Antineoplastic Agents. 511. Direct Phosphorylation of Phenpanstatin and Pancratistatin

Article abstract of DOI:10.1021/np030299+

Selective phosphorylation of phenpanstatin (3a) with tetrabutylammonium dihydrogen phosphate and dicyclohexylcarbodiimide in pyridine followed by cation-exchange chromatographic procedures was found to provide an efficient route to a new series (3b-3d) of promising 3,4-O-cyclic phosphate prodrugs designated phenpanstatin phosphates. Application of analogous reaction conditions to pancratistatin (1a) led to a mixture of monophosphate derivatives where sodium pancratistatin 4-O-phosphate (4a) was isolated and the structure confirmed by X-ray crystallography. Modification of the reaction conditions allowed direct phosphorylation of pancratistatin followed by cation-exchange chromatography to afford sodium pancratistatin 3,4-O-cyclic phosphate (5a), which was selected for preclinical development.

Full text of DOI:10.1021/np030299+

3
22
J . Nat. Prod. 2004, 67, 322-327
An tin eop la stic Agen ts. 511. Dir ect P h osp h or yla tion of P h en p a n sta tin a n d
P a n cr a tista tin †
George R. Pettit,* Noeleen Melody, and Delbert L. Herald
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, PO Box 872404,
Tempe, Arizona 85287-2404
Received J une 30, 2003
Selective phosphorylation of phenpanstatin (3a ) with tetrabutylammonium dihydrogen phosphate and
dicyclohexylcarbodiimide in pyridine followed by cation-exchange chromatographic procedures was found
to provide an efficient route to a new series (3b-3d ) of promising 3,4-O-cyclic phosphate prodrugs
designated phenpanstatin phosphates. Application of analogous reaction conditions to pancratistatin (1a )
led to a mixture of monophosphate derivatives where sodium pancratistatin 4-O-phosphate (4a ) was
isolated and the structure confirmed by X-ray crystallography. Modification of the reaction conditions
allowed direct phosphorylation of pancratistatin followed by cation-exchange chromatography to afford
sodium pancratistatin 3,4-O-cyclic phosphate (5a ), which was selected for preclinical development.
In 1984, we first succeeded in isolating and elucidating
the structure (by X-ray employing the 7-methoxy deriva-
2
tive) of (+)-pancratistatin (1a ), the principal anticancer
constituent of the Amaryllidaceae tropical spider lily Pan-
cratium littorale, which was later reidentified as Hymeno-
3
2b
callis littoralis. Because of the early promise of 1a as a
4
new type of anticancer and antiviral (RNA viruses) drug,
various phases of preclinical development5 have been
underway for over 17 years. Meanwhile, we and others
have been increasingly successful in developing the avail-
3
ability of pancratistatin (1a ) by horticultural and synthetic
approaches as well as further defining SAR requirements.
6
7
When the preclinical drug formulation of 1a began to
present another serious challenge owing to its sparing (53
2
µg/mL in H O) solubility, we began to investigate structural
modifications5 that were expected to greatly increase
aqueous solubility while serving as a successful delivery-
5
c
type prodrug. Those studies led to useful syntheses (four
5
steps) of sodium pancratistatin 7-O-phosphate (1b) with
considerably improved aqueous solubility (20 mg/mL).5b
While the 7-O-phosphate salt 1b proved to have attrac-
tive aqueous solubility properties, the yield-penalizing
synthetic steps from 1a required us to continue parallel
efforts to directly, but selectively, phosphorylate 1a . The
necessity of discovering more efficient techniques for
converting 1a to very effective phosphate prodrugs has
been accelerating with the recent realization that the long
elusive key mechanism of action by 1a against in vivo
neoplastic disease is cancer antiangiogenesis/vascular tar-
8
geting. Furthermore, pancratistatin (1a ) has also recently
been found to display remarkable activity against mi-
crospirochesis,9 another potentially lethal challenge for
some cancer patients.
Toward the objective of efficiently phosphorylating pan-
cratistatin (1a ), the more abundant but closely related
1
0
Amaryllidaceae biosynthetic product narciclasine (2a ),
which also required conversion to a phosphate prodrug for
anticancer preclinical development, served as a model for
most of the exploratory phosphorylation11 experiments.
Eventually reaction conditions were found for efficient
†
Dedicated to Professor Carl Djerassi, a great pioneer in medicinal,
natural products, and organic chemistry, on the occasion of his 80th
birthday.
*
To whom correspondence should be addressed. Tel: 480-965-3351.
Fax: 480-965-8558.
1
0.1021/np030299+ CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/16/2004

Antineoplastic Agents
J ournal of Natural Products, 2004, Vol. 67, No. 3 323
phosphorylation of 2a with tetrabutylammonium dihydro-
gen phosphate using dicyclohexylcarbodiimide (DCCI) in
pyridine containing p-toluenesulfonic acid to yield the very
useful 3,4-O-cyclic phosphate 2b as a pyridinium salt that
separated from the reaction mixture.12 By application of
ion-exchange methods, the pyridinium salt was easily
converted to a series of metal and ammonium cation
derivatives.12
2
mixture from H O-MeOH yielded (3.7%) pancratistatin
4-O-phosphate (4a , 31P NMR δ 4.73). The second and third
crops of crystals and the mother liquor residue were found
3
1
to be mixtures ( P δ 6.81, 6.00, 4.90, and 3.26) of
phosphates.
