Antineoplastic agents. 443. Synthesis of the cancer cell growth inhibitor hydroxyphenstatin and its sodium diphosphate prodrug

Article abstract of DOI:10.1021/jm000045a

A structure - activity relationship (SAR) study of the South African willow tree (Combretum caffrum) antineoplastic constituent combretastatin A-4 (3b) led to the discovery of a potent cancer cell growth inhibitor designated phenstatin (5a). This benzophenone derivative of combretastatin A-4 showed remarkable antineoplastic activity, and the benzophenone derivative of combretastatin A-1 was therefore synthesized. The benzophenone, designated hydroxyphenstatin (6a), was synthesized by coupling of a protected bromobenzene and a benzaldehyde to give the benzhydrol with subsequent oxidation to the ketone. Hydroxyphenstatin was converted to the sodium phosphate prodrug (6e) by a dibenzyl phosphite phosphorylation and subsequent benzyl cleavage (6a → 6d → 6e). While hydroxyphenstatin (6a) was a potent inhibitor of tubulin polymerization with activity comparable to that of combretastatin A-1 (3a), the phosphorylated derivative (6e) was inactive.

Full text of DOI:10.1021/jm000045a

J . Med. Chem. 2000, 43, 2731-2737
2731
An tin eop la stic Agen ts. 443. Syn th esis of th e Ca n cer Cell Gr ow th In h ibitor
Hyd r oxyp h en sta tin a n d Its Sod iu m Dip h osp h a te P r od r u g
George R. Pettit,*,† Matthew P. Grealish,† Delbert L. Herald,† Michael R. Boyd,‡ Ernest Hamel,§ and
Robin K. Pettit†
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University,
P.O. Box 872404, Tempe, Arizona 85287-2404, and Laboratory of Drug Discovery Research and Development and
Screening Technologies Branch, DTP/ DCTD, National Cancer Institute, Frederick Cancer Research and Development Center,
Frederick, Maryland 21702-1201
Received February 4, 2000
A structure-activity relationship (SAR) study of the South African willow tree (Combretum
caffrum) antineoplastic constituent combretastatin A-4 (3b) led to the discovery of a potent
cancer cell growth inhibitor designated phenstatin (5a ). This benzophenone derivative of
combretastatin A-4 showed remarkable antineoplastic activity, and the benzophenone derivative
of combretastatin A-1 was therefore synthesized. The benzophenone, designated hydroxyphen-
statin (6a ), was synthesized by coupling of a protected bromobenzene and a benzaldehyde to
give the benzhydrol with subsequent oxidation to the ketone. Hydroxyphenstatin was converted
to the sodium phosphate prodrug (6e) by a dibenzyl phosphite phosphorylation and subsequent
benzyl cleavage (6a f 6d f 6e). While hydroxyphenstatin (6a ) was a potent inhibitor of tubulin
polymerization with activity comparable to that of combretastatin A-1 (3a ), the phosphorylated
derivative (6e) was inactive.
Ch a r t 1
Podophyllum, the roots and rhizomes of Podophyllum
species such as peltatum L. (Podophyllaceae, May
Apple), found important uses, including cancer and
antiviral applications, in the traditional medicine of
early Americans and in India.2a Indeed, it was an
important component of the United States Pharma
copoeia from 1820 to 19422b (the derived resin has been
found to contain up to 38% podophyllotoxin2c (1a , Chart
1)) and was the first terrestrial plant anticancer agent
developed to clinical trials by the U.S. National Cancer
Institute some 50 years ago.2c Subsequently, podophyl-
lotoxin was converted to the glycoside derivative known
as etoposide (1b), now widely used in human cancer
treatment.3a
In 1958, we initiated a SAR investigation3b,c focused
on the trimethoxy and methylenedioxy diarylmethylene
unit of podophyllotoxin (1a ). While not detected at the
time,3b,c owing to limitations of the early antineoplastic
evaluation options, we later found the diaryl ketone (2)
to significantly inhibit growth of the P388 lymphocytic
leukemia cell line with an ED50 value of 2.6 µg/mL. By
1978, we were investigating cancer cell growth inhibi-
tion by extracts of the African willow tree Combretum
caffrum Kuntze (Combretaceae) and later discovered
three potentially important constituents, designated
combretastatins A-1 (3a ), A-2 (4), and A-4 (3b).4 Com-
bretastatin A-4, as the water soluble prodrug (3c), has
so far reached the most advanced stage of preclinical
and clinical development. More recently, we found the
diaryl ketone named phenstatin (5a ),5 structurally
related to podophyllotoxin (1a ) and combretastatin A-4
(3b), to be a very strong anticancer substance compa-
rable to the stilbene 3b (Table 1). These and other
results6 encouraged us to undertake synthesis and
evaluation of diphenol 6a .
