Antineoplastic agents. 550. Synthesis of 10b(S)-epipancratistatin from (+)-narciclasine

Article abstract of DOI:10.1021/np068046e

By means of a five-step reaction sequence, narciclasine (2a), isolated from Narcissus sp., was converted to 10b(S)-epipancratistatin (3a) in 5.7% overall yield. The key step entailed a radical-initiated 10b,1 C-O cleavage employing tributyltin hydride to yield a B/C cis ring juncture (3b). Biological evaluation of 10b(S)-epipancratistatin (3a) provided evidence that antineoplastic activity was reduced by a factor of 10 when the B/C trans juncture was replaced with a B/C cis ring juncture.

Full text of DOI:10.1021/np068046e

J. Nat. Prod. 2007, 70, 417-422
417
,1
Antineoplastic Agents. 550. Synthesis of 10b(S)-Epipancratistatin from (+)-Narciclasine
George R. Pettit,* Noeleen Melody, Delbert L. Herald, John C. Knight, and Jean-Charles Chapuis
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State UniVersity, P.O. Box 872404,
Tempe, Arizona 85287-2404
ReceiVed September 15, 2006
By means of a five-step reaction sequence, narciclasine (2a), isolated from Narcissus sp., was converted to 10b(S)-
epipancratistatin (3a) in 5.7% overall yield. The key step entailed a radical-initiated 10b,1 C-O cleavage employing
tributyltin hydride to yield a B/C cis ring juncture (3b). Biological evaluation of 10b(S)-epipancratistatin (3a) provided
evidence that antineoplastic activity was reduced by a factor of 10 when the B/C trans juncture was replaced with a
B/C cis ring juncture.
In 1984 we reported isolation of a new antineoplastic isocar-
bostyril, designated pancratistatin (1), from the Amaryllidaceae plant
Hymenocallis littorale2a (a.k.a. Pancratium littorale). Initial in ViVo
evaluations of pancratistatin (1a) against the murine P388 lym-
phocytic leukemia and M5076 ovary sarcoma cell lines led to
significant inhibition of these U.S. National Cancer Institute in ViVo
systems that exhibited 38-106% (0.75-12.5 mg/kg dose range)
and 53-84% (0.38-3.0 mg/kg dose) life extensions, respectively.
Because of the sparingly soluble properties and resistance to stable
salt formation based on the phenol group, preclinical development
of pancratistatin (1a) was delayed until we were able to synthesize
promising phosphate prodrug modifications.2b-e Meanwhile, a variety
of biological studies focused on pancratistatin have revealed an
important selection of promising activities that include potent cancer
vascular disruption (vascular shutdown complete within 2 h of
treatment),3 RNA antiviral,4 antimicrosporidium,5 and good stability
(at least 15 days at rt) in human serum.6 Such encouraging properties
have led us and others to pursue efficient syntheses7 and structural
modifications8 of pancratistatin (1a).
Results and Discussion
As part of our research focused on utilizing the more readily
available (from Narcissus sp.) narciclasine (2a) for a practical
synthetic conversion to isocarbostyril 1a7 and for SAR studies, we,
herein, summarize its conversion to the structural modification 10b-
(S)-epipancratistatin (3a). Initially, the synthetic approach pursued
was the deoxygenation of the tertiary benzylic alcohol group of
10b(R)-hydroxypancratistatin (4).8c Conventional synthetic ap-
proaches involving the dehydration of the 10b-tertiary alcohol
followed by hydrogenation were expected to introduce the pos-
sibility of altering the configuration of the hydroxyl group at C-1.
Other methods involving the reduction of a suitable alcohol
derivative would be affected by steric hindrance and the possibility
of elimination to the dehydro derivative. Therefore, radical-
promoted deoxygenation of the 10b-tertiary alcohol was chosen as
an attractive route to (+)-pancratistatin. Instead, this led to formation
of the cis ring juncture isomer of pancratistatin (3a), which proved
useful in our overall SAR evaluations.
Based on the pioneering investigations of Barton,9 radical
deoxygenation of the cyclic thiocarbonate (5a)8c was planned using
Bu3SnH. The more stable radical should be formed, followed by
deoxygenation at the tertiary position. Attempts at synthesizing the
cyclic thionocarbonate from the partially acetylated diol (4a) by
Dedicated to the late Dr. Kenneth L. Rinehart of the University of
Illinois at Urbana-Champaign for his pioneering work on bioactive natural
products.
* To whom correspondence should be addressed. Tel: (480) 965-3351.
Fax: (480) 965-8558. E-mail: bpettit@asu.edu.
treatment with thiocarbonyldiimidazole10 in THF was challenging
in that the 7-O-acetate proved to be unstable. Difficult-to-separate
10.1021/np068046e CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/09/2007

418 Journal of Natural Products, 2007, Vol. 70, No. 3
Pettit et al.
