Total synthesis and biological activities of (+)- and (-)-boscialin and their 1'-epimers

Article abstract of DOI:10.1021/np970517x

Natural (-)-boscialin [(-)-1] has recently been described as one of the constituents of various medicinal plants. To obtain more material for investigations of its biological activities, we carried out the synthesis of (-)-1 and its isomers. Starting from the chiral building block 2, the key steps of the synthesis involved a regioselective reduction and a nucleophilic addition. The enantiomer of the natural product, (+)-boscialin [(+)-1], could be obtained via acid-catalyzed epimerization of hydroxyketone 4 to (+)-3. Starting the synthesis with (-)-3 led to (-)-boscialin [(-)-1] with the natural absolute configuration. In addition to (+)- and (-)-boscialin, the corresponding 1'-epimers (+)- and (-)-epiboscialin were also obtained. In vitro assays with (-)-boscialin [(-)-1] and its three stereoisomers were carried out to test for activity against microbes, parasites, and human fibroblasts. The investigations revealed activity against various microbes and against Trypanosoma brucei rhodesiense and also revealed cytotoxicity against human cancer cells.

Full text of DOI:10.1021/np970517x

J . Nat. Prod. 1998, 61, 591-597
591
Tota l Syn th esis a n d Biologica l Activities of (+)- a n d (-)-Boscia lin a n d Th eir
1′-Ep im er s
J oachim Busch,† Yvonne Grether,‡ Dietmar Ochs,§ and Urs Se´quin*,†
Institut fu¨r Organische Chemie, Universita¨t Basel, St. J ohanns-Ring 19, CH-4056 Basel, Switzerland,
Schweizerisches Tropeninstitut, Socinstrasse 57, Postfach, CH-4002 Basel, Switzerland, and
Ciba Chemikalien GmbH, Ko¨chlinstrasse 1, D-79639 Grenzach-Wyhlen, Germany
Received November 17, 1997
Natural (-)-boscialin [(-)-1] has recently been described as one of the constituents of various
medicinal plants. To obtain more material for investigations of its biological activities, we
carried out the synthesis of (-)-1 and its isomers. Starting from the chiral building block 2,
the key steps of the synthesis involved a regioselective reduction and a nucleophilic addition.
The enantiomer of the natural product, (+)-boscialin [(+)-1], could be obtained via acid-catalyzed
epimerization of hydroxyketone 4 to (+)-3. Starting the synthesis with (-)-3 led to (-)-boscialin
[(-)-1] with the natural absolute configuration. In addition to (+)- and (-)-boscialin, the
corresponding 1′-epimers (+)- and (-)-epiboscialin were also obtained. In vitro assays with
(-)-boscialin [(-)-1] and its three stereoisomers were carried out to test for activity against
microbes, parasites, and human fibroblasts. The investigations revealed activity against various
microbes and against Trypanosoma brucei rhodesiense and also revealed cytotoxicity against
human cancer cells.
Due to increasing interest in volatile aroma compo-
nents, many C13-norisoprenoids have recently been
isolated and identified from various plants.1 As some
of these plants are used in traditional medicine,2,3 there
is also much interest in the biological properties of their
constituents. As an example, some â-ionone derivatives,
isolated from Cinnamomum cassia Blume, showed
potent antiulcerogenic activity.4
Resu lts a n d Discu ssion
Regioselective reduction of diketone 2 with H2/Raney-
nickel (Scheme 1) afforded a mixture of the hydroxy-
ketones (-)-3 and 4 with preferential formation of 4;
the diastereomers could be separated by MPLC. Acid-
catalyzed epimerization of 4 gave access to (+)-3, the
enantiomer of hydroxyketone (-)-3. Protection of the
alcoholic function of both enantiomeric hydroxyketones
with the TBDMS-group led to the ketones (+)- and (-)-
5, the substrates for the attachment of the side chain.
The synthesis of these enantiomeric cyclohexanone
building blocks was the key to the synthesis of both
enantiomeric boscialin isomers.
Reactivity and stereoselectivity of nucleophilic addi-
tions to hindered cyclohexanones are limited by steric
effects.11 Reactions with acetylenic nucleophiles take
place exclusively under axial attack, whereas mixtures
are obtained with vinyllithium derivatives.12
The natural â-ionone derivative (-)-boscialin [(-)-1]
and its â-D-glucopyranoside were first isolated from the
methanolic extract of the leaves of the African tree
Boscia salicifolia Oliv.5 The constitutions and relative
configurations of these compounds were assigned from
spectroscopic measurements, especially from NOE ex-
periments. Later, Pe´rez et al. were able to determine
the absolute configuration. They obtained natural (-)-
boscialin [(-)-1] through selective oxidation of (3S,5R,-
6S,9R)-3,6-dihydroxy-5,6-dihydro-â-ionol,2 a natural prod-
uct isolated from Apollonias barbujana.
Nucleophile 9, the side chain synthon (Scheme 2), was
built up starting with the acetylenic alcohol 6, which
was trimethylsilylated to 7 and subsequently hydrostan-
nated with Bu3SnH-AIBN. During hydrostannation,
a maximum E/Z ratio of 87:11 could be achieved by
thermodynamic reaction control; the product ratio was
In many African countries, extracts from aerial parts
of B. salicifolia Oliv. are used in traditional medicine
for wound healing.6 Alcoholic extracts of the bark and
leaves were shown to exhibit antibacterial and antifun-
gal activity.7,8 Recently, boscialin [(-)-1] and its glu-
coside were also obtained from Dendranthema shi-
wogiku (Kitam.) Kitamara,9 Prunus prostrata Labill.,3
and Averrhoa carambola L.10 To our knowledge, no
investigation of the biological activities of boscialin has
so far been reported. To get more material for in vitro
tests, we developed a synthesis of 1, which, in addition,
led to some novel stereoisomers.
1
determined by GC. In the H NMR spectrum, the vinyl
protons of the main product showed a coupling constant
of J ) 19 Hz, which proved the E-configuration of this
compound. Because separation of the two isomers was
not possible, the mixture was used for the next reaction
step. Transmetalation of 8 with n-BuLi at -78 °C lead
to the vinyllithium derivative 9. Subsequent nucleo-
philic addition of 9 to (-)-5 (Scheme 3) resulted in the
formation of the diastereomeric alcohols (-)-10 and (-)-
11 in a ratio of approximately 2:5. The diastereomeric
mixture was then subjected to oxidation with pyri-
dinium fluorochromate, which deprotected the allylic
alcohol and oxidized it in one step without cleavage of
* To whom correspondence should be addressed. Tel.: 41-61-
2671110. Fax: 41-61-2671103. E-mail: sequin@ubaclu.unibas.ch.
† Universita¨t Basel.
‡ Schweizerisches Tropeninstitut.
§ Ciba Chemikalien GmbH.
S0163-3864(97)00517-X CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/11/1998

592 J ournal of Natural Products, 1998, Vol. 61, No. 5
Busch et al.
Sch em e 1a
Sch em e 3a
a
Key: (i) THF, -78 °C; (ii) pyridinium fluorochromate, 16 h;
a
Key: (i) H2, Raney-Ni, rt, MeOH, 1 h; (ii) HCl, CH2Cl2; (iii)
(iii) Bu4NF.
TBDMSCl, DMF, imidazole, 2 h.