The general procedure we reported3c in 1962 for
obtaining ketone (2) was attempted first. Coupling
reactions between 2,3-bis(tert-butyldimethylsilyloxy)-4-
methoxybromobenzene (7b) and N-(3,4,5-trimethoxy-
benzoyl)morpholine (8a ) (Scheme 1) utilizing either
* To whom correspondence should be addressed. Tel: 480-965-3351.
Fax: 480-965-8558.
† Arizona State University.
‡ LDDRD, NCI.
§ STB, NCI.
10.1021/jm000045a CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/24/2000

2732 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Pettit et al.
Ta ble 1. Evaluation of Hydroxyphenstatin (6a ), the Sodium Diphosphate Prodrug (6e), Related Derivatives, and Synthetic
Intermediates compared to Combretastatin A-1 (3a ), A-4 (3b) and Phosphate Prodrugs (3c and 3e) against a Series of Human Cancer
Cell Lines and the Murine P388 Lymphocytic Leukemia Cell Line
a
GI50
,
µg/mL
13
>10
14.2
cell type
cell line
P388
BXPC-3
RPMI-7951
SF-295
NCI-H460
KM20L2
DU-145
6a
6c
6d
2.55
>10
>10
>10
>10
5.9
>10
6e
3a
3b
3c
3e
leukemia
pancreas-a
melanoma
CNS
lung-NSC
colon
0.315
3.5
>10
>10
>10
>10
>10
ND
0.0336
5.3
0.3
4.4
ND
0.0003
0.39
ND
>0.001
0.0006
0.34
0.0004
ND
ND
0.036
0.029
0.53
<0.01
1.5
ND
0.036
0.038
0.58
0.04
0.21
NDa
0.048
ND
ND
23.1
19.0
ND
0.23
0.35
33.5
0.3
ND
0.74
0.061
0.17
prostate
>10
32.5
0.0008
ND
0.034
a
ND, no data available.
n-butyl- or tert-butyllithium were unsuccessful. Chang-
ing the acylating agent to a benzoyl chloride was also
not productive. Presumably, the bulky TBDMS substit-
uents caused enough steric hindrance to prevent nu-
cleophilic attack of the lithium-benzene complex on the
carbonyl group. Thus, we next chose the smaller meth-
oxymethyl ether (MOM) protecting group.7 However,
formation of the benzophenone using the MOM-pro-
tected bromobenzene (7c) and either the morpholine
amide (8a ) or the benzoyl chloride (8b) met with limited
success, affording 24% and 20% yields, respectively.
Further attempts to prepare protected diphenol (6c)
using Grignard reactions, Weinreb amides,8 or dimethyl-
amides also afforded low yields (14-44%). Application
striking is the obvious out-of-plane twist adopted by the
lower ring in relation to the common trimethoxyphenyl
ring of each compound. The conformations of these two
compounds are strikingly similar, possibly explaining
in part the similar antineoplastic activity exhibited by
these substances.
Hydroxyphenstatin (6a ) was found to potently inhibit
tubulin polymerization, and its activity appeared to be
somewhat greater than that of combretastatin A-1 (3a )
(Table 2). Nevertheless, 6a was somewhat less active
than 3a as an inhibitor of the binding of [3H]colchicine
to tubulin (Table 2). The reason for the apparent
difference in relative activities between the catalytic
assembly assay and the stoichiometric colchicine bind-
ing assay is not understood. However, it has been
observed with other colchicine site drugs,12 and an
analogous pattern was also observed when phenstatin
(5a ) and combretastatin A-4 (3b) were compared.5
9
of organometallic reagents such as La(OTf)3 , Bu3P,10
and Fe(acac)311 did not lead to improved yields of ketone
(6c).
To determine if the protecting groups were interfer-
ing, ketone formation was evaluated starting with 2,3,4-
trimethoxybromobenzene (9) and morpholine amide
(8a ). The resulting yields were found to range from 17%
to 20%. These results indicated that the protecting
groups used in the preceding reaction may not have
significantly influenced the poor yields. However, we
found that condensing the bromobenzene (9) with 3,4,5-
trimethoxybenzaldehyde led to the formation of ben-
zhydrol 14 in 86% yield. Subsequent oxidation with
pyridinium dichromate (PDC) to benzophenone 13 was
accomplished at 83% yield. Those favorable results led
us to utilize the reaction between an efficient aldehyde
and an organolithium reagent to prepare a benzhydrol
derivative of ketone 6a . That approach was realized
when the lithium derivative of MOM-protected bro-
mobenzene 7c and 3,4,5-trimethoxybenzaldehyde were
condensed to afford protected benzhydrol 15 in 92%
yield. Oxidation by PDC produced protected hydroxy-
phenstatin (6c) in good yield (96%) and MOM cleavage
(acidic) afforded hydroxyphenstatin (6a ) in 97% yield.