Table 1. Human Cancer Cell Line and Murine P-388 Lymphocytic Leukemia Inhibitory Activities of Structures 1, 2a-c, 3a,b, 4a-c,
5b, 6, and 7b
ED50 (µg/ml)
GI50 (µg/mL)
leukemia
P-388
pancreas-a
BXPC-3
breast
MCF-7
CNS
SF268
lung-NSC
NCIH460
colon
KM20L2
prostrate
DU-145
compound
1
0.017
0.0012
0.027
0.013
0.14
>1
0.02
0.0035
0.16
0.011
0.32
>1
0.023
0.0032
0.088
0.060
0.27
>1
2.0
5.2
0.014
0.0031
0.055
0.16
0.26
>1
0.032
0.0084
0.28
0.033
0.23
>1
0.025
0.0032
0.043
0.051
0.31
>1
0.015
0.0032
0.023
0.028
0.21
>1
2a
2b
2c
3a
3b
3c
4a
4b
4c
5b
6
0.20
1.7
1.8
3.0
1.3
1.8
1.4
14.4
>10
>10
>1
9.9
>10
>10
>10
>1
3.6
4.3
>10
>10
>10
>1
>10
>10
>1
>10
>10
>1
>10
>10
>1
8.9
>1
1.5
3.1
4.9
7.5
2.9
2.8
1.5
7b
>10
>10
>10
>10
>10
>10
>10
Scheme 1
mixtures of the tetraacetate (5a) and triacetate (5b) were obtained.
Because the tetraacetate of narciclasine (2b) was found to cause a
contact dermatitis, it was decided to use narciclasine triacetate (2c)
prepared by acetylating narciclasine employing 3.25 equiv of acetic
anhydride and separating it from the resulting mixture of acetates
using column chromatography. As part of the study, triacetate (2c)
was also dihydroxylated using OsO4 to yield diol 4b.
Attempted synthesis of the cyclic thionocarbonate (5b) from 4b
was next carried out in various solvents, such as tetrahydrofuran,
dimethylformamide, and dioxane, but the reaction did not go to
completion and/or a number of products were obtained. The best
results were realized with the method developed by Kutney and
colleagues11 using butan-2-one as the solvent and heating to 80 °C
to afford thionocarbonate (5b) in 72% yield. Recrystallization from
dichloromethane-ethanol yielded crystals suitable for X-ray analy-
sis, and the resulting crystal structure was used for structure
confirmation. When cyclic thionocarbonate (5b) was treated with
Bu3SnH and AIBN in toluene at reflux, the cis isomer (3b) was
obtained in 50% yield, along with the cyclic carbonate (6) in 22%
yield. The carbonate, recrystallized from dichloromethane-ethanol,
yielded crystals suitable for X-ray crystallography, which was
employed to confirm its structure and stereochemistry.
product was a mixture of oxalates, and trioxalate (7b) was separated
by crystallization (29% yield). When the methyl oxalate mixture
was passed through a silica gel column using a mixture of 9:1
1
dichloromethane-methanol as eluent, an H NMR of the fraction
recovered revealed a mixture predominately consisting of the tertiary
monomethyloxalate derivative (68% yield). Bu3SnH reduction of
the pure trioxalate or of the oxalate mixtures proceeded to yield
the cis isomer (3c) as the major product. The dehydro product (8)
and a trace amount of the pancratistatin tetraacetate derivative (1b)
were also observed in the reaction product by 1H NMR. In a final
HPLC comparison experiment using pure pancratistatin tetraacetate
as a reference compound, the presence of that compound in the
reaction product was confirmed. When an excessive amount of
AIBN was added to the reaction mixture, the yield of cis ring
juncture increased, the amount of dehydro decreased considerably
to a trace quantity, and no pancratistatin tetraacetate could be
detected in the proton NMR of the reaction products. Further
experiments directed at improving the yield of pancratistatin
tetraacetate from the potentially very efficient conversion of (+)-
narciclasine to natural pancratistatin should eventually be devised.
Evaluation of 10b(S)-epipancratistatin (3a) against the P388
lymphocytic leukemia cell line and a minipanel of human cancer
cell lines (Table 1) showed that the epimerization at ring juncture
position 10b reduced antineoplastic activity by a factor of 10.13a
Preliminary evaluation13b of isomer 3a and a selection of synthetic
intermediates for antibacterial and/or antifungal activity did not
provide any significant results.
A potential synthetic route to natural (+)-pancratistatin (1a) was
also evaluated and involved reduction of the tertiary methyloxalate12
(7a) using Bu3SnH (AIBN as radical initiator) in toluene. Here it
was assumed that methyl oxalyl chloride would react with the
hindered alcohol of 4c to form methyl oxalate (7a). However, the

Synthesis of 10b(S)-Epipancratistatin from (+)-Narciclasine
Journal of Natural Products, 2007, Vol. 70, No. 3 419
Scheme 2
Experimental Section
silica gel (eluent: gradient 97:3 f 9:1 DCM-CH3OH) yielded a white
solid (0.92 g, 85% yield). Recrystallization from MeOH gave colorless
General Experimental Procedures. Solvents were purified by
redistillation, and in addition, tetrahydrofuran was distilled from sodium
benzophenone and pyridine was dried over KOH pellets and redistilled
prior to use. Organic extracts of aqueous solutions were dried using
anhydrous Na2SO4. Thin-layer chromatography was performed on
Analtech silica gel GHLF plates eluting with the solvents indicated.
Visualization was provided by UV lamp and development with a ceric
sulfate spray/heat. Flash column chromatography was conducted with
Merck silica gel 60 slurry packed in columns with the initiating solvent.
