Antineoplastic agents 440. Asymmetric synthesis and evaluation of the combretastatin A-1 SAR probes (1S,2S)- and (1R,2R)-1,2-dihydroxy-1-(2',3'-dihydroxy-4'-methoxyphenyl)-2-(3'' ,4'',5''-trimethoxyphenyl)-ethane

Article abstract of DOI:10.1021/np0000623

The synthetic (E)-isomer (3b) of natural combretastatin A-1 (1a) isolated from the African bushwillow Combretum caffrum was the focus of chiral hydroxylation (Sharpless) reactions as part of a structure-activity relationship study. The resulting (R,R)- and (S,S,)-diols (6 and 7) and synthetic intermediates were evaluated against a series of cancer cell lines, microorganisms, and tubulin. Chiral diols 6 and 7 showed increased activity against the P-388 murine lymphocytic leukemia cell line with ED₅₀ values of 3.9 and 2.9 μg/mL, respectively, when compared to the precursor (E)-stilbene 3b. In contrast, (E)-stilbene 3b exhibited more potent antibiotic activity than the chiral diols (6 and 7). Both diols, (R,R)-6 and (S,S)-7, displayed less cancer cell growth inhibition and less antibiotic activity than did natural combretastatin A-1 (1a) (P-388 ED₅₀ 0.25 μg/mL).

Full text of DOI:10.1021/np0000623

J . Nat. Prod. 2000, 63, 969-974
969
An tin eop la stic Agen ts 440. Asym m etr ic Syn th esis a n d Eva lu a tion of th e
Com br eta sta tin A-1 SAR P r obes (1S,2S)- a n d (1R,2R)-1,2-Dih yd r oxy-
1-(2′,3′-d ih yd r oxy-4′-m eth oxyp h en yl)-2-(3′′,4′′,5′′-tr im eth oxyp h en yl)-eth a n e‡
George R. Pettit,* J ohn W. Lippert III, Delbert L. Herald, Ernest Hamel,† and Robin K. Pettit
Cancer Research Institute and Department of Chemistry Arizona State University, Tempe, Arizona 85287-2404, and Screening
Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201
Received February 7, 2000
The synthetic (E)-isomer (3b) of natural combretastatin A-1 (1a ) isolated from the African bushwillow
Combretum caffrum was the focus of chiral hydroxylation (Sharpless) reactions as part of a structure-
activity relationship study. The resulting (R,R)- and (S,S,)-diols (6 and 7) and synthetic intermediates
were evaluated against a series of cancer cell lines, microorganisms, and tubulin. Chiral diols 6 and 7
showed increased activity against the P-388 murine lymphocytic leukemia cell line with ED50 values of
3.9 and 2.9 µg/mL, respectively, when compared to the precursor (E)-stilbene 3b. In contrast, (E)-stilbene
3b exhibited more potent antibiotic activity than the chiral diols (6 and 7). Both diols, (R,R)-6 and (S,S)-
7, displayed less cancer cell growth inhibition and less antibiotic activity than did natural combretastatin
A-1 (1a ) (P-388 ED50 0.25 µg/mL).
By 1979, our study of cancer cell growth inhibitory
constituents of the African willow tree Combretum caffrum
Kuntze (Combretaceae)1a was well under way, and, in 1982,
we reported the isolation and structure of combretastatin,1b,c
the first member of a series of biologically active bibenzyls,
stilbenes, and phenanthrenes.2a-c In 1987 and 1989, we
reported combretastatins A-1 (1a )2c and A-4 (1b)2d respec-
tively, which structurally resemble the antimitotic and cell
growth inhibitors colchicine3,5 and podophyllotoxin,4,5 and
represent two of the most promising antineoplastic con-
stituents of C. caffrum. Monophenol 1b proved to be a
potent inhibitor of microtubule assembly and exhibited an
ED50 value of 7 nM (0.007 µM) against the murine L1210
leukemia cell line,2a-d while diphenol 1a , which was equally
effective as an inhibitor of microtubule assembly (IC50 2-3
µM), was much less potent as an inhibitor of L1210 cell
growth (ED50 0.6 µM).
Presumably the reduced cytotoxicity to cancer cells
shown by diphenol 1a is due to the presence of its vicinal
phenolic groups, which can be easily oxidized to the 1,2-
quinone.2a,b,5,6 Such an interpretation was supported by the
fact that acetylation of the hydroxyl groups enhanced
cytotoxicity 10-fold, while significantly reducing the in vitro
inhibition of tubulin polymerization and colchicine binding.2a
Despite these superficial negative aspects, some biological
properties exhibited by diphenol 1a make it attractive. For
example, diphenol 1a may be the most potent antagonist
of colchicine binding known, with nearly a 99% inhibition
of binding at equal concentrations of inhibitor and [3H]
colchicine.1d,5 In addition, diphenol 1a was found to be more
potent than monophenol 1b in its ability to increase
intracellular daunorubicin concentrations in multidrug
resistant cancer cell lines.7 Most importantly, tubulin-
binding stilbenes 1a and 1b selectively elicit irreversible
vascular shutdown within solid tumors.8a The degree of
reduction ranged from 50% with 1a to 70% with 1b,8a while
the combretastatin A-4 prodrug (1c), a sodium phosphate
derivative of monophenol 1b, induced a complete vascular
shutdown within metastatic tumors at doses one-tenth of
the maximum tolerated dose.8b These very encouraging
results indicated that the A-series of combretastatin stil-
benes should be further investigated in respect to their
antiangiogenic and other anticancer properties. Hence, we
initiated a structure-activity relationship (SAR) study of
diphenol 1a to parallel a long-term investigation of the
combretastatin A-4 prodrug (1c).9-11
Resu lts a n d Discu ssion
Previous SAR analyses of the combretastatin A-series
indicated that the cis configuration of the stilbene unit is
the most important factor for inhibition of cancer cell
growth.2 With the corresponding (E)-stilbenes, inhibitory
effects on cancer cell growth and tubulin polymerization
drop precipitously from effects exhibited by the correspond-
ing (Z)-isomers.9 Initially, both the trans isomers 3b and
3d were found to have moderate inhibitory effects on cancer
cell growth. Later studies using trans-stilbene 3d revealed
that freshly prepared solutions in dimethyl sulfoxide were
inactive and gained activity only with the passage of time,
suggesting that the trans isomer is slowly converted to the
active cis isomer on storage.11 Bibenzyls 2a and 2b, which
contain an sp3-hybridized freely rotating ethane bridge,
exhibit a decrease in antineoplastic activity when compared
to the corresponding stilbenes. Recently, we improved the
anticancer activity of stilbene 1a and bibenzyl 2a , by
conversion to the sodium phosphate prodrugs 1d and 2b.12
Both the combretastatin A-1 (1d ) and B-1 (2b) prodrugs
exhibit approximately a tenfold increase in cancer cell
growth inhibition over their parent compounds, 1a and 2a
respectively.