Verification of the structure of hydroxyphenstatin (6a )
was established via single-crystal X-ray crystallography.
The unit cell contained four molecules of the parent
compound, each asymmetric unit consisting of two
independent molecules of hydroxyphenstatin. In addi-
tion, adjacent molecules of hydroxyphenstatin are linked
via intermolecular hydrogen bonding between the O10
carbonyl and O7 hydroxyl group, as shown in Figure 1.
A comparative view of the solid-state conformations
exhibited by hydroxyphenstatin (6a ) and the combret-
astatin A-4 prodrug (3c) is shown in Figure 2. Note-
worthy, is the distinct “cis” conformational arrangement
of the two aromatic rings in both compounds. Even more
Due to the improved therapeutic properties of the
combretastatin A-4 sodium phosphate prodrug (3c) vs
the parent phenol (3b),14 the corresponding hydroxy-
phenstatin prodrug (6e) was synthesized (6a -e, Scheme
2). The previous phosphorylation techniques4c,d,15 we
used for such syntheses, based on pentavalent and
trivalent phosphorus precursors, were evaluated. They
proved to be substantially less effective than employ-
ment of the dibenzyl phosphite approach.4c,d,16 The
prodrug was synthesized in three steps from hydroxy-
phenstatin by phosphorylation of phenol (6a ) utilizing
dibenzyl phosphite (under basic conditions in acetoni-
trile),16 followed by cleavage of the benzyl groups (6d )
with trimethylsilyl iodide (formed in situ) and reaction
of the resulting phosphoric acid with sodium methoxide
in ethanol to afford the sodium phosphate prodrug (6e)
in 92% overall yield. As expected, 6e was not active as
an inhibitor of tubulin polymerization (IC50 > 40 µM;
Table 2), as has been the case with other phosphorylated
derivatives in the combretastatin series. However, its
activity as an inhibitor of cancer cell growth (Table 1)
was significant.
Comparative testing of 6a and 6e in the NCI 60-cell
screen revealed a differential cytotoxicity profile and
potency (e.g. mean-panel GI50 ) 1.67 ( 0.24 × 10-7 M)
that were essentially indistinguishable from each other
or from those of combretastatin A-4. The antimicrobial
activities of the Combretum caffrum constituents com-
bretastatins A-1 and A-4 have been reported.13 While
several precursors to the related compound sodium
hydroxyphenstatin diphosphate (6e) exhibited antifun-
gal and/or antibacterial action (Table 3), the prodrug

Antineoplastic Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2733
Sch em e 1
(6e) did not. Compounds 6a , 6c, 7b, 7c, 8a , 13, 14, and
15 were also available in sufficient quantity for antibi-
otic screening. At 100 µg/disk, none of these compounds
inhibited growth of the two fungal and eight bacterial

2734 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Pettit et al.
Sch em e 2. Synthesis of Hydroxyphenstatin Prodrug
(6e)
F igu r e 1. Crystal structure of hydroxyphenstatin (6a ), show-
ing intermolecular H-bonding between the carbonyl oxygen O7
and the hydroxyl hydrogen on O10.
+
6e, M = Na , n = 2-4
+
f, M = Li , n = 2-4
+
g, M = K , n = 2-4
+ +
=
h, M Ca , n = 1-2
Ta ble 3. Antimicrobial Activities of Sodium Hydroxy-
phenstatin Phosphate Synthetic Intermediates
minimum inhibitory
compd
microbe(s) inhibited
Micrococcus luteus
Cryptococcus neoformans
Stenotrophomonas maltophilia
C. neoformans
concentration (µg/disk)
6d
10
50-100
25-50
50-100
6.25-12.5
12.5-25
50-100
25-50
12.5-25
50-100
12.5-25
25-50
50-100
25-50
<6.25
11
Candida albicans
Escherichia coli
Neisseria gonorrhoeae
C. neoformans
12
7a
C. albicans
N. gonorrheae
F igu r e 2. Comparative view of the solid-state conformations
of combretastatin A-4 prodrug 3c (left) and hydroxyphenstatin
(6a ) (right).
