New tools for studying vesicular-mediated protein trafficking: Synthesis and evaluation of ilimaquinone analogs in a non-radioisotope-based antisecretory assay

Article abstract of DOI:10.1021/jo962292l

Structural variants of the marine sponge metabolite ilimaquinone, with comparable biological activity, have been prepared. These analogs, as well as related natural products, were screened for their effects on the Golgi apparatus through a novel, non-radioisotope-based secretion assay. The assay has identified a variant of ilimaquinone that contains a versatile linker group yet retains the natural product's cellular activity. This functional ilimaquinone analog will be a valuable tool for studying intracellular protein trafficking.

Full text of DOI:10.1021/jo962292l

J . Org. Chem. 1997, 62, 2823-2831
2823
New Tools for Stu d yin g Vesicu la r -Med ia ted P r otein Tr a ffick in g:
Syn th esis a n d Eva lu a tion of Ilim a qu in on e An a logs in a
Non -Ra d ioisotop e-Ba sed An tisecr etor y Assa y
Heike S. Radeke, Cheryl A. Digits,1 Steven D. Bruner,2 and Marc L. Snapper*
Eugene F. Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167-3860
Received December 9, 1996X
Structural variants of the marine sponge metabolite ilimaquinone, with comparable biological
activity, have been prepared. These analogs, as well as related natural products, were screened
for their effects on the Golgi apparatus through a novel, non-radioisotope-based secretion assay.
The assay has identified a variant of ilimaquinone that contains a versatile linker group yet retains
the natural product’s cellular activity. This functional ilimaquinone analog will be a valuable tool
for studying intracellular protein trafficking.
In tr od u ction
Ilimaquinone3 (1) and related sesquiterpene quinones,
such as avarone4 (2), are reported to have interesting
biological effects including antimicrobial, anti-HIV, anti-
inflammatory, and antimitotic activity.5 More recently,
ilimaquinone’s ability to inhibit the cytotoxicity of ricin
and diphtheria toxin,6 as well as to reversibly disrupt the
Golgi apparatus, has also been noted.7 Considering the
proven value of using natural products as probes of
cellular processes,8 our plan is to exploit ilimaquinone’s
F igu r e 1. Modifications to ilimaquinone.
unique activity for the study of intracellular vesicular
trafficking.
contains a versatile linker group. Such a compound will
allow us to probe vesicular trafficking through a variety
of protein isolation techniques including affinity chro-
matography, photoaffinity labeling, and ligand blotting
experiments.9 In this regard, our studies on the synthe-
sis of (-)-ilimaquinone and related compounds are re-
ported herein. In addition, the development of a novel,
non-radioisotope-based antisecretion assay to evaluate
the ilimaquinone analogs is also described. The com-
bined synthetic and biochemical effort has allowed for
the identification of an active analog possessing the
functionality required to study ilimaquinone’s role in
vesicular trafficking.
A crucial element in our plan is the design and
preparation of an active variant of ilimaquinone that
Resu lts a n d Discu ssion
* Corresponding author: phone, (617) 552-8096; fax, (617) 552-0230;
e-mail, marc.snapper@BC.edu.
Syn th esis. Given that the shortest route to a com-
pound with the functionality required for our studies
might be realized through direct modification of ili-
maquinone, our initial chemical efforts focused on de-
rivatizing the natural product. This strategy was sup-
ported by the fact that several variants of ilimaquinone’s
quinone moiety have already been reported (Figure 1, 3
and 4).3a,5a Nevertheless, to increase the likelihood of
identifying a useful compound, additional analogs were
sought. To this end, (-)-ilimaquinone was oxidized with
SeO2/t-BuOOH to introduce a hydroxyl at the C3 position
X Abstract published in Advance ACS Abstracts, April 1, 1997.
(1) Department of Education GAANN Fellow.
(2) Boston College Undergraduate Research Fellow.
(3) (a) Luibrand, R. T.; Erdman, T. R.; Vollmer, J . J .; Scheuer, P.
J .; Finer, J .; Clardy, J . C. Tetrahedron 1979, 35, 609. (b) Capon, R. J .;
MacLeod, J . K. J . Org. Chem. 1987, 52, 5060.
(4) Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lett. 1974, 38,
3401.
(5) (a) Loya, S.; Tal, R.; Kashman, Y.; Hizi, A. Antimicrob. Agents
Chemother. 1990, 34, 2009. (b) Kushlan, D. M.; Faulkner, D. J .;
Parkanyi, L.; Clardy, J . C. Tetrahedron 1989, 45, 3307. (c) Bourguet-
Kondracki, M.-L.; Longeon, A.; Morel, E.; Guyot, M. Int. J . Immunop-
harmacol. 1991, 13, 393. (d) Schro¨der, H. C.; Wenger, R.; Gerner, H.;
Reuter, P.; Kuchino, Y.; Sladic, D.; Mu¨ller, W. E. G. Cancer Res. 1989,
49, 2069. (e) Sarin, P. S.; Sun, D.; Thornton, A.; Mu¨ller, W. E. G. J .
Natl. Cancer Inst. 1987, 78, 663. (f) Belisario, M. A.; Maturo, M.; Pecce,
R.; De, R. S.; Villani, G. R. D. Toxicology 1992, 72, 221. (g) Mu¨ller, W.
E. G.; Dogovic, N.; Zahn, R. K.; Maidhof, A.; Diehl, S. B.; Bedker, C.;
Sachsse, W.; Gasic, M. J .; Schroeder, H. C. Basic Appl. Histochem.
1985, 29, 321.
(6) Nambiar, M. P.; Wu, H. C. Exp. Cell Res. 1995, 219, 671.
(7) (a) Talizawa, P. A.; Yucel, J . K.; Veit, B.; Faulkner, D. J .;
Deerinck, T.; Soto, G.; Ellisman, M.; Malhotra, V. Cell 1993, 73, 1079.
(b) Veit, B.; Yucel, J . K.; Malhotra, V. J . Biol. Chem. 1993, 122, 1197.
(8) (a) Schreiber, S. L.; Albers, M. W.; Brown, E. J . Acc. Chem. Res.
1993, 26, 412. (b) Schreiber, S. L. Chem. Eng. News 1992, 70 (Oct.
26), 22.
(9) For example, see: (a) J ones, K. Am. Biotechnol. Lab. 1990, 8,
26. (b) Fretz, H.; Albers, M. W.; Galat, A.; Standaert, R. F.; Lane, W.
S.; Burakoff, S. J .; Bierer, B. E.; Schreiber, S. L. J . Am. Chem. Soc.
1991, 113, 1409. (c) Wilchek, M.; Bayer, E. A. Methods Enzymol. 1990,
184, 243. (d) Wade, D. P.; Knight, B. L.; Soutar, A. K. Biochem. J .
1985, 229, 785. (e) Morris, D. I.; Robbins, J . D.; Ruoho, A. E.;
Sutkowski, E. M.; Seamon, K. B. J . Biol. Chem. 1991, 266, 13377. For
additional experiments, see: (f) Handschumacher, R. E.; Harding, M.
W.; Rice, J .; Drugge, R. J .; Speicher, D. W. Science 1984, 226, 544. (g)
Shen, G. K.; Zukoski, C. F.; Montgomery, D. W. Int. J . Immunophar-
macol. 1992, 14, 63. (h) Siekierka, J . J .; Staruch, M. J .; Hung, S. H.;
Sigal, N. H. J . Immunol. 1989, 143, 1580.
S0022-3263(96)02292-X CCC: $14.00 © 1997 American Chemical Society

2824 J . Org. Chem., Vol. 62, No. 9, 1997
Radeke et al.
Sch em e 1a
Sch em e 2a
a
Key: (a) Li0, NH3, 2-chloro-3,5-dimethoxybenzyl bromide
(75%); (b) Ph3PdCH2, DMSO (71%); (c) H2, PtO2, CH2Cl2 (60%);
(d) PCC, CH2Cl2 (95%); (e) Ph3PdCH2, DMSO (82%); (f) CAN,
CH3CN, H2O (72%); (g) Pd(Ph3P)4, THF, H2O, NaHCO3 (33%).
involved a migration of the ∆4,11 olefin.14 In retrospect,
this is not surprising given the strong acidic conditions
of the Thiele-Winter acetoxylation (H2SO4).
When the acidity problem was circumvented by treat-
ing 9 with sodium acetate in acetic acid, clean addition
of acetate into the quinone system was observed. How-
ever, subsequent acetylation of this acetate addition
product (10a ) did not yield compound 3, an intermediate
derived from the natural product. A NOE study of the
quinone/aromatic protons in the unidentified product, as
well as in compound 9, indicated that the compound
obtained in the acetate addition is 10a , not the required
acetate regioisomer 10b. Apparently, steric factors over-
ride the electronic biases in the addition of an acetate
into the quinone functionality of compound 9.
Fortunately, as reported in a preliminary communica-
tion, modifications to the quinone formation strategy
provided a synthesis of the desired natural product.15 The
solution is realized through the incorporation of a chlo-
rine onto the dimethoxyaryl moiety (Scheme 2, 6 f 12).
Compound 13 is prepared from 12 in a similar fashion
to our initial studies (cf. Scheme 1). Interestingly, this
dimethoxychloroarene 13 undergoes a regioselective oxi-
dation to an appropriately functionalized chloroquinone
which is then hydrolyzed to provide ilimaquinone (13 f
1).
This successful pathway to (-)-ilimaquinone can now
be altered at various intermediates to yield a series of
potentially useful structural analogs. For example, as
illustrated in Scheme 3, the preparation of analog 16
originates with the synthetic intermediate 12. Hydrobo-
ration of the ∆8,13 olefin in 12 followed by oxidative
workup yields a 2:1 ratio of epimeric hydroxymethyl-
containing substrates (14:17) in 74% yield. Attachment
of a linker group to the newly formed C13 hydroxyl group
of 14 provides compound 15. The subsequent steps then
follow the reaction sequence developed for the natural
product (f16). An analogous sequence was carried out
on the epimeric hydroxymethyl product (17 f 19). In a
similar fashion, compound 13 can be stereoselectively
hydroborated in 88% yield to provide 20. After attach-
ment of a linker (20 f 21) the quinone functionality is
introduced in a straightforward manner to yield ili-
maquinone analog 22.
a
Key: (a) Li0, NH3, 3,5-dimethoxybenzyl bromide (57%); (b)
Ph3PdCH2, DMSO (60%); (c) H2, Pd/C (84%); (d) PCC, CH2Cl2
(94%); (e) Ph3PdCH2, DMSO (71%); (f) CrO3 (68%); (g) AcOH,
NaOAc, H2O (86%); (h) FeCl3, H2O.
(94% yield). The C3 hydroxyl group was then esterified
with BOC-protected 6-aminohexanoic acid (84% yield) to
provide compound 5. If active, this variant of ili-
maquinone (5) with a tethered linker group should be
an effective tool for our studies. Unfortunately, our
activity assay (see assay results) indicated that 5 retains
none of ilimaquinone’s influence on protein trafficking
(i.e., vesicular-mediated protein secretion). While ad-
ditional modifications to ilimaquinone are possible, insuf-
ficient supplies of the natural product precluded further
synthetic investigations.