Optical rotation values were recorded using a Perkin-Elmer 241
polarimeter. HPLC was performed with an analytical ZorbaxSB-C18
column (4.6 mm × 25 cm) and a semipreparative ZorbaxSB-C18
column (9.4 × 25 cm) using a Waters Co. Delta 600 HPLC with dual
UV detectors. Melting points are uncorrected and were observed using
an Electrothermal 9100 capillary and Fisher-Johns melting point
apparatus. Nuclear magnetic resonance spectra were acquired at either
300 or 500 MHz for 1H and 75 and 125 MHz for 13C employing Varian
Gemini 300 MHz and Varian Unity 500 MHz instruments. The mass
spectra were determined using a JEOL LCmate magnetic sector in APCI
mode with a polyethylene glycol reference. Analytical combustion
analyses were performed by Galbraith Laboratories, Inc., Knoxville,
TN. The X-ray crystal structure data were determined with a Bruker
SMART 6000 diffractometer.
23
crystals: mp 183 °C; [R]D +94.2 (c 1.24, CH3OH); HRMS (APCI)
calcd for C20H22NO12 [M + H]+, 468.1142; found [M + H]+ 468.1142;
1H NMR (DMSO-d6, 300 MHz) δ 12.65 (1H, s, OH-7), 8.76 (1H, s,
NH), 6.75 (1H, s, H-10), 6.06-6.05 (2H, m, -O2CH2), 5.72 (1H, OH-
10b), 5.43 (1H, t, J ) 12 Hz, H-2), 5.34 (1H, m), 5.23 (1H, dd, J )
3.7, 10.3 Hz), 5.09 (1H, d, J ) 8.1 Hz, OH-1), 3.88 (1H, J ) 3 Hz,
H-4a), 3.46 (1H, t, J ) 8.4 Hz, H-1), acetates; 13C NMR (DMSO, 75
MHz) δ 170.5, 170.2, 162.9, 152.5, 145.1, 144.5, 140.7, 133.2, 104.9,
102.7, 100.6, 73.2 72.8, 71.2, 69.9, 68.9, 56.8, 21.5, 21.3, 21.1; anal.
C 49.46%, H 4.82%, N 2.70%, calcd for C20H21NO12‚2CH3OH, C
49.77%, H 5.46%, N 2.64%.
10b,1-Cyclic-thionocarbonate-2,3,4-tri-O-acetylepipancratista-
tin (5b). A solution of 10b(R)-hydroxy-2,3,4-tri-O-acetylpancratistatin
(4b) (0.3 g, 0.64 mmol) and N,N′-thiocarbonydiimidazole (90%, 0.25
g, 1.26 mmol, 1.97 equiv) in 2-butanone (16 mL) was heated to 80 °C
under argon for 24 h. An 1H NMR of the crude reaction mixture showed
approximately 40% conversion to product at this point. Thus, N,N′-
thiocarbonyldiimidazole (90%, 0.27 g, 1.36 mmol, 2 equiv) in 2-bu-
tanone (20 mL) was added dropwise (addition funnel) to the reaction
mixture. After 48 h, the reaction was approximately 75% complete.
The mixture was concentrated to minimum volume, and ethyl acetate
(50 mL) was added. The solution was washed with 1 N HC1 (2 × 25
mL), saturated NaCl (1 × 25 mL), and NaHCO3 (saturated 25 mL),
dried, and evaporated to dryness. Flash chromatography on silica gel
(eluent 96:4 DCM-CH3OH) gave 0.236 g (72%), which recrystallized
from DCM-EtOH as needles, which were examined by X-ray
2,3,4,-Tri-O-acetylnarciclasine (2c). To a solution of narciclasine
(2a) (5 g, 0.016 mol)8c in pyridine (38 mL) was added acetic anhydride
(5 mL, 0.053 mol, 3.25 equiv), and the reaction mixture was stirred at
rt under argon for 24 h. Ethyl acetate (400 mL) was added to the
reaction mixture and the solution washed with water (4 × 100 mL),
dried, and concentrated to a brown foam. The triacetate was isolated
as a colorless, crystalline powder (2.05 g, 29%) following column
chromatography on silica gel (eluent: DCM-CH3OH 3%). Recrys-
tallization from CH2Cl2-MeOH gave needles: mp 221 °C; [R]D25 +152
23
crystallography: mp 151-153 °C; [R]D -124.8 (c 1.09, CH2Cl2);
HRMS (APCI)+ calcd for C21H20O12NS, 510.0651 [M + H]+; found
1
510.0706 [M + H]+; H NMR (CDCl3, 300 MHz) δ 12.42 (1H, s,
OH-7), 6.49 (1H, s, H-10), 6.27 (1H, m, NH), 6.16, 6.14 (each 1H, d,
J ) 1.2 Hz, OCH2O), 5.56 (1H, t, J ) 3.3 Hz, H-3), 5.36 (1H, t, J )
3 Hz, H-2), 5.31 (1H, d, J ) 1.8 Hz, H-1), 5.04 (1H, dd, J ) 2.7, 10.8
Hz, H-4), 4.35 (1H, dd, J ) 4.1, 10.7, H-4a), 2.19 (3H, s, OCOCH3),
2.10 (3H, s, OCOCH3), 2.03 (3H, s, OCOCH3); 13CNMR (CDCl3, 100
MHz) δ 187.2, 169.6, 169.4, 168.4, 167.5, 153.2, 146.9, 136.8, 123.9,
107.4, 103.3, 98.5, 84.1, 80.2, 67.6, 67.3, 66.4, 52.4, 20.6, 20.54, 20.48;
anal. C 49.21%, H 4.46%, N 2.36%, calcd for C21H19NO12S‚CH3CH2-
OH, C 49.73%, H 4.54%, N 2.52%.