The specific purpose of the present investigation was to
explore possible conversion of the inactive (E)-isomer of
combretastatin A-1 into a more active derivative. Recently,
we converted the (E)-isomer of combretastatin A-4 (3d ) to
chiral diols (R,R)-4 and (S,S)-5, where the (E)-olefin unit
was replaced by a freely rotating sp3-hybridized chiral
ethanediol.9a These experiments have now been extended
to include the (E)-isomer of combretastatin A-1, generating
* To whom correspondence should be addressed. Tel.: (480) 965-3351.
Fax: (480) 965-8558.
† Bldg. 469, Room 237, NCI-FCRDC, P.O. Box B, Frederick, MD 21702.
‡ Dedicated to Professor Norman R. Farnsworth on the occasion of his
70th birthday.
10.1021/np0000623 CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/30/2000

970 J ournal of Natural Products, 2000, Vol. 63, No. 7
Pettit et al.
chiral diols (R,R)-6 and (S,S)-7. Asymmetric dihydroxyla-
tion13 of trans-stilbene 3a 2c,12 with AD-mix-R (Sharpless
reagent: (DHQ)2PHAL, K3Fe(CN)6, K2CO3, K2OsO4‚2H2O)
afforded (S,S)-diol 8. Photoisomerization of the (E)-stilbene
was prevented using an aluminum foil-wrapped reaction
vessel.
F igu r e 1. Crystal structure (excluding hydrogens) of (S,S)-diol (8).
diol 8 with acetic anhydride afforded (S,S)-diacetate 10.
Selective desilylation of (S,S)-diacetate 10 with TBAF
proved to be difficult, yielding several products and sug-
gesting concurrent oxidation.14 Other methods for desily-
lation15 were investigated, but none was able to displace
TBAF as the desilyation agent of choice based on its
efficient reaction time (ca. 20 min). The (R,R)-diols silyl
ether 9, diacetate 11, and tetraol 6 were prepared analo-
gously using AD-mix-â (DHQD).
A comparison of the diols generated from (E)-olefin 3a
revealed a decrease in cancer cell growth inhibition from
that of combretastatin A-1 (1a ), which is consistent with
results obtained from the parallel set of diols prepared from
the (E)-isomer of combretastatin A-4. Interestingly (S,S)-
diol 5, a derivative of the trans isomer of combretastatin
A-4, is significantly more active than (R,R)-diol 4 in the
cancer cell lines employed (Table 1). In the combretastatin
A-1 series, on the other hand, the difference noted in cancer
cell line inhibition was negligible, and both (S,S)-diol 7 and
(R,R)-diol 6 were substantially less active than (S,S)-diol
5 from the A-4 series.
In previous studies,2d combretastatins A-4 (1b) and A-1
(1a ) had equivalent activities as inhibitors of tubulin
polymerization, while combretastatin A-1 appeared to be
somewhat more active as an inhibitor of the binding of
colchicine to tubulin. Precise analysis of the difference
between the two natural products was not possible at that
time, and the quantitative results obtained were uncertain
because of the marked instability of combretastatin A-1.
The synthesis of diols 4-7 made possible a reevaluation,
albeit indirect, of the relative importance of the second
hydroxyl group in the B-ring of the combretastatins. In
addition, we have resynthesized combretastatin A-1 and
have reevaluated its interactions with tubulin. Table 2
summarizes results obtained with the four diols as inhibi-
tors of tubulin assembly, with a contemporaneous com-
parison with combretastatins A-4 and A-1. The reaction
system currently used16 yields lower IC50 values than that
previously used2d but minimizes the occurrence of aberrant
polymerization reactions that often occur with agents that
bind to tubulin.17
As a check that the correct chirality was being induced
in the preparation of diols (R,R)-6 and (S,S)-7, a single-
crystal X-ray structure determination was conducted on
one of the intermediate silyl-protected products formed in
the AD-mix reactions. Thus, treatment of the trans-stilbene
3a with AD-mix-R yielded diol 8, which was expected to
have 1S,2S absolute stereochemistry. X-ray structure
determination of this product (which occurred as the
hemihydrate in the solid state) confirmed the expected
1S,2S stereochemistry. A computer-generated perspective
of the silylated diol 8 is shown in Figure 1. Although the
absolute stereochemistry of 8 was certain (Flack ø absolute
structure parameter ) 0.1080 with esd ) 0.0492), it was
apparent that considerable disorder was present in the
solid state of this compound, as evidenced by multiple
splitting occurrences for a number of the atoms present in
the unit cell. This resulted in the rather poor standard
crystallographic residual (R1 of 0.1496) obtained for this
structure determination. No attempt was made to further
refine the molecules over the multiple occupancy sites,
inasmuch as our main objective of confirming the absolute
stereochemistry had been met.