A more plentiful source of analogs should be available
through synthesis from readily available starting materi-
als. Our initial approach to (-)-ilimaquinone, which was
modeled after Sarma’s preparation of (()-avarone10 (2),
is illustrated in Scheme 1. Unfortunately, reactions to
install the necessary quinone functionality did not pro-
vide the expected results.11
We anticipated that a Thiele-Winter acetoxylation of
912 followed by treatment of the resulting triacetoxyaryl
moiety with LAH and mild oxidation would provide (-)-
ilimaquinone.13 Unfortunately, the acetoxylation reac-
tion resulted in low mass recovery which, in part,
(10) (a) Sarma, A. S.; Gayen, A. K. Tetrahedron Lett. 1983, 24, 3385.
(b) Sarma, A. S.; Chattopadhyay, P. J . Org. Chem. 1982, 47, 1727. (c)
Sharma, A. S.; Gayen, A. K. Tetrahedron 1985, 41, 4581.
(11) For successful preparations of the quinone portion, see: (a)
Danheiser, R. L.; Cha, D. D. Tetrahedron Lett. 1990, 31, 1527. (b)
Yadav, J . S.; Upender, V.; Rao, A. V. R. J . Org. Chem. 1992, 57, 3242.
(c) Reinaud, O.; Capdevielle, P.; Maumy, M. Tetrahedron 1987, 43,
4167.
(12) McOmie, J . F. W.; Blatchly, J . M. Org. React. 1972, 19, 199.
(13) For recent examples of this sequence in related systems, see:
(a) Sargent, M. V.; Wangchareontrakul, S. J . Chem. Soc. Perkin Trans.
I 1990, 1429. (b) Almeida, W. P.; Correia, C. R. D. Tetrahedron Lett.
1994, 35, 1367.
Efforts were also directed toward the synthesis of
avarone (2) and related analogs to investigate the role
of ilimaquinone’s functionality on vesicular trafficking,
as well as to determine if related sesquiterpene quinones
(14) Capon, R. J . J . Nat. Prod. 1990, 53, 753.
(15) Bruner, S. D.; Radeke, H. S.; Tallarico, J . A.; Snapper, M. L. J .
Org. Chem. 1995, 60, 1114.

Vesicular-Mediated Protein Trafficking
J . Org. Chem., Vol. 62, No. 9, 1997 2825
Sch em e 3a
Ch a r t 1. P h osp h a ta se Activity ver su s
Ilim a qu in on e Con cen tr a tion (µM)
fashion, direct CAN oxidation of compound 24 provides
isoavarone (25), a hybrid structure comprised of ili-
maquinone’s decalin system and avarone’s quinone func-
tionality.
Activity Assa y. Since structural modifications may
annul ilimaquinone’s unique effects on the Golgi ap-
paratus and protein trafficking, an assay to screen
structural variants for antisecretory activity is required.
A common method for examining the effect of agents on
secretory function is through pulse-chase radiolabeling
experiments.18 Choosing to minimize the use of radioac-
tive compounds for safety and environmental reasons, we
sought instead to employ an enzymatic assay. Specifi-
cally, we adapted a system originally designed in the
Crabtree and Schreiber laboratories to study signal
transduction events.19
In this system a plasmid encoding for secreted alkaline
phosphatase (AP) under the control of the interleukin-2
NFAT enhancer region is introduced into J urkat T-
lymphocytes.20 These transiently transfected cells can
then be selectively activated to express and secrete
alkaline phosphatase upon treatment with phorbol ester
(PMA) and ionomycin. The amount of alkaline phos-
phatase secreted is determined spectroscopically by
measuring AP-catalyzed hydrolysis of p-nitrophenyl phos-
phate. With this secreted reporter gene system, the
effects of various compounds on protein secretion can
provide a convenient assessment of secretory function.
Chart 1 represents the measured activity of secreted
alkaline phosphatase from activated J urkat cells treated
with various concentrations of (-)-ilimaquinone. The
lack of cytotoxicity at the ilimaquinone concentrations
tested (e250 µM) suggests that general inhibition of
protein synthesis is not a factor and that these levels of
phosphatase activity reflect ilimaquinone’s noncytotoxic
inhibition of vesicle-mediated trafficking. According to
these results, 50% inhibition of AP activity (IC50) is
observed at 19 µM ((3 µM) ilimaquinone.
a
Key: (a) BH3‚DMS, H2O2, NaOH; (b) HO2C(CH2)5NHBOC,
EDCI, DMAP; (c) PCC, CH2Cl2; (d) Ph3PdCH2, DMSO; (e) CAN,
CH3CN, H2O; (f) Pd(Ph3P)4, THF, H2O, NaHCO3.
Sch em e 4a
a
In a fashion similar to the study with ilimaquinone,
other compounds were examined for inhibition of secreted
alkaline phosphatase activity. Chart 2 displays the 50%
inhibition concentration (IC50) for ilimaquinone (1), as
well as variants of this natural product. The structures
of these analogs are illustrated in Figure 2. It is not
Key: (a) Ph3PdCH2, DMSO (87%); (b) RhCl3‚H2O, EtOH,
CHCl3 (84%); (c) CAN, H2O, AcOH.
share ilimaquinone’s cellular activity. While the prepa-
ration of compound 23 (Scheme 4) has been described in
Sarma’s synthesis of 2, a shortened, enantioselective
route is realized with the sequence illustrated in Scheme
4.16 The desired natural product (2) is prepared through
a three-step sequence from 23. Wittig olefination of 23
yields compound 24; olefin isomerization17 of 24 followed
by CAN oxidation then delivers avarone (2). In a related
(18) For example, see: (a) Ulmer, J . B.; Palade, G. E. Proc. Natl.
Acad. Sci. U.S.A. 1989, 86, 6992. (b) Misumi, Y.; Misumi, Y.; Miki, K.;
Takatsuki, A.; Tamura, G.; Ikehara, Y. J . Biol. Chem. 1986, 261, 11398.
(c) Sampath, D.; Varki, A.; Freeze, H. H. J . Biol. Chem. 1992, 267,
4440.
(19) Spencer, D. M.; Wandless, T. J .; Schreiber, S. L.; Crabtree, G.
R. Science 1993, 262, 1019.
(16) Also see: An, J .; Wiemer, D. F. J . Org. Chem. 1996, 61, 8775.
(17) Andrieux, J .; Barton, D. H. R.; Patin, H. J . Chem. Soc. Perkin
Trans. I 1977, 359.
(20) The J urkat cells were modified to constituitively express the
T-antigen. This allows for replication of the plasmid through its SV40
promoter (Mr. Peter Belshaw, Harvard University).

2826 J . Org. Chem., Vol. 62, No. 9, 1997
Radeke et al.
F igu r e 2. Structural variants of ilimaquinone and related natural products.
Ch a r t 2. Con cen tr a tion s (µM) of An a logs
Requ ir ed To In h ibit Alk a lin e P h osp h a ta se
Secr etion by 50%
the applicability of total synthesis toward the construc-
tion of ilimaquinone structural variants is demonstrated.
Second, the development of a non-radioisotope-based
secretion assay to identify agents that effect intracellular
protein trafficking is described. Finally, the combination
of analog syntheses with the employment of a novel
secretion assay has identified a tether-containing ili-
maquinone analog (22) with all the antisecretory activity
of the natural product. With this versatile analog in
hand, our future studies are directed at isolating and
identifying the target responsible for ilimaquinone’s
cellular activities.
Exp er im en ta l Section
Gen er a l. Starting materials and reagents were purchased
from commercial suppliers and used without further purifica-
tion except the following. THF and Et2O were distilled over
sodium and benzophenone under N2. DMSO, CH2Cl2, and
CH3CN were distilled from CaH2 under N2. Cerium am-
monium nitrate (CAN, (NH4)2Ce(NO3)6) was recrystallized
from dilute nitric acid. Hexanes and EtOAc were distilled
before use.
surprising that two of the more active compounds tested
are chloroquinone 26 (IC50 7 µM) and ilimaquinone
monoacetate 3 (IC50 22 µM), since both of these com-
pounds can readily serve as in vivo precursors to ili-
maquinone. Because of this possibility, it is unclear
whether these variants will lead to useful probes of
ilimaquinone’s cellular function.
All oxygen- or moisture-sensitive reactions were carried out
under N2 or Ar in oven-dried (140 °C, g4 h) glassware. Air-
or moisture-sensitive liquids were transferred by syringe or
cannula and introduced into the reaction flasks through rubber
septa. Air- or moisture-sensitive solids were transferred in a
glovebag. Unless otherwise stated, reactions were stirred with
a Teflon-covered stir bar and carried out at rt. Concentration
refers to the removal of solvent using a Bu¨chi rotatory
evaporator at 40-100 Torr followed by use of a vacuum pump
at approximately 1 Torr. Silica gel column chromatography
was performed using Baxter brand silica gel 60 Å (230-400
Mesh ASTM). The term “brine” refers to saturated NaCl.
Proton nuclear magnetic resonance spectra (1H NMR) were
measured at 300 MHz on a Varian Unity-300 instrument or
at 400 MHz on a Varian Gemini-400 instrument. Chemical
shifts are reported in ppm downfield from tetramethylsilane.
Infrared spectra (IR) were measured on a Nicolet 510 FT-IR
spectrometer and are reported in wavenumbers (cm-1). Opti-
cal rotations were measured on a Perkin-Elmer 241 polarim-
eter using a 1 dm cell. UV-vis absorbances were measured
on a Varian Cary 1 E and are reported as λ (ꢀ), nm.
More pertinent to the goal of our studies, however, are
the effects of compound 22 (IC50 8 µM).21 This analog,
with all of ilimaquinone’s activity and a protected amine
group attached through a tether, can serve well as a
versatile tool for studying ilimaquinone’s cellular interac-
tions. For example, deprotection of the amine group
followed by attachment to biotin, a fluorescing group, or
a photoaffinity label will provide useful tools for studying
ilimaquinone’s cellular effects. In fact, preliminary af-
finity chromatography experiments with 22 immobilized
on a solid support have already yielded selective cellular
interactions.22
Con clu sion s
This work presents three key advancements in a
program to decipher ilimaquinone’s cellular effects. First,
(21) Analog 22 has also been shown to vesiculate the Golgi ap-
Elemental analyses were performed by Robertson Microlit
Laboratories, Inc., Madison, NJ . Mass spectral analyses were
performed by the Mass Spectrometry Laboratory, University
of Illinois at Urbana-Champaign.
paratus in
a manner similar to ilimaquinone (C. J amora and V.
Malhotra, UCSD).
(22) Radeke, H. S.; Bruner, S. D.; Digits, C. A.; Snapper, M. L.
Manuscript in preparation.