Detailed NMR analysis was carried out in an attempt
to establish the position of the phosphate (4a ). The ring
protons were assigned using a COSY spectrum where
H-10b (δ 2.96) showed strong correlation signals with H-4a
Resu lts a n d Discu ssion
(δ 3.74) and H-1 (δ 4.24). In turn, H-4a exhibited strong
correlation signals with H-4 (δ 4.29). The H-3 and H-2
resonances were assigned to the narrow multiplet at δ 3.95
integrating for two hydrogens. The correlation peaks
associated with this multiplet were in accord with those
When the preceding direct phosphorylation reaction was
applied to pancratistatin-1-benzoate,6a renamed phenpan-
statin (3a , another of the pancratistatin series we have in
preclinical development that required a suitable phosphate
derivative), the main product was again the 3,4-O-cyclic
phosphate. The crude tetrabutylammonium salt was con-
verted by an ion-exchange and LH-20 Sephadex chromato-
graphic separation series to the corresponding sodium salt
designated sodium phenpanstatin (3b) and subsequently
predicted. A D
nation of the four signals at δ 13.34 (phenolic OH), 10.22
NH), 4.74-4.72 (OH), and 3.47 (OH). The OH resonance
2
O exchange NMR experiment led to elimi-
(
at δ 4.73 gave a strong correlation peak in the COSY
spectrum with the resonance assigned to H-1. Therefore,
this signal was assigned to OH-1. A 31P NMR spectrum
displayed one signal at δ 4.73, implying one phosphorus
atom per molecule. An X-ray crystal structure determina-
tion was required to confirm the structure of 4a . Recrys-
+
+
to the Li and K cation derivatives 3c and 3d . The 3,4-
O-cyclic phosphate structure (3b) was well supported by
the NMR spectra, with the 31P NMR shift downfield at 13.0
ppm close to that of 3,4-O-cyclic phosphate 2b (δ 20.3),
whose structure had been confirmed by X-ray crystal
structure determination,12 as well as by other evidence that
tallization of 4a from H
suitable for crystallography.
2
O-MeOH provided a crystal
now follows.
The cyclic phosphate of pancratistatin (5a ) was eventu-
ally prepared following a series of exploratory experiments
by decreasing the tetrabutylammonium dihydrogen phos-
phate from 8 equiv to 1.9 equiv and maintaining an excess
of dicyclohexylcarbodiimide. The p-toluenesulfonic acid was
also decreased from 3 to 1.3 equiv. The reaction goes to
completion overnight at 80 °C to yield one product by H
NMR. This method was found to be low yielding, as the
workup caused decomposition of the cyclic phosphate to a
Detailed analysis of the H, 13C, COSY, HMQC, and D
1
2
O
exchange experiments was carried out in an effort to assign
the carbon and proton spectrum. A downfield shift of the
ring protons H-3 and H-4 was observed when compared
1
with the H spectrum of the starting material 3a . There
1
were no OH-3 and OH-4 signals observed in the spectrum.
The signal for the OH-2 proton was observed downfield at
δ 6.13 as a broad singlet, which disappeared when a D O
2
2
2 2
:1 ratio of 5a :4a . Recrystallization from MeOH-CH Cl
exchange experiment was performed.
yielded pure cyclic phosphate (28%); however, further
purification of the mother liquor, which contains a mixture
of 5a , 4a , and sodium tosylate, was not achieved. The cyclic
phosphate (5a ) was eventually prepared in high yield (48%
Analysis of the COSY spectrum indicated the H-1 signal
was downfield at δ 5.73 as expected, showing strong
correlation peaks with the signals at δ 4.19 and 3.3 for H-2
and H-10b, respectively. The H-2 signal in turn correlated
with the signal at δ 4.32 (H-3). The H-10b signal correlated
with the signal at δ 4.41 (H-4a). The signal at δ 4.25 was
assigned to H-4. The OH-2 signal at δ 6.13 showed a
correlation peak with the signal assigned to H-2 at δ 4.19.
following recrystallization from H
2
2 2
O-MeOH-CH Cl ) when
the p-toluenesulfonic acid was excluded and the reaction
was allowed to proceed for 48 h with additional amounts
of dicyclohexylcarbodiimide and tetrabutylammonium di-
hydrogen phosphate being added after 24 h.
The D
of the H
H-10b at δ 3.27 (D
2
O exchange experiment resulted in a downfield shift
O peak, which allowed one to see the signal for
O/DMSO-d ) as a broad doublet. The
C spectrum was examined using HMQC. There was a
noticeable downfield shift of the C-3 and C-4 signals from
Detailed NMR analyses using H, 13C, COSY, HMQC,
1
2
2
and D O exchange experiments were carried out to confirm
the 3,4 position of the cyclic phosphate. Analysis of the
product by 31P NMR shows a phosphorus peak downfield
at δ 13.22 (DMSO-d ), which indicated the presence of a
6
2
cyclic phosphate group. A D O exchange experiment showed
2
6
1
3
δ 68.8 in the 1 C spectrum of 3a to 74.8 (C-3) and 74.9 (C-
3
4
) in 3b, further proof that the cyclic phosphate had been
exchangeable peaks to be at δ 13.25, 8.10, 5.42, and 4.69,
OH-7, N-H, OH-2, and OH-1, respectively. The COSY
spectrum revealed strong correlation peaks between the
multiplets at δ 2.85 (H10b), 4.27 (H-1), and 4.09 (H-4a).
The peak at δ 4.02 was assigned to H-2 due to the
correlation peaks observed with H-1 and the OH peak at δ
prepared.
The lithium and potassium salts (3b and 3c) were
prepared by passing the sodium salt through an ion-
exchange column of the respective cation.