Ta ble 1. Activity of Compounds (+)-1, (+)-16, (-)-1, and
(-)-16 Against Trypanosoma brucei rhodesiense and Human
HT-29 Cells
Sch em e 2a
T. b. rhodesiense HT-29 cells
selectivity
indexa
MICb
IC50
MICb IC50
b
b
melarsoprol
0.011 0.0004
11
56
3.6
8.1
9000
0.3
1.0
23
(+)-1 [(+)-boscialin]
100
31
18
(+)-16 [(+)-epiboscialin] 100
>100 17
(-)-1 [(-)-boscialin]
11
3.7
1.9
0.9
>100 43
(-)-16 [(-)-epiboscialin]
56
8.5
9.4
a
b
IC50 (HT-29 cells)/IC50 (T. b. rhodesiense). In µg/mL.
a
Key: (i) hexamethyldisilazane, 110 °C, 16 h; (ii) Bu3SnH,
AIBN, 130 °C, 16 h; (iii) n-BuLi, THF, -78 °C, 1.5 h.
µg/mL and an IC50 of 0.9 µg/mL against T. b. rhod-
esiense. No inhibition of the growth of T. cruzi could
be observed up to 100 µg/mL for all four compounds
tested. Against Leishmania donovani, only compound
(+)-16 showed any activity with an IC50 value of 77 µg/
mL; the other compounds were not active. All four
compounds showed cytotoxic activity against the HT-
29 cell line. To assess the cytotoxicity in relation to the
activity against the parasites, selectivity indices were
calculated by dividing the IC50 values for mammalian
cells by the IC50 values for the parasites. The selectivity
index for T. b. rhodesiense was 23 for compound (-)-1
and 9.4 for compound (-)-16. In contrast, melarsoprol,
one compound of choice against T. b. rhodesiense infec-
tions, has a selectivity index of 9000, which is signifi-
cantly better than the values determined here for (-)-1
and (-)-16. All four compounds tested proved to be
more toxic against the human HT-29 cells than against
T. cruzi and L. donvani. All this indicates the rather
high general toxicity of these compounds.
the tert-butyldimethylsilyl (TBDMS) group. After sepa-
ration of the two epimers on Si gel, the TBDMS group
was finally removed using Bu4NF to give (-)-boscialin
[(-)-1] and (-)-epiboscialin [(-)-16]. The spectral data
of (-)-boscialin [(-)-1] were identical with those of the
natural product. Its optical rotation corroborated the
absolute configuration of the natural enantiomer to be
(1′S,4′S,6′R). Using the same synthetic steps as out-
lined above, but starting from the hydroxyketone (+)-
5, the unnatural (+)-boscialin [(+)-1] and its epimer (+)-
16 were readily obtained.
Compounds (+)-1, (+)-16, (-)-1, and (-)-16 were
tested against some protozoan parasites and against a
human cancer cell line (Table 1). Whereas (+)-1 and
(+)-16 were found to inhibit the growth of the blood-
stream forms of Trypanosoma brucei rhodesiense only
at a high concentration (100 µg/mL for 100% inhibition)
and therefore were considered as inactive, the natural
boscialin [(-)-1] and its epimer (-)-16 gave more
interesting results. For compound (-)-1, a minimum
inhibitory concentration (MIC) of 11 µg/mL and a 50%
inhibitory concentration (IC50) of 1.9 µg/mL were found.
The most active compound, (-)-16, showed a MIC of 3.7
Compounds (+)-1, (+)-16, (-)-1, and (-)-16 were also
tested by agar diffusion method for their antimicrobial
activity against Gram-positive and Gram-negative bac-
teria and the yeast Candida albicans (Table 2). The
test organisms particularly represent the microorgan-

Synthesis and Activity of Boscialin
J ournal of Natural Products, 1998, Vol. 61, No. 5 593
Ta ble 2. Microbiostatic Activity of Compounds (+)-1, (+)-16,
(-)-1, and (-)-16
(4R ,6R )-4-H yd r oxy-2,2,6-t r im e t h ylcycloh e xa -
n on e (4): colorless needles (Et2O-hexane); mp 45.8-
46.6 °C (lit.14 45.0-47.5 °C); [R]25 -110.1 (c 0.70,
inhibition zone (in mm)
D
MeOH); [lit.14 -108.9 (c 1.06, MeOH)]; IR (KBr) νmax
(+)-1 (-)-1 (+)-16 (-)-16
3700-3100 (OH), 2966, 2929 (C-H), 1698 (CdO) cm-1
;
Staphylococcus aureus ATCCa 9144 10.5 2.5
3
0
10
8
1H NMR (300 MHz, CDCl3) δ 4.22 (1H, m, H-4), 3.18
Staphylococcus epidermidis
8
0
(1H, m, H-6), 2.14 (1H, m, Heq-5), 2.00 (1H, m, Heq-3),
ATCC 12228
Corynebacterium xerosis ATCC 373 13
2
9.5
3
5
12
20
1.93 (1H, s, OH), 1.80 (1H, m, Hax-3), 1.69 (1H, m, Hax
-
Corynebacterium minutissimum
ATCC 23348
18
5), 1.36 (3H, s, CH3-2), 1.05 (3H, s, CH3-2), 1.04 (3H, d,
J ) 6.6 Hz, CH3-6); 13C NMR (75 MHz, CDCl3) δ 217.7
(C-1), 66.1 (C-4), 47.5 (C-3), 44.6 (C-2), 42.7 (C-5), 35.5
(C-6), 26.3 (CH3eq-C-2), 28.5 (CH3ax-C-2), 14.7 (CH3-C-
6); EIMS m/z 156 [M]+ (11), 138 (6, [M-H2O]+), 110 (4),
95 (10), 83 (100), 69 (48), 57 (62), 41 (49). Anal. C,
69.19%; H, 10.24%; O, 20.56%; calcd for C9H16O2: C,
69.19%; H, 10.32%; O, 20.48%.
Escherichia coli NCTCb 8196
Candida albicans ATCC 10231
2
4.5
1
5
1
4
1.5
15.5
a
b
ATCC ) American Type Culture Collection. NCTC ) Na-
tional Collection of Type Cultures.
isms of the resident skin flora (Staphylococci and
Corynebacteria) and typical organisms of the transient
skin flora (Escherichia coli and Candida albicans). All
isomers showed a broad-spectrum antimicrobial activity.
The strongest efficacy was observed against the Gram-
positive Corynebacteria strains, whereas the activity
against the Gram-negative Escherichia coli was the
weakest. In general, (+)-boscialin [(+)-1] and (-)-
epiboscialin [(-)-16] showed a better activity than (-)-
boscialin [(-)-1] and (+)-epiboscialin [(+)-16]. The
latter two epimers were not active against Staphylo-
coccus epidermidis in the test method used.
Ep im er iza tion of Hyd r oxyk eton e 4. (4R,6R)-4-
Hydroxy-2,2,6-trimethylcyclohexanone (4, 15.99 g, 102.4
mmol) was dissolved in CH2Cl2 (180 mL), and 37% HCl
(7.5 mL) was added. After 70 min of stirring at room
temperature, saturated aqueous NaHCO3 solution (90
mL) was added. The organic layer was separated, and
the aqueous layer was washed with CH2Cl2 (2 × 25 mL).
The organic layers were evaporated under reduced
pressure, and the product was purified by MPLC (SiO2,
n-hexane-Et2O 1:1) to give 12.99 g (83.17 mmol, 81.2%)
of
(4R,6S)-4-hydroxy-2,2,6-trimethylcyclohexanone
Exp er im en ta l Section
[(+)-3]: colorless needles (Et2O-n-hexane); mp 51.8-
Gen er a l Exp er im en ta l P r oced u r es. All solvents
were dried and distilled before use. The chemicals were
obtained either from Fluka or Aldrich. Melting points
were measured on a Kofler hot stage and are uncor-
rected. Optical rotations were determined with a Per-
kin-Elmer polarimeter 141. UV spectroscopy was
carried out with a Beckmann UV 25 spectrometer. IR
spectra were obtained on a FT-IR Perkin-Elmer 1600.