Vesicular-Mediated Protein Trafficking
J . Org. Chem., Vol. 62, No. 9, 1997 2827
(+)-3r-(6-BOC-a m in oh exa n oic a cid ester )ilim a qu in o-
n e (5). Ilimaquinone (1) (5.0 mg, 0.014 mmol) was dissolved
in dry CH2Cl2 (1 mL). SeO2 (0.77 mg, 0.007 mmol) and
t-BuOOH (3.0 µL, 0.028 mmol, 90% in H2O) were added to the
stirring solution. The reaction mixture was stirred for 6 h at
25 °C, upon which it was diluted with 1 mL of saturated NH4-
Cl. The organic layer was washed with saturated NH4Cl (3×)
and brine, dried over Na2SO4, filtered through Celite, and
concentrated to yield a crude oil. Silica gel chromatography
(98:2 Et2O:AcOH) afforded 3-hydroxyilimaquinone (5.0 mg,
94%) as a clear yellow oil: 1H NMR (CDCl3, 300 MHz) δ 7.48
(1H, s), 5.86 (1H, s), 4.80 (1H, d, J ) 1.5 Hz), 4.73 (1H, d, J )
1.5 Hz), 4.23 (1H, s), 3.86 (3H, s), 2.59 (1H, d, J ) 13.6 Hz),
2.48 (1H, d, J ) 13.7 Hz), 2.00 (1H, d, J ) 11.9 Hz), 1.81-
1.58 (4H, m), 1.38 (2H, m), 1.25 (6H, s), 0.98 (3H, d, J ) 6.4
Hz), 0.88 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 181.4, 179.8,
162.7, 159.8, 150.1, 128.8, 101.3, 99.4, 55.5, 50.0, 48.7, 44.1,
41.5, 38.1, 37.9, 31.2, 30.5, 28.6, 27.7, 26.0, 22.2, 18.5; IR (thin
film, NaCl) 3436, 2925, 2855, 1653, 1644, 1611, 1456, 1378,
1351, 1260, 1238, 1094, 1039 cm-1; HRMS calcd for C22H30O5
374.2090, found 374.2093; MS (70 eV) m/ z (rel int) 374(M+,
4), 356(5), 319(3), 295(3), 281(3), 221(6), 205(7), 189(55), 168-
(100), 147(13), 133(18), 119(29), 95(47), 83(37), 69(53), 57(62);
(1H, ddd, J ) 17.4, 5.9, 2.6 Hz), 1.98 (1H, ddd, J ) 13.3, 6.3,
2.2 Hz), 1.67 (2H, m), 1.34-1.22 (4H, m), 1.12 (3H, s), 0.96
(3H, s); 13C NMR (CDCl3, 100 MHz) δ 216.3, 160.3, 141.1,
121.1, 108.4, 108.2, 98.3, 79.3, 55.0, 45.2, 43.8, 35.1, 34.1, 30.1,
24.0, 23.2, 22.6, 11.5; IR (thin film, NaCl) 3484, 2949, 2867,
2842, 1703, 1665, 1602, 1457, 1425, 1319, 1205, 1148, 1067
cm-1; [R]25 ) +15 (c 5.0, EtOAc). Anal. Calcd for C21H30O4:
D
C, 72.80; H, 8.73. Found: C, 72.80; H, 8.73.
(+)-tr a n s-Octah ydr o-1r-[(3,5-dim eth oxyph en yl)m eth yl]-
1â,4a â-d im eth yl-5â-h yd r oxy-2(1H)-n a p h th a len e (7). So-
dium hydride (60% oil dispersion; 2.8 g, 0.070 mol) was washed
with several portions of anhydrous Et2O to remove the mineral
oil. The flask was purged with N2 after each washing. The
system was then equipped with a rubber septum, a reflux
condenser, and a magnetic stir bar. Dry DMSO (56 mL) was
introduced, and the reaction mixture was heated at 75 °C for
1 h. The resulting aqua blue methylsulfinyl carbanion was
cooled to 0-5 °C. CH3PPh3Br (32.2 g, 0.09 mol) in warm
DMSO (152 mL) was added through a cannula. The resulting
dark yellow solution of the methyltriphenylphosphorane (Ph3-
PCH2) was stirred at 25 °C for 1 h before being used.
Ph3PCH2 (63 mL, 0.4 M, 28 mmol) was transferred with a
cannula into a two-neck round bottom flask fitted with a water
condenser and a Schlenk adapter. The ketone 6a (0.95 g, 2.8
mmol) was added dropwise to the ylide at rt. Upon completion
of addition, the solution was stirred at 75 °C for 4 h and
monitored by TLC (1:1 hexane:EtOAc). The reaction was
quenched with NH4Cl (20 mL), and the aq layer was extracted
with Et2O (3×). The combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated. The
crude product was purified by silica gel chromatography (4:1
hexane:EtOAc) to yield compound 7 (510 mg, 60%) as a yellow
oil: 1H NMR (CDCl3, 400 MHz) δ 6.31 (3H, s), 4.89 (1H, s),
4.64 (1H, s), 3.74 (6H, s), 3.18 (1H, dd, J ) 10.2, 5.0 Hz), 2.73
(1H, d, J ) 13.6 Hz), 2.56 (1H, d, J ) 13.6 Hz), 2.24 (2H, m),
1.75 (1H, dt, J ) 12.5, 2.9 Hz), 1.60 (2H, m), 1.51 (2H, m),
1.39-1.17 (3H, m), 0.99 (3H, s), 0.91 (3H, s); 13C NMR (CDCl3,
100 MHz) δ 160.4, 153.7, 141.7, 109.7, 109.4, 98.5, 81.5, 55.8,
[R]25 ) -15 (c 0.1, CHCl3).
D
The 3-hydroxyilimaquinone (5 mg, 0.014 mmol) was dis-
solved in CH2Cl2 (1 mL). DMAP (2.7 mg, 0.014 mmol), 6-BOC-
aminohexanoic acid (3.2 mg, 0.014 mmol), and EDCI (2.7 mg,
0.014 mmol) were added to the stirring solution. The reaction
mixture was allowed to stir for 2 days, the reaction quenched
with Et2O/H2O, and the mixture acidified with AcOH. The
organic solution was separated, washed with saturated NH4-
Cl and brine, and dried over Na2SO4. The crude mixture of
product and starting material was purified by using silica gel
chromatography (gradient of hexanes, Et2O, and AcOH) to
yield 5 (1.7 mg, 84% yield at 75% conversion): 1H NMR (CDCl3,
300 MHz) δ 7.49 (1H, s), 5.86 (1H, s), 4.96 (1H, d, J ) 1.4 Hz),
4.80 (1H, d, J ) 1.5 Hz), 4.73 (1H, d, J ) 1.3 Hz), 4.55 (1H, br
s), 3.86 (3H, s), 3.12 (2H, dt, J ) 6.8, 7.3 Hz), 2.59 (1H, d, J )
13.7 Hz), 2.48 (1H, d, J ) 13.9 Hz), 2.36 (2H, t, J ) 7.5 Hz),
1.99 (2H, d, J ) 10.8 Hz), 1.80 (2H, d, J ) 10.8 Hz), 1.67-
1.52 (9H, m), 1.43 (9H, s), 1.24 (3H, s), 0.98 (3H, d, J ) 6.2
Hz), 0.88 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 183.7, 181.2,
174.3, 161.1, 159.0, 153.7, 142.2, 113.8, 107.7, 100.2, 79.7, 67.9,
66.6, 56.6, 56.0, 53.8, 50.0, 46.1, 43.1, 39.6, 39.3, 37.3, 35.9,
34.4, 29.6, 28.2, 26.3, 22.2, 24.2, 17.8, 14.8; IR (thin film, NaCl)-
3351, 2926, 2855, 1713, 1698, 1650, 1608, 1557, 1538, 1505,
1456, 1261, 1166 cm-1; HRMS calcd for C33H50NO8 588.3536,
found 588.3537; MS (70 eV) m/ z (rel int) 588(M+, <1),
477(<1), 374(7), 356(6), 319(6), 207(5), 189(64), 168(100),
47.7, 47.5, 43.7, 39.8, 38.1, 31.0, 29.7, 24.8, 23.4, 15.3 cm-1
;
IR (thin film, NaCl) 3434, 2935, 2862, 1596, 1460, 1428, 1344,
1321, 1293, 1151; [R]25 ) +18 (c 3.5, EtOAc). Anal. Calcd
D
for C22H32O3: C, 76.70; H, 9.36. Found: C, 76.41; H, 9.13.
(+)-tr a n s-Octah ydr o-5r-[(3,5-dim eth oxyph en yl)m eth yl]-
5â,6â,8a â-t r im et h yl-1â-n a p h t h a len ol (7c). Compound 7
(10.0 mg, 0.29 mmol), dissolved in CH2Cl2 (0.5 mL), was
syringed into a flask containing PtO2 (61 mg, 0.27 mmol). A
Schlenk adapter was fitted with a double balloon. The flask
was evacuated and flushed with N2 (3×) and then with H2.
Hydrogenation was complete in 2 days as determined by NMR.
The solution was filtered through a silica gel plug (3 cm).
Silica gel chromatography (4:1 hexane:EtOAc) was used to
separate the 13:1 mixture of diastereoisomers to provide the
major product 7c (8.4 mg, 84%): 1H NMR (CDCl3, 400 MHz)
δ 6.33 (1H, s), 6.26 (2H, s), 3.77 (6H, s), 3.00 (1H, dd, J ) 10.9,
3.5 Hz), 2.63 (1H, d, J ) 14.1 Hz), 2.47 (1H, d, J ) 14.1 Hz),
1.82 (2H, m), 1.61 (3H, m), 1.32 (6H, m), 1.13 (1H, d, J ) 13.8
Hz), 1.12 (1H, d, J ) 15.3 Hz), 0.98 (3H, d, J ) 6.2 Hz), 0.88
(3H, s), 0.80 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 160.0, 141.1,
109.9, 97.7, 80.6, 55.2, 45.8, 43.2, 40.6, 39.4, 36.8, 35.5, 29.9,
26.7, 23.9, 21.6, 17.7, 16.7, 12.6; IR (thin film, NaCl) 3459,
140(12), 119(26), 59(26); [R]25 ) +70 (c 0.1, CHCl3).
D
(+)-tr a n s-Octah ydr o-1r-[(3,5-dim eth oxyph en yl)m eth yl]-
1â,4a â-d im eth yl-5r-h yd r oxy-2(1H)-n a p h th a len on e (6a ).
Liquid NH3 (100 mL) was distilled from a flask containing Li0
into a 250 mL two-neck round bottom flask equipped with a
dry ice condenser. Li0 (0.212 g, 30.5 mmol) was added to the
NH3, and the resulting blue solution was refluxed for 30 min.
The keto alcohol 6 (1.49 g, 7.5 mmol), dissolved in THF (10
mL), was added dropwise over 30 min to the blue solution.