When the tetrabutylammonium dihydrogen phosphate
reaction was applied to phosphorylation of pancratistatin
5
.42. The multiplet at 4.21 was assigned to H-3 due to
(1a ) using the procedure that provided 3,4-O-cyclic phos-
cross-peaks observed with the H-2 peak at δ 4.02. The
multiplet at δ 4.08-4.04 was assigned to the H-4 signal.
phates 2b and 3b, the result was quite different, presum-
ably owing to the unprotected 1-hydroxy group. The
reaction was performed at 80 °C over 5 days and appeared
complete by 300 MHz NMR analysis at that point. The
mixture of pancratistatin phosphate salts was converted
to the sodium salts for ease of separation using an ion-
exchange (Dowex 50WX8-200, Na+ form) and Sephadex
G-10 sequence. The phosphates were retained in preference
to other components on the G-10 Sephadex column, and
further separation by recrystallization of the phosphate
1
1
The H NMR spectrum of 5a was compared with the H
NMR spectrum of 1a . Downfield shifts of the H-3, H-4, and
H-4a signals by 0.38, 0.36, and 0.36, respectively, were
observed. The downfield shifts observed for H-1 and H-2
were relatively minor, 0.01 and 0.08 ppm, respectively. This
proves that the cyclic phosphate was in the 3,4 position.
The lithium and potassium salts (5b and 5c) were
prepared by passage through an ion-exchange column

3
24 J ournal of Natural Products, 2004, Vol. 67, No. 3
Pettit et al.
Ta ble 1. Human Cancer Cell Line and Murine P-388 Lymphocytic Inhibitory Activities of Compounds 1a ,b, 2a ,b, 3a ,b, 4a -l, and 5a
ED50 (µg/mL)
GI50 (µg/mL)
leukemia
P-388
pancreas-a
BXPC-3
breast
MCF-7
CNS
SF268
lung-NSC
NCI-H460
colon
KM20L2
prostate
DU-145
compound
a,b
1
1
2
2
3
3
4
4
4
4
4
4
4
4
4
4
4
4
5
a 2
0.017
0.24
0.02
0.20
0.0035
0.069
0.0019
0.25
0.18
0.36
0.30
0.45
0.40
0.25
0.27
0.31
0.29
0.25
0.23
0.34
3.3
0.023
0.20
0.014
0.079
0.0031
0.047
0.00055
0.17
0.12
0.35
0.28
0.39
0.36
0.26
0.38
0.27
0.38
0.21
0.28
0.27
0.032
0.19
0.0084
0.058
0.0001
0.029
0.38
0.42
0.35
0.40
0.42
0.27
0.42
0.35
0.46
0.25
0.31
0.36
3.8
0.025
0.17
0.0032
0.06
0.00037
0.13
0.24
0.33
0.25
0.30
0.36
0.23
0.30
0.24
0.37
0.13
0.22
0.23
3.7
0.015
0.026
0.0032
0.031
0.00021
0.13
0.94
0.20
0.20
0.22
0.22
0.12
0.17
0.21
0.18
0.13
0.15
0.20
5
b
0
2
a
b
a
b
a
1
0.013
0.012
0.0016
0.061
0.018
0.047
0.036
0.025
0.020
0.21
0.17
0.019
0.27
0.063
0.066
0.21
0.0032
0.059
0.00031
0.041
0.18
0.43
0.39
0.46
0.44
0.36
0.47
0.38
0.40
0.33
0.35
0.38
2.9
1
6
b
a
b
c
d
e
f
g
h
i
j
k
l
a
3.33
2.9
2.3
containing the respective ion. When the cyclic phosphate
pyridine preceding distillation was dried over potassium
hydroxide pellets. The pancratistatin derivatives were visible
as green-blue fluorescent spots on TLC plates under long-wave
ultraviolet light. Dowex 50WX8-200 cation-exchange resin (H
2
form) was washed with MeOH, 1 N HCl, and deionized H O.
The cation forms of the resin were obtained by washing with
a 1 N solution of the appropriate base followed by deionized
water.
Sod iu m P h en p a n sta tin 3,4-O-Cyclic P h osp h a te (3b).
Phenpanstatin (3a , 0.20 g, 0.26 mmol) was dissolved in
pyridine (10 mL) and the solution heated to 80 °C under argon
before addition of tetrabutylammonium dihydrogen phosphate
(1.25 g, 3.68 mmol, 8 equiv), dicyclohexylcarbodiimide (1.20
+
5
a was passed through an ion-exchange column (H form)
in an effort to prepare the cyclic phosphoric acid, it
decomposed to the 4-O-phosphoric acid (4b). This develop-
ment led to the preparation of the 4-O-phosphate salts 4c-
+
4
l.
The lithium salt (4c) was prepared by passage of 4b
through a column containing Dowex 50WX8-200 cation-
exchange resin containing that cation. The potassium salt
was then prepared from the lithium salt by dissolving 4c
2
in H O and passing it through a column containing Dowex
0WX8-200 cation-exchange resin (K+ form).
5
g, 5.8 mmol, 12.6 equiv), and p-toluenesulfonic acid (0.26 g,
The magnesium (4d ), calcium (4e), and zinc (4f) salts
1
1
.36 mmol, 3 equiv). The reaction was monitored by H NMR
were obtained by dissolving 4b in MeOH and adding 1
equiv of the respective metal acetate. The resulting opaque
solutions were stirred for several days as the salt precipi-
tated from solution. The mixture was then concentrated,
and the salts were washed with MeOH. A selection of
ammonium salts were prepared by allowing 4b to react
with the respective amine at room temperature. The
reaction mixture was concentrated and the solid washed
with a suitable solvent to give the ammonium salts 4h -
(DMSO-d
6
), and after 24 h the NMR spectrum showed a 50:
5
0 mixture of starting material to product. Dicyclohexylcar-
bodiimide (0.425 g) was added, and the reaction continued for
a total of 48 h.
The mixture was cooled, and the dicyclohexylurea (DCU)
precipitate was collected and washed with H O. Additional
2
H
2
O (100 mL) was added to the mother liquor and the DCU
again collected. The mother liquor was extracted with butanol
2 × 40 mL), and the butanol fractions were combined and
concentrated to a light brown oil. A solution of the oil in H
(
2
O
4
l.