1H and 13C NMR spectra were recorded on a Varian
Gemini 300, and chemical shifts are in parts per million
using Me4Si as internal standard. MS were measured
on a Hewlett-Packard 5970A GC/MS at 70 eV.
Regioselective Red u ction of Dik eton e 2. (R)-
2,2,6-Trimethyl-1,4-cyclohexanedione (2, 5.86 g, 38.0
mmol) was dissolved in MeOH (50 mL) and Raney-Ni
(W2-activity) was added. After evacuation, the suspen-
sion was hydrogenated under shaking for 1 h. After
filtration through Celite, the MeOH solution was evapo-
rated. The product mixture was separated by MPLC
(SiO2, Et2O-n-hexane 1:1) to give (-)-3 (1.01 g, 6.45
mmol, 17%) and 4 (4.92 g, 31.5 mmol, 83%).
52.8 °C (lit.14 48.0-50.0 °C); [R]25 +91.3° (c 0.88,
D
MeOH) [lit.14 [R]25 +95.0° (c 0.88, MeOH)]; H NMR
1
D
(300 MHz, CDCl3) δ 4.33 (1H, m, H-4), 2.72 (1H, m, H-6),
2.27 (1H, m, Heq-5), 2.07 (1H, m, Heq-3), 1.84 (1H, s, OH),
1.58 (1H, m, Hax-3), 1.42 (1H, m, Hax-5), 1.21 (3H, s,
CH3-2), 1.07 (3H, s, CH3-2), 1.04 (3H, d, J ) 6.6 Hz,
CH3-6); 13C NMR (75 MHz, CDCl3) δ 215.7 (C-1), 65.6
(C-4), 49.4 (C-3), 44.6 (C-2, C-5), 37.9 (C-6), 26.4 (CH3eq
C-2), 25.8 (CH3ax-C-2), 14.8 (CH3-C-6).
-
P r otection of Hyd r oxyk eton e (+)-3. (4R,6S)-4-
Hydroxy-2,2,6-trimethylcyclohexanone [(+)-3, 7.78 g,
49.8 mmol], imidazole (6.80 g, 99.9 mmol), and (tert-
butyl)dimethylsilyl chloride (14.41 g, 90.7 mmol) were
dissolved in DMF (200 mL) and stirred for 90 min. The
reaction mixture was hydrolyzed by adding H2O (180
mL). The layers were separated, and the aqueous layer
was washed three times with Et2O. The organic layer
was washed with brine and evaporated under reduced
pressure. Purification of the product was carried out
by vacuum distillation (138 °C/12-14 mbar) to give
(+)-5 (11.50 g, 42.6 mmol, 85.5%): colorless oil; mp 4-5
°C; [R]25 +58.2 (c 0.99, MeOH) [lit.14 [R]25 +58.0 (c
(4S ,6R )-4-H y d r o x y -2,2,6-t r im e t h y lc y c lo h e x -
a n on e [(-)-3]: colorless needles (Et2O-hexane); mp
D
D
51.9-52.6 °C (lit.13 51.5-52.5 °C); [R]25 -52.5 (c 0.75,
0.98, MeOH)]; IR (NaCl) νmax 2957, 2929, 2885, 2857
(C-H), 1712 (CdO), 1256 (Si-C), 1086 (Si-O) cm-1; 1H
NMR (300 MHz, CDCl3) δ 4.27 (1H, m, H-4), 2.70 (1H,
m, H-6), 2.14 (1H, m, Heq-5), 1.92 (1H, m, Heq-3), 1.60
(1H, m, Hax-3), 1.43 (1H, m, Hax-5), 1.19 (3H, s, CH3-2),
1.05 (3H, s, CH3-2), 1.02 (3H, d, J ) 6.5 Hz, CH3-6),
0.90 [9H, s, CH3(tert-butyl)], 0.10 (6H, s, CH3-Si); 13C
NMR (75 MHz, CDCl3) δ 215.6 (C-1), 66.3 (C-4), 49.9
(C-3), 44.7 (C-5), 44.3 (C-2), 38.1 (C-6), 26.4 (CH3eq-C-
2), 25.8 (CH3ax-C-2, CH3-(tert-butyl), 18.1 ((CH3)3C-Si),
14.8 (CH3-C-6), -4.7 (CH3-Si); EIMS m/z 213
[M-C4H9]+ (28), 171 (100), 157 (15), 143 (49), 121 (11),
75 (42), 59 (8), 41 (11). Anal. C, 66.65%; H, 11.18%;
calcd for C15H30O2Si: C, 66.61%; H, 11.18%.
D
MeOH; ee ca. 50%) [Lit.13 -105 (c 0.8, MeOH)]; IR (KBr)
νmax 3700-3100 (OH), 2966, 2929 (C-H), 1698 (CdO)
1
cm-1; H NMR (300 MHz, CDCl3) δ 4.32 (1H, m, H-4),
2.72 (1H, m, H-6), 2.28 (1H, m, Heq-5), 2.21 (1H, s, OH),
2.07 (1H, m, Heq-3), 1.58 (1H, m, Hax-3), 1.41 (1H, m,
ax-5), 1.21 (3H, s, CH3-2), 1.07 (3H, s, CH3-2), 1.03 (3H,
H
d, J ) 6.6 Hz, CH3-6); 13C NMR (75 MHz, CDCl3) δ 215.8
(C-1); 65.6 (C-4); 49.4 (C-3); 44.6 (C-2, C-5); 37.9 (C-6);
26.4 (CH3eq-C-2); 25.8 (CH3ax-C-2); 14.8 (CH3-C-6);
EIMS m/z 156 [M]+ (13), 138 [M-H2O]+ (6), 110 (5), 95
(11), 83 (100), 69 (51), 57 (64), 41 (60). Anal. C, 69.15%;
H, 10.32%; O, 20.52%; calcd for C9H16O2: C, 69.19%;
H, 10.32%; O, 20.48%.

594 J ournal of Natural Products, 1998, Vol. 61, No. 5
Busch et al.
P r otection of Hyd r oxyk eton e (-)-3. As for (+)-3,
but with (4S,6R)-4-hydroxy-2,2,6-trimethylcyclohex-
anone [(-)-3, 4.06 g, 26.0 mmol], imidazole (3.55 g, 52.2
mmol), and (tert-butyl)dimethylsilyl chloride (7.52 g,
47.4 mmol). Purification of the product was carried out
by vacuum distillation (135-137 °C/12 mbar) to give
cycloh exa n -1-ol [(+)-10, 380 m g, 0.918 m m ol, 25%]:
obtained as a colorless oil; [R]25 +6.1 (c 0.94, CHCl3);
D
IR (NaCl) νmax 3620 (OH), 2957, 2858 (C-H), 1082 (C-
1
O) cm-1; H NMR (300 MHz, CDCl3) δ 5.71-5.65 (1H,
m, H-2′), 5.52-5.45 (1H, m, H-1′), 4.34 (1H, qui, J )
5.4 Hz, H-3′), 3.84 (1H, m, H-4), 1.88 (1H, m, H-6), 1.69-
1.26 (5H, m, H-5, H-3, OH), 1.23 (3H, d, J ) 6.3 Hz,
CH3-4′), 0.96 (3H, s, CH3-2), 0.94-0.74 (3H, m, CH3-6,
CH3-2), 0.89 [9H, s, CH3(tert-butyl)], 0.11 (9H, s, CH3-
SiTMS), 0.06 (6H, s, CH3-SiTBDMS); 13C NMR (75 MHz,
CDCl3) δ 134.5 (C-1′), 131.9, 131.7 (C-2′), 76.8 (C-1), 68.9
(C-3′), 67.3 (C-4), 45.8, 45.7 (C-3), 39.6 (C-5), 39.5 (C-2),
34.2, 34.1 (C-6), 25.9, 25.1, 25.0 24.6 [C-4′, CH3(tert-
butyl), CH3eq-C-2, CH3ax-C-2], 18.2 [(CH3)3C-Si], 16.0,
15.9 (CH3-C-6), 0.1 (CH3-SiTMS), -4.6 (CH3-SiTBDMS);
EIMS m/z 399 ([M-CH3]+ (<1), 381 (<1), 357 (<1), 309
(<1), 282 (1), 267 (2), 225 (1), 201 (43), 175 (12), 143
(15), 117 (25), 95 (19), 75 (78), 73 (100), 69 (10), 55 (12),
43 (20). Anal. C, 63.51%; H, 10.95%; calcd for C22H46O3-
Si2: C, 63.71%; H, 11.18%.