The data presented in Table 2 demonstrate that all four
diols are much less potent than 1a and 1b as inhibitors of
tubulin polymerization, in agreement with their much
reduced cytotoxicity toward human cancer cell lines. The
apparent preference of tubulin for (S,S)-diols 5 and 7 versus
Protected diol (S,S)-8 was next desilyated with tetrabu-
tylammonium fluoride (TBAF) to obtain the tetraol (S,S)-
diol 7 in moderate yield (59%). Reaction of silyl ether (S,S)-

Antineoplastic Agents 440
J ournal of Natural Products, 2000, Vol. 63, No. 7 971
Ta ble 1. Human Cancer Cell Line and Murine P-388 Lymphocytic Leukemia Inhibitory Activity of Combretastatins A-1, A-4, B-1,
and Synthetic Modifications
leukemia
P-388
compound ED50 µg/mL
pancreas-ca neuroblast thyroid ca lung-NSC pharynx-sq prostate
ovarian
CNS
renal
A498
colon
KM20L2
BXPC-3
SK-N-SH
SW1736
NCI-H460
FADU
DU-145
OVCAR-3 SF-295
GI50 µg/mL
1a a
0.2
4.4
0.19
3.1
0.74
0.23
0.17
1b
1c
1d
2a
0.0003
0.0004
<0.01
1.7
0.3
19.0
2.0
0.39
0.0003
0.0007
0.0006
0.029
0.038
0.0007
0.0008
<0.001 <0.001
0.061
0.34
0.53
0.023
0.024
0.036
0.036
0.041
1.5
0.034
2b
>10
>10
3.9
>10
>10
3.3
4.4
2.5
7.9
>10
2.7
3.5
2.1
>10
>10
2.0
4.5
0.38
7.2
>10
2.3
5.7
0.52
2.6
2.9
>10
8.2
0.52
7.0
(R,R)-4
(S,S)-5
(R,R)-6
(S,S)-7
1.5
0.28
3.1
6.1
1.8
>10
>10
2.1
0.60
4.9
>10
0.91
>10
>10
3.9
2.9
3.8
>10
>10
a
Resynthesis of diphenol 1a afforded an increase in biological activity from that previously reported in 1987.12
Ta ble 2. Inhibition of Tubulin Polymerization by Diols 4-7
and Combretastatin A-4 (1b)a
Ta ble 3. Antimicrobial Activity of Combretastatins A-1, A-4,
B-1, and Synthetic Modifications
inhibition of
colchicine binding
minimum inhibitory
concentration (µg/disk)
compound
microbe(s) inhibiteda
1a 9a
S. aureus
50-100
25-50
inhibitor:colchicine
inhibition of tubulin
(% inhibition ( SD)
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
S. aureus
S. pneumoniae
N. gonorrhoeae
S. maltophilia
C. albicans
polymerization
1b9a
1c9a
2a 9a
3b
25-50
compound
IC50 (µM) ( SD
0.2:1
1:1
50-100
12.5-25
50-100
50-100
6.25-12.5
50-100
50-100
50-100
1a
1b
(R,R)-4
(S,S)-5
(R,R)-6
(S,S)-7
1.1 ( 0.07
1.0 ( 0.05
>40
89 ( 3
81 ( 4
99.6 ( 0.7
98 ( 1
14 ( 2
20 ( 2
10 ( 2
a
In the polymerization experiments, the tubulin concentration
was 10 µM. In the colchicine binding experiments, the tubulin
concentration was 1.0 µM, and the [3H]-colchicine concentration
was 5.0 µM. See Verdier-Pinard et al.16 for further details. All data
with 1a were obtained with solutions prepared immediately before
the experiments were performed.
C. neoformans
3d 9a
b
(R,R)-49a
(S,S)-59a
(R,R)-6
(S,S)-7
S. pneumoniae
50-100
25-50
S. pneumoniae
b
N. gonorrhoeae
25-50
a
Each compound was screened against eight bacteria and two
fungi as described in the Experimental Section. Only microbes
inhibited by <100 µg/disk are listed. At 100 µg/disk, there was
no inhibition of the 10 bacteria and fungi tested.
the analogous (R,R)-diols 4 and 6 is clear. The (S,S)-
enantiomer is at least twice as inhibitory as the (R,R)-
enantiomer. Specifically addressing the question of the
importance of the second hydroxyl group in the combret-
astatin B ring, the results with both the (R,R) pair and
the (S,S) pair support the concept that the strength of the
combretastatin interaction with tubulin is enhanced by the
presence of the additional hydroxyl moiety. Curiously, the
addition of this hydroxyl enhances the interaction with
tubulin of the (R,R)-diol somewhat more (6 at least twice
as active as 4) than of the (S,S)-diol (7 only 40% more active
than 5). The greater difference between 4 and 5 than
between 6 and 7 in their interactions with tubulin may also
account for the greater difference between the former pair
and the latter in the cytotoxicity results. However, relative
affinities of the four diols for tubulin do not account for
the observation that 5 is the most cytotoxic of the four
compounds.