The new pancratistatin phosphate prodrug series was
(
2
minimum amount) was purified through a Dowex 50WX8-
00 ion-exchange column (Na form) (14 cm × 2 cm). The UV-
+
evaluated against a minipanel of human cancer cell lines
and the murine P388 lymphocytic leukemia cell line.
Results of the cancer cell line evaluations compared to the
parent pancratistatin (1a ) and phenpanstatin (3a ) appear
in Table 1 and confirm retention of cancer cell line
inhibitory activity comparable to the respective anticancer
drug.
active fractions were combined and freeze-dried to yield a
white solid, which was further purified in methanol on a
Sephadex LH-20 column (70 cm × 2 cm, eluent MeOH at 1.8
mL/min): 0.145 g, 60%, mp 268 °C dec, [R] -51.8° (c 0.5, CH -
OH), R
NMR (400 MHz, DMSO-d
D
3
1
f
) 0.69 (BuOH-MeOH-H
2
O-NH
4
OH, 4:3:2:1);
H
6
) δ 13.25 (s, 1H), 8.52 (s, 1H), 8.1
(
d, J ) 7.6 Hz, 1H), 7.59 (t, J ) 7.4 Hz, 1H), 7.41 (t, J ) 7.6
Hz, 2H), 6.30 (s, 1H), 6.13 (bs, 1H), 6.01 (s, 1H), 5.96 (s, 1H),
Exp er im en ta l Section
5.73 (s, 1H), 4.41 (dd, J ) 8.4, 13.2 Hz, 1H), 4.32-4.21 (m,
2
d
1
3
H), 4.11 (bs, 1H), 3.33 (m, 1H); 13C NMR (100 MHz, DMSO-
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined using a Fisher-J ohns melting point apparatus and
are uncorrected. TLC was performed on Analtech silica gel
GHLF plates. HRMS was provided by the Washington Uni-
versity Mass Spectrometry Resource. All H NMR spectra were
initially obtained using a Varian Gemini 300 MHZ instrument
6
) δ 169.2, 165.4, 152.1, 145.8, 133.4, 133.3, 132.3, 130.2 (2C),
29.2, 128.7, 107.4, 101.9, 95.8, 74.9, 74.8, 69.1, 66.3, 66.1, 51.3,
31
5.7 ppm; P NMR (162 MHz, DMSO-d
6
) δ 12.99; HRFAB m/z
-
calcd for [C21
H17NO11P] 490.0539Z, found 490.0540.
1
Gen er a l P r oced u r e for P r ep a r a tion of th e P h en p a n -
sta tin P h osp h a te P r od r u gs (3c-3d ). Sodium phenpansta-
1
3
1
1
1
13
unless otherwise noted. The C, H- H COSY, H- C HMBC,
tin 3,4-cyclic phosphate (6 mg) was dissolved in H O (0.5 mL)
2
1
13
31
H- C HMQC, and P NMR experiments were conducted
employing Inova 400 and Varian Unity 500 MHz instruments.
+)-Pancratistatin (1a ) was isolated from Hymenocallis
and eluted with H O through a column (1 cm × 5 cm) of Dowex
2
50WX8-200 ion-exchange resin (respective cation). The UV-
active fractions were combined and freeze-dried to give the
corresponding phenpanstatin phosphate prodrug.
(
3
littoralis as previously described. Reagents were purchased
from Acros Chemical Company unless otherwise noted and
used as received. Solvents were distilled prior to use, and
Lit h iu m P h en p a n st a t in 3,4-Cyclic P h osp h a t e (3c):
1
glassy solid, 4 mg, mp 280 °C (dec); H NMR (300 MHz, CD
3
-

Antineoplastic Agents
J ournal of Natural Products, 2004, Vol. 67, No. 3 325
OD) δ 8.05 (d, J ) 7.2 Hz, 2H), 7.53 (t, J ) 7.35 Hz, 1H), 7.42
(
4
t, J ) 7.35 Hz, 2H), 6.39 (s, 1H), 5.96 (s, 1H), 5.90 (s, 1H),
.61-4.51 (m, 3H), 4.33 (bs, 1H), 3.44 (m, 1H).
P ota ssiu m P h en p a n sta tin 3,4-Cyclic P h osp h a te (3d ):
1
white solid, 3.9 mg, mp 245 °C (dec); H NMR (300 MHz, CD
3
-
OD) δ 8.05 (d, J ) 7.2 Hz, 2H), 7.53 (t, J ) 7.35 Hz, 1H), 7.42
(
4
t, J ) 7.65 Hz, 2H), 6.39 (s, 1H), 5.96 (s, 1H), 5.90 (s, 2H),
.62-4.51 (m, 3H), 4.33 (bs, 1H), 3.46-3.41 (m, 1H).
Sod iu m P a n cr a tista tin 4-O-P h osp h a te (4a ). Pancrat-
istatin (1a , 0.2 g, 0.615 mmol) was added to pyridine (10 mL),
and the solution was heated. Next, tetrabutylammonium
dihydrogen phosphate (1.67 g, 4.92 mmol, 8 equiv), dicyclo-
hexylcarbodiimide (1.01 g, 4.92 mmol, 8 equiv), and p-tolu-
enesulfonic acid (0.35 g, 1.84 mmol, 3 equiv) were added. The
resulting solution was stirred under argon and monitored by
1
H NMR at 80 °C for 2 days. As starting material was still
present in the reaction mixture, additional DCCI (0.5 g) was
added. The mixture was stirred and heated (80 °C) for a total
of 5 days. After addition of H
2
O (100 mL), dicyclohexylurea
F igu r e 1. X-ray thermal ellipsoid plot (50% probability) of sodium
pancratistatin 4-O-phosphate (4a ) as the dihydrate.