S. maltophilia
E. coli
Staphylococcus aureus
N. gonorrhoeae
Ta ble 2. Interactions with Tubulin of Hydroxyphenstatin (6a ),
Its Diphosphate Derivative (6e), Combretastatin A-1 (3a ),
Combretastatin A-4 (3b), and Its Prodrug (3c)
13C NMR spectra were determined using a Varian Gemini 300
MHz instrument with CDCl3 (TMS internal reference) as
solvent, unless otherwise noted. The 31P NMR spectra were
measured in CDCl3 with 85% H3PO4 as an external standard
employing a Varian Unity 500 MHz instrument. The X-ray
crystal structure data collection was performed on an Enraf-
Nonius CAD4 diffractometer. Elemental analyses were deter-
mined by Galbraith Laboratories, Inc., Knoxville, TN.
inhibition of tubulin
polymerization
(IC50 ( SD, µM)
% inhibition of
colchicine
binding ( SD
compd
6a
6e
3a
3b
3c
0.82 ( 0.2
>40
1.1 ( 0.07
1.0 ( 0.05
>40
77 ( 4
NDa
99.6 ( 0.7
98 ( 1
ND
2-Acetoxy-3-m eth oxyben za ld eh yd e (10). To a solution
of o-vanillin (10.1 g) and a catalytic quantity (0.8 g) of
(dimethylamino)pyridine in N,N-diisopropylethylamine (23
mL) at 0 °C was added acetic anhydride (8 mL). The solution
was stirred overnight, poured into 2 N hydrochloric acid (100
mL), and extracted with dichloromethane, and the solvent was
removed in vacuo to afford a yellow solid. Recrystallization
from ethanol yielded yellow crystals (10.9 g, 85%): mp 75.4-
76.2 °C, lit.17 mp 76 °C; EIMS m/z 194 (M+), 152, 106, 43.
2-Acetoxy-3-m eth oxy-6-br om oben za ld eh yd e (11). To a
solution of potassium bromide (40 g) in H2O (250 mL) was
added bromine (6.8 mL). To the dark red solution was added
aldehyde 10 (20.2 g). The turbid orange solution was stirred
overnight, filtered, rinsed with ethyl acetate, and recrystallized
from EtOAc/hexane to afford yellow crystals (22.4 g, 79%): mp
121.6-123.4 °C, lit.18 mp 119-120 °C; EIMS m/z 274 (M+,
a
Not done.
strains tested. Further biological evaluations, including
animal studies, of the hydroxyphenstatin series are
planned including the sodium (6e), lithium (6f), potas-
sium (g), calcium (h ) and ammonium cation prodrugs.
Exp er im en ta l Section
All solvents were redistilled. Both the course of and products
from reactions were monitored by thin-layer chromatography
using Analtech silica gel GHLF uniplates. Solvent extracts of
aqueous solutions were dried over anhydrous sodium sulfate
unless otherwise noted. Flash column chromatography was
performed using silica gel (230-400 mesh ASTM).
1
81Br), 272 (M+, 79Br), 232, 230, 186, 184, 43; H NMR δ 10.26
Melting points were recorded employing an Electrothermal
9100 digital melting point apparatus and are uncorrected. The
IR spectra were obtained using a Mattson FTIR model 2020
instrument. Low resolution mass spectral data were collected
using a Varian MAT 312 instrument (EIMS). The high-
resolution FAB spectra were obtained at the Midwest Center
for Mass Spectrometry employing a Kratos MS-50 mass
spectrometer, University of Nebraska, Lincoln NE. All 1H and
(1H, s, CHO), 7.51 (1H, d, J ) 9.0 Hz, H5), 7.05 (1H, d, J ) 9.0
Hz, H4), 3.85 (3H, s, OCH3), 2.38 (3H, s, COCH3); 13C NMR
(75.5 MHz) δ 190.38, 168.71, 151.78, 140.37, 131.41, 126.49,
117.69, 116.35, 56.40, 20.44. Anal. (C10H9O4Br) C,H.
2-Hyd r oxy-3-m eth oxy-6-br om oben za ld eh yd e (12). To
the aldehyde 11 (17.7 g) in aqueous methanol (125 mL) was
added sodium bicarbonate (7.6 g, 1.1 equiv), and the turbid
bright yellow solution was stirred for 2 h. The solution was

Antineoplastic Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2735
acidified and extracted with dichloromethane and the solvent
removed in vacuo to afford a yellow solid. The product was re-
crystallized from ethyl acetate/hexane to afford yellow crystals
(14.7 g, 98%): mp 105.6-106.4 °C, lit.19 mp 102-103 °C; EIMS
m/z 232 (M+, 81Br), 230 (M+, 79Br), 186, 107, 79, 54, 32.