In addition, the data in Table 2 confirm the previously
observed relative activities of 1a and 1b. The differences
between the compounds are not great, but 1a appears to
be slightly more active as an inhibitor of colchicine binding.
Against microbe panels, the (E)-isomer of combretastatin
A-1 (3b) was the most active compound, presumably due
to its photoisomerization to the active cis isomer (Table 3).
The difference in antimicrobial activity between parent
compounds 1a and 1b and their diol derivatives was
unremarkable (Table 2).
b
marized may have important biological properties that will
eventually be ascertained.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Pyridine, acetic
anhydride, ethyl acetate (EtOAc), hexane, and CH2Cl2 (DCM)
were redistilled. TBAF, 4-(dimethylamino)pyridine (DMAP),
diisopropylethylamine, and tert-butyl alcohol were purchased
from the Fisher-Acros Chemical Co., and all other reagents
were purchased from the Sigma-Aldrich Chemical Co. Solvent
extracts of aqueous solutions were dried over anhydrous
sodium sulfate. Column chromatography was performed using
either flash Si gel (230-400 mesh ASTM) or gravity Si gel
(70-230 mesh ASTM). Analtech Si gel GHLF plates were used
for TLC, and all compounds were visualized with fluorescent
short-wave light (254 nm).
Melting points were recorded employing an Electrothermal
9100 apparatus and are uncorrected. The 1H NMR spectra
were determined by means of a Varian VXR-300 instrument
and are referenced to TMS as the internal standard. Mass
spectral data were recorded using a Varian MAT 312 instru-
ment (EIMS), or Vestec Lasertec Research mass spectrometer
incorporating a Laser Sciences nitrogen laser (337 nm light
pulses of 3-ns duration, with 4-hydroxybenzylidenemalononi-
trile as the matrix and cytochrome c as the external standard
for calibration purposes TOFMS).18 The IR spectra were
obtained with a Mattonson Instruments 2020 Galaxy Series
FTIR instrument. Optical rotation values were recorded
employing a Perkin-Elmer 241 polarimeter. The X-ray crystal
structure data collection was performed using an Enraf-Nonius
The premise that the (Z)-olefin geometry of the combre-
tastatin stilbenes plays an integral role in the overall
biological activity was further supported by the results of
this study. In view of the significant astrocyte reversal (9
ASK system) exhibited by the first member of this series,
(-)-combretastatin (2c),1a the chiral diols herein sum-

972 J ournal of Natural Products, 2000, Vol. 63, No. 7
Pettit et al.
Sch em e 1
Reagents and conditions: (a) AD-mix-â, CH3SO2NH2, t-BuOH-H2O, 0 °C; (b) AD-mix-R, CH3SO2NH2, t-BuOH-H2O, 0 °C; (c) TBAF, THF; (d) Ac2O, pyridine,
DMAP, DMF.
CAD4 diffractometer. Elemental analyses were determined by
Galbraith Laboratories, Inc., Knoxville, TN.
J ) 8.1 Hz, H-5′), 7.05 (1H, d, J ) 8.7 Hz, H-6′); EIMS m/z
594 (M+, 5), 519 (95), 397 (100), 339 (10), 198 (95); anal. C
59.70%, H 8.50%, calcd for C30H50O8Si2‚1/2H2O, C 59.80%, H
8.50%.
(1R,2R)-1,2-Dih yd r oxy-1-[2′,3′-d i(ter t-bu tyld im eth ylsi-
lyloxy)-4′-m eth oxyph en yl]-2-(3′′,4′′,5′′-tr im eth oxyph en yl)-
eth a n e (9). To a 100-mL foil-wrapped round-bottom flask
containing a magnetic stirbar was added distilled water (14
mL), tert-butyl alcohol (14 mL), AD-mix-â (3.8 g; 1.40 g per
mmol), and methanesulfonamide (0.27 g; 2.85 mmol). Before
being cooled to 0 °C, the mixture was stirred at room
temperature for several min. Some of the compounds began
to precipitate, and 2′,3′-bis(tert-butyldimethylsilyloxy)-3,4,4′,5-
tetramethoxy-(E)-stilbene (3a )12 (1.4 g; 2.54 mmol) was added
at once; the slurry was then stirred vigorously at 0 °C for 8 h
and at room temperature for 24 h. Sodium sulfite (3.9 g; 30.9
mmol; 12 eq) was added and the mixture stirred for an
additional 0.5 h. The product was extracted with EtOAc (4 ×
50 mL), and the combined organic phase was washed with 2
N KOH (aqueous) and dried. Evaporation of the solvent in
vacuo produced an off-yellow solid that was absorbed onto Si
gel and subjected to flash column chromatography (eluent
gradient: 9:1 to 1:1 hexane-EtOAc) to afford a clear oil, which
crystallized from hexane as a colorless fluffy solid (0.94 g; 74%
based on 0.23 g of 3a recovered): mp 122-123 °C; Rf 0.50 (1:
(1R,2R)-1,2-Dih yd r oxy-1-(2′,3′-d ih yd r oxy-4′-m et h oxy-
p h en yl)-2-(3′′,4′′,5′′-tr im eth oxyp h en yl)-eth a n e (6). To a
solution of (R,R)-diol 9 (0.24 g; 0.397 mmol) in anhydrous THF
(4 mL), was added TBAF (0.84 µL; 0.84 mmol; 2.1 eq; 1 M in
THF solution) in a previously flame-dried flask. After 20 min
the reaction was terminated with ice-cold 6 N HCl and
extracted with EtOAc (4 × 20 mL). The combined organic
phase was washed with saturated NaCl (aqueous) and dried.