precipitated. The mixture was stirred for 1 h, and the solution
filtered and concentrated to a brown oil. A solution of the oil
in the minimum amount of H
exchange column (Dowex 50WX8-200, 12 cm × 3.5 cm, sodium
form) and eluted with H O. The UV-active fractions were
combined according to TLC mobility (n-BuOH-CH OH-H O-
NH OH, 4:3:2:1) to give a crude white solid with considerable
impurities (0.83 g). After further separation on a G-10 Sepha-
dex column (60 cm × 2 cm), eluting with H O (at 10 mL/8 min),
54 fractions were collected and combined according to the
OH, 4:3:2:1). Sodium
2
O was passed through an ion-
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 refine-
ment process. In addition to the parent sodium phosphate salt,
two additional molecules of water solvent were also present
in the asymmetric unit. The final standard residual R value
1
for the model shown in Figure 1 converged to 0.0438 (for
observed data) and 0.0453 (for all data). The corresponding
2
3
2
4
2
2
TLC data (n-BuOH-CH
3
OH-H
2
O-NH
4
pancratistatin 4-O-phosphate was found in fractions 41-88
and crystallized from a concentrated solution of these fractions
2
Sheldrick R values were wR of 0.1205 and 0.1221, respec-
tively, and the GOF ) 1.062 for all data. The difference Fourier
in H
2
O-MeOH to afford 4-O-phosphate (4a ) as a colorless
map showed residual electron density, the largest difference
3
solid, insoluble in MeOH: 9.2 mg (3.7% yield), mp 280 °C dec;
peak and hole being +0.682 and -0.404 e/Å , respectively. The
1
H NMR (400 MHz, DMSO-d
6
) δ 13.34 (s, 1H, Ph-OH), 10.22
Flack absolute structure parameter for the model in Figure 1
refined to -0.04(4), indicating that the absolute structure for
the enantiomer shown is correct. Final bond distances and
angles were all within acceptable limits.
(bs, 1H, NH), 6.45 (s, 1H, Ar-H), 6.03 (s, 1H), 5.99 (s, 1H),
4
2
.74-4.72 (m, 1H, OH), 4.29-4.24 (m, 2H), 3.95 (narrow m,
H), 3.74 (dd, J ) 13.4 Hz, 10.2 Hz, Hz, H-4a), 3.47 (bm, 2H,
1
3
OH), 2.96 (m, 1H, H-10b); C (120 MHz, DMSO-d
1
6
and third crops (21 mg) were found to be mixtures of several
phosphates when analyzed by P NMR, and the mother liquor
was found to be a mixture (39 mg) of two, in a total of 79.6 mg
6
) δ 168.6,
P a n cr a tista tin 4-O-P h osp h or ic Acid (4b). Sodium pan-
cratistatin 3,4-cyclic phosphate (5a , 0.0165 g, 0.04 mmol) was
dissolved in H O (1 mL) and eluted through a column contain-
2
51.6, 145.3, 135.6, 131.6, 107.9, 101.5, 97.6, 72.7, 72.1, 69.9,
3
1
8.5, 50.2, 39.8; P (162 MHz, DMSO-d
6
) δ 4.73. The second
ing Amberlite IR 120 (H+) resin. The UV-active fractions were
3
1
combined and freeze-dried, 0.011 g, 69%, mp 150 °C (dec). The
1
material decomposed over several days. H NMR (DMSO-d
6
,
(
32%). The remaining material on the column visible by UV
300 MHz) δ 12.9 (s, 1H), 8.12 (s, 1H), 6.49 (s, 1H), 6.04-6.02
(m, 2H), 4.43 (m, 1H), 4.30 (m, 1H), 3.98-3.87 (m, 3H), 3.07-
3.01 (m, 1H).
Lith iu m P a n cr a tista tin 4-O-P h osp h a te (4c). Sodium
pancratistatin 3,4-cyclic phosphate (5a , 0.144 g, 0.35 mmol)
long-wave light was eluted using a gradient of MeOH-H
2
O
to MeOH and was identified by NMR as a narciprimine (6)
derivative (27 mg) pointing to aromatization of the cyclitol ring
during this phosphorylation reaction.
X-r a y Cr ysta l Str u ctu r e Deter m in a tion . Sodium pan-
cratistatin 4-O-phosphate (4a ) hydrate: long, colorless, rod-
shaped crystal (∼0.58 × 0.16 × 0.13 mm), grown from a
2
was dissolved in H O (1 mL) and eluted on an IR-120 (H+)
ion-exchange resin to convert to the monophosphoric acid, and
the active fractions were then directly eluted through a Dowex
+
MeOH-H
2
O solution, was mounted on the tip of a glass fiber.
50WX8-200 ion-exchange resin (Li ) form. The UV-active
Cell parameter measurements and data collection were per-
formed at 123 ( 2 K on a Bruker SMART 6000 diffractometer.
Final cell constants were calculated from a set of 8435
reflections from the actual data collection. Frames of data were
collected in the θ range 5.59-69.67° (-8 e h e 8, -17 ek e
fractions were combined and freeze-dried to yield a white solid,
which was washed with hot MeOH, and the solution was
1
filtered to yield 0.083 g, 62% yield, mp 265 °C; H NMR
(DMSO-d
6
, 300 MHz) δ 13.3 (s, 1H), 10.3 (s, 1H), 6.44 (s, 1H),
6.02 (s, 1H), 5.98 (s, 1H), 4.76 (m, 1H), 4.26-4.23 (m, 2H), 3.94
1
3
1
7, -18 e l e 19) using 0.396° steps in ω such that a
(m, 2H), 3.74 (m, 1H), 2.98-2.93 (m, 1H); C NMR (DMSO-
, 100 MHz) δ 168.6, 151.6, 145.3, 135.5, 131.6, 107.9, 101.5,
comprehensive coverage of the sphere of reflections was
performed. After data collection, an empirical absorption
correction was applied with the program SADABS.13 Subse-
quent statistical analysis of the complete reflection set using
d
6
3
1
97.6, 76.7, 72.1, 69.9, 68.5, 50.2, 48.6; P NMR (DMSO-d
MHz) δ 2.57; HRESI m/z calcd for [C14
found m/z 404.0383.