After the addition was complete, the reaction mixture was
refluxed for an additional 30 min. 2,5-Dimethoxybenzyl
bromide (17.0 g, 73.0 mmol) dissolved in THF (15 mL) was
added to the stirring solution quickly. The white slurry stirred
for 30 min followed by addition of saturated NH4Cl (50 mL)
and dilution with Et2O (150 mL). The dry ice condenser was
replaced with a water condenser, and the NH3 was allowed to
evaporate. Et2O and HCl (5% aq) were added to the remaining
crude solid, the layers were separated, and the aq layer was
extracted with Et2O (3×). The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concen-
trated. The resulting orange oil was purified by silica gel
chromatography (4:1 hexane:EtOAc) to afford 6a (1.58 g, 57%)
as a yellow oil: 1H NMR (CDCl3, 400 MHz) δ 6.30 (1H, s), 6.21
(2H, s), 3.74 (6H, s), 3.42 (1H, d, J ) 8.2 Hz), 3.32 (1H, d, J )
13.6 Hz), 3.05 (1H, dd, J ) 10.2, 5.0 Hz), 2.84 (1H, br s), 2.68
(1H, d, J ) 13.0 Hz), 2.51 (1H, ddd, J ) 18.6, 12.0, 7.2 Hz),
2.44 (1H, dd, J ) 9.3, 5.1 Hz), 2.38 (1H, d, J ) 13.6 Hz), 2.30
2917, 1596, 1457, 1382, 1287, 1073, 954, 828 cm-1; [R]25
)
D
+2.3 (c 1.5, EtOAc). Anal. Calcd for C22H34O3: C, 76.26; H,
9.89. Found: C, 76.01; H, 10.17.
(-)-tr a n s-Octah ydr o-5r-[(3,5-dim eth oxyph en yl)m eth yl]-
5â,6â,8a â-tr im eth yl-1(2H)-n a p h th a len on e (7d ). Alcohol 7c
(167 mg, 0.482 mmol) dissolved in CH2Cl2 (5 mL) was added
dropwise to a stirring solution of PCC (0.103 g, 0.482 mmol)
in CH2Cl2 (25.0 mL) at 0 °C. The reaction was monitored by
TLC (1:1 hexane:EtOAc) for completion, and after 12 h silica
gel was added. The reaction mixture was filtered and con-
centrated. The resulting oil was purified by passing it through
a plug of silica gel (1 cm) that was presaturated with Et2O to
afford compound 7d (152 mg, 92%) as a clear oil: 1H NMR
(CDCl3, 400 MHz) δ 6.30 (1H, s), 6.20 (2H, s), 3.74 (6H, s),
2.63 (1H, d, J ) 13.9 Hz), 2.52 (1H, d, J ) 13.9 Hz), 2.11 (3H,
m), 1.81 (4H, dd, J ) 12.8, 3.0 Hz), 1.35 (5H, m), 1.14 (3H, s),

2828 J . Org. Chem., Vol. 62, No. 9, 1997
Radeke et al.
0.99 (3H, d, J ) 7.2 Hz), 0.89 (3H, s); 13C NMR (CDCl3, 100
MHz) δ 216.4, 160.9, 140.9, 123.2, 109.4, 98.1, 55.5, 47.4, 43.7,
42.1, 37.7, 35.5, 32.8, 26.8, 25.8, 22.0, 19.3, 18.3, 16.8; IR (thin
film, NaCl) 2962, 1709, 1596, 1596, 1464, 1426, 1187, 1162
(-)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m eth yl]-5â,8a â-d im eth yl-6â-(h yd r oxym eth yl)-1-
n a p h th a len ol (17). Olefin 12 (616 mg, 1.63 mmol) dissolved
in THF (5 mL) was cooled to 0 °C. BH3‚SMe2 (0.96 mL, 10 M,
8.8 mmol) was added dropwise to the cooled solution over 30
min. Once the borane addition was complete, the reaction
mixture was heated to reflux. After 3 h the reaction mixture
was allowed to cool to rt. H2O (2.5 mL) and EtOH (10 mL)
were added; then after 30 min H2O2 (30%, 3.4 mL) and NaOH
(39 mL, 1 M) were added. After an additional 1 h, K2CO3 (100
mg) was added. The reaction mixture was washed with Et2O
(3×), and the organic layer was dried over Na2SO4 and
concentrated. The diastereomers were separated by silica gel
chromatography (3:2 hexanes:EtOAc) to provide 14 and 17 in
an overall yield of 74% (14:17 ) 2:1).
cm-1; [R]25 ) -24 (c 1.1, EtOAc). Anal. Calcd for C22H32O3:
D
C, 76.70; H, 9.36. Found: C, 77.80; H, 9.24.
(-)-tr a n s-Octah ydr o-5r-[(3,5-dim eth oxyph en yl)m eth yl]-
5â,6â,8a â-tr im eth yl-1(2H)-n a p h th a len e (8). Compound 7d
(80 mg, 0.23 mmol), dissolved in DMSO (1.0 mL), was added
dropwise to Ph3PCH2 (4.0 mL, 0.4 M, 1.6 mmol). The solution
was refluxed at 80 °C for 15 h and monitored by TLC (4:1
hexane:EtOAc). Purification with Ag-impregnated silica gel
(17%, w/w) chromatography (98:2 hexanes:EtOAc) afforded
compound 8 as a clear oil (57 mg, 71%): 1H NMR (CDCl3, 400
MHz) δ 6.31 (1H, s), 6.25 (2H, s), 4.41 (1H, t, J ) 1.4 Hz), 4.39
(1H, t, J ) 1.7 Hz), 3.76 (6H, s), 2.64 (1H, d, J ) 13.6 Hz),
2.46 (1H, d, J ) 13.6 Hz), 2.32 (2H, td, J ) 13.8, 5.0 Hz), 2.09
(1H, d, J ) 11.8 Hz), 1.95 (2H, m), 1.59 (1H, qd, J ) 13.7, 3.2
Hz), 1.50-1.29 (2H, m), 1.06 (3H, s), 0.99 (3H, d, J ) 5.7 Hz),
0.83 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 160.1, 160.7, 141.8,
109.5, 103.5, 98.4, 55.9, 48.1, 44.1, 42.2, 40.8, 37.3, 36.4, 33.7,
28.7, 28.0, 23.5, 21.4, 18.4, 17.8; IR (thin film, NaCl) 2924,
17: 1H NMR (CDCl3, 300 MHz) δ 6.42 (1H, d, J ) 2.7 Hz),
6.33 (1H, d, J ) 2.7 Hz), 4.13 (1H, d, J ) 11.4 Hz), 3.87 (3H,
d, J ) 1.2 Hz), 3.80 (3H, d, J ) 1.2 Hz), 3.23 (1H, dd, J )
10.3, 5.3 Hz), 3.06 (1H, d, J ) 10.5 Hz), 2.93 (1H, d, J ) 13.7
Hz), 2.75 (1H, d, J ) 14.1 Hz), 1.92-1.65 (6H, m), 1.51-1.17
(7H, m), 1.07 (1H, d, J ) 11.5 Hz), 0.93 (3H, s), 0.84 (3H, s);
13C NMR (CDCl3, 100 MHz) δ 158.5, 156.4, 139.4, 109.8, 98.6,
81.1, 66.1, 56.7, 56.1, 47.7, 45.7, 41.9, 41.7, 40.1, 36.5, 30.6,
24.4, 22.9, 22.3, 18.6, 13.2, 13.1; IR (thin film, NaCl) 3404,
2936, 2863, 1591, 1453, 1415, 1202, 1163 cm-1; MS (70 eV)
m/ z (rel int) 398(7, M2+), 396(16, M+), 359(3), 314(4), 290(6),
270(3), 251(4), 193(25), 186(100), 175(30), 163(14), 151(14),
133(25), 119(27), 109(58), 95(65), 81(83), 67(54), 55(84),
2861, 1585, 1457, 1426, 1211, 1155, 1067, 897, 834 cm-1; [R]25
D
) -40 (c 0.1, EtOAc). Anal. Calcd for C21H30O2: C, 80.21; H,
9.62. Found: C, 80.46; H, 9.62.
(-)-2-Meth oxy-6-[(tr a n s-octah ydr o-5â,6â,8aâ-tr im eth yl-
1(2H )-n a p h t h a len yl)m et h yl]-2,5-cycloh exa d ien e-1,4-d i-
on e (9). CrO3 (36.0 mg, 0.36 mmol) dissolved in H2O (100
µL) and AcOH (600 µL) was added dropwise to compound 8
(36.3 mg, 0.114 mmol) in AcOH (600 µL) at 0 °C. The reaction
mixture was monitored by TLC (4:1 hexanes:EtOAc). After
30 min, ice water was added, and the aq layer was extracted
with EtOAc (7×). The combined organic layers were washed
with (saturated) NH4Cl, dried over MgSO4, filtered, and
concentrated. Purification through silica gel chromatography
(4:1 hexanes:EtOAc) afforded compound 9 (21.0 mg, 68%): 1H
NMR (CDCl3, 400 MHz) δ 6.38 (1H, d, J ) 4.0 Hz), 5.87 (1H,
d, J ) 4.0 Hz), 4.43 (1H, t, J ) 2.5 Hz), 4.47 (1H, t, J ) 1.9
Hz), 3.81 (3H, s), 2.56 (1H, d, J ) 13.5 Hz), 2.4 (1H, d, J )
13.5 Hz), 2.30 (2H, td, J ) 16.0, 4.0 Hz), 2.09 (2H, d, J ) 12.1
Hz), 1.88 (2H, d, J ) 12.1 Hz), 1.54 (2H, d, J ) 11.0 Hz), 1.41-
1.14 (4H, m), 1.05 (3H, s), 0.94 (3H, d, J ) 6.6 Hz), 0.88 (3H,
s); 13C NMR (CDCl3, 100 MHz) δ 209.9, 196.3, 160.7, 145.8,
137.7, 122.1, 108.0, 104.1, 57.3, 50.1, 43.8, 41.3, 38.1, 37.7, 36.0,
33.8, 29.0, 28.4, 23.6, 21.6, 18.7, 17.8; IR (thin film, NaCl) 2936,
41(78); [R]25 ) -9.8 (c 12.0, CHCl3).
D
1
14: H NMR (CDCl3, 300 MHz) δ 6.59 (1H, d, J ) 2.6 Hz),
6.35 (1H, d, J ) 2.6 Hz), 4.02 (1H, dd, J ) 9.9, 3.3 Hz), 3.81
(3H, s), 3.74 (3H, s), 3.18 (1H, d, J ) 13.9 Hz), 3.15 (1H, dd, J
) 10.9, 4.1 Hz), 2.36 (1H, d, J ) 13.9 Hz), 2.26 (2H, br s), 1.73-
1.66 (3H, m), 1.56 (2H, m), 1.59-1.33 (5H, m), 0.92 (3H, s),
0.91 (3H, s) (partial); 13C NMR (CDCl3, 100 MHz) δ 158.6,
156.2, 140.0, 109.7, 108.3, 98.0, 81.6, 62.1, 56.7, 56.0, 47.6, 44.9,
40.9, 40.4, 39.1, 32.5, 30.7, 24.8, 22.2, 21.1, 19.5, 15.3; IR (thin
film, NaCl) 3400, 2960, 2872, 1596, 1458, 1344, 1208, 1162,
1041, 932, 748 cm-1; MS (70 eV) m/ z (rel int) 398(2, M2+),
396(7, M+), 362(1), 221(1), 211(5), 205(24), 193(28), 186(100),
175(32), 163(8), 152(25), 133(12), 121(18), 109(31), 95(26),
81(30), 69(18), 55(29), 43(22); [R]25 ) +7.7 (c 19.0, CHCl3).