Removal of the organic solvent yielded a dark oil, which was
subjected to column chromatography (66:33:1 EtOAc-hexane-
HOAc) to afford a light brown oil that crystallized from
EtOAc-hexane as a colorless solid (75 mg; 52%): mp 64-66
°C; Rf 0.36 (66:33:1, hexane-EtOAc-HOAc); [R]25D +56° (c 1.1,
CHCl3); IR (film) νmax 3425, 2939, 1593, 1454, 1327, 1234, 1145
1
cm-1; H NMR (300 MHz, CDCl3) δ 3.70 (6H, s, 2 × OCH3),
3.79 (3H, s, OCH3), 3.80 (3H, s, OCH3), 4.67 (1H, d, J ) 7.8
Hz, CH), 4.83 (1H, d, J ) 7.2 Hz, CH), 6.12 (1H, d, J ) 8.7
Hz, H-5′), 6.24 (1H, d, J ) 9.0 Hz, H-6′), 6.33 (2H, s, H-2′′,
H-6′′); EIMS m/z 348 (M+ - H2O, 60), 319 (95), 196 (100), 167
(10), 153 (25); anal. C 57.59%, H 6.18%, calcd for C18H22O8‚
1/2H2O, C 57.41%, H 6.25%.
1, hexane-EtOAc); [R]25 +46° (c 1.2, CHCl3); IR (Nujol) νmax
D
3526, 1595, 1251, 1124, 1097, 831, 781 cm-1
;
1H NMR (300
MHz, CDCl3) δ -0.16 (3H, s, SiCH3), -0.050 (3H, s, SiCH3),
0.040 (3H, s, SiCH3), 0.17 (3H, s, SiCH3), 0.89 (9H, s, C4H9),
1.01 (9H, s, C4H9), 2.45 (1H, d, J ) 1.7 Hz, OH, D2O
exchanged), 2.89 (1H, d, J ) 1.2 Hz, OH, D2O exchanged), 3.72
(6H, s, 2 × OCH3), 3.75 (3H, s, OCH3), 3.76 (3H, s, OCH3),
4.66 (1H, dd, J ) 7.5 Hz, J ) 2.4 Hz, CH), 5.06 (1H, dd, J )
7.8 Hz, J ) 3.3 Hz, CH), 6.33 (2H, s, H-2′′, H-6′′), 6.59 (1H, d,
(1S,2S)-1,2-Dih yd r oxy-1-[2′,3′-d i(ter t-bu tyld im eth ylsi-
lyloxy)-4′-m eth oxyph en yl]-2-(3′′,4′′,5′′-tr im eth oxyph en yl)-
eth a n e (8). The asymmetric dihydroxylation reaction condi-
tions described above for preparation of (R,R)-diol 9 were
applied to (E)-olefin 3a (1.56 g; 2.78 mmol) using AD-mix-R to
afford (S,S)-diol 8 as a colorless fluffy solid (1.2 g; 75%): mp
122-123 °C; Rf 0.50 (1:1, hexane-EtOAc); [R]25 -46° (c 1.3,
D

Antineoplastic Agents 440
J ournal of Natural Products, 2000, Vol. 63, No. 7 973
CHCl3); IR (Nujol) νmax 3524, 2361, 1593, 1251, 1124, 1097,
(1R,2R)-1,2-Dia cetoxy-1-[2′,3′-d i(ter t-bu tyld im eth ylsi-
lyloxy)-4′-m eth oxyph en yl]-2-(3′′,4′′,5′′-tr im eth oxyph en yl)-
eth a n e (11). To a solution of (R,R)-diol 9 (0.21 g; 0.351 mmol)
in anhydrous DCM (2 mL) was added acetic anhydride (26 mL;
2.7 mmol; 7.8 eq), pyridine (0.20 mL; 2.35 mmol; 6.7 eq), and
a catalytic amount of DMAP (5 mg). After 3 h, the reaction
was terminated with ice-water and extracted with EtOAc (4
× 25 mL). The combined organic phase was washed with 2 N
HCl followed by 10% NaHCO3 (aqueous) and dried. Removal
(reduced pressure) of solvent yielded a clear oil that was
separated by flash column chromatography (1:1 hexane-
EtOAc) to afford a crude product, which crystallized from
hexane as a colorless solid (0.24 g; 99%): mp 128-129 °C; Rf
954, 831 cm-1 1H NMR (300 MHz, CDCl3) δ -0.16 (3H, s,
;
SiCH3), -0.05 (3H, s, SiCH3), 0.04 (3H, s, SiCH3), 0.17 (3H, s,
SiCH3), 0.89 (9H, s, C4H9), 1.01 (9H, s, C4H9), 2.44 (1H, d, J )
3.3 Hz, OH, D2O exchanged), 2.87 (1H, d, J ) 1.2 Hz, OH,
D2O exchanged), 3.72 (6H, s, 2 × OCH3), 3.75 (3H, s, OCH3),
3.76 (3H, s, OCH3), 4.66 (1H, dd, J ) 7.8 Hz, J ) 2.1 Hz, CH),
5.06 (1H, dd, J ) 7.5 Hz, J ) 3.3 Hz, CH), 6.33 (2H, s, H-2′′,
H-6′′), 6.59 (1H, d, J ) 8.7 Hz, H-5′), 7.06 (1H, d, J ) 8.4 Hz,
H-6′); EIMS m/z 594 (M+, 10), 519 (95), 397 (100), 339 (20),
198 (100); anal. C 59.70%, H 8.50%, calcd for C30H50O8Si2‚
1/2H2O, C 59.66%, H 8.61%.