6
, 400
-
H15NO11P] 404.0391,
1
4
the XPREP program indicated the space group was P2
1
2
1
2
1
.
P ota ssiu m P a n cr a tista tin 4-O-P h osp h a te (4d ). Lithium
pancratistatin 4-O-phosphate (0.08 mg) was eluted through
Cr ystal data: C14
H
15NNaO11P‚2H
2
O (hydrate), a ) 7.35630-
3
+
(
10) Å, b ) 14.6555(2) Å, c ) 15.8310(2) Å, V ) 1706.74(4) Å ,
an IR-120 (K ) ion-exchange resin, and the UV-active fractions
-
3
1
λ ) (Cu KR) ) 1.54178 Å, F
c
) 1.803 g cm for Z ) 4 and fw
were collected and freeze-dried, wt 0.006 g, mp 230 °C;
NMR (DMSO-d /D
H
)
436.26, F(000) ) 960. A total of 12 108 reflections were
6
2
O) δ 6.42 (s, 1H), 5.99-5.96 (m, 2H), 4.20
(m, 2H), 3.99 (m, 1H), 3.94 (m, 1H), 3.84 (m, 1H), 2.99-2.93
collected, of which 3026 were unique (Rint ) 0.0366), and 2918
were considered observed (I > 2σ(I )). These were used in the
subsequent structure solution and refinement with SHELXTL-
o
o
(m, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e P a n -
cr a tista tin 4-O-P h osp h a te Diva len t Ca tion Sa lts (4e-4g).
The phosphoric acid (4b) (0.036 g, 0.086 mmol) was taken up
in MeOH (3 mL). A 1 mL aliquot of this solution was added to
a round bottom flask containing 1 equiv of the corresponding
1
4
V5.1. All non-hydrogen atoms for 4a were located using the
default settings of that program. Hydrogen atoms were placed
in calculated positions and assigned thermal parameters equal
to either 1.2 or 1.5 (depending upon chemical type) of the Uiso

3
26 J ournal of Natural Products, 2004, Vol. 67, No. 3
Pettit et al.
metal acetate. The opaque solutions were allowed to stir for
several days before concentrating to a residue, which was
washed with MeOH and dried.
6.48 (s, 1H), 6.03-6.02 (m, 2H), 5.42 (bs, 1H), 4.69 (d, J ) 5
Hz, 1H), 4.28 (m, 1H), 4.22 (m, 1H), 4.09-4.03 (m, 3H), 2.85
(m, 1H); 13C NMR (DMSO-d
125 MHz) δ 169.2, 152.1, 145.4,
6
Ma gn esiu m P a n cr a tista tin 4-O-P h osp h a te (4e): beige
135.2, 131.7, 107.4, 101.7, 97.6, 75.7, 75.3, 69.4, 67.7, 50.5, 37.0;
3
1
-
solid, 0.010 g, mp 255 °C (dec), insoluble in DMSO-d
6
and D
2
O;
P NMR δ 13.22; HRFAB m/z found 386.0267 (M - Na) ,
-
-
HRESI m/z calcd for [C14
H
15NO11P] 404.0391, found m/z
C
14
H
13
O
10NP requires 386.02771 (M - Na) .
4
04.0368.
Ca lciu m P a n cr a tista tin 4-O-P h osp h a te (4f): gray solid,
.011 g, mp 290 °C; H NMR (DMSO-d , 300 MHz) δ 13.2 (s,
6
H), 9.65 (s, 1H), 6.45 (s, 1H), 6.02-5.99 (m, 2H), 4.96 (m, 1H),
.34-4.26 (m, 2H), 3.96 (m, 2H), 3.85-3.73 (m, 1H), 3.02-
.97 (m, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of P a n cr a ti-
sta tin -3,4-Cyclic P h osp h a te P r od r u gs (5b,5c). Sodium
pancratistatin 3,4-cyclic phosphate (5a , 20 mg) was dissolved
1
0
1
4
2
in H
2
O and the solution passed through a column (1 × 20 cm)
of Dowex 50WX8-200 bearing the respective cation. The UV-
active fractions were combined and freeze-dried to give the
corresponding pancratistatin 3,4-cyclic phosphate salt as a
solid.
Zin c P a n cr a tista tin 4-O-P h osp h a te (4g): crystalline
powder, 0.0085 g, mp 270 °C (dec), insoluble in DMSO-d
6
and
-
D
2
O; HRESI m/z calcd for [C14
04.0372.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e P a n -
H
15NO11P] 404.0391, found m/z
Lit h iu m P a n cr a t ist a t in 3,4-Cyclic P h osp h a t e (5b ):
1
4
23.9 mg, mp 240 °C; H NMR (DMSO-d
6
, 300 MHz) δ 13.25
(s, 1H), 8.09 (s, 1H), 6.48 (s, 1H), 6.03-6.01 (m, 2H), 5.40 (bs,
1H), 4.69 (d, J ) 5.1 Hz, 1H), 4.27-4.22 (m, 2H), 4.11-4.03
(m, 3H), 2.87-2.83 (m, 1H).
cr a t ist a t in 4-O-P h osp h a t e Am m on iu m Sa lt s (4h -4l).