(-)-5 (6.24 g, 23.1 mmol, 89.0%): colorless oil; [R]25
D
1
-36.8 (c 1.18, MeOH; ee ca. 50%); H NMR (300 MHz,
CDCl3) δ 4.26 (1H, m, H-4), 2.69 (1H, m, H-6), 2.14 (1H,
m, Heq-5), 1.92 (1H, m, Heq-3), 1.60 (1H, m, Hax-3), 1.44
(1H, m, Hax-5), 1.19 (3H, s, CH3-2), 1.05 (3H, s, CH3-2),
1.02 (3H, d, J ) 6.5 Hz, CH3-6), 0.90 [9H, s, CH3(tert-
butyl)], 0.10 (6H, s, CH3-Si); 13C NMR (75 MHz, CDCl3)
δ 215.6 (C-1); 66.3 (C-4); 49.9 (C-3); 44.7 (C-5); 44.3 (C-
2); 38.1 (C-6); 26.4 (CH3eq-C-2); 25.8 [CH3ax-C-2, CH3-
(tert-butyl)]; 18.1 [(CH3)3C-Si]; 14.8 (CH3-C-6); -4.7
(CH3-Si).
(R/S)-3-(Tr im eth ylsiloxy)-1-bu tyn e (7). To (R/S)-
3-butyn-2-ol (6, 10.25 g, 0.1466 mol) was added under
argon hexamethyldisilazane (12.19 g, 75.58 mmol) and
the mixture refluxed for 16 h at 110 °C. After cooling,
the reaction mixture was filtered through SiO2 to give
7 (20.63 g, 0.1450 mol, 99%): colorless liquid (bp: 110
°C); the spectroscopic data of the product were identical
with those in the literature.15
(1S,4R,6S)-4-[(ter t-Bu t yl)d im et h ylsiloxy]-2,2,6-
tr im eth yl-1-[(E,R/S)-3′-(tr im eth ylsiloxy)-1′-bu ten yl]-
cycloh exa n -1-ol [(+)-11, 835 m g, 2.02 m m ol, 55%]:
obtained as a colorless oil; [R]25 +26.5 (c 0.48, CHCl3);
D
IR (NaCl) νmax 3512 (OH); 2958, 2859 (C-H); 1251, 1038
(C-O) cm-1; 1H NMR (300 MHz, CDCl3) δ 5.90 (1H, d,
J ) 15.6 Hz, H-1′), 5.69 (1H, m, H-2′), 4.36 (1H, qui, J
) 6.2 Hz, H-3′), 3.93 (1H, m, H-4), 1.95 (1H, m, H-6),
1.75-1.42 (5H, m, H-5, H-3), 1.29 (s, OH), 1.23, 1.23
(3H, 2d, J ) 6.3 Hz, CH3-4′), 1.05 (3H, s, CH3-2), 0.89
[9H, s, CH3(tert-butyl)], 0.81-0.74 (3H, d, CH3-6, CH3-
2), 0.12, 0.12 (9H, 2s, CH3-SiTMS), 0.06 (6H, s, CH3-
SiTBDMS); 13C NMR (75 MHz, CDCl3) δ 135.1 (C-1′), 128.1
(C-2′), 78.0 (C-1), 69.4 (C-3′), 67.3 (C-4), 47.4, 47.3 (C-
3), 41.9, 41.8 (C-5), 39.2, 39.1 (C-2), 35.7, 35.5 (C-6), 26.2
(C-4′), 25.9 [CH3(tert-butyl)], 25.3, 25.1 (CH3eq-C-2), 22.8
(CH3ax-C-2), 18.2 [(CH3)3C-Si], 15.7 (CH3-C-6), 0.2
(CH3-SiTMS), -4.6 (CH3-SiTBDMS); EIMS m/z 414 [M]+
(<1), 399 [M-CH3]+ (<1), 357 (<1), 324 (<1), 267 (2),
225 (1), 201 (40), 171 (7), 143 (7), 119 (27), 95 (17), 75
(84), 73 (100), 69 (15), 55 (13), 43 (22). Anal. C, 63.53%;
H, 11.04%; calcd for C22H46O3Si2: C, 63.71%; H, 11.18%.
H yd r ost a n n a t ion of Alk yn e 7. To (R/S)-3-(tri-
methylsiloxy)-1-butyne (7, 18.81 g, 132.2 mmol) was
added under argon tri-n-butylstannane (45 mL, 0.17
mol) and azobis(isobutyronitrile) (AIBN, 150 mg) and
the mixture refluxed for 16 h at 130 °C. After cooling
to room temperature, the excess of tri-n-butylstannane
was removed at 75-80 °C/0.05 mbar. The product
mixture was distilled at 126-130 °C/0.04 mbar to give
stannane 8: colorless liquid (54.95 g, 126.8 mmol,
86.5%); GC/EIMS m/z E-isomer: 377 [M-C4H8]+ (100),
369 (3), 321 (6), 291 (3), 249 (5), 235 (4), 209 (28), 193
(49), 175 (15), 143 (20), 121 (18), 73 (42), 41 (14); GC/
EIMS m/z Z-isomer: 377 [M-C4H8]+ (18), 365 (2), 323
(100), 319 (42), 305 (33), 281 (5), 267 (69), 249 (13), 207
(62), 193 (23), 177 (15), 135 (13), 121 (17), 73 (12), 41
(25). Anal. C, 52.60%; H, 9.62%; calcd for C19H42
OSiSn: C, 52.67%; H, 9.77%.
-
(1R,4R,6S)-4-[(ter t-Bu t yl)d im et h ylsiloxy]-2,2,6-
t r im et h yl-1-[(E,R/S)-3′-h yd r oxy-1′-b u t en yl]cyclo-
h exa n -1-ol (12, 25 m g, 0.73 m m ol): obtained as a
colorless oil; IR (NaCl) νmax 3650-3100 (OH), 2958, 2857
(C-H), 1071 (C-O) cm-1; 1H NMR (300 MHz, CDCl3) δ
5.80-5.73 (1H, m, H-2′), 5.58-5.53 (1H, d, J ) 15.7 Hz,
H-1′), 4.38 (1H, m, H-3′), 3.84 (1H, m, H-4), 1.90 (1H,
m, H-6), 1.70-1.18 (8H, m, H-5, H-3, OH, CH3-4′), 0.97,
0.95 (3H, 2s, CH3-2), 0.89 [9H, s, CH3(tert-butyl)], 0.88-
0.77 (6H, m, CH3-6, CH3-2), 0.06 (6H, s, CH3-Si); 13C
NMR (75 MHz, CDCl3) δ 134.3 (C-1′), 133.1, 133.0 (C-
2′), 76.8 (C-1), 68.6 (C-3′), 67.2 (C-4), 45.7 (C-3), 39.6
(C-5), 39.4 (C-2), 34.1 (C-6), 25.9 [CH3(tert-butyl)], 25.8,
25.1, 24.6, 23.9 (C-4′, CH3eq-C-2, CH3ax-C-2), 18.2
[(CH3)3C-Si], 15.9 (CH3-C-6), -4.6 (CH3-Si). EIMS
m/z 267 (3), 238 (5), 215 (2), 201 (100), 185 (8), 173 (64),
159 (12), 135 (38), 119 (21), 109 (30), 95 (63), 75 (99),
57 (21), 43 (69). Anal. C, 66.64%; H, 11.04%; calcd for
C19H38O3Si2: C, 66.61%; H, 11.18%.