(hexanes/ethyl acetate 19:1) and yielded the title compound21
as a yellow oil (5.9 g, 78%): EIMS m/z 234 ((M+ - CH3, 81Br),
232 (M+ - CH3, 79Br), 107, 95, 69, 58, 44; 1H NMR (CDCl3,
300 MHz) δ 7.21 (1H, d, J ) 9.0 Hz, H6), 6.58 (1H, d, J ) 9.0
Hz, H5), 3.91 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.85 (3H, s,
OCH3).
2′,3,3′,4,4′,5-Hexa m eth oxyben zop h en on e (13). To a solu-
tion of bromobenzene 9 (0.21 g) in dry THF (5 mL) cooled to
-78 °C was added n-butyllithium (0.38 mL, 2.5 M, 1.1 equiv).
The solution was stirred for 30 min, 3,4,5-trimethoxybenzoyl
chloride (0.2 g) in anhydrous tetrahydrofuran was added, and
the solution was stirred for 28 h. The reaction was stopped
with water and extracted with ethyl acetate and the solvent
removed (in vacuo) to give a yellow oil. Separation by flash
column chromatography (hexanes/ethyl acetate 3:1) afforded
a colorless solid (0.06 g, 20.5%). The solid was recrystallized
twice from ethyl acetate/hexane: mp 124.6-125.9 °C, lit.22 mp
121 °C; EIMS m/z 362 (M+), 345, 317, 181, 169, 151; 1H NMR
δ 7.11 (1H, d, J ) 9.0 Hz, H6′), 7.08 (2H, s, H2,6), 6.72 (1H, d,
J ) 9.0 Hz, H5′), 3.94 (3H, s, OCH3), 3.93 (3H, s, OCH3), 3.90
(3H, s, OCH3), 3.85 (6H, s, OCH3 3,5), 3.80 (3H, s, OCH3).
1-Br om o-2,3-d ih yd r oxy-4-m eth oxyben zen e (7a ). Alde-
hyde 12 (23 g) was suspended in 2% sodium hydroxide (300
mL), and a solution of 30% hydrogen peroxide (15.8 g, 1.4
equiv) was added. After 2 h, another portion of 30% hydrogen
peroxide (1.4 equiv) was added and the solution stirred
overnight. The reaction mixture was acidified, extracted with
dichloromethane, and washed with sodium thiosulfate, and the
solvent was removed in vacuo to afford a tan solid. The solid
was recrystallized from methanol to afford colorless crystals
(14 g, 64%): mp 122-124 °C, lit.20 mp 124-126 °C; EIMS m/z
220 (M+, 81Br), 218 (M+, 79Br), 205, 203, 177, 175, 95; 1H NMR
δ 6.99 (1H, d, J ) 9.0 Hz, H5), 6.42 (1H, d, J ) 9.0 Hz, H4),
5.56 (1H, s, OH), 5.51 (1H, s, OH), 3.88 (3H, s, OCH3); 13C
NMR (75.5 MHz) δ 146.59, 140.95, 133.47, 122.21, 104.35,
101.55, 56.31.
1-Br om o-2,3-b is(ter t-b u t yld im et h ylsilyloxy)-4-m et h -
oxyben zen e (7b). To a solution of diphenol 7a (0.51 g) in dry
dimethylformamide (10 mL) was added successively diisopro-
pylethylamine (1.25 mL, 3.1 equiv) and tert-butyldimethylsilyl
chloride (0.78 g, 2.2 equiv), and the mixture was stirred at
room temperature under argon for 3 h. (HCl evolution was
noted.) The reaction was terminated by adding ice. After
extraction with DCM, the combined solvent was washed with
water, saturated sodium bicarbonate, and water, and dried.
Removal of solvent gave an oil which solidified on trituration
with ether. The solid was recrystallized from methanol and
afforded colorless crystals (0.92 g, 90%): mp 68.9-69.6 °C;
EIMS m/z 448 (M+, 81Br), 446 (M+, 79Br), 443, 431, 391, 389,
167; IR (Kbr, cm-1) νmax 2934, 2859, 1576, 1472, 1254, 1092,
845, 671; 1H NMR δ 7.06 (1H, d, J ) 8.7 Hz, H6), 6.42 (1H, d,
J ) 8.7 Hz, H5), 3.75 (3H, s, OCH3), 1.06 (9H, s, C(CH3)3), 0.99
(9H, s, C(CH3)3), 0.19 (6H, s, Si(CH3)2), 0-10 (6H, s, Si(CH3)2);
13C NMR 75.5 MHz) δ 151.74, 145.32, 138.06, 124.33, 108.36,
105.64, 55.04, 26.46, 26.12, 18.75, 18.73, -3.10, -3.85. Anal.