Pancratistatin 4-O-phosphoric acid 4b (0.012 g, 0.03 mmol)
was dissolved in MeOH (1 mL), and the amine (1.2 equiv) was
added with stirring at room temperature. The reaction was
stirred for 4 days before concentrating to a residue, which was
washed with MeOH, and the solid was filtered and dried.
P ota ssiu m P a n cr a tista tin 3,4-Cyclic P h osp h a te (5c):
1
17.8 mg, mp 238-248 °C; H NMR (DMSO-d
6
, 300 MHz) δ
13.25 (s, 1H), 8.11 (s, 1H), 6.48 (s, 1H), 6.03-6.01 (m, 2H),
5.41 (bs, 1H), 4.27-4.20 (m, 2H), 4.08-4.02 (m, 3H), 2.87-
2.83 (m, 1H).
P ip er a zin iu m P a n cr a tista tin 4-O-P h osp h a te (4h ): off-
1
white solid, 0.011 g, mp 180 °C; H NMR (DMSO-d
6
, 300 MHz)
δ 13.3 (s, 1H), 10.1 (s, 1H), 6.44 (s, 1H), 6.02-5.99 (m, 2H),
Ack n ow led gm en t. We are pleased to acknowledge finan-
cial support provided by Outstanding Investigator Grant
CA44344-03-12 and RO1 CA90441-01-03 awarded by the
Division of Cancer Treatment and Diagnosis, National Cancer
Institute, DHHS; the Arizona Disease Control Research Com-
mission; the Robert B. Dalton Endowment Fund; the Caitlin
Robb Foundation; Gary L. and Diane R. Tooker; Polly J .
Trautman; J ohn C. Budzinski; the Eagles Art Ehrmann
Cancer Fund; and the Ladies Auxiliary to the Veterans of
Foreign Wars. Other helpful assistance was from Drs. Fiona
Hogan, J ohn C. Knight, Venugopal J . R. V. Mukku, J ean-
Charles Chapuis, and J ean M. Schmidt, Linda A. Richert, and
Lee Williams, as well as Washington University Mass
Spectrometry Resource, a NIH Research Resource (Grant
P41RR0954).
4
9
.26-4.24 (m, 2H), 3.94 (m, 2H), 3.73 (m, 1H), 2.98-2.88 (m,
+
H); HRESI m/z calcd for C18
found 492.1383 (M + H) .
27 11 3
H O N P (M + H) 492.1383,
+
Mor p h olin iu m P a n cr a tista tin 4-O-P h osp h a te (4i): hy-
droscopic solid, 0.01 g, mp 130 °C; H NMR (DMSO-d , 300
6
1
MHz) δ 13.2 (bs, 1H), 9.93 (s, 1H), 6.45 (s, 1H), 6.03-5.99 (m,
2
3
H), 5.19 (bm, 3H), 4.29-4.24 (m, 2H), 3.95 (nm, 2H), 3.75-
.63 (m, 5H), 2.99-2.86 (m, 5H).
Im id a zoliu m P a n cr a tista tin 4-O-P h osp h a te (4j): crys-
1
talline solid, 0.007 g, mp 125 °C; H NMR (DMSO-d
6
, 300 MHz)
δ 13.2 (s, 1H), 9.5 (bs, 1H), 7.93 (s, 2H), 7.21 (bs, 4H), 6.45 (s,
1
H), 6.03-5.99 (m, 2H), 4.57-4.25 (m, 2H), 3.95 (m, 2H), 3.8
(m, 1H), 3.01-2.97 (m, 1H).
Qu in iu m P a n cr a tista tin 4-O-P h osp h a te (4k ): beige
1
solid, 0.015 g, mp 173 °C; H NMR (DMSO-d
6
, 300 MHz) δ
1
1
2
3.19 (s, 1H), 9.9 (s, 1H), 8.71 (m, 1H), 7.92 (d, 1H), 7.58 (m,
Su p p or tin g In for m a tion Ava ila ble: Tables of X-ray crystal-
lographic data for compound 4a are available free of charge via the
Internet at http://pubs.acs.org.
H), 7.48 (m, 1H), 7.39 (d, 1H), 6.45 (s, 1H), 6.02-5.99 (m,
H), 5.82 (m, 2H), 5.6-4.92 (m, 3H), 4.29-4.24 (m, 2H), 3.95
(m, 4H), 3.79 (m, 2H), 3.4 (m, 2H), 3.15 (s, 1H), 3.01 (m, 4H),
2
.5 (m, 1H), 1.9 (m, 2H), 1.71 (m, 1H), 1.44 (m, 1H).
Refer en ces a n d Notes
Qu in id iu m P a n cr a tista tin 4-O-P h osp h a te (4l): off-
(
1) For Antineoplasic Agents Part 510, consult: Pettit, G. R.; Xu, J .-P.;
1
white solid, 0.017 g, mp 180 °C (dec); H NMR (DMSO-d
6
300
Doubek, D. L.; Chapuis, J .-C.; Schmidt, J . M. J . Nat. Prod., accepted.
MHz) δ 13.22 (s, 1H), 9.9 (s, 1H), 8.71-8.69 (m, 1H), 7.94-
(2) (a) Pettit, G. R.; Gaddamidi, V.; Cragg, G. M. J . Nat. Prod. 1984, 47,
018-1020. (b) Pettit, G. R.; Gaddamidi, V.; Cragg, G. M.; Herald,
D. L.; Sagawa, Y. J . Chem. Soc., Chem. Commun. 1984, 1693-1694.
c) Pettit, G. R.; Gaddamidi, V.; Herald, D. L.; Singh, S. B.; Cragg, G.
M.; Schmidt, J . M.; Boettner, F. E.; Williams, M.; Sagawa, Y. J . Nat.
Prod. 1986, 49, 995-1002.