Tr a n sm eta la tion to 9 a n d Su bsequ en t Nu cleo-
p h ilic Ad d ition to (+)-5. Vinylstannane 8 (3.20 g,
7.38 mmol) was dissolved in THF (10 mL) and cooled
to -78 °C; n-BuLi (1.6 M in hexane, 4.60 mL, 7.36
mmol) was added slowly, and the reaction mixture was
stirred for 90 min at -78 °C. (4R, 6S)-4-[(tert-Butyl)-
dimethylsiloxy]-2,2,6-trimethylcyclohexanone ((+)-5, 0.995
g, 3.67 mmol) was dissolved in THF (15 mL) and added
within 30 min to the reaction flask. After stirring for 2
h at -78 °C, the solution was allowed to warm to 0 °C
and was quenched with saturated aqueous NH4Cl
solution (10 mL). The layers were separated, and the
aqueous layer was washed with Et2O (3 × 10 mL). The
combined organic layers were dried over Na2SO4. The
product ratio was determined by GC. After evaporation
of the solvent, the diastereomers (+)-10 and (+)-11 were
separated by MPLC (SiO2-n-hexane-Et2O gradient).
During chromatography on SiO2, the TMS group was
cleaved off to some extent and the allylic alcohols 12
and 13 were also obtained.
(1S,4R,6S)-4-[(ter t-Bu t yl)d im et h ylsiloxy]-2,2,6-
t r im et h yl-1-[(E,R/S)-3′-h yd r oxy-1′-b u t en yl]cyclo-
h exa n -1-ol (13, 22 m g, 0.064 m m ol): obtained as a
(1R,4R,6S)-4-[(ter t-Bu t yl)d im et h ylsiloxy]-2,2,6-
tr im eth yl-1-[(E,R/S)-3′-(tr im eth ylsiloxy)-1′-bu ten yl]-

Synthesis and Activity of Boscialin
J ournal of Natural Products, 1998, Vol. 61, No. 5 595
colorless oil; IR (NaCl) νmax 3600-3100 (OH), 2958, 2859
(C-H), 1037 (C-O) cm-1; 1H NMR (300 MHz, CDCl3) δ
6.01-5.95 (1H, m, H-1′), 5.80-5.73 (1H, m, H-2′), 4.40
(1H, m, H-3′), 3.93 (1H, m, H-4), 1.96 (1H, m, H-6),
1.76-1.20 (8H, m, H-5, H-3, OH, CH3-4′), 1.05 (3H, s,
CH3-2), 0.89 [9H, s, CH3(tert-butyl)], 0.81-0.76 (6H, m,
CH3-6, CH3-2), 0.06 (6H, s, CH3-Si); 13C NMR (75 MHz,
CDCl3) δ 134.8 (C-1′), 129.2 (C-2′), 78.0 (C-1), 68.9 (C-
3′), 67.2 (C-4), 47.3, 47.2 (C-3), 41.8 (C-5), 39.0 (C-2),
35.6 (C-6), 26.1 (C-4′), 25.9 [CH3(tert-butyl)], 24.0, 23.9
(CH3eq-C-2), 22.7 (CH3ax-C-2), 18.2 [(CH3)3C-Si], 15.7
(CH3-C-6), -4.7 (CH3-Si); EIMS m/z 324 [M-H2O]+
(1), 308 (1), 293 (2), 267 (8), 238 (6), 211 (11), 201 (70),
175 (9), 159 (8), 137 (11), 123 (20), 109 (51), 95 (41), 85
(33), 75 (100), 69 (51), 55 (35), 43 (83). Anal. C, 66.35%;
H, 10.90%; calcd for C19H38O3Si2: C, 66.61%; H, 11.18%.
(3H, d, J ) 6.4 Hz, CH3-6′), 0.07 (6H, s, CH3-Si); 13C
NMR (75 MHz, CDCl3) δ 197.8 (C-2), 146.9 (C-3), 130.8
(C-4), 78.9 (C-1′), 67.0 (C-4′), 47.3 (C-3′), 41.8 (C-5′), 39.4
(C-2′), 36.3 (C-6′), 28.1 (C-1), 26.3 (CH3eq-C-2′), 25.9
[CH3(tert-butyl)], 22.8 (CH3ax-C-2′), 18.2 [(CH3)3C-Si],
15.8 (CH3-C-6′), -4.6 (CH3-Si); EIMS m/z 340 [M]+
(<1), 325 (1), 307 (1), 283 (26), 227 (16), 209 (18), 191
(15), 173 (12), 143 (57), 133 (30), 111 (34), 75 (100), 73
(60), 59 (24), 55 (21), 43 (80). Anal. C, 67.16%; H,
10.56%; calcd for C19H36O3Si: C, 67.01%; H, 10.65%.
Dep r otection of (+)-14. (E)-4-{(1′R,4′R,6′S)-4-[(tert-
Butyl)dimethylsiloxy]-1′-hydroxy-2′,2′,6′-trimethylcyclo-
hex-1-yl}-3-buten-2-one [(+)-14, 53.0 mg, 0.156 mmol]
was dissolved in THF (5 mL), and Bu4NF (350 mg, 1.11
mmol) dissolved in THF (6 mL) was added. After 16 h
of stirring at room temperature, H2O (8 mL) was added,
and the layers were separated. The aqueous layer was
washed exhaustively with Et2O (10 × 8 mL). The
combined organic layers were evaporated under reduced
pressure, and the residue was purified by MPLC (SiO2-
n-hexane-EtOAc gradient) to yield (E)-4-[(1′R,4′R,6′S)-
1′,4′-dihydroxy-2′,2′,6′-trimethylcyclohexyl]-3-buten-2-
one ((+)-boscialin) [(+)-1, 32.7 mg, 0.145 mmol, 92.8%]:
Oxid a t ion of (+)-10. (1R,4R,6S)-4-[(tert-Butyl)di-
methylsiloxy]-2,2,6-trimethyl-1-[(E,R/S)-3′-(trimethylsi-
loxy)-1′-butenyl]cyclohexane-1-ol [(+)-10, 0.132 g, 0.319
mmol] was dissolved in THF (10 mL) and pyridinium
fluorochromate (0.50 g, 2.5 mmol) was added. The
suspension was stirred for 16 h at room temperature.