(C19H35BrO3Si2) C, H.
1-Br om o-2,3-b is(m et h oxym et h yloxy)-4-m et h oxyb en -
zen e (7c). To a solution of 1-bromo-2,3-dihydroxy-4-methoxy-
benzene (5.0 g) and anhydrous THF (20 mL) at 0 °C under
argon was added diisopropylethylamine (8.0 mL). The solution
was stirred for 15 min, methyloxymethyl chloride (3.5 mL) was
added, and the reaction mixture stirred for 3 h. The solution
was poured into water (250 mL) and extracted with DCM and
the solvent removed in vacuo to provide an orange oil. The oil
was purified by flash column chromatography (hexane/EtOAc
2:1) to yield a clear oil (6.4 g, 91%): EIMS m/z 308 (M+, 81Br),
306 (M+, 79Br), 232, 230, 45; IR (neat, cm-1) νmax 2963, 2836,
1221, 1159, 1084, 966; 1H NMR δ 7.25 (1H, d, J ) 9.0 Hz, H6),
6.61 (1H, d, J ) 9.0 Hz, H5), 5.20 (2H, s, OCH2), 5.13 (2H, s,
OCH2), 3.84 (3H, s, OCH3), 3.66 (3H, s, OCH3), 3.59 (3H, s,
OCH3); 13C NMR (75.5 MHz) δ 153.31, 148.24, 140.05, 127.49,
2′,3,3′,4,4′,5-Hexa m eth oxyd ip h en ylca r bin ol (14). The
preceding experiment (cf. 10) was repeated using bromoben-
zene 9 (0.55 g), anhydrous tetrahydrofuran (15 mL), and
n-butyllithium (0.93 mL, 2.5 M, 1.05 equiv). A solution of 3,4,5-
trimethoxybenzaldehyde (0.44 g) was added and the solution
stirred for 16 h. The resulting oily product was separated by
flash column chromatography (hexanes/ethyl acetate 9:1) to
give a clear oil (0.70 g, 86%): EIMS m/z 364 (M+), 331, 315,
195, 181, 169; IR (neat, cm-1) νmax 3462, 2940, 2837, 1593,
1
1464, 1234, 1127, 1015; H NMR δ 6.90 (1H, d, J ) 8.7 Hz,
H6′), 6.64 (1H, d, J ) 8.7 Hz, H5′), 6.61 (2H, s, H2,6), 5.88 (1H,
d, J ) 3.9 Hz, CH), 3.86 (3H, s, OCH3), 3.85 (3H, s, OCH3),
3.83 (3H, s, OCH3), 3.82 (6H, s, OCH3 × 2), 3.76 (3H, s, OCH3);
13C NMR (75.5 MHz) δ 153.37, 152.98, 151.20, 142.03, 139.55,
136.91, 129.63, 122.15, 106.98, 103.46, 72.03, 60.85, 60.74,
60.61, 56.01, 55.88. Anal. (C19H24O7) C, H.
2′,3′-Bis(m e t h oxym e t h yloxy)-3,4,4′,5-t e t r a m e t h oxy-
d ip h en ylca r bin ol (15). To a solution of protected bromoben-
zene 7c (0.91 g, 2.95 mmol) in anhydrous tetrahydrofuran (5.0
mL) cooled to -78 °C was added n-butyllithium (1.21 mL, 2.44
M, 2.95 mmol). The solution was stirred for 1 h, 3,4,5-
triethoxybenzyaldehyde (0.58 g, 2.95 mmol) was added, and
the solution was stirred for 4 h. The reaction was ended by
adding water, and the mixture was extracted with ethyl
acetate. Removal of solvent (in vacuo) led to a yellow oil that
was separated by flash column chromatography (hexanes/ethyl
acetate 9:1) to afford a clear oil that solidified upon standing
(1.15 g, 92%). The solid was recrystallized from methanol and
yielded colorless plates: mp 79.9-81.8 °C; HRMS 424.1749
C
21H28O9; EIMS m/z 424 (M+), 362, 347, 331, 317, 289, 181;
IR (KBr, cm-1) νmax 3407, 3001, 2942, 2836, 1236, 1155, 1123,
1063; 1H NMR δ 6.71 (1H, d, J ) 9.0 Hz, H6′), 6.68 (2H, s,
H
2,6), 6.63 (1H, d, J ) 9.0 Hz, H5′), 6.09 (1H, d, J ) 3.3 Hz,
CH), 5.20 (2H, dd, J ) 6.0, 10.5 Hz, OCH2), 5.13 (2H, s, OCH2),
3.85 (3H, s, OCH3), 3.84 (6H, s, OCH3 × 2), 3.82 (3H, s, OCH3),
3.61 (3H, s, OCH3), 3.58 (3H, s, OCH3); 13C NMR (75.5 MHz)
δ 153.23, 153.05, 149.73, 138.17, 138.12, 136.80, 131.16,
123.52, 108.04, 104.32, 103.53, 100.03, 98.59, 69.79, 60.85,
57.74, 57.37, 56.07, 55.93. Anal. (C21H28O9) C, H.