(c 0.45, CHCl3) [lit.14a mp 200-201 °C, lit.14b [R]D +202 (c 0.44,
23
1
CHCl3)]; H NMR (CDCl3, 300 MHz) δ 12.44 (1H, s, OH-7), 6.65
(1H, s, H-10), 6.22 (1H, s, NH), 6.15 (1H, nm, H-1), 6.08-6.06 (2H,
m, O2CH3), 5.45 (1H, m), 5.35-5.32 (1H, m), 5.22 (1H, dd, J ) 2.1,
9.6 Hz, H-4), 4.62 (1H, m, H-4a), 2.13 (3H, s, OCOCH3), 2.11 (3H, s,
OCOCH3), 2.01 (3H, s, OCOCH3); 13C NMR (CDCl3, 300 MHz) δ
169.8, 169.1, 169.0, 168.6, 152.5, 145.3, 134.7, 132.7, 129.9, 117.7,
105.5, 102.1, 96.0, 70.7, 67.9, 67.4, 49.8, 20.3 (2 20.2).
X-ray Crystal Structure Determination of 10b(R)-1-Cyclic-
thiocarbonate-2,3,4-tri-O-acetylepipancratistatin (5b). A thin, color-
less, needle-shaped crystal (∼0.39 × 0.03 × 0.02 mm), grown from a
dichloromethane-methanol solution, was mounted on the tip of a glass
fiber. Cell parameter measurements and data collection were performed
at 123 ( 2 K on a Bruker SMART 6000 diffractometer. Final cell
constants were calculated from a set of 2237 reflections from the actual
data collection. Frames of data were collected in the θ range of 6.47-
65.86° (-14 e h e 14, -8 e k e 8, -16 e l e 16) using 0.396°
steps in ω such that a comprehensive coverage of the sphere of
reflections was performed. After data collection, an empirical absorption
correction was applied with the program SADABS.15 Subsequent
10b(R)-Hydroxy-2,3,4-tri-O-acetylepipancratistatin (4b). To a 250
mL round-bottom flask were added dimethylformamide (9 mL) and
water (1 mL) followed in succession by (DHQ)2PHAL (0.1 g, 0.128
mmol), 4-N-methylmorpholine N-oxide (0.6 mL, 60 wt % in H2O),
and osmium tetroxide (4 wt % in H2O, 0.1 mL, 0.0157 mmol). The
pale green solution was stirred for 2 h, and narciclasine triacetate (2c,
1 g, 2.3 mmol) was then added. The reaction was stopped following
22 h of stirring by the addition of sodium metabisulfite (4 g) followed
by an equal volume of acetone and Na2SO4 (2 g). The mixture was
stirred for 3 h, the precipitate collected, and the solvent concentrated
to a yellow residue. Separation by flash column chromatography on

420 Journal of Natural Products, 2007, Vol. 70, No. 3
Pettit et al.
Figure 1. X-ray asymmetric unit content of the cyclic thiocarbonate
compound 5b. Thermal ellipsoids are displayed at the 50%
probability level.
statistical analysis of the complete reflection set using the XPREP15
program indicated the monoclinic space group P21.
Figure 2. X-ray structure of the cyclic carbonate 6. Atoms are
displayed as 20% thermal probability ellipsoids.
Crystal data: C21H19NO12S‚CH3OH, a ) 12.2768(8) Å, b ) 7.1383-
(5) Å, c ) 13.6850(11) Å, â ) 90.219(4)°, V ) 1199.28(15) Å3, λ )
(Cu KR) ) 1.54178 Å, µ ) 1.849 mm-1, Fc ) 1.499 g cm-3 for Z )
2 and fw ) 541.47, F(000) ) 564. A total of 3648 reflections were
collected, of which 2674 were unique (Rint ) 0.0886) and considered
observed (Io > 2σ(Io)). These were used in the subsequent structure
solution and refinement with SHELXTL-V5.1.15 All non-hydrogen
atoms for 5b were located using the default settings of that program.
Hydrogen atoms were placed in calculated positions, assigned thermal
parameters equal to either 1.2 or 1.5 (depending upon chemical type)
times the Uiso value of the atom to which they were attached, then
both coordinates and thermal values were forced to ride that atom during
final cycles of refinement. All non-hydrogen atoms were refined
anisotropically in a full-matrix least-squares refinement process. The
final standard residual R1 value for the model shown in Figure 1,
consisting of a single molecule of the thionocarbonate and single
molecule of disordered MeOH solvent in the asymmetric unit,
converged to 0.0834 (for observed data) and 0.1062 (for all data). The
corresponding Sheldrick R values were wR2 ) 0.2018 and 0.2124,
respectively, and the GOF ) 0.969 for all data. The difference Fourier
map showed small residual electron density; the largest difference peak
and hole were +0.880 and -0.415 e/Å3, respectively. Final bond
distances and angles were all within acceptable limits.