(3) Pettit, G. R.; Pettit, G. R., III; Groszek, G.; Backhaus, R. A.; Doubek,
D. L.; Barr, R. J .; Meerow, A. W. J . Nat. Prod. 1995, 58, 756-759.
1
7
6
2
1
.91 (d, 1H), 7.57 (m, 1H), 7.40-7.37 (m, 2H), 6.45 (s, 1H),
.02-5.99 (m, 3H), 5.73 (s, 1H), 5.16-5.13 (m, 3H), 4.25 (m,
H), 3.94 (m, 4H), 3.77 (m, 4H), 3.35 (m, 4H), 3.01-2.97 (m,
(
H), 2.19 (m, 1H), 1.83 (m, 1H), 1.67 (m, 2H), 1.13 (m, 1H);
+
HRESI m/z calcd for C34
H
41
N
3
O
13P (M + H) 730.2377, found
+
7
30.2378 (M + H) .
Sod iu m P a n cr a tista tin 3,4-Cyclic P h osp h a te (5a ). Pan-
cratistatin (1a ) (0.2 g, 0.615 mmol) was dissolved in pyridine
8 mL) and heated to 80 °C under argon before adding
(
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) (a) Pettit, G. R.; Freeman, S.; Simpson, M. J .; Thompson, M. A.; Boyd,
M. R.; Williams, M. D.; Pettit, G. R., III; Doubek, D. L. Anti-Cancer
Drug Des. 1995, 10, 243-250. (b) Pettit, G. R.; Orr, B.; Ducki, S. Anti-
Cancer Drug Des. 2000, 15, 389-395. (c) Toki, B. E.; Cerveny, C. G.;
Wahl, A. F.; Senter, P. D. J . Org. Chem. 2002, 67, 1866-1872.
6) (a) Pettit, G. R.; Melody, N.; Herald, D. L. J . Org. Chem. 2001, 66,
2583-2587. (b) Mutsuga, M.; Kojima, K.; Yamashita, M.; Ohno, T.;
Ogihara, Y.; Inoue, M. Biol. Pharm. Bull. 2002, 25, 223-228.
7) (a) Hudlicky, T.; Rinner, U.; Golzalez, D.; Akgun, H.; Schilling, S.;
Siengalewicz, P.; Martinot, T. A.; Pettit, G. R. J . Org. Chem. 2002,
tetrabutylammonium dihydrogen phosphate (0.3 g, 1.47 equiv)
and dicyclohexylcarbodiimide (0.92 g, 4.44 mmol, 7.25 equiv).
1
The reaction was stirred at 80 °C for 24 h. H NMR of a crude
sample of the reaction mixture showed a 50:50 mixture of 5a
and starting material. Tetrabutylammonium dihydrogen phos-
phate (0.15 g) and dicyclohexylcarbodiimide (0.5 g) were added,
and the reaction continued for a further 24 h. The reaction
(
(
2
mixture was cooled and H O (100 mL) was added. The
6
7, 8726-8743. (b) McNulty, J .; Mao, J .; Gibe, R.; Mo, R.; Wolf, S.;
dicyclohexylurea (DCU) precipitate was filtered off and the
mother liquor concentrated to a white residue. Water was
added to effect solution, and the material was eluted on a
Pettit, G. R.; Herald, D. L.; Boyd, M. R. Bioorg. Med. Chem. Lett.
2001, 11, 169-172. (c) Pettit, G. R.; Melody, N.; O’Sullivan, M.;
Thompson, M. A.; Herald, D. L.; Coates, B. J . Chem. Soc., Chem.
Commun. 1994, 2725-2726.
+
Dowex 50WX8-200 (Na form) ion-exchange resin. The UV-
(
8) Bibby, M. C.; Holwell, S. E.; Pettit, G. R. Anti-Vascular and Anti-
Tumour Effects of the Novel Agent Pancratistatin Phosphate. Biologi-
cal Basis for Antiangiogenic Therapy Conference, Milan, Italy,
November 8-10, 1999.
active fractions were combined and concentrated to a beige
crystalline solid, 0.244 g, recrystallized from H
DCM to yield 5a , 120 mg, 48%, mp >300 °C, [R]
.54 H ), R ) 0.63 (n-BuOH-MeOH-H O-NH OH, 4:3:2:1);
H NMR (DMSO-d , 500 MHz) δ 13.25 (s, 1H), 8.11 (s, 1H),
2
O-MeOH-
D
25
-3.3° (c
(
9) Ouarzane-Amara, M.; Franetich, J .-F.; Mazier, D.; Pettit, G. R.;
Meijer, L.; Doerig, C.; Desportes-Livage, I. Antimicrob. Agents
Chemother. 2002, 45, 3409-3415.
0
2
f
2
4
1
6

Antineoplastic Agents
J ournal of Natural Products, 2004, Vol. 67, No. 3 327
(
10) Cerotti, G. Nature 1967, 213, 595-596.
(14) SHELXTL-Version 5.1, an integrated suite of programs for the
determination of crystal structures from diffraction data; Bruker AXS,
Inc.: Madison, WI 53719, 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).
(
11) (a) Khorana, H. G.; Todd, A. R. J . Chem. Soc. 1953, 2257-2260. (b)
Khorana, H. G. J . Am. Chem. Soc. 1954, 76, 3517-3522. (c) Dekker,
C. A.; Khorana, H. G. J . Am. Chem. Soc. 1954, 76, 3522-3527. (d)
Tener, G. M. J . Am. Chem. Soc. 1961, 83, 159-168.
(12) Pettit, G. R.; Melody, N.; Simpson, M.; Thompson, M.; Herald, D. L.;
Knight, J . C. J . Nat. Prod. 2003, 66, 92-96.
(
13) Blessing, R. Acta Crystallogr. 1995, A51, 33-8.
NP030299+