108.94, 108.73, 99.39, 98.73, 58.14, 57.46, 56.11. Anal. (C11H15
BrO5) C, H.
-
N-(3,4,5-Tr im eth oxyben zoyl)m or p h olin e (8a ). Morpho-
line (0.8 mL) was slowly added to a solution composed of
toluene (10 mL) and 3,4,5-trimethoxybenzoyl chloride (1.1 g).
The reaction was accompanied by evolution of heat and
precipitation of morpholine hydrochloride. After a 3 h period,
the solution was filtered and concentrated in vacuo to afford
a white solid which was recrystallized from ethanol to afford
colorless needles (1.2 g, 86%): mp 119.8-120.7 °C, lit.3c mp
120-121 °C; EIMS m/z 281 (M+), 266, 195; 1H NMR δ 6.63
(2H, s, H2,6), 3.87 (6H, s, OCH3 × 2), 3.86 (3H, s, OCH3), 3.70
(8H, bs, CH2 × 4).
2′,3′-Bis(m eth oxym eth yloxy)-3,4,4′,5-tetr a m eth oxyben -
zop h en on e (6c). To a stirred solution of diphenylcarbinol 15
(6.85 g) in dichloromethane (250 mL) was added 4 Å molecular
sieves (9 g) and pyridinium dichromate (9.1 g, 1.5 equiv). The
black solution was stirred overnight, filtered through Celite,
rinsed with methanol and the solvent removed in vacuo to
afford a black residue. The mixture was separated by flash
column chromatography (hexanes-ethyl acetate, 4:1) to pro-
vide a clear oil that solidified upon standing (6.5 g, 96%). The
solid was recrystallized twice from ethyl acetate-hexane to
afford colorless crystals: mp 70.2-71.7 °C; HRMS 422.1577
1-Br om o-2,3,4-tr im eth oxyben zen e (9). Pyrogallol trimeth-
yl ether (5.1 g) was suspended in CCl4 (60 mL), and N-
bromosuccinimide (6.5 g, 1.2 equiv) was added. The reaction
mixture was heated at reflux for 20 h. The succinimide was
collected and the filtrate concentrated in vacuo to a brown oil.
The oil was separated by gravity column chromatography
C
21H26O9; EIMS m/z 422 (M+), 346, 195, 181; IR (KBr, cm-1
)
νmax 3005, 2944, 2845, 1649, 1583, 1231, 1125, 1071; 1H NMR
δ 7.16 (1H, d, J ) 8.5 Hz, H6′), 7.12 (2H, s, H2,6), 6.78 (1H, d,

2736 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Pettit et al.
J ) 8.5 Hz, H5′), 5.18 (2H, s, OCH2 2′), 5.01 (2H, s, OCH2),
3.93 (3H, s, OCH3), 3.92 (3H, s, OCH3), 3.85 (6H, s, OCH3 3,5),
3.62 (3H, s, OCH3), 3.26 (3H, s, OCH3); 13C NMR (75.5 MHz)
δ 193.87, 155.93, 152.77, 149.48, 142.42, 138.80, 133.07,
127.35, 125.48, 107.64, 107.36, 99.76, 98.66, 60.92, 57.41,
57.32, 56.22, 56.05. Anal. (C21H26O9) C, H.