D
(+)-t r a n s-De ca h yd r o-5r-[(2-ch lor o-3,5-d im e t h oxy-
p h en yl)m eth yl]-5â,8a â-d im eth yl-6â-(h yd r oxym eth yl)-1â-
n a p h th a len ol 6-(N-t-BOC-a m in o)h exa n oa te (18). Alcohol
17 (165 mg, 0.42 mmol), 6-(N-t-BOC-amino)hexanoic acid (96
mg, 0.42 mmol), and DMAP (61 mg, 0.50 mmol) were placed
in a flask and cooled (0-5 °C). EDCI (120 mg, 0.63 mmol)
dissolved in CH2Cl2 (4.5 mL) was then added dropwise to the
flask. The reaction mixture was stirred for 2 h at 0 °C and
then allowed to warm to 25 °C. After 12 h the reaction mixture
was quenched with saturated NH4Cl. The aqueous layer was
washed (2×) with CH2Cl2. The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated. The
crude product was purified by silica gel chromatography (7:3
hexanes:EtOAc) to yield ester 18 (169 mg, 66%) as a clear
yellow oil: 1H NMR (CDCl3, 300 MHz) δ 6.41 (1H, d, J ) 2.6
Hz), 6.31 (1H, d, J ) 2.7 Hz), 4.56 (1H, s), 4.42 (1H, dd, J )
10.4, 2.2 Hz), 3.87 (2H, m), 3.86 (3H, s), 3.78 (3H, s), 3.11 (2H,
dt, J ) 6.6, 6.2 Hz), 3.05 (1H, dd, J ) 10.8, 4.0 Hz), 2.90 (1H,
d, J ) 14.3 Hz), 2.78 (1H, d, J ) 14.3 Hz), 2.30 (2H, t, J ) 7.5
Hz), 1.81-1.47 (11H, m), 1.43 (9H, s), 1.35 (4H, m), 1.07 (2H,
d, J ) 9.7 Hz), 0.92 (3H, s), 0.90 (3H, s); 13C NMR (CDCl3, 100
MHz) δ 174.7, 158.2, 156.6, 156.5, 139.1, 116.7, 109.6, 98.6,
81.2, 68.1, 56.9, 56.2, 47.8, 42.2, 41.7, 41.6, 41.1, 41.0, 40.1,
36.3, 34.9, 30.7, 30.5, 29.1, 27.0, 25.4, 24.5, 23.1, 22.5, 18.9,
13.1; IR (thin film, NaCl) 3403, 2937, 2865, 1730, 1727, 1707,
1678, 1646, 1596, 1457, 1388, 1325, 1224, 1055, 897 cm-1
;
HRMS calcd for C22H30O3 342.2195, found 342.2180; MS (70
eV) m/ z (rel int) 342(6, M+), 191(37), 175(12), 151(51),
135(22), 108(28), 95(100), 79(27); [R]25 ) -15 (c 2.0, EtOAc).
D
(-)-3-Acetyl-6-[(tr a n s-d eca h yd r o-5â,6â,8a â-tr im eth yl-
1(2H )-n a p h t h a len yl)m et h yl]-2-m et h oxyh yd r oq u in on e
(10a ). Quinone 9 (8.0 mg, 0.048 mmol) was dissolved in AcOH
(400 µL) saturated with NaOAc. The reaction mixture was
stirred at rt under N2 and monitored by TLC (6:4 hexanes:
EtOAc, product Rf ) 0.57) for consumption of quinone. After
7 days, the reaction mixture was diluted with Et2O (1 mL)
and H2O (1 mL), and the reaction was quenched with saturated
NH4Cl (1 mL) and 5% citric acid. The organic layer was
separated, dried over MgSO4, filtered, and concentrated.
Purification through silica gel chromatography (3:2 hexanes:
EtOAc) provided compound 10a as a clear oil (6.0 mg, 86%):
1H NMR (CDCl3, 400 MHz) δ 6.4 (1H, s), 5.54 (1H, s), 5.30
(1H, s), 4.42 (2H, t, J ) 1.9 Hz), 4.38 (1H, t, J ) 1.3 Hz), 3.87
(3H, s), 2.64 (1H, d, J ) 14.3 Hz), 2.47 (1H, d, J ) 14.5 Hz),
2.32 (3H, s), 2.04 (3H, t, J ) 5.9 Hz), 1.85 (2H, m), 1.81 (2H,
d, J ) 1.5 Hz), 1.46 (1H, d, J ) 13.0 Hz), 1.40 (1H, d, J ) 8.1
Hz), 1.25 (2H, m), 1.04 (3H, s), 0.97 (3H, d, J ) 5.7 Hz), 0.87
(1H, m), 0.82 (3H, s); 13C NMR (CDCl3, 400 MHz) δ 175.4,
160.6, 135.8, 120.7, 117.5, 103.2, 61.7, 48.7, 42.4, 40.7, 37.2,
37.1, 36.7, 33.5, 30.1, 28.8, 28.2, 23.4, 21.4, 21.1, 18.1, 17.9;
IR (thin film, NaCl) 3427, 2936, 2856, 1772, 1659, 1514, 1464,
1376, 1332, 1193, 1332, 1056, 1023, 973, 891 cm-1; HRMS calcd
for C24H34O5 402.2406, found 402.2406; MS (70 eV) m/ z (rel
int) 402(12, M+), 360(7), 212(66), 191(26), 170(100), 135(15),
121(20), 95(59), 79(13), 67(11).
1692, 1590, 1454, 1201, 1166 cm-1; HRMS calcd for C33H51
-
NO7Cl 608.3354, found 608.3352; MS (70 eV) m/ z (rel int)
610(42, M2+), 608(5, M+), 553(88), 555(51), 517(100), 499(71),
472(59), 458(64), 406(25), 192(30), 132(18), 91(23); [R]25D ) +18
(c 11.0, CHCl3).
(-)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m eth yl]-6â,8a â-d im eth yl-6r-(h yd r oxym eth yl)-1â-
n a p h th a len ol 6-(N-t-BOC-a m in o)h exa n oa te (15b): see
1
compound 18 for similar experimental; H NMR (CDCl3, 300

Vesicular-Mediated Protein Trafficking
J . Org. Chem., Vol. 62, No. 9, 1997 2829
MHz) δ 6.52 (1H, d, J ) 2.7 Hz), 6.35 (1H, d, J ) 2.7 Hz), 4.65
(1H, br s), 4.55 (1H, dd, J ) 11.2, 4.2 Hz), 4.23 (1H, t, J )
10.3 Hz), 3.81 (3H, s), 3.74 (3H, s), 3.25 (1H, d, J ) 14.4 Hz),
3.20 (1H, dd, J ) 11.2, 4.2 Hz), 3.06 (2H, dt, J ) 6.5, 6.4 Hz),
2.32 (2H, t, J ) 6.8 Hz), 2.29 (1H, d, J ) 14.6 Hz), 1.94 (2H,
dd, J ) 8.8, 3.7 Hz), 1.63-1.44 (9H, m), 1.39 (9H, s), 1.35-
1.22 (7H, m), 0.94 (3H, s), 0.90 (3H, s); 13C NMR (CDCl3, 100
MHz) δ 174.2, 158.8, 156.4, 139.6, 116.9, 108.1, 98.4, 81.4, 64.5,
56.8, 56.1, 47.5, 41.5, 41.0, 40.6, 39.0, 34.9, 32.2, 30.8, 30.6,
30.2, 29.1, 28.4, 26.9, 25.3, 22.4, 22.2, 20.8, 19.9, 15.3; IR (thin
film, NaCl) 3327, 2924, 2848, 1734, 1652, 1552, 1451, 1262,
m), 1.19 (1H, d, J ) 10.2 Hz), 1.08 (3H, s), 0.92 (3H, s): 13C
NMR (CDCl3, 100 MHz) δ 174.7, 160.0, 158.5, 156.4, 139.1,
109.4, 103.9, 98.7, 68.1, 56.8, 56.2, 49.4, 42.5, 42.2, 42.8, 41.1,
40.7, 36.2, 35.0, 33.6, 30.4, 29.1, 28.8, 27.1, 25.9, 25.3, 22.7,
22.5, 19.4, 19.3; IR (thin film, NaCl) 3390, 2934, 2873, 1720,
1592, 1456, 1251, 1173 cm-1; HRMS calcd for C34H52NO6Cl
605.3479, found 605.3483; MS (70 eV) m/ z (rel int) 607(4, M2+),
605(10, M+), 459(5), 375(7), 320(21), 189(100), 186(33),
176(23), 147(8), 133(15), 119(10), 107(13), 95(41), 93(12),
69(10), 57(36) 55(8); [R]25 ) +58 (c 15.0, CHCl3).
D
(-)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m et h yl]-5â,8a â-d im et h yl-6r-(h yd r oxym et h yl)-
1(2H)-n a p h th a len yl 6-(N-t-BOC-a m in o)h exa n oa te (15d ):
see compound 18d for similar experimental; 1H NMR (CDCl3,
300 MHz) δ 6.62 (1H, d, J ) 2.4 Hz), 6.38 (1H, d, J ) 2.5 Hz),
4.66 (1H, dd, J ) 11.0, 2.1 Hz), 4.52 (1H, s), 4.50 (1H, s), 4.29
(1H, t, J ) 11.2 Hz), 3.85 (3H, s), 3.78 (3H, s), 3.31 (1H, d, J
) 14.4 Hz), 3.09 (2H, dt, J ) 6.6, 6.1 Hz), 2.35 (1H, d, J )
15.1 Hz), 2.32 (1H, d, J ) 14.6 Hz), 1.94 (2H, dd, J ) 8.8, 3.7
Hz), 1.63-1.44 (9H, m), 1.39 (9H, s), 1.35-1.22 (6H, m), 0.94
1155 cm-1; [R]25 ) -20 (c 2.0, CHCl3).
D
(-)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m eth yl]-5â,8a â-d im eth yl-6â-(h yd r oxym eth yl)-1â-
n a p h th a len on e 6-(N-t-BOC-a m in o)h exa n oa te (18c). To
a stirring solution of PCC (58.8 mg, 0.26 mmol) in CH2Cl2 (8.8
mL) was added alcohol 18 (107.2 mg, 0.18 mmol) dissolved in
CH2Cl2 (8.8 mL) dropwise. The reaction mixture was stirred
for 3 h. The brown mixture was filtered through silica gel,
which was prewashed with 1:1 hexanes:EtOAc, to yield the
ketone 18c (104 mg, 97%) as a clear yellow oil: 1H NMR
(CDCl3, 300 MHz) δ 6.40 (1H, d, J ) 2.6 Hz), 6.26 (1H, d, J )
2.7 Hz), 4.56 (1H, br s), 4.41 (1H, dd, J ) 10.8, 2.4 Hz), 3.86
(2H, m), 3.85 (3H, s), 3.75 (3H, s), 3.11 (2H, dt, J ) 6.2, 6.0
Hz), 2.89 (1H, d, J ) 14.4 Hz), 2.83 (1H, d, J ) 14.5 Hz), 2.60
(1H, dt, J ) 14.1, 7.1 Hz), 2.31 (2H, t, J ) 7.5 Hz), 2.2 (1H,
dd, J ) 14.5, 4.8 Hz), 2.05 (2H, m), 1.75 (2H, m), 1.62 (4H, m),
1.51-1.31 (6H, m), 1.43 (9H, s), 1.17 (3H, s), 0.99 (3H, s); 13C
NMR (CDCl3, 75 MHz) δ 216.1, 174.7, 158.6, 156.7, 138.4,
116.4, 109.7, 98.4, 79.8, 74.7, 67.6, 56.9, 56.1, 49.7, 48.7, 42.7,
42.1, 41.3, 41.1, 38.0, 34.9, 32.1, 30.5, 29.1, 27.0, 25.9, 25.4,
22.8, 22.6, 19.4, 19.3; IR (thin film, NaCl) 3380, 2932, 2865,
1708, 1577, 1451, 1158 cm-1; HRMS calcd for C33H50NO7Cl
607.3272, found 607.3276; MS (70 eV) m/ z (rel int) 609(6, M2+),
607(14, M+), 533(7), 471(9), 377(7), 348(5), 322(84), 191(41),
186(100), 185(29), 173(30), 163(13), 151(13), 149(31), 147(16),
140(11), 135(28), 132(21), 114(20), 107(16), 85(15), 69(25),
(3H, s), 0.90 (3H, s); 13
C NMR (CDCl3, 100 MHz) δ 174.2, 160.4,
159.0, 156.4, 139.8, 108.0, 103.2, 98.4, 64.4, 57.0, 56.2, 49.6,
41.9, 41.4, 41.2, 41.1, 40.9, 38.8, 35.0, 33.5, 30.9, 30.3, 29.1,
27.0, 25.3, 23.0, 22.8, 20.5, 20.2; IR (thin film, NaCl) 3400,
2960, 2872, 1730, 1710, 1596, 1458, 1344, 1208, 1162, 1041,
932, 748 cm-1
; [R]25 ) -60 (c 2.0, CHCl3).