After a crude filtration through SiO2, the product was
purified by MPLC (SiO2-n-hexane-Et2O gradient) and
crystallized from Et2O-n-hexane 4:1 to give (E)-4-
{(1′R,4′R,6′S)-4′-[(tert-butyl)dimethylsiloxy]-1′-hydroxy-
2′,2′,6′-trimethylcyclohexyl}-3-buten-2-one [(+)-14, 0.090
g, 0.264 mmol, 83.0%]: colorless crystals; mp 98.8-99.0
colorless amorphous solid; [R]25 +22.0 (c 0.70, CHCl3);
D
UV (EtOH) λmax (log ꢀ) 232 (4.01); IR (NaCl) νmax 3700-
3050 (OH), 2935 (C-H), 1671, 1625 (CdO), 1040 (C-
O) cm-1; 1H NMR (300 MHz, CDCl3) δ 6.76 (1H, d, J )
15.8 Hz, H-4), 6.37 (1H, d, J ) 15.7 Hz, H-3), 3.90 (1H,
m, H-4′), 2.28 (3H, s, H-1), 2.07 (1H, m, H-6′), 1.87-
1.23 (6H, m, H-5′, H-3′, 2 OH), 1.04 (3H, s, CH3-2′), 0.88
(3H, s, CH3-2′), 0.81 (3H, d, J ) 6.8 Hz, CH3-6′); 13C
NMR (75 MHz, CDCl3) δ 197.6 (C-2), 150.6 (C-3), 130.3
(C-4), 77.8 (C-1′), 66.4 (C-4′), 45.0 (C-3′), 39.9 (C-2′), 38.9
(C-5′), 33.9 (C-6′), 28.2 (C-1), 25.1 (CH3eq-C-2′), 24.5
(CH3ax-C-2′), 15.9 (CH3-C-6′); EIMS m/z 208 [M-H2O]+
(8), 170 (30), 140 (4), 126 (35), 111 (100), 98 (26), 82 (22),
71 (21), 55 (31), 43 (95). Anal. C, 69.15%; H, 9.90%; O,
21.03%; calcd for C13H22O3: C, 68.99%; H, 9.80%; O,
21.21%.
°C; [R]25 +19.7 (c 0.64, CH3OH); IR (KBr) νmax 3600-
D
3250 (OH), 2952, 2856 (C-H), 1679, 1624 (CdO), 1150,
1
1093 (C-O) cm-1; H NMR (300 MHz, CDCl3) δ 6.74
(1H, d, J ) 15.8 Hz, H-4), 6.38 (1H, d, J ) 15.8 Hz, H-3),
3.86 (1H, m, H-4′), 2.27 (3H, s, H-1), 2.03 (1H, m, H-6′),
1.72-1.24 (5H, m, H-5′, H-3′, OH), 1.03 (3H, s, CH3-2′),
0.89 [9H, s, CH3(tert-butyl)], 0.86 (3H, s, CH3-2′), 0.78
(3H, d, J ) 6.8 Hz, CH3-6′), 0.06 (6H, s, CH3-Si); 13C
NMR (75 MHz, CDCl3) δ 197.6 (C-2), 150.5 (C-3), 130.2
(C-4), 77.8 (C-1′), 66.9 (C-4′), 45.5 (C-3′), 39.8 (C-2′), 39.3
(C-5′), 33.9 (C-6′), 28.3 (C-1), 25.9 [CH3(tert-butyl)], 25.2
(CH3eq-C-2′), 24.6 (CH3ax-C-2′), 18.2 [(CH3)3C-Si], 16.0
(CH3-C-6′), -4.6 (CH3-Si); EIMS m/z 284 [M-C4H8]+
(13), 265 (3), 227 (11), 208 (27), 191 (22), 173 (27), 159
(8), 149 (14), 135 (17), 111 (55), 93 (28), 75 (85), 73 (50),
59 (20), 43 (100). Anal. C, 67.12%; H, 10.54%; calcd
for C19H36O3Si: C, 67.01%; H, 10.65%.
Dep r otection of (+)-15. As for (+)-14 but with (E)-
4-{(1′S,4′R,6′S)-4-[(tert-butyl)dimethylsiloxy]-1′-hydroxy-
2′,2′,6′-trimethylcyclohex-1-yl}-3-buten-2-one [(+)-15, 93.0
mg, 0.274 mmol] in THF (5 mL) and Bu4NF (690 mg,
2.18 mmol) in THF (6 mL). (E)-4-[(1′S,4′R,6′S)-1′,4′-
dihydroxy-2′,2′,6′-trimethylcyclohexyl]-3-buten-2-one ((+)-
epi-boscialin) [(+)-16, 49.1 mg, 0.217 mmol, 79.3%] was
Oxid a t ion of (+)-11. (1S,4R,6S)-4-[(tert-Butyl)di-
methylsiloxy]-2,2,6-trimethyl-1-[(E,R/S)-3′-(trimethyl-
siloxy)-1′-butenyl]cyclohexane-1-ol [(+)-11, 0.252 g, 0.609
mmol] was dissolved in THF (10 mL) and pyridinium
fluorochromate (0.50 g, 2.5 mmol) was added. The
suspension was stirred for 16 h at room temperature.
After a quick first separation on SiO2 the product was
purified by MPLC (SiO2-n-hexane-Et2O gradient) and
crystallized from Et2O-n-hexane 4:1 to give (E)-4-
{(1′S,4′R,6′S)-4′-[(tert-butyl)dimethylsiloxy]-1′-hydroxy-
2′,2,′6′-trimethylcyclohexyl}-3-buten-2-one [(+)-15, 0.163
g, 0.479 mmol, 78.7%]: colorless crystals; mp 115.5-
obtained: colorless crystals; mp 151.9-152.3 °C; [R]25
D
+49.7 (c 0.92, CHCl3); UV (EtOH) λmax (log ꢀ) 232 (4.08);
IR (KBr) νmax 3700-3100 (OH), 2951, 2860 (C-H), 1667,
1643 (CdO), 1029 (C-O) cm-1 1H NMR (300 MHz,
;
CDCl3) δ 7.11 (1H, d, J ) 15.7 Hz, H-4), 6.41 (1H, d, J
) 15.7 Hz, H-3), 4.02 (1H, m, H-4′), 2.30 (3H, s, H-1),
2.05 (1H, m, H-6′), 1.98-1.23 (6H, m, H-5′, H-3′, 2OH),
1.10 (3H, s, CH3-2′), 0.83 (3H, s, CH3-2′), 0.82 (3H, d, J
) 6.7 Hz, CH3-6′); 13C NMR (75 MHz, CDCl3) δ 197.7
(C-2), 146.5 (C-3), 130.8 (C-4), 78.9 (C-1′), 66.3 (C-4′),
46.8 (C-3′), 41.4 (C-5′), 39.4 (C-2′), 36.2 (C-6′), 28.2 (C-
1), 26.2 (CH3eq-C-2′), 22.8 (CH3ax-C-2′), 15.7 (CH3-C-
6′); EIMS m/z 208 [M-H2O]+ (6), 193 (1), 170 (19), 156
(7), 152 (4), 137 (4), 126 (32), 111 (96), 98 (32), 82 (24),
71 (24), 57 (16), 55 (35), 43 (100). Anal. C, 69.11%; H,
10.01%; O, 21.25%; calcd for C13H22O3: C, 68.99%; H,
9.80%; O, 21.21%.
116.0 °C; [R]25 +44.8 (c 0.76, CH3OH); IR (KBr) νmax
D
3600-3200 (OH), 2954, 2859 (C-H), 1685, 1615 (CdO),
1
1078 (C-O) cm-1; H NMR (300 MHz, CDCl3) δ 7.11
(1H, d, J ) 15.9 Hz, H-4), 6.38 (1H, d, J ) 15.7 Hz, H-3),
3.95 (1H, m, H-4′), 2.29 (3H, s, H-1), 2.00 (1H, m, H-6′),
1.80-1.26 (5H, m, H-5′, H-3′, OH), 1.08 (3H, s, CH3-2′),
0.90 [9H, s, CH3(tert-butyl)], 0.80 (3H, s, CH3-2′), 0.79
(E)-4-{(1′S,4′S,6′R)-4′-[(ter t-Bu tyl)dim eth ylsiloxy]-

596 J ournal of Natural Products, 1998, Vol. 61, No. 5
Busch et al.