X-r a y Cr ysta l Str u ctu r e of (1S,2S)-1,2-Dih yd r oxy-1-
[2′,3′-bis(ter t-bu tyldim eth ylsilan yloxy)-4′-m eth oxyph en yl]-
2-(3′′,4′′,5′′-tr im eth oxyp h en yl)-eth a n e h em ih yd r a te (8). A
thin plate, 0.40 × 0.40 × 0.06 mm, obtained from a MeOH-
hexane solution, was mounted on the tip of a glass fiber with
Super Glue. Data collection was performed at 27 ( 1° for a
monoclinic system, with all reflections corresponding to slightly
more than a complete quadrant (2θ e 130°) being measured
using an ω/2θ scan technique. Friedel reflections were also
collected, whenever possible, immediately after each original
reflection. Subsequent statistical analysis of the complete
reflection data set using the XPREP19 program indicated the
space group was P21. Each asymmetric unit of the cell was
found to contain four independent molecules of the parent
molecule, as well as two molecules of water. Crystal data:
0.63 (2:1, hexane-EtOAc); [R]25 -22° (c 1.4, CHCl3); EIMS
D
m/z 678 (M+, 5), 621 (40), 439 (60), 397 (100), 73 (30); anal. C
60.14%, H 8.01%, calcd for C34H54O10Si2, C 60.38%, H 8.09%.
(1S,2S)-1,2-Dia cet yoxy-[2′,3′-d i-(ter t-b u t yld im et h ylsi-
lyloxy)-4′-m eth oxyph en yl]-2-(3′′,4′′,5′′-tr im eth oxyph en yl)-
eth a n e (10). The silyl ether (S,S)-diol 8 (0.25 g; 0.42 mmol)
was acylated as described above for the synthesis of (R,R)-
diacetate 11 to afford a colorless solid (0.28 g; 98%): mp 128-
129 °C; Rf 0.63 (2:1, hexane-EtOAc); [R]25D +20° (c 1.3, CHCl3);
TOFMS m/z 717, (M + K)+; anal. C 60.14%, H 8.01%, calcd
for C34H54O10Si2, C 60.08%, H 8.08%.
Tu bu lin Assa ys. The tubulin polymerization and colchicine
binding experiments were performed as described previously.16
However, in the polymerization assays Beckman DU7400/7500
spectrophotometers equipped with “high-performance” tem-
perature controllers were used. These instruments are micro-
processor controlled, and assays required use of programs
provided by MDB Analytical Associates, South Plainfield, NJ .
An tim icr obia l Su scep tibility Testin g. Compounds were
screened against the bacteria Stenotrophomonas maltophilia,
Micrococcus luteus, Staphylococcus aureus, Escherichia coli,
Enterobacter cloacae, Enterococcus faecalis, Streptococcus pneu-
moniae, and Neisseria gonorrhoeae, and the fungi Candida
albicans and Cryptococcus neoformans, according to estab-
lished disk susceptibility testing protocols.22
C
30H50O8‚1/2H2O, a ) 13.303(2), b ) 32.475(7), c ) 19.960(4)
Å, V ) 7179(3) Å3, λ(Cu KR) ) 1.54178 Å, Fc ) 1.117 g cm-3
for Z ) 8 and fw ) 603.89, F(000) ) 2616. After Lorentz and
polarization corrections, merging of equivalent reflections and
rejection of systematic absences, 19 017 unique reflections
[R(int) ) 0.1195] remained, of which 13 433 were considered
observed [Io > 2σ(Io)] and were used in the subsequent
structure solution and refinement. Linear and anisotropic
decay corrections were applied to the intensity data as well
as an empirical absorption correction (based on a series of
ψ-scans).20 Structure determination was accomplished with
SHELXS.19 All non-hydrogen atoms for 8, including the water-
solvate atoms, were located using the default settings of that
program. The remaining hydrogen atom positions were cal-
culated at optimal positions. The latter atoms were assigned
thermal parameters equal to 1.2 or 1.5 (depending upon
structural atom type) of the Uiso value of the atom to which
they were attached, and then both coordinates and thermal
values were forced to ride that atom during final cycles of
refinement. All non-hydrogen atoms were refined anisotropi-
cally in a full-matrix least-squares refinement process with
SHELXL19 in the SHELXTL-PC software package. The final
standard residual R value for the model shown in Figure 1
was 0.1484 for observed data (13 433 reflections) and 0.1850
for all data (19 017 reflections). The corresponding Sheldrick
R values were wR2 of 0.3706 and 0.4031, respectively. The
Flack absolute structure parameter ø was 0.05 (5) for the
model depicted in Figure 1, thus indicating that the absolute
stereochemistry shown (and expected) for 8 is correct (i.e., 1S,
2S). A final difference Fourier map showed residual electron
density attributed solely to the silicon atoms; the largest
difference peak and hole being 0.61 and -0.55 e/Å3, respec-
tively. Final bond distances and angles were all within
acceptable limits.21
Ack n ow led gm en t. Thanks and appreciation for support
of this research are directed to Outstanding Investigator Grant
CA 44344-6-11 with the Division of Cancer Treatment and
Diagnosis, NCI, DHHS; the Arizona Disease Control Research
Commission; Diane Cummings Halle; Gary L. and Diane R.