D
(+)-2-Me t h oxy-5-ch lor o-6-[[t r a n s-oct a h yd r o-5â,8a â-
d im eth yl-6â-[[[6-(N-t-BOC-a m in o)h exa n oyl]oxy]m eth yl]-
1(2H )-n a p h t h a len yl]m et h yl]-1,4-cycloh exa d ien e-1,4-d i-
on e (18e). Arene 18d (9.1 mg, 0.015 mmol) was dissolved in
acetonitrile (0.63 mL). CAN (73.6 mg, 0.13 mmol) dissolved
in H2O (0.13 mL) was added dropwise to the stirring solution
over 1.25 h. The reaction mixture was allowed to stir for 4 h;
then the reaction mixture was partitioned between H2O and
Et2O. The aqueous layer was separated and washed with Et2O
(3×). The combined organic layers were washed with brine,
filtered, and concentrated. The resulting oil was purified by
silica gel chromatography (1:1 hexanes:EtOAc) to yield the
quinone 18e (8.5 mg, 94%) as a bright yellow oil: 1H NMR
(CDCl3, 300 MHz) δ 6.03 (1H, br s), 4.60 (1H, s), 4.51 (2H, d,
J ) 5.7 Hz), 4.24 (1H, dd, J ) 11.2, 2.2 Hz), 3.84 (3H, s), 3.70
(2H, dd, J ) 10.5, 9.0 Hz), 3.12 (2H, dt, J ) 7.6, 6.0 Hz), 2.84
(1H, d, J ) 13.4 Hz), 2.79 (1H, d, J ) 13.0 Hz), 2.35 (1H, dd,
J ) 13.9, 8.2 Hz), 2.28 (2H, t, J ) 7.3 Hz), 2.11 (1H, d, J ) 9.2
Hz), 1.83 (2H, m), 1.66-1.48 (7H, m), 1.43 (9H, s), 1.35-1.25
(6H, m), 1.06 (3H, s), 0.89 (3H, s); 13C NMR (CDCl3, 100 MHz)
δ 180.7, 179.4, 174.5, 160.0, 159.8, 156.7, 144.4, 144.0, 110.2,
107.4, 104.2, 79.7, 67.9, 57.4, 53.6, 44.81, 44.77, 41.1, 39.6, 36.4,
34.9, 33.4, 30.5, 30.4, 29.1, 29.0, 27.0, 25.3, 24.1, 21.0, 18.0;
IR (thin film, NaCl) 3398, 2929, 2861, 1359, 2341, 1716, 1683,
1653, 1629, 1259, 1163 cm-1; HRMS calcd for C33H50NO7Cl
607.3273, found 607.3275; MS (70 eV) m/ z (rel int) 609(<1,
57(92), 55(18); [R]25 ) -3.5 (c 13.0, CHCl3).
D
(+)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m et h yl]-5â,8a â-d im et h yl-6r-(h yd r oxym et h yl)-
1(2H )-n a p h t h a len on e
6-(N-t-BOC-a m in o)h exa n oa t e
1
(15c): see compound 18e for similar experimental; H NMR
(CDCl3, 300 MHz) δ 6.55 (1H, d, J ) 2.6 Hz), 6.40 (1H, d, J )
2.5 Hz), 4.61 (1H, dd, J ) 11.2, 5.8 Hz), 4.62 (1H, br s), 4.14
(1H, t, J ) 11.8 Hz), 3.84 (2H, m), 3.78 (3H, s), 3.28 (1H, d, J
) 14.3 Hz), 3.09 (2H, dt, J ) 6.1, 6.0 Hz), 2.54 (1H, dd, J )
10.0, 6.0 Hz), 2.38 (1H, d, J ) 14.6 Hz), 2.32 (2H, t, J ) 7.0
Hz), 2.23 (1H, d, J ) 14.0 Hz), 2.00 (2H, br s), 1.78-1.56 (10H,
m), 1.42 (9H, s), 1.33 (5H, m), 1.33 (3H, s), 0.99 (3H, s); 13C
NMR (CDCl3, 75 MHz) δ 215.3, 174.1, 159.0, 156.6, 139.2,
108.2, 98.6, 64.3, 56.8, 56.2, 50.0, 48.2, 42.1, 41.4, 41.1, 39.0,
37.8, 37.7, 34.9, 30.4, 30.3, 29.0, 28.8, 27.5, 27.0, 26.2, 25.2,
22.2, 21.7, 21.1, 19.7; IR (thin film, NaCl) 3300, 2961, 2930,
1734, 1641, 1598, 1353, 1200, 1133 cm-1; [R]25D ) +2.6 (c 16.0,
CHCl3).
M
2+), 607(1, M+), 533(2), 507(9), 469(2), 375(9), 320(12), 189-
(100), 133(19), 95(46), 56(54); [R]25 ) +11 (c 10, CHCl3).
D
(-)-2-Me t h oxy-5-ch lor o-6-[[t r a n s-oct a h yd r o-5â,8a â-
d im eth yl-6r-[[[6-(N-t-BOC-a m in o)h exa n oyl]oxy]m eth yl]-
1(2H )-n a p h t h a len yl]m et h yl]-1,4-cycloh exa d ien e-1,4-d i-
on e (15e): see compound 18e for similar experimental; 1H
NMR (CDCl3, 300 MHz) δ 6.52 (1H, d, J ) 2.7 Hz), 6.35 (1H,
d, J ) 2.7 Hz), 4.65 (1H, br s), 4.55 (2H, dd, J ) 11.2, 4.2 Hz),
4.23 (1H, t, J ) 10.3 Hz), 3.81 (3H, s), 3.74 (3H, s), 3.25 (1H,
d, J ) 14.4 Hz), 3.20 (1H, dd, J ) 11.2, 4.2 Hz), 3.06 (2H, dt,
J ) 6.5, 6.4 Hz), 2.32 (2H, t, J ) 6.8 Hz), 2.29 (1H, d, J ) 14.6
Hz), 2.26 (1H, m), 1.94 (2H, dd, J ) 8.8, 3.7 Hz), 1.63-1.44
(9H, m), 1.39 (9H, s), 1.35-1.22 (7H, m), 0.94 (3H, s), 0.90 (3H,
s); 13C NMR (CDCl3, 100 MHz) δ 174.2, 158.8, 156.4, 139.6,
116.9, 108.1, 98.4, 81.4, 64.5, 56.8, 56.1, 47.5, 41.5, 41.0, 40.6,
39.0, 34.9, 32.2, 30.8, 30.6, 30.2, 29.1, 28.4, 26.9, 25.3, 22.4,
22.2, 20.8, 19.9, 15.3; IR (thin film, NaCl) 3327, 2924, 2848,
(+)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h e n yl)m e t h yl]-5â,8a â-d im e t h yl-6â-(h yd r oxym e t h yl)-
1(2H)-n a p h th a len yl 6-(N-t-BOC-a m in o)h exa n oa te (18d ).
A solution of Ph3PCH2 (0.4 M, 1.53 mmol, 3.84 mL) was placed
in a round bottom flask. Ketone 18c (103.7 mg, 0.17 mmol)
dissolved in DMSO (1.8 mL) was added dropwise to the stirring
ylide solution. The reaction mixture was heated to 75 °C for
30 min and then the reaction quenched with saturated NH4-
Cl. The crude reaction mixture was extracted several times
with Et2O (3×). The combined organic layers were washed
with saturated NH4Cl and brine, dried over Na2SO4, and
concentrated. The crude product was purified by silica gel
chromatography (7:3 hexanes:EtOAc) to afford olefin 18d (30.0
mg, 75%) as a clear oil: 1H NMR (CDCl3, 300 MHz) δ 6.39
(1H, d, J ) 3.3 Hz), 6.28 (1H, d, J ) 2.9 Hz), 4.56 (1H, br s),
4.47 (1H, s), 4.43 (1H, s), 4.41 (1H, dd, J ) 10.8, 2.2 Hz), 3.89
(2H, m), 3.85 (3H, s), 3.76 (3H, s), 3.11 (2H, d, J ) 6.6, 7.3
Hz), 2.88 (1H, d, J ) 14.3 Hz), 2.76 (1H, d, J ) 14.5 Hz), 2.30
(2H, t, J ) 7.5 Hz), 2.38 (1H, dd, J ) 12.7, 5.0 Hz), 2.11 (1H,
d, J ) 13.0 Hz), 1.91 (2H, d, J ) 9.7 Hz), 1.75 (1H, dd, J )
12.6, 4.0 Hz), 1.64-1.50 (5H, m), 1.43 (9H, s), 1.38-1.25 (8H,
1734, 1652, 1552, 1451, 1262, 1155 cm-1; [R]25 ) -9 (c 2.0,
D
CHCl3).
(+)-2-Meth oxy-5-h yd r oxy-6-[[tr a n s-octa h yd r o-5â,8a â-
d im eth yl-6r-[[[6-(N-t-BOC-a m in o)h exa n oyl]oxy]m eth yl]-
1(2H )-n a p h t h a len yl]m et h yl]-1,4-cycloh exa d ien e-1,4-d i-
on e (16). Pd(PPh)3 (7.9 mg, 6.9 µmol) was placed in a flask
with THF (0.6 mL) and H2O (0.6 mL). Chloroquinone 15e

2830 J . Org. Chem., Vol. 62, No. 9, 1997
Radeke et al.