1′-h yd r oxy-2′,2′,6′-tr im eth ylcycloh exyl}-3-bu ten -2-
on e ((-)-14) a n d (E)-4-{(1′R,4′S,6′R)-4′-[(ter t-Bu tyl)-
d im e t h y ls ilo x y ]-1′-h y d r o x y -2′,2′,6′-t r im e t h y l-
cycloh ex-1-yl}-3-bu ten -2-on e [(-)-15]. Vinylstan-
nane 8 (26.0 g, 59.9 mmol) was dissolved in THF (60
mL) and cooled to -78 °C. Then n-butyllithium (1.6 M
in hexane, 37.5 mL, 60.0 mmol) was added dropwise.
After 2 h of stirring at -78 °C, (4S, 6R)-4-[(tert-butyl)-
dimethylsilyloxy]-2,2,6-trimethylcyclohexanone [(-)-5,
6.24 g, 23.0 mmol] dissolved in THF (18 mL) was added
within 30 min. The solution was stirred for another 2
h at -78 °C, before it was allowed to warm to 0 °C and
quenched with saturated aqueous NH4Cl solution (60
mL). The layers were separated, and the aqueous layer
was washed with Et2O (4 × 50 mL). The combined
organic layers were dried over Na2SO4. After evapora-
tion of the solvent, the product mixture was prepurified
by column chromatography on SiO2 (n-hexane-Et2O
gradient).
combined organic layers were evaporated under reduced
pressure, and the residue was purified by MPLC (SiO2-
n-hexane-EtOAc gradient) to give (E)-4-[(1′S,4′S,6′R)-
1′,4′-dihydroxy-2′,2′,6′-trimethylcyclohexyl]-3-buten-2-
one [(-)-boscialin] [(-)-1, 0.698 g, 3.09 mmol, 88%]:
colorless amorphous solid; [R]25 -18.6 (c 0.84, CHCl3),
D
[lit.5 [R]25 -19 ( 5 (c 0.14, CHCl3)];2 -21.1 (c 0.31,
D
1
CHCl3); H NMR (300 MHz, CDCl3) δ 6.76 (1H, d, J )
15.9 Hz, H-4), 6.38 (1H, d, J ) 15.9 Hz, H-3), 3.90 (1H,
m, H-4′), 2.28 (3H, s, H-1), 2.07 (1H, m, H-6′), 2.05 (1H,
s, OH), 1.87-1.24 (5H, m, H-5′, H-3′, OH), 1.04 (3H, s,
CH3-2′), 0.88 (3H, s, CH3-2′), 0.81 (3H, d, J ) 6.8 Hz,
CH3eq-6′); 13C NMR (75 MHz, CDCl3) δ 197.7 (C-2), 150.7
(C-3), 130.3 (C-4), 77.7 (C-1′), 66.3 (C-4′), 45.0 (C-3′), 39.9
(C-2′), 38.8 (C-5′), 33.9 (C-6′), 28.1 (C-1), 25.1 (CH3-C-
2′), 24.5 (CH3ax-C-2′), 15.9 (CH3-C-6′). Anal. C, 68.70%;
H, 9.93%; O, 21.09%; calcd for C13H22O3: C, 68.99%; H,
9.80%; O, 21.21%.
Dep r otection of (-)-15. As above for (-)-14 but
with (E)-4-{(1′R,4′S,6′R)-4-[(tert-butyl)dimethylsiloxy]-
1′-hydroxy-2′,2′,6′-trimethylcyclohex-1-yl}-3-buten-2-
one [(-)-15, 2.08 g, 6.11 mmol] dissolved in THF (40
mL) and Bu4NF (20.0 g, 63.5 mmol) dissolved in THF
(100 mL) to yield (E)-4-[(1′R,4′S,6′R)-1′,4′-dihydroxy-
2′,2′,6′-trimethylcyclohexyl]-3-buten-2-one [(-)-epibos-
cialin] [(-)-16, 1.10 g, 4.87 mmol, 79.7%]: colorless
The mixture of (-)-10 and (-)-11 thus obtained (32.79
g, 23 mmol) was dissolved in CH2Cl2 (300 mL), pyri-
dinium fluorochromate (24.66 g, 123.3 mmol) was added,
and the suspension was stirred for 20 h. The reaction
mixture was filtered through SiO2, and the solvent was
evaporated. Further purification and separation was
carried out by MPLC (SiO2-n-hexane-Et2O gradient).
(E)-4-{(1′S,4′S,6′R)-4′-[(tert-Butyl)dimethylsiloxy]-1′-hy-
droxy-2′,2′,6′-trimethylcyclohexyl}-3-buten-2-one [(-)-
14, 1.54 g, 4.53 mmol, 19.7% from (-)-5] was obtained
crystals; mp 152.0-152.3 °C; [R]25 -62.6 (c 0.91,
D
1
CHCl3); H NMR (300 MHz, CDCl3) δ 7.11 (1H, d, J )
15.7 Hz, H-4), 6.41 (1H, d, J ) 15.7, Hz H-3), 4.02 (1H,
m, H-4′), 2.30 (3H, s, H-1), 2.05 (1H, m, H-6′), 1.98-
1.23 (6H, m, H-5′, H-3′, 2OH), 1.10 (3H, s, CH3-2′), 0.83
(3H, s, CH3-2′), 0.82 (3H, d, J ) 6.7 Hz, CH3-6′); 13C
NMR (75 MHz, CDCl3) δ 197.8 (C-2), 146.5 (C-3), 130.8
(C-4), 78.9 (C-1′), 66.3 (C-4′), 46.8 (C-3′), 41.4 (C-5′), 39.4
(C-2′), 36.2 (C-6′), 28.2 (C-1), 26.2 (CH3eq-C-2′), 22.8
(CH3ax-C-2′), 15.7 (CH3-C-6′). Anal. C, 68.89%; H,
10.00%; O, 21.21%; calcd for C13H22O3: C, 68.99%; H,
9.80%; O, 21.21%.
as colorless crystals: mp 98.8-99.0 °C; [R]25 -15.5 (c
D
1
0.88, CH3OH); H NMR (300 MHz, CDCl3) δ 6.74 (1H,
d, J ) 15.9 Hz, H-4), 6.38 (1H, d, J ) 15.8 Hz, H-3),
3.86 (1H, m, H-4′), 2.27 (3H, s, H-1), 2.03 (1H, m, H-6′),
1.67-1.34 (5H, m, H-5′, H-3′, OH), 1.03 (3H, s, CH3-2′),
0.89 [9H, s, CH3(tert-butyl)], 0.86 (3H, s, CH3-2′), 0.78
(3H, d, J ) 6.8 Hz, CH3-6′), 0.06 (6H, s, CH3-Si); 13C
NMR (300 MHz, CDCl3) δ 197.6 (C-2), 150.5 (C-3), 130.1
(C-4), 77.8 (C-1′), 66.9 (C-4′), 45.5 (C-3′), 39.8 (C-2′), 39.3
(C-5′), 33.9 (C-6′), 28.3 (C-1), 25.9 [CH3(tert-butyl)], 25.2
(CH3eq-C-2′), 24.6 (CH3ax-C-2′), 18.2 [(CH3)3C-Si], 15.9
(CH3-C-6′), -4.6 (CH3-Si).