Synthesis of 10b(S)-2,3,4-Tri-O-acetylpancratistatin (3b) and 10b-
(R)-1-Cyclic-carbonate-2,3,4-tri-O-acetylepipancratistatin (6). To a
three-necked round-bottom flask under argon was added toluene (dry)
(9 mL). The toluene was heated to reflux before Bu3SnH (0.09 mL,
0.087 g, 0.3 mmol, 2 equiv) was added followed by AIBN. A solution
of 5b (0.075, 0.15 mmol) in toluene (5 mL) was then added dropwise
slowly over time (1.5 h), and the reaction was monitored by TLC (97:3
DCM-MeOH). The reaction mixture was stirred at 80 °C for 16 h,
then cooled and concentrated to remove toluene. Acetonitrile (10 mL)
was added and extracted with hexane (3 × 15 mL). The acetonitrile
fraction was then concentrated to a glass, which was separated by
column chromatography to yield crude carbonate 6 (16 mg) and alcohol
3b (33 mg, 49.6%). The products from several experiments were
combined for characterization purposes. Recrystallization of 6 from
CH2Cl2-EtOH yielded needles, which were examined by X-ray
crystallography: mp 253-255 °C, [R]25D -108.8 (c 0.33, CH2Cl2); 1H
NMR (CDCl3, 300 MHz) δ 12.4 (1H, s, OH-7), 6.52 (1H, s, H-10),
6.14-6.13 (2H, m, OCH2O), 5.5 (1H, t, J ) 3.3 Hz, H-3), 5.33 (1H,
t, J ) 3.3 Hz, H-2), 5.18 (1, bd, J ) 2.4 Hz, H-1), 5.08 (1H, dd, J )
10.4, 3.3 Hz, H-4), 4.32 (1H, dd, J ) 10.5, 4.2 Hz, H-4a), 2.17 (3H,
s, OCOCH3), 2.08 (3H, s, OCOCH3), 2.01 (3H, s, OCOCH3); 13C NMR
(CDCl3, 75 MHz) δ 169.7, 169.4, 168.5, 167.6, 153.2, 151.0, 146.9,
140.3, 136.6, 107.4, 103.3, 98.3, 79.6, 76.7, 67.5, 67.3, 66.7, 52.9, 20.6,
20.5 (2); HRAPCI+ m/z 494.0926 (calcd for C21H20NO13 (M + H)+,
494.0934); anal. C 50.65%, H 3.93%, N 2.79%, calcd for C21H19NO13,
C 51.12%, H 3.88%, N 2.84%.
133.9, 133.6, 106.1, 102.4, 102.3, 72.0, 71.9, 69.2, 68.6, 53.0, 42.2,
20.9, 20.8, 20.6; HRMS (APCI+) calcd for C20H22NO11 (M + H)+,
452.1193; found 452.1193; anal. C 51.09%, H 5.34%, N 2.80%, calcd
for C20H21NO11‚H2O, C 51.17%, H 4.93%, N 2.98%.
X-ray Crystal Structure Determination of 10b(R)-1-Cyclic-
carbonate-2,3,4-tri-O-acetylepipancratistatin (6). In a procedure
analogous to that described for the X-ray data collection of the cyclic
thiocarbonate compound 5b, a colorless, plate-shaped crystal (∼0.36
× 0.13 × 0.10 mm), grown from a CH2Cl2-MeOH, was used in the
structure determination of 6. Frames of data were collected in the θ
range of 6.47-65.86° (-15 e h e 15, -15 e k e 15, -32 e l e 30).
The XPREP16 program indicated the rather unusual tetragonal space
group P43212 (#96).
Crystal data: C21H19NO13, a ) 12.8186(7) Å, b ) 12.8186(7) Å, c
) 27.0417(19) Å, V ) 4443.4(5) Å3, λ ) (Cu KR) ) 1.54178 Å, µ )
1.085 mm-1, Fc ) 1.475 g cm-3 for Z ) 8 and fw ) 493.37, F(000)
) 2048. A total of 4127 reflections were collected, of which 2367
were unique (Rint ) 0.0486) and considered observed (Io > 2σ(Io)).
The final standard residual R1 value for the model shown in Figure 2,
consisting of a single molecule of the cyclic carbonate 6 in the
asymmetric unit, converged to 0.0611 (for observed data) and 0.0905
(for all data). The corresponding Sheldrick R values were wR2 ) 0.1622
and 0.1759, respectively, and the GOF ) 0.894 for all data. The
difference Fourier map showed minimal residual electron density; the
largest difference peak and hole were 0.349 and -0.181 e/Å3,
respectively. Final bond distances and angles were all within acceptable
limits.
10b(S)-Epipancratistatin (3a). To a 10% aqueous MeOH-CH2-
Cl2 mixture (1:1, 6 mL) were added acetate 3b (0.05 g, 0.11mmol)
and K2CO3 (0.015 g, 0.1 mmol, 1 equiv). The yellow solution was
stirred at rt and monitored by TLC (96:4 DCM-CH3OH 4%) for 16 h.