Tooker; Polly Trautman; the Caitlin Robb Foundation, and the
Robert B. Dalton Endowment. We are pleased to thank for
other assistance Drs. Cherry L. Herald, Fiona Hogan, J ean
M. Schmidt, and J . Charles Chapuis, as well as Laura Crews
and Lee Williams.
Su p p or tin g In for m a tion Ava ila ble: X-ray data for 8. This
material is available free of charge via the Internet at http://
acs.pubs.org.
Refer en ces a n d Notes
(1) (a) Pettit, G. R.; Singh, S. B.; Cragg, G. M. J . Org. Chem. 1985, 50,
3404-3406 and Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt,
J . M.; Lohavanijaya, P. Can. J . Chem. 1982, 60, 1374-1376. (b)
Hutchings, A.; Scott, A. H.; Lewis, G.; Cunningham, A. B. Zulu
Medicinal Plants, an Inventory; University of Natal Press: South
Africa, 1996; p 214. (c) Pettit, G. R.; Cragg, G. M.; Singh, S. B. J .
Nat. Prod. 1987, 50, 386-391. (d) Pettit, R. K.; Hamel, E.; Hazen,
K.; Pettit, G. R.; Crews, L. C. Antimicrob. Agents Chemother., in
preparation.
(2) (a) Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.;
Schmidt, J . M.; Hogan, F. J . Med. Chem. 1995, 38, 1666-1672. (b)
Hamel, E. Med. Res. Rev. 1996, 16, 207-231. (c) Pettit, G. R.; Singh,
S. B.; Niven, M. L.; Hamel, E.; Schmidt, J . M. J . Nat. Prod. 1987, 50,
119-131. (d) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts,
D. S.; Garcia-Kendall, D. Experientia 1989, 45, 209-211, and Lin,
C. M.; Singh, S. B.; Ping, S. C.; Dempcy, R. O.; Schmidt, J . M.; Pettit,
G. R.; Hamel, E. Mol. Pharm. 1988, 34, 200-208. (e) Pettit, G. R.;
Singh, S. B.; Schmidt, J . M.. Nat. Prod. 1988, 51, 517-527. (f) Nandy,
P.; Samitendu, B.; Gao, H.; Hui, M. B. V.; Lien, E. J . Pharm. Res.
1991, 8, 776-781. (g) Roberson, R. W.; Tucker, B.; Pettit, G. R. Mycol.
Res. 1998, 102, 378-382.
(1S,2S)-1,2-Dih ydr oxy-1-(2′,3′-dih ydr oxy-4′-m eth oxyph e-
n yl)-2-(3′′,4′′,5′′-tr im eth oxyp h en yl)-eth a n e (7). Silyl ether
(S,S)-diol 8 (0.36 g; 0.610 mmol) was desilylated as described
above for the synthesis of (R,R)-diol 6 and afforded (S,S)-diol
7 as a colorless solid (0.13 g; 59%): mp 64-66 °C; Rf 0.36 (66:
33:1 hexane-EtOAc-HOAc); [R]25 -52° (c 0.99, CHCl3); IR
D
(film) νmax 3418, 2939, 1593, 1510, 1462, 1327, 1290, 1234, 1124
1
cm-1; H NMR (300 MHz, CDCl3) δ 3.73 (6H, s, 2 × OCH3),
3.81 (3H, s, OCH3), 3.83 (3H, s, OCH3), 4.70 (1H, d, J ) 7.2
Hz, CH), 4.88 (1H, d, J ) 7.2 Hz, CH), 6.15 (1H, d, J ) 8.1
Hz, H-5′), 6.27 (1H, d, J ) 8.1 Hz, H-6′), 6.37 (2H, s, H-2′′,
H-6′′); EIMS m/z 348 (M+ - H2O, 20), 319 (15), 196 (100), 168
(20), 153 (10); anal. C 57.59%, H 6.18%, calcd for C18H22O8‚
1/2H2O, C 57.61%, H 6.28%.
(3) Boye, O.; Brossi, A. In The Alkaloids; Brossi, A., Cordell, G. A., Eds.;
Academic: New York, 1992; Vol. 41, pp 125-178.
(4) Bush, E. J .; J ones, D. W. J . Chem. Soc., Perkin Trans. 1 1995, 151-
155.

974 J ournal of Natural Products, 2000, Vol. 63, No. 7
Pettit et al.
(5) Sackett, D. L. Pharmacol. Ther. 1993, 59, 163-228.
(f) Xie, L.; Takeuchi, Y.; Cosentino, L. M.; Lee, K. H. Biorg. Med.
Chem. Lett. 1998, 8, 2151-2156. (g) Trost, B. M.; Lee, C. B. J . Am.
Chem. Soc. 1998, 120, 6818-6819. (h) Sinha, S. C.; Sinha, A.; Sinha,
S. C.; Keinan, E. J . Am. Chem. Soc. 1998, 120, 4017-4018. (i)
Marshall, J . A.; Hinkle, K. W. Tetrahedron Lett. 1998, 39, 1303-
1306.
(6) (a) Pelizzoni, F.; Verotta, L.; Rogers, C. B.; Colombo, R.; Pedrotti, B.;
Balconi, G.; Erba, E.; D’Incalci, M. Nat. Prod. Lett. 1993, 1, 273-
280. (b) Orsini, F.; Pelizzoni, F.; Bellini, B.; Miglierini, G. Carbohydr.
Res. 1997, 301, 95-109.
(7) McGown, A. T.; Fox, B. W. Cancer Chemother. Pharmacol. 1990, 26,
79-81.
(14) Haines, A. H. In Methods for Oxidation of Organic Compounds;
Academic: New York, 1988; Vol. 2, pp 305-323, 438-447.