(16.7 mg, 0.03 mmol) in THF (0.6 mL) and NaHCO3 (1 M, 61
µL) were added dropwise to the flask. After completion of the
addition, the reaction mixture was heated to 80 °C for 10 min;
then the reaction was quenched with HCl (5%). The aqueous
layer was extracted with Et2O (3×), and the combined organic
layers were washed with brine, dried over Na2SO4, and
concentrated. The crude product was purified by silica gel
chromatography (1:1:0.001 hexanes:Et2O:AcOH) to yield quino-
ne 16 (1.2 mg, 7%) as a yellow oil: 1H NMR (CDCl3, 300 MHz)-
δ 5.87 (1H, s), 4.71 (2H, d, J ) 10.5 Hz), 4.56 (1H, br s), 4.51
(1H, s), 4.49 (1H, s), 4.33 (1H, t, J ) 11.2 Hz), 3.87 (3H, s),
3.13 (2H, dt, J ) 6.5, 6.0 Hz), 2.76 (1H, d, J ) 13.0 Hz), 2.27
(2H, t, J ) 8.1 Hz), 2.26 (1H, d, J ) 13.2 Hz), 2.08 (2H, br s),
1.86-1.52 (10H, m), 1.48 (9H, s), 1.35-1.20 (6H, m), 1.12 (3H,
s), 0.94 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 180.9, 179.5,
174.4, 159.8, 156.6, 145.0, 144.3, 110.2, 107.4, 103.9, 79.7, 64.9,
57.4, 49.2, 44.2, 41.6, 41.4, 36.8, 35.0, 33.4, 33.1, 30.7, 30.4,
29.4, 28.8, 27.0, 24.8, 22.8, 22.1, 21.8, 21.0; IR (thin film, NaCl)-
3423, 2963, 2931, 1762, 1684, 1643, 1629, 1589, 1465, 1441,
21 (74 mg, 94%) as a clear yellow oil: 1H NMR (CDCl3, 300
MHz) δ 6.41 (1H, d, J ) 2.6 Hz), 6.37 (1H, d, J ) 2.6 Hz), 4.51
(1H, br s), 4.18 (1H, d, J ) 5.5 Hz), 4.15 (1H, dd, J ) 11.3, 5.1
Hz), 3.97 (2H, dd, J ) 10.7, 8.6 Hz), 3.87 (3H, s), 3.80 (3H, s),
3.10 (2H, dt, J ) 6.8, 6.6 Hz), 2.79 (1H, d, J ) 13.9 Hz), 2.73
(1H, d, J ) 14.5 Hz), 2.25 (2H, t, J ) 7.3 Hz), 1.79 (1H, br d,
J ) 7.2 Hz), 1.76-1.59 (7H, m), 1.43 (9H, s), 1.40-1.30 (9H,
m), 1.08 (3H, s), 0.92 (3H, d, J ) 6.0 Hz), 0.83 (3H, s); 13C
NMR (CDCl3, 100 MHz) δ 174.6, 158.3, 156.3, 140.1, 116.8,
109.7, 98.2, 80.0, 66.2, 56.9, 56.2, 50.6, 50.4, 43.2, 42.4, 41.1,
38.9, 37.9, 37.3, 35.0, 30.5, 29.1, 28.2, 27.03, 26.97, 26.3, 25.3,
23.8, 20.3, 19.1, 18.2, 15.7; IR (thin film, NaCl) 3371, 2917,
2855, 1730, 1712, 1688, 1590, 1537, 1503, 1453 cm-1; HRMS
calcd for C34H54NO6Cl 607.3639, found 607.3634; MS (70 eV)
m/ z (rel int) 609(<1, M2+), 607(<1, M+), 515(1), 446(<1),
365(4), 321(50), 190(78), 151(28), 121(35), 95(34); [R]25D ) -30
(c 3.0, CHCl3).
(+)-2-Meth oxy-5-ch lor o-6-[[tr a n s-octa h yd r o-5â,6â,8a â-
tr im eth yl-1â-[[[6-(N-t-BOC-6-am in o)h exan oyl]oxy]m eth yl]-
5r-n a p h th yl]m eth yl]-1,4-cycloh exa d ien e-1,4-d ion e (21e).
Arene 21 (95 mg, 0.16 mmol) was dissolved in CH3CN (7.5
mL). CAN (683 mg, 1.25 mmol) dissolved in water (1.25 mL)
was added dropwise to the stirring CH3CN solution over 1.25
h. After 2 h the reaction mixture was diluted with Et2O and
extracted (5×). The organic solution was washed with brine,
dried over Na2SO4, and concentrated. The crude product was
purified by silica gel chromatography (1:1 hexanes:Et2O) to
yield chloroquinone 21e (85 mg, 92%) as a yellow oil: 1H NMR
(CDCl3, 300 MHz) δ 6.04 (1H, s), 4.51 (1H, br s), 4.21 (1H, dd,
J ) 10.4, 3.3 Hz), 3.86 (3H, s), 3.69 (2H, dd, J ) 10.6, 3.7 Hz),
3.10 (2H, dt, J ) 7.3, 6.0, Hz), 2.79 (1H, d, J ) 12.8 Hz), 2.72
(1H, d, J ) 12.8 Hz), 2.27 (2H, t, J ) 7.3 Hz), 1.80 (2H, br d,
J ) 10.8 Hz), 1.69-1.53 (8H, m), 1.43 (9H, s), 1.34-1.08 (8H,
m), 0.85 (3H, s), 0.83 (3H, s), 0.81 (3H, d, J ) 6.4 Hz); 13C
NMR (CDCl3, 100 MHz) δ 180.8, 179.6, 174.5, 160.1, 156.3,
145.6, 140.1, 107.3, 66.1, 57.4, 54.7, 50.5, 45.9, 41.1, 40.2, 40.1,
39.1, 38.3, 35.0, 30.4, 29.1, 28.4, 27.2, 27.0, 26.3, 25.3, 24.4,
19.5, 17.2, 15.6; IR (thin film, NaCl) 3402, 2930, 2867, 1731,
1713, 1678, 1632, 1456, 1366, 1226, 1168 cm-1; HRMS calcd
for C33H50ClNO7 607.3275, found 607.3278; MS (70 eV) m/ z
(rel int) 609(1, M2+), 607(2, M+), 551(3), 515(4), 507(18),
497(4), 471(36), 377(5), 366(18), 358(3), 342(76), 322(100),
299(10), 191(40), 175(8), 135(9), 121(11), 109(13), 95(20),
1230 cm-1; [R]25 ) +10 (c 0.3, CHCl3).
D
(-)-2-Meth oxy-5-h yd r oxy-6-[[tr a n s-octa h yd r o-5â,8a â-
d im eth yl-6â-[[[6-(N-t-BOC-a m in o)h exa n oyl]oxy]m eth yl]-
1(2H )-n a p h t h a len yl]m et h yl]-1,4-cycloh exa d ien e-1,4-d i-
1
on e (19): see quinone 16 for similar experimental; H NMR
(CDCl3, 400 MHz) δ 5.87 (1H, s), 4.58 (1H, br s), 4.51 (1H, d,
J ) 10.1 Hz), 4.50 (1H, s), 4.47 (1H, s), 4.22 (1H, t, J ) 5.9
Hz), 3.86 (3H, s), 3.13 (2H, dt, J ) 6.7, 5.2 Hz), 2.65 (1H, d, J
) 14.1 Hz), 2.45 (1H, d, J ) 13.8 Hz), 2.32 (2H, t, J ) 7.3 Hz),
2.16 (3H, m), 1.77-1.58 (4H, m), 1.44 (9H, s), 1.41-1.20 (8H,
m), 1.07 (3H, s), 0.90 (3H, s); 13C NMR (CDCl3, 100 MHz) δ
181.7, 179.8, 174.2, 159.8, 156.8, 145.8, 145.6, 107.8, 105.7,
100.9, 66.6, 64.1, 57.8, 51.8, 44.2, 44.0, 41.8, 41.4, 40.0, 39.8,
38.0, 36.2, 30.1, 28.4, 27.1, 26.8, 25.8, 24.1, 23.8, 22.0; IR (thin
film, NaCl) 3417, 2925, 2866, 1734, 1613, 1353, 1254, 1184,
1120 cm-1; [R]25 ) -10 (c 0.1, CHCl3).
D
(-)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m eth yl]-5â,6â,8a â-tr im eth yl-1â-(h yd r oxym eth yl)-
1(2H)-n a p h th a len e (20). Olefin 13 (77 mg, 0.20 mmol)
dissolved in THF (1 mL) was cooled to 0 °C. BH3‚SMe2 (10
M, 0.031 mL, 0.31 mmol) was added dropwise to the cooled
solution over 30 min. After completion of addition the reaction
mixture was allowed to stir at 25 °C for 3 h; then NaOH (0.2
mL, 6 M) and EtOH (1 mL) were added. Reaction mixture
was cooled again to 0 °C, and 30% H2O2 (0.2 mL) was added.
After completion of addition the mixture was heated to reflux
for 1 h. The reaction was then quenched with water and ice,
and the aqueous portion was extracted with Et2O (3×). The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated. The crude oil was purified using
silica gel chromatography (3:1 hexanes:EtOAc) to yield alcohol
20 (74 mg, 92%) as a clear oil: 1H NMR (CDCl3, 300 MHz) δ
6.40 (1H, d, J ) 2.7 Hz), 6.37 (1H, d, J ) 2.8 Hz), 3.86 (3H, s),
3.78 (3H, s), 3.44 (1H, dd, J ) 10.6, 8.1 Hz), 3.20 (1H, dd, J )
10.4, 8.4 Hz), 2.79 (1H, d, J ) 14.1 Hz), 2.72 (1H, d, J ) 14.1
Hz), 1.77 (4H, m), 1.61 (2H, m), 1.41-1.19 (8H, m), 0.98 (3H,
s), 0.92 (3H, d, J ) 6.4 Hz), 0.81 (3H, s); 13C NMR (CDCl3, 100
MHz) δ 158.3, 156.3, 140.1, 109.7, 98.2, 64.3, 56.8, 56.2, 54.4,
50.6, 39.0, 37.92, 37.87, 28.4, 28.3, 27.1, 26.1, 24.2, 24.1, 23.9,
19.2, 18.2, 15.8; IR (thin film, NaCl) 3383, 2936, 2872, 1587,
1454, 1324, 1201, 1161, 1087, 1039, 756 cm-1; HRMS calcd
for C23H35O3Cl 394.2275, found 394.2279; MS (70 eV) m/ z (rel
int) 396(M2+, 5), 394(M+, 13), 209(27), 186(100), 151(12),
81(8), 69(12), 57(23), 43(14); [R]25 ) +7 (c 5.0, CHCl3); UV-
D
vis (MeOH) 203 (4.62), 288 (4.16) nm.
(+)-2-Met h oxy-5-h yd r oxy-6-[[tr a n s-oct a h yd r o-5â,6â,-
8a â-t r im e t h yl-1â-[[[6-(N-t-BOC-a m in o)h e xa n oyl]oxy]-
m eth yl]-5r-n a p h th yl]m eth yl]-1,4-cycloh exa d ien e-1,4-d i-
on e (22). Pd(Ph3P)4 (20.7 mg, 0.018 mmol) was placed in a
flask with THF (2.5 mL) and H2O (2.5 mL). Chloroquinone
21e (43.6 mg, 0.072 mmol) in THF (2.5 mL) and NaHCO3 (1
M, 0.16 mL) were added dropwise to the flask. After comple-
tion of the addition the reaction mixture was heated to 80 °C
for 10 min; then the reaction quenched with HCl (5%). The
aqueous layer was extracted with Et2O (3×), and the combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated. The crude product was purified by silica gel
chromatography (1:1:0.001 hexanes:Et2O:AcOH) to yield quino-
ne 22 (5.2 mg, 16%, 75% conversion) as a yellow oil: 1H NMR
(CDCl3, 300 MHz) δ 7.56 (1H, br s), 5.88 (1H, s), 4.51 (1H, br
s), 4.22 (1H, d, J ) 10.2 Hz), 3.88 (3H, s), 3.64 (2H, dd, J )
10.5, 8.2 Hz), 3.10 (2H, dt, J ) 6.7, 6.6 Hz), 2.55 (1H, d, J )
13.7 Hz), 2.45 (1H, d, J ) 13.5 Hz), 2.25 (2H, t, J ) 7.1 Hz),
2.00 (2H, d, J ) 11.0 Hz), 1.80 (2H, br d, J ) 11.7 Hz), 1.60
(4H, m), 1.43 (9H, s), 1.34-1.16 (10H, m), 0.95 (3H, d, J ) 6.2
Hz), 0.84 (3H, s), 0.80 (3H, s); 13C NMR (CDCl3, 100 MHz) δ
180.6, 179.8, 173.2, 159.7, 156.4, 146.6, 139.1, 107.3, 65.3, 58.1,
54.6, 51.1, 48.1, 44.0, 42.1, 40.2, 39.7, 38.0, 35.7, 30.4, 28.8,
26.2, 26.4, 24.5, 23.8, 23.2, 22.2, 19.1, 18.2, 17.5; IR (thin film,
NaCl) 3348, 2931, 2862, 1731, 1714, 1694, 1660, 1608, 1453,
135(35), 121(20), 95(28), 67(16); [R]25 ) -15 (c 7.0, CHCl3).