The mixture was neutralized with acetic acid and concentrated to an
off-white residue, which was dissolved in 4:1 DCM-CH3OH and
filtered through a silica gel plug. The filtrate was concentrated to a
white, amorphous solid (23 mg, 64%) and recrystallized from MeOH
to afford a colorless, crystalline solid, which was characterized as
25
1
follows: mp >300 °C; [R]D +72 (c 0.2, CH3OH); H NMR (300
MHz, DMSO-d6) δ 12.85 (s, 1H), 8.32 (s, 1H), 6.4 (s, 1H), 6.10 (s,
1H), 6.01-6.00 (m, 2H), 5.06 (s, 1H), 4.62 (m, 2H), 3.92 (s, 1H), 3.66
(s, 1H), 3.41 (m, 2H), 3.04 (m, 1H), 2.73-2.70 (m, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 169.8, 151.0, 144.9, 137.0, 132.1, 106.1, 102.4,
101.7, 73.5, 73.2, 70.6, 69.4, 55.1, 40.7; HRFAB calcd for C14H16NO8
(M + H)+, 326.0876; found (M + H)+, 326.0887.
10b(R)-Hydroxy-1,2,3,4,7-penta-O-acetylpancratistatin (4c). To
a solution of 10b(R)-hydroxy-2,3,4,7-tetra-O-acetylnarciclasine (4a, ref
8c) (1.55 g, 3.04 mmol) in pyridine (7 mL) was added acetic anhydride
(7 mL) and the solution stirred at rt under anhydrous conditions for
6 h. Ice-water was added, and the aqueous layer was extracted with
CH2Cl2 (3 × 50 mL). The dried extract was concentrated (under
vacuum) to a foam, which was dissolved in toluene with heating.
Crystallization occurred overnight at 0 °C to yield colorless crystals
(1.38 g, 82%): mp 236 °C; Rf 0.53 (hexane-acetone, 1:1); [R]D +109
(c 0.41, CHCl3); EIMS m/z (%) 551 [M + 2.8], 509 (100), 347 (12.9),
The alcohol 3b was crystallized from CH2Cl2-EtOH: mp 216 °C;
1H NMR (CDCl3, 300 MHz) δ 12.02 (1H, s), 6.87 (s, 1H), 6.45 (s,
1H), 6.01 (m, 2H), 5.53 (t, J ) 3 Hz, H-4), 5.39 (t, J ) 10 Hz, H-2),
5.29 (s, 1H, OH-1), 5.23 (dd, J ) 3, 10 Hz, H-3), 3.96 (t, J ) 3 Hz,
H-4a), 3.84 (t, J ) 10 Hz, H-1), 3.02 (1H, dd, J ) 3, 10 Hz, H-10b);
13C NMR (CDCl3, 125 MHz) δ 170.9, 170.5, 170.1, 169.5, 152.4, 146.4,

Synthesis of 10b(S)-Epipancratistatin from (+)-Narciclasine
Journal of Natural Products, 2007, Vol. 70, No. 3 421
287 (49.3), 258 (30), 222 (84), 165 (5), 115 (2.8); 1H NMR (300 MHz,
CDCl3) δ 6.95 (s, 1H), 6.08 (m, 2H), 5.69 (m, 1H), 5.5 (t, J ) 3.3 Hz,
1H), 5.38 (m, 2H), 4.0 (d, 1H), 3.3 (s, 1H), 2.33 (s, 3H), 2.16 (s, 3H),
1.99 (s, 3H), 1.97 (s, 3H), 1.95 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 169.9, 169.8, 169.1, 168.9, 168.7, 162.3, 152.5, 140.6, 137.5, 133.8,
112.9, 103.1(2C), 73.2, 72.9, 69.5, 69.0, 67.7, 56.9, 20.8, 20.7, 20.6,
20.5, 20.3; HRFAB calcd for C24H26NO14 [M + H]+, 552.1353; found
[M + H]+, 552.1365; anal. C 50.77%, H 4.80%, N 2.46%, calcd for
C24H25NO14‚H2O, C 50.62%, H 4.78%, N 2.46%.
Acknowledgment. We are very pleased to acknowledge most
helpful discussions concerning the radical reaction with the late (and
great) Professor Sir Derek H. R. Barton. For financial assistance we
acknowledge with thanks Grant R01 CA-90441-01-05 from the Division
of Cancer Treatment and Diagnosis, NCI, DHHS; Dr. Alec D. Keith,
The Arizona Disease Control Commision; The Robert B. Dalton
Endowment Fund; and Gary L. and Diane Tooker. In addition, we wish
to thank for technical assistance Drs. F. Hogan, J. Lieman, and V.
Mukku, as well as F. Cracienusccu, M. Dodson, Dr. R. K. Pettit, C.
Webber, and L. Williams.
10b(R),N,7-O-Methyloxalyl-1,2,3,4-tetra-O-acetylpancratistatin
(7b). To a solution of alcohol 4c (0.3 g, 0.5 mmol) in pyridine (3 mL)
was added methyl oxalyl chloride (0.5 mL, 5.4 mmol, 10.8 equiv).