(15) (a) Sinhababu, A. K.; Kawase, M.; Borchardt, R. T. Synthesis 1988,
710-711. (b) Nelson, T. D.; Crouch, D. Synthesis 1996, 1031-1069.
(16) Verdier-Pinard, P.; Lai, J .-Y.; Yoo, H.-D.; Yu, J .; Marquez, B.; Nagle,
D. G.; Nambu, M.; White, J . D.; Falck, J . R.; Gerwick, W. H.; Day, B.
W.; Hamel, E. Mol. Pharmacol. 1998, 53, 62-76.
(17) (a) Hamel, E.; Blokhin, A. V.; Nagle, D. G.; Yoo, H.-D.; Gerwick, W.
H. Drug Dev. Res. 1995, 34, 110-120. (b) Bai, R.; Durso, N. A.;
Sackett, D. L.; Hamel, E. Biochemistry 1999, 38, 14302-14310.
(18) Brune, D. C.; Cheung, J . Proceedings of the 45th ASMS Conference
on Mass Spectrometry and Allied Topics, Palm Springs, CA, J une
1-5, 1997.
(19) SHELXTL-PC, Version 5.1, 1997, an integrated software system for
the determination of crystal structures from diffraction data, Bruker
Analytical X-ray Systems, Inc.: Madison, WI, 1997. This package
includes, among others: XPREP, an automatic space group determi-
nation program; XS, the Bruker SHELXS module for the solution of
X-ray crystal structures from diffraction data; XL, the Bruker
SHELXL module for structure refinement; XP, the Bruker interactive
graphics display module for crystal structures.
(8) (a) Chaplin, D. J .; Pettit, G. R.; Parkins, C. S.; Hill, S. A. Br. J . Cancer
1996, 74, S86-S88. (b) Dark, G. G.; Hill, S. A.; Prise, V. E.; Tozer, G.
M.; Pettit, G. R.; Chaplin, D. J . Cancer Res. 1997, 57, 1829-1834.
(9) (a) Pettit, G. R.; Toki, B. E.; Herald, D. L.; Boyd, M. R.; Hamel, E.;
Pettit, R. K.; Chapuis, J .-C. J . Med. Chem. 1999, 42, 1459-1465. (b)
Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Chaplin, D. J .; Stratford,
M. R. L.; Hamel, E.; Pettit, R. K.; Chapuis, J .-C.; Oliva, D. Anti-Cancer
Drug Des. 1998, 13, 981-993. (c) Pinney, K. G.; Bounds, A. D.;
Dingeman, K. M.; Mocharla, V. P.; Pettit, G. R.; Bai, R.; Hamel, E.
Bioorg. Med. Chem. Lett. 1999, 9, 1081-1086.
(10) (a) Hatanaka, T.; Fujita, K.; Ohsumi, K.; Nakagawa, R.; Fukuda, Y.;
Nihei, Y.; Suga, Y.; Akiyama, Y.; Tsuji, T. Bioorg. Med. Chem. Lett.
1998, 8, 3371-3374. (b) Ohsumi, K.; Hatanaka, T.; Fujita, K.;
Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.;
Akiyama, Y.; Tsuji, T. Bioorg. Med. Chem. Lett. 1998, 8, 3153-3158.
(c) Ohsumi, K.; Nakagawa, R.; Yumiko, F.; Hatanaka, T.; Morinaga,
Y.; Nihei, Y.; Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. J . Med.
Chem. 1998, 41, 3022-3032. (d) Medarde, M.; Ramos, A.; Caballero,
E.; Pelaez-Lamamie de Clairac, R.; Lopez, J . L.; Gravalos, D. G.;
Feliciano, A. S. Eur. J . Med. Chem. 1998, 33, 71-77.
(11) (a) Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.;
Lin, C. M.; Hamel, E. J . Med. Chem. 1991, 34, 2579-2588. (b)
Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H.-M.; Lin, C. M.;
Hamel, E. J . Med. Chem. 1992, 35, 2293-2306.
(12) Pettit, G. R.; Lippert, J . W., III. Anti Cancer Drug Des. in press.
(13) (a) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev.
1994, 94, 2483-2547. (b) Sharpless, K. B.; Amberg, W.; Bennani, Y.
L.; Crispino, G. A.; Hartung, J .; J eong, K.-S.; Kwong, H.-L.; Morikawa,
K.; Wang, Z.-M.; Xu, D.; Zhang, X.-L. J . Org. Chem. 1992, 57, 2768-
2771. (c) Harcken, C.; Bruckner, R.; Rank, E. Chem.-Eur. J . 1998,
4, 2342-2352. (d) Hajamis, U. D.; Gadre, J . N.; Pednekar, S. Indian
J . Chem., Sect. B 1998, 37, 925-928. (e) Gypser, A.; Michel, D.;
Nirschl, D. S.; Sharpless, K. B. J . Org. Chem. 1998, 63, 7322-7327.
(20) North, A. C.;Phillips, D. C.; Mathews, F. S. Acta Crystallogr. 1968,
A24, 351-359.
(21) Crystallographic data for the structure(s) reported in this paper have
been deposited with the Cambridge Crystallographic Data Centre.
Copies of the data can be obtained, free of charge, on application to
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
+44-(0)1223-336033 or e-mail: deposit@ccdc,cam.ac.uk).
(22) National Committee for Clinical Laboratory Standards 1997. Perfor-
mance standards for antimicrobial disk susceptibility tests-sixth
edition: Approved Standard M2-A6, NCCLS, Wayne, PA.
NP0000623