P a r a site Str a in s. Trypanosoma brucei rhodesiense
STIB 900 is a cloned population isolated in 1982, from
a patient in Tanzania. The strain was adapted to grow
under axenic culture conditions according to Baltz et
al.16 The minimum essential medium (MEM) was
supplemented with 15% heat-inactivated horse serum.
Stock cultures were kept in 24-well tissue culture plates
at 37 °C in an atmosphere of 5% CO2 in air. Trypano-
soma cruzi MHOM/Br/OO/Y was cultivated in MEM
supplemented with 10% heat-inactivated fetal calf
serum in L6 myoblasts (ECACC 92102118). Stock
cultures were maintained in T-12.5 flasks at 37 °C in
an atmosphere of 5% CO2 in air. Leishmania donovani
MHOM/ET/67/L82 was maintained in hamsters.
(E)-4-{(1′R,4′S,6′R)-4′-[(ter t-Bu tyl)dim eth ylsiloxy]-
1′-h yd r oxy-2′,2′,6′-tr im eth ylcycloh exyl}-3-bu ten -2-
on e [(-)-15, 2.72 g, 8.00 m m ol, 34.8% fr om (-)-5]:
obtained as colorless crystals; mp 115.5-116.0 °C; [R]25
D
1
-30.2 (c 0.86, CH3OH); H NMR (300 MHz, CDCl3) δ
7.11 (1H, d, J ) 15.9 Hz, H-4), 6.38 (1H, d, J ) 15.7
Hz, H-3), 3.96 (1H, m, H-4′), 2.29 (3H, s, H-1), 2.00 (1H,
m, H-6′), 1.77-1.29 (5H, m, H-5′, H-3′, OH), 1.08 (3H,
s, CH3-2′), 0.90 [9H, s, CH3(tert-butyl)], 0.80 (3H, s, CH3-
2′), 0.79 (3H, d, J ) 6.4 Hz, CH3-6′), 0.07 (6H, s, CH3-
Si); 13C NMR (75 MHz, CDCl3) δ 197.8 (C-2), 146.9 (C-
3), 130.8 (C-4), 78.9 (C-1′), 67.0 (C-4′), 47.3 (C-3′), 41.8
(C-5′), 39.4 (C-2′), 36.3 (C-6′), 28.1 (C-1), 26.3 (CH3eq-C-
2′), 25.9 [CH3(tert-butyl)], 22.9 (CH3ax-C-2′), 18.2 [(CH3)3C-
Si], 15.8 (CH3-C-6′), -4.7 (CH3-Si).
Dep r otection of (-)-14. (E)-4-{(1′S,4′S,6′R)-4-[(tert-
Butyl)dimethylsiloxy]-1′-hydroxy-2′,2′,6′-trimethylcyclo-
hex-1-yl}-3-buten-2-on [(-)-14, 1.19 g, 3.50 mmol] was
dissolved in THF (25 mL) and Bu4NF (12.0 g, 38.1
mmol) dissolved in THF (60 mL) was added. After 18
h stirring at room temperature, H2O (50 mL) was added,
and the layers were separated. The aqueous layer was
washed exhaustively with Et2O (10 × 50 mL). The
Hu m a n Cell Lin e. The human cell line HT-29
derived from an isolated primary case of adenocarci-
noma was obtained from the American Type Culture
Collection (HTB 38). Cells were cultivated in MEM
supplemented with 10% heat-inactivated fetal calf
serum in T-25 flasks at 37 °C in an atmosphere of 5%
CO2 in air.
Bioa ssa ys. The test compounds were dissolved in
DMSO as a 100-fold stock solution leading to a final
DMSO concentration of 1% in the assay system. The
highest concentration of the test compounds was 100
µg/mL (500 µg/mL for tests with HT-29 cells), with

Synthesis and Activity of Boscialin
J ournal of Natural Products, 1998, Vol. 61, No. 5 597
dilution steps of three in the assays. The T. b. rhod-
esiense and HT-29 assays were performed as previously
described by Ra¨z et al.17 and Kaminsky et al.,18 respec-
tively. Briefly, trypanasomes (200 cells/100 µL) or HT-
29 cells (5000 cells/100 µL) were incubated in the
presence of various drug concentrations in 96-well
microtiter plates. After 72 h of drug exposure, the
minimum inhibitory concentration (MIC) was deter-
mined microscopically. MIC was defined as the lowest
concentration at which no cells with normal morphology
or motility could be found. Additionally, 50% inhibitory
concentrations (IC50) were determined by adding the
fluorochrome Alamar Blue (Laboserve). The plate was
incubated for further 2 h, then the fluorescence was
quantified with a Cyto Fluor plate reader (model 2300;
Millipore, Bedford, MA) and IC50 values of fluorescence
were calculated by linear interpolation.19 For the T.
cruzi assay, L6 cells (50 000 cells/500 µL) were incu-
bated in 48-well microtiter plates. After 24 h of incuba-
tion, the cells were infected with trypomastigotes from
the supernatant of stock cultures at a ratio of five
parasites per cell and incubated for another 48 h. The
parasites were then exposed to serial drug dilutions for
5 d in total, with a medium change with drug after 3 d.
A MIC was determined microscopically. The L. dono-
vani assay was performed as decribed by Neal et al.20
Isolated mouse peritoneal macrophages were cultivated
in tissue chamber slides (50 000 cells/500 µL) in RPMI
1640 plus 10% heat inactivated fetal calf serum. After
24 h of incubation they were infected with a suspension
of amastigotes from hamster spleen at a ratio of 10
amastigotes per macrophage. Again 24 h later, the
amastigote suspension was replaced by medium con-
taining serial dilutions of drug. After 5 d of drug
exposure with a medium change with drug after 3 d,
the macrophages were fixed with MeOH and stained
with Giemsa. The proportion of infected to uninfected
macrophages was determined microscopically and IC50
values were calculated.
105 colony-forming units per mL. The inoculated agar
(6 mL) was equally distributed on the surface of sterile
casein soymeal peptone agar plates (Petri dishes with
a diameter of 9 cm). After solidification of the agar
medium, circular holes with a diameter of 1 cm were
cut out, and 100 µL of the test substances (1 mg/mL in
H2O) were given in each hole. The plates were incu-
bated at 37 °C for 24 h. For the assessment of the
microbiostatic activity, the diameter of the inhibition
zone around the holes was measured.
Ack n ow led gm en t. The authors wish to thank the
Swiss National Science Foundation (grant No. 20-
41857.94) and the UNDP/World Bank/WHO Special
Program for Research and Training in Tropical Diseases
(TDR) for financial support. In addition, we are grateful
to Dr. R. Brun, Swiss Tropical Institute, Basel, Swit-
zerland, for letting us perform the measurements of
antiparasitic activity and cytotoxicity in his laboratory.
Refer en ces a n d Notes
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Aga r Diffu sion Test.21 Gram-positive bacteria (Sta-
phylococcus aureus ATCC 9144, Staphylococcus epider-
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and Corynebacterium minutissimum ATCC 23348),
Gram-negative bacteria (Escherichia coli NCTC 8196),
and a yeast (Candida albicans ATCC 10231) were used
as test organisms for a qualitative assessment of the
antimicrobial activity of the boscialin isomers. Over-
night cultures of the respective test strains in casein
peptone soymeal peptone medium (Merck) were diluted
in sterile 0.9% NaCl in distilled H2O and subsequently
added to molten casein peptone soymeal peptone agar
(Merck) resulting in a final concentration of 5 × 104-
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(17) Ra¨z, B.; Iten, M.; Grether-Bu¨hler, Y.; Kaminsky, R.; Brun, R.
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(19) Hills, M.; Hudson, C.; Smith, P. G. WHO Document WHO/ No.
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York, 1972.
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