The reaction mixture turned brown, and an immediate precipitate
separated. Pyridine (4 mL) was added, and the reaction mixture was
stirred at rt under argon for 6 h, whereupon TLC examination (9:1
DCM-CH3OH) indicated starting material had been consumed. The
reaction mixture was cooled and placed in an ice-water bath, ice-
water was added, and the resultant precipitate was collected. The
precipitate was washed with H2O, which removed the brown color.
The remaining white precipitate was dissolved in CH2Cl2, washed with
H2O (20 mL), and dried, and the solution was filtered and concentrated
to a white residue. The residue was dissolved in hexane (10 mL) and
acetone added until the product dissolved with heating. Crystallization
occurred overnight at 0 °C: 120.6 mg, 28.9%, mp 265 °C; [R]D -11.3
(c 1.04, CH2Cl2); Rf 0.81 (19:1 DCM-CH3OH); 1H NMR (500 MHz,
CDCl3) δ 7.38 (s, 1H), 6.57 (d, 1H), 6.20 (m, 2H), 5.74 (d, J ) 6.9
Hz, 1H), 5.30 (t, J ) 1.5 Hz, 1H), 5.22 (t, J ) 1.5 Hz, 1H), 5.05 (dd,
J ) 6.9 Hz, 1.8 Hz, 1H), 3.99 (s, 3H) 3.88 (s, 3H), 3.19 (s, 3H), 2.19
(s, 3H), 2.17 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 169.6, 169.0, 168.4, 167.7, 162.0, 161.0, 158.9, 156.8, 156.4,
155.3, 154.1, 152.8, 142.5, 133.3, 128.5, 114.9, 109.2, 102.3 (2C), 79.4,
67.8, 67.4, 67.1, 67.1, 54.0, 53.7, 53.0, 51.7, 30.9, 20.6, 20.5, 20.2;
EIMS m/z (%) 767 (M+, 13.3), 695 (18.9), 636 (25.2), 622 (100), 594
(28.7), 550 (44.8), 374 (27.9), 287 (35.7), 248 (41.9); anal. C 49.20%,
H 4.25%, N 2.02%, calcd for C31H29O22N, C 48.51%, H 3.81%, N
1.82%.
10b(S)-1,2,3,4-Tetra-O-acetylepipancratistatin (3c). To a solution
of methyloxalate (7b) (0.05 g, 0.065 mmol) in dry toluene (20 mL),
under argon at reflux, were added tributyltin hydride (150 mL, 8.57
equiv) and AIBN (20 mg). The reaction mixture was heated at reflux
for 2.5 h, and TLC (19:1, DCM-CH3OH) showed complete consump-
tion of starting material. The solution was concentrated to a white,
gelatinous solid, which crystallized from toluene-hexane to yield an
amorphous solid upon standing at 0 °C for 16 h. After washing with
hexane to remove tributyltin hydride and drying, the solid obtained
(32 mg) was found to consist mainly of acetate (3c), with four minor
impurities (one being pancratistatin tetraacetate by 1H NMR). The
purification of acetate (3c) proved difficult using standard silica gel
column chromatography techniques. However, purification of a small
quantity (7 mg) was accomplished via separation of the mixture using
References and Notes
(1) For the preceding part in this series, see Antineoplastic Agents 549:
Pinney, K. G.; Jelinek, C.; Edvardsen, K.; Chaplin, D. J.; Pettit, G.
R. Anticancer Agents Nat. Prod. 2005, 23-46.
(2) (a) Pettit, G. R.; Gaddamidi, V.; Cragg, G. M.; Herald, D. L.; Sagawa,
Y. J. Chem. Soc., Chem. Commun. 1984, 1693-1694. (b) Pettit, G.
R.; Gaddamidi, V.; Herald, D. L.; Singh, S. B.; Cragg, G. M.;
Schmidt, J. M. J. Nat. Prod. 1986, 49, 995-1002. (c) Pettit, G. R.;
Melody, N.; Herald, D. L. J. Nat. Prod. 2004, 67, 322-327. (d) Pettit,
G. R.; Ducki, S.; Orr, B. Antineoplastic Agents 453. Synthesis of
Pancratistatin Prodrugs. Anti-Cancer Drug Des. 2000, 15, 389-396.
(e) Pettit, G. R.; Freeman, S.; Simpson, M. J.; Thompson, M. A.;
Boyd, M. R.; Williams, M. D.; Pettit, G. R., III; Doubek, D. L.
Anticancer Drug Des. 1995, 10, 243-250.
(3) Bibby, M. C.; Holwell, S. E.; Pettit, G. R. Anti-Vascular & Anti-
Tumour Effects of the Novel Agent Pancratistatin Phosphate.
Abstract. Biological Basis for Antiangiogenic Therapy Conference,
Milan, Italy, November 8-10, 1999.
(4) Gabrielsen, B.; Monath, T. P.; Huggins, J. W.; Kefauver, D. F.; Pettit,
G. R.; Groszek, G.; Hollingshead, M.; Kirsi, J. J.; Shannon, W. M.;
Schubert, E. M.; Dare, J.; Ugarkar, B.; Ussery, M. A.; Phelan, M. J.
J. Nat. Prod. 1992, 55, 1569-1581.
(5) Ouarzane-Amara, M.; Franetich, J-F.; Mazier, D.; Pettit, G. R.; Meijer,
L.; Doerig, C.; Desportes-Livage, I. Antimicrob. Agents Chemother.