D
(-)-t r a n s-Oc t a h yd r o-5r-[(2-c h lor o-3,5-d im e t h oxy-
p h en yl)m eth yl]-5â,6â,8a â-tr im eth yl-1â-(h yd r oxym eth yl)-
n a p h th yl 6-(N-t-BOC-a m in o)h exa n oa te (21). Alcohol 20
(51 mg, 0.13 mmol), 6-(N-t-BOC-amino)hexanoic acid (30.0 mg,
0.13 mmol), and DMAP (24 mg, 0.20 mmol) were placed in a
flask and cooled to 0 °C. EDCI (37.0 mg, 0.20 mmol) dissolved
in CH2Cl2 (1.5 mL) was added dropwise. The mixture was
stirred at 0 °C for 2 h and then allowed to warm to 25 °C.
After 12 h the reaction was quenched with saturated NH4Cl.
The aqueous layer was extracted with CH2Cl2 (2×). The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated. The crude product was purified
by silica gel chromatography (7:3 hexanes:EtOAc) to yield ester
1367, 1238, 1170 cm-1; [R]25 ) +10 (c 0.2, CHCl3); UV-vis
D
(MeOH) 212 (4.24), 287 (3.98) nm (ilimaquinone: 210 (4.35),
288 (4.06) nm).3a Anal. Calcd for C33H51NO8: C, 67.21; H,
8.72; N, 2.37. Found: C, 67.10; H, 8.69; N, 2.38.
(-)-tr a n s-Octah ydr o-5r-[(2,5-dim eth oxyph en yl)m eth yl]-
5â,6â,8a â-tr im eth yl-1-m eth ylen en a p h th a len e (24). Ke-

Vesicular-Mediated Protein Trafficking
J . Org. Chem., Vol. 62, No. 9, 1997 2831
tone 23 (356 mg, 1.0 mmol) in DMSO (5 mL) was added
dropwise to a stirring solution of Ph3PCH2 (22.5 mL, 0.4 M, 9
mmol) in a 100 mL round bottom flask. The reaction mixture
was heated to 75 °C for 5 h and cooled to 25 °C, the reaction
quenched with H2O, and the mixture extracted with Et2O (3×).
The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated. The resulting oil was purified
by silica gel chromatography (99:1 hexanes:Et2O) to afford 24
(310 mg, 90%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ
6.70 (1H, d, J ) 3.0 Hz), 6.67 (1H, s), 6.64 (1H, d, J ) 2.9 Hz),
4.40 (1H, t, J ) 2.4 Hz), 4.36 (1H, t, J ) 2.1 Hz), 3.75 (3H, s),
3.71 (3H, s), 2.65 (1H, d, J ) 14.1 Hz), 2.58 (1H, d, J ) 13.9
Hz), 2.32 (1H, td, J ) 14.0, 5.2 Hz), 2.08 (2H, d, J ) 16.0 Hz),
1.89 (1H, m), 1.49-1.12 (8H, m), 1.05 (3H, s), 1.00 (3H, d, J )
6.4 Hz), 0.84 (3H, s); 13C NMR (CDCl3, 400 MHz) δ 160.9,
153.5, 153.3, 129.3, 119.4, 111.9, 111.4, 103.3, 55.3, 55.1, 48.7,
42.8, 40.9, 37.8, 37.2, 36.9, 33.8, 29.0, 28.4, 23.8, 21.3, 18.4,
18.2; IR (thin film, NaCl) 2924, 1495, 1457, 1218, 1048, 885,
803, 797, 715 cm-1; mp 74-75 °C; [R]25D ) -50 (c 2.4, EtOAc).
Anal. Calcd for C23H34O2: C, 80.65; H, 10.00. Found: C,
80.29; H, 10.16.
Isoa va r on e (25). CAN (35.5 mg, 0.648 mmol), dissolved
in H2O (500 µL), was added dropwise to compound 24 (8.0 mg,
0.024 mmol) in CH3CN (500 µL). The reaction was monitored
hourly by TLC (95:5 hexanes:Et2O), and after 5 h, the reaction
mixture was diluted with H2O (2 mL) and Et2O (2 mL). The
reaction mixture was extracted with Et2O (2 mL, 3×), and the
combined organic layers were dried over MgSO4, filtered, and
concentrated. Purification by silica gel chromatography (98:2
hexanes:Et2O) yielded compound 25 as a red oil (2.8 mg, 57%):
1H NMR (CDCl3, 400 MHz) δ 6.76 (1H, s), 6.68 (1H, s), 6.55
(1H, s), 4.45 (2H, br s), 2.58 (1H, d, J ) 13.8 Hz), 2.42 (1H, d,
J ) 1.4 Hz), 2.31 (2H, t, J ) 13.0 Hz), 2.08 (2H, d, J ) 12.6
Hz), 1.89 (2H, d, J ) 11.2 Hz), 1.77 (2H, m), 1.25 (3H, s), 0.94
(3H, d, J ) 13.9 Hz), 0.86 (3H, br s); 13C NMR (CDCl3, 400
MHz) δ 160.3, 148.1, 137.8, 137.2, 128.9, 124.5, 110.6, 104.0,
99.3, 49.6, 37.3, 35.9, 33.5, 30.4, 28.7, 28.1, 23.3, 21.3, 17.6
(partial carbon); IR (thin film, NaCl) 2930, 2867, 1665, 1652,
1476, 1382, 1255, 1092, 890 cm-1; HRMS calcd for C21H28O2
312.2089, found 312.2076; MS (70 eV) m/ z (rel int) 191(51),
149(35), 109(31), 95(100), 69(41), 59(51), 57(56).
Secr etion Assa y. All plasticware and glassware were
presterilized or purchased sterile. Cell culture media was
filtered through Nalgene sterile filters with 0.2 µm nylon
membrane filters before use. The plasmid encoding for
secreted AP was obtained from the Schreiber laboratory at
Harvard University and used with permission. Incubation
refers to storing cells in tissue culture flasks with canted necks
and vent caps in a CO2 incubator at 37 °C, 5.0% CO2, and 100%
humidity. Centrifugation, pelleting, or spinning down refers
to using a swinging bucket centrifuge at room temperature at
1000 rpm for 10 min and then aspirating the spent media.
Cell counts were obtained using a hemacytometer with cells
stained with trypan blue (0.4%); counts were done in duplicate
and averaged. Cell morphology was monitored using a phase-
contrast cell culture light microscope at 40×.
J urkat cells were incubated in RPMI 1640 media (without
phenol red), 10% fetal bovine serum (FBS), and 5% penicillin/
streptomycin. For normal cell maintenance, cells were grown
until saturation (1.5-2.5 × 106 cells/mL) and then diluted 10-
fold with fresh media. To run the assay, 1 × 107 cells were
pelleted (amount required for two compounds assayed in
duplicate). Cells were rinsed with RPMI 1640 media and
resuspended in 500 µL of RPMI 1640 media. The cells were
pipetted into a 4 mm cuvette containing plasmid (10 µg).
Equilibration (20 min, rt) was then followed by electroporation
(960 µF, 300 V, rt). The cells were reequilibrated (20 min,
rt), then diluted with unselected media (10.0 mL, RPMI 1640,
10% FBS), and incubated for 48 h. Under these conditions, a
50% viability of cells was obtained. The cells were then
pelleted and resuspended in fresh media (4 mL, RPMI 1640,
10% FBS, 5% penicillin/streptomycin).
A range of compound concentrations was introduced into
96-well plates. Unactivated cells (100 µL, 1.2 × 106 cells/mL)
were pipetted into the wells. To activate the cells, ionomycin
and PMA (2 µM and 100 ng/mL concentrations, respectively)
were added. The total volume in each well was diluted to 200
µL with fresh media. The plate was incubated overnight (37
°C, 5% CO2, 100% humidity) and then heated (68 °C, 1.5 h) to
denature sensitive phosphatases. A p-nitrophenyl phosphate
solution (100 µL, 1 mg/mL of p-nitrophenyl phosphate in 2 M
diethanolamine bicarbonate, pH 10.0) was added to the wells
of a new plate. The lyzed cells (100 µL) were added to the
corresponding p-nitrophenyl phosphate-containing wells and
incubated for 12-24 h. The absorption (410 nm) of each well
was measured using a microplate spectrophotometer.
Ava r on e (2). The olefin 24 (13 mg, 0.038 mmol) was
dissolved in ethanol (0.1 mL, 95%) and chloroform (0.1 mL).
RhCl3‚H2O (1 mg, 0.004 mmol) was added to the stirring
solution. After the addition the reaction mixture was heated
at reflux under N2 for 2 days. The reaction mixture was cooled
to 25 °C, diluted with Et2O, and filtered through silica gel (2
cm). The filtrate was concentrated to yield avarol dimethyl
ether as a clear oil (11 mg, 85%).
Ackn owledgm en t. The National Institutes of Health
(CA 66617) and the Massachusetts Department of
Public Health are gratefully acknowledged for their
support. In addition, we thank Prof. G. Crabtree and
Prof. S. L. Schreiber for a gift of their plasmid, as well
as Mr. P. Belshaw for assistance in establishing the
antisecretory assay. We also thank Prof. S. M. Hecht
for supplying three of the natural products examined
(27-29) and Prof. J . C. Clardy and Prof. P. J . Scheuer
for samples of ilimaquinone. Helpful discussions with
Dr. R. B. Wellner and Prof. V. Malhotra are also
acknowledged.
CrO3 (257 mg, 0.47 mmol) in H2O (1.7 mL) was added
dropwise to avarol dimethyl ether (14.4 mg, 0.042 mmol) in
THF (1.7 mL) at rt. The reaction was monitored by TLC (95:5
hexanes:EtOAc), and after 1 h H2O (0 °C) was added. The
reaction mixture was extracted with Et2O (3×), washed with
(saturated) NH4Cl, dried over MgSO4, filtered, and concen-
trated. Purification with Ag-impregnated silica gel (20%, w/w)
chromatography (95:5 hexanes:EtOAc) afforded avarone (2)
(7.5 mg, 57%) that was identical in all aspects to the natural
product:23 1H NMR (CDCl3, 400 MHz) δ 6.77 (2H, m), 6.51 (1H,
br s), 5.14 (2H, br s), 2.64 (1H, d, J ) 13.4 Hz), 2.43 (1H, d, J
) 13.6 Hz), 2.02 (1H, m), 1.84 (3H, q, J ) 9.7 Hz), 1.66 (1H, t,
J ) 3.3 Hz), 1.62 (1H, t, J ) 3.3 Hz), 1.53 (3H, s), 1.38 (2H, dt,
J ) 9.9, 3.3 Hz), 1.00 (3H, s), 0.93 (3H, d, J ) 7.1 Hz), 0.85
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
all new compounds (28 pages). This material is contained in
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, and can be ordered from the
ACS; see any current masthead page for ordering information.
(3H, s); IR (thin film, NaCl) 2911, 1651, 1654 cm-1
.
(23) Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lett. 1974, 38,
3401.
J O962292L