mL) and CH2Cl2 (35 mL) to
combined fractions (35 mL), separating the organic layer, and
removing the solvents at room temperature with no fluorescent
lighting in a flask painted black.
Refer en ces a n d Notes
(1) Robinson, W. E.; Reinecke, M. G.; Abdel-Malek, S.; J ia, Q.; Chow, S.
A. Proc. Natl. Acad. Sci. USA 1996, 93, 6326-6331.
(2) Amoros, M.; Lurton, E.; Boustie, J .; Girre, L. J . Nat. Prod. 1994, 57,
644-647.
(3) Hashimoto, T.; Tori, M.; Asakawa, Y. Z. Naturforsch. 1988, 43, 470-
472.
(4) Grunberger, D.; Banerjee, R.; Eisinger, K.; Oltz, E. M.; Efros, L.;
Caldwell, M.; Estevez, V.; Nakanishi, K. Experientia 1988, 44, 230-
232.
(5) Allison, K. W.; Child, J . The Liverworts of New Zealand; University
of Otago Press: Dunedin, 1975.
(6) Asakawa, Y.; Toyota, M.; Nakaishi, E.; Tada, Y. J . Hattori Bot. Lab.
1996, 80, 271-295.
(7) Asakawa, Y. Prog. Chem. Org. Nat. Prod. 1995, 65, 1-525.
(8) Toyota, M.; Asakawa, Y.; Takemoto, T. Phytochemistry 1981, 20,
2359-2366.
(9) Toyota, M.; Asakawa, Y. Phytochemistry 1988, 27, 2155-2159.
(10) Asakawa, Y.; Toyota, M.; Ueda, A.; Tori, M.; Fukazawa, Y. Phyto-
chemistry 1991, 30, 3037-3040.
(11) Spjut, R. W.; Suffness, M.; Cragg, G. M.; Norris, D. H. Econ. Bot.
1986, 40, 310-338.
Na vicu lyl ca ffea t e (1): colorless oily gum; [R]28 +28°,
D
+12°, [R]28
+4°, [R]28
-68°, [R]28
-76°
28
[R]
577 nm
546 nm
435 nm
405 nm
(c 0.2, MeOH); UV (MeOH) λmax (log ꢀ) 244 (3.66), 300
(shoulder), 326 (3.88) nm; IR (film) νmax 3500-3100, 2927,
1678, 1600, 1443, 1276, 1150 cm-1; 1H and 13C NMR, see Table
1; FABMS m/z 384.2326 (20, calcd for C24H32O4, 384.2301), 205
(100); P-388 GI50, see Table 2.
Isola tion of n a vicu lol (2). A bulk extract of collection
940127-04 (25 g) was prepared by Soxhlet extraction with
CHCl3 (200 mL, 20 h). The solvent was removed to give a green
gum (1.14 g). The crude extract was subjected to Si gel flash
chromatography (1.13 g precoated on 2.2 g, loaded on a 20-g
column), developed in steps from cyclohexane to EtOAc. A
fraction eluted with 8:2 cyclohexane-EtOAc (90 mg) was
subjected to reversed-phase HPLC (as above). Compound 2 (14
mg) had a retention time of 13.2 min.
(12) Lorimer, S. D.; Barns, G.; Evans, A. C.; Foster, L. M.; May, B. C. H.;
Perry, N. B.; Tangney, R. S. Phytomedicine 1996, 2, 327-333.
(13) Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91-109.
(14) Ibrahim, R.; Barron, D. Phenylpropanoids. In Methods in Plant
Biochemistry: Plant Phenolics; Harborne, J . B., Ed.; Academic:
London, 1989; Vol. 1, pp 75-111.
(15) Skrzypek, Z.; Wysokinska, H.; Swiatek, L.; Wroblewski, A. E. J . Nat.
Prod. 1991, 62, 127-129.
(16) Toyota, M.; Nagashima, F.; Asakawa, Y. Phytochemistry 1989, 28,
1661-1665.
Na vicu lol (2): colorless gum; [R]20D +44° (c 0.2, CHCl3); IR
(17) Tori, M.; Arbiyanti, H.; Taira, Z.; Asakawa, Y. Phytochemistry 1993,
32, 335-348.
(film) νmax 3320 (broad), 2927, 1450, 1009 cm-1 1H and 13C
;
NMR as previously reported.16
(18) Bauer, R. Echinacea: Biological effects and active principles. In
Phytomedicines of Europe: Chemistry and Biological Activity; Lawson,
L. D., Bauer, R., Eds.; American Chemical Society: Washington, DC,
1998; pp 140-157.
Syn th esis of 3-Meth ylbu t-2-en yl Ca ffea te. A solution of
caffeic acid (Aldrich, 57 mg, 0.32 mmol), 3-methylbut-2-enol
(Merck, 37 mg, 0.42 mmol), and DCC (Aldrich, 66 mg, 0.32
mmol) in dry THF (2 mL) was stood at room temperature in
NP990492X