2001, 45, 3409-3415.
(6) Khan, P.; Abbas, S.; Pettit, G. R.; Caffrey, R.; Megram, V.; McGown,
A. J. Chromatogr. B 1999, 726, 249-254.
(7) For leading references, refer to: Pettit, G. R.; Melody, N.; Herald,
D. H. J. Org. Chem. 2001, 66, 2583-2587.
(8) (a) Hudlicky, T.; Rinner, U.; Gonzalez, D.; Akgun, H.; Schilling,
S.; Siengalewicz, P.; Martinot, T. A.; Pettit, G. R. J. Org. Chem.
2002, 67, 8726-8743. (b) McNulty, J.; Mao, J.; Gibe, R.; Mo, R.;
Wolf, S.; Pettit, G. R.; Herald, D. L.; Boyd, M. R. Bioorg. Med.
Chem. Lett. 2001, 11, 169-172. (c) Pettit, G. R.; Melody, N.; Herald,
D. L.; Schmidt, J. M.; Pettit, R. K.; Chapuis, J.-C. Heterocycles 2002,
56, 139-155.
(9) (a) Barton, D. H. R; McCombie, S. W. J. Chem. Soc., Perkin Trans.
1 1975, 1574-1585. (b) Barton, D. H. R.; Subramanian, R. J. Chem.
Soc., Chem. Comm. 1976, 867-868. (c) Barton, D. H. R.; Hartwig,
W.; Hay Motherwell, R. S.; Motherwell, W. B.; Stange, A.
Tetrahedron Lett. 1982, 23, 2019-2022.
(10) Pullukat, T. J.; Urry, G. Tetrahredron Lett. 1967, 21, 1953-1954.
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(13) (a) Rinner, U.; Siengalewicz, P.; Hudlicky, T. Org. Lett. 2002, 4,
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(14) (a) Okamoto, T.; Torii, Y.; Isogai, Y. Chem. Pharm. Bull. 1968, 16,
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(15) Blessing, R. Acta Crystallogr. 1995, A51, 33-8.
(16) SHELXTL-Version 5.1, an integrated suite of programs for the
determination of crystal structures from diffraction data; Bruker AXS,
Inc.: Madison, WI, 1997. This package includes, among others,
XPREP (an automatic space group determination program), SHELXS
(a structure solution program via Patterson or direct methods), and
SHELXL (structure refinement software).
(17) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paul, K.;
Vestica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J.
Natl. Cancer Inst. 1991, 83, 757-755.
C18 reversed-phase HPLC (ZorbaxSB-C18, 3:2 H2O-CH3OH to CH3-
OH): mp 169 °C; EIMS m/z (%) 493 [M+, 15.5], 433 (5), 313 (18.9),
271 (100), 206 (17.2); HRFAB calcd for C22H24O12N [M + H]+,
494.1298; found [M + H]+, 494.1296; 1H NMR (500 MHz, CDCl3) δ
11.97 (s, 1H), 6.27 (s, 1H), 6.03 (m, 2H), 5.52-5.47 (m, H-4, H-2),
5.30 (dd, J ) 11,3 Hz, H-3), 5.25 (t, J ) 11 Hz, H-1), 4.01 (m, H-4a),
3.15 (m, H-10b), 2.19 (s, 3H), 2.04 (S, 3H), 2.03 (s, 3H), 1.95 (s, 3H);
13C NMR (125 Hz, CDCl3) δ 170.2, 170.2, 170.1, 169.4, 169.0, 152.4,
146.6, 134.1, 132.4, 106.4, 102.5, 101.0, 72.0, 69.4, 69.2, 68.3, 52.9,
40.0, 20.7, 20.6, 20.6, 20.5.
Cancer Cell Line Procedures. Inhibition of human cancer cell
growth was assessed using the National Cancer Institute’s standard
sulforhodamine B assay as previously described.17 Briefly, cells in a
5% fetal bovine serum/RPMI1640 medium solution were inoculated
in 96-well plates and incubated for 24 h. Serial dilutions of the
compounds were then added. After 48 h, the plates were fixed with
trichloroacetic acid, stained with sulforhodamine B, and read with an
automated microplate reader. A growth inhibition of 50% (GI50 or the
drug concentration causing a 50% reduction in the net protein increase)
was calculated from optical density data with Immunosoft software.
Mouse leukemia P388 cells18 were incubated 24 h in a 10% horse
serum/Fisher medium solution followed by a 48 h incubation with serial
dilutions of the compounds. Cell growth inhibition (ED50) was then
calculated using a Z1 Beckman/Coulter particle counter.
(18) Suffness, M.; Douros, J. In Methods in Cancer Research, Vol. XVI,
Cancer Drug DeVelopment Part A; Devita, V. T., Jr., Bush, H., Eds.;
Academic Press: New York, 1979; pp 73-126.

422 Journal of Natural Products, 2007, Vol. 70, No. 3
Pettit et al.
(19) Crystallographic data for the structures reported in this paper have
been deposited as CIF files 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) 223-336033 or e-mail: deposit@
ccdc.cam.ac.uk].
NP068046E