(7.8 mL, 3.0 equiv) was added and the solution was stirred
for 16 h under argon at -10 °C and then brought to room
temperature. The reaction was terminated with 0.5 M KH2-
PO4 and extracted with ethyl acetate, and the combined
solvent was washed with brine and dried. Removal of solvent
(in vacuo) afforded an orange oil which was separated by flash
column chromatography (hexanes/ethyl acetate 1:1 to 0:1) to
provide a white solid (9.7 g, 96%). The solid was recrystallized
twice from ethyl acetate/hexane: mp 86.9-87.4 °C; EIMS m/z
854 (M+), 656, 576, 514, 486, 91; IR (KBr, cm-1) νmax 2967,
2945, 2841, 1659, 1298, 1020, 951; 1H NMR δ 7.44 (1H, d, J )
9.0 Hz, H6′), 7.27 (18H, m, Ar-H), 7.11 (2H, s, H2,6), 7.08 (2H,
m, Ar-H), 6.92 (1H, d, J ) 9.0 Hz, H5′), 5.23 (2H, s, CH2Bn),
5.21 (2H, s, CH2Bn), 4.77 (2H, dd, J ) 4.5, 6.9 Hz, CH2Bn),
4.64 (2H, dd, J ) 4.5, 6.9 Hz, CH2Bn), 3.84 (3H, s, OCH3),
3.80 (6H, s, OCH3 3,5), 3.77 (3H, s, OCH3); 13C NMR (75.5 MHz)
δ 191.73, 171.11, 154.84, 152.84, 142.20, 135.81, 135.74,
135.24, 135.18, 132.77, 128.42, 128.37, 128.29, 127.96, 127.67,
127.58, 125.90, 109.19, 107.55, 69.98, 69.93, 69.87, 69.82,
60.80, 60.36, 56.30, 56.23; 31P NMR (DMSO, decoupled,
-202.35 MHz) δ -5.01, -5.79. Anal. Calcd for C45H44O13P2:
C, 63.23; H, 5.19. Found: C, 62.81; H, 5.58.
Hyd r oxyp h en sta tin [(2′,3′-Dih yd r oxy-4-m eth oxyp h en -
yl)(3,4,5-tr im eth oxyp h en yl)m eth a n on e] (6a ). To a stirred
solution of MOM-protected hydroxyphenstatin 6c (0.120 g,
0.284 mmol) in methanol (10.0 mL) was added 1 N HCl (0.57
mL) and the solution was stirred for 2 h. The reaction mixture
was poured into water and, extracted with dichloromethane
and the solvent evaporated in vacuo to yield a yellow solid (0.09
g, 97%). The solid was recrystallized (twice) from methanol:
mp 171.1-171.9 °C; HRMS 334.1052 C17H18O7; EIMS m/z 334
(M+), 303, 195, 168, 153; IR (KBr, cm-1) νmax 3273, 3100, 3001,
1
2944, 1636, 1574, 1121, 1063; H NMR δ 12.23 (1H, s, OH),
7.27 (1H, d, J ) 8.7 Hz, H6′), 6.92 (2H, s, H2,6), 6.51 (1H, d, J
) 8.7 Hz, H5′), 5.57 (1H, s, OH), 3.99 (3H, s, OCH3), 3.94 (3H,
s, OCH3), 3.90 (6H, s, OCH3 × 2); 13C NMR (75.5 MHz) δ
199.58, 152.93, 152.08, 150.94, 141.33, 133.67, 133.13, 125.59,
113.99, 106.80, 102.53, 60.98, 56.32, 56.24. Anal. (C17H18O7)
C, H.
Sod iu m Hyd r oxyp h en sta tin Dip h osp h a te (6e). A mix-
ture of the phosphorylated hydroxyphenstatin 6d (9.0 g) and
sodium iodide (6.3 g, 4.0 equiv) in anhydrous acetonitrile (30
mL) was stirred (under argon) and trimethylsilyl chloride (5.4
mL, 4.0 equiv) was added. The solution was stirred for 2 h
and the reaction was stopped with water. After extraction with
ethyl acetate, the aqueous layer was concentrated to a light
brown foam. To the residue in ethanol (75 mL) was added
sodium methoxide (2.3 g, 4.0 equiv) and the solution stirred
for 12 h. The reaction mixture was concentrated and the
residue crystallized from water/acetone to yield an amphorous
solid (5.6 g, 92%) which was recrystallized (three times) from
water/acetone: mp 145-147; LRFAB m/z 583.1 (M + H+),
calcd 583.2; IR (KBr, cm-1) νmax 3009, 2947, 2843, 1643, 1343,
1289, 1182, 1123, 990; 1H NMR (D2O, 500 MHz) δ 7.07 (2H, s,
H2,6), 6.98 (1H, d, J ) 5.1 Hz, H6′), 6.75 (1H, d, J ) 5.1 Hz,
H5′), 3.80 (3H, s, OCH3), 3.75 (6H, s, OCH3 × 2), 3.74 (3H, s,
OCH3); 13C NMR (D2O, reference to CDCl3, 75.5 MHz) δ 197.15,
156.15, 152.24, 145.03, 141.38, 135.70, 133.99, 126.37, 125.49,
108.72, 106.95, 61.16, 56.40, 56.30; 31P NMR (D2O, decoupled,
-202.35 MHz) δ 0.05, -1.49. The solubility of sodium hydroxy-
phenstatin diphosphate was found to be 100 mg/mL in distilled
water at 25 °C.
Lith iu m Hyd r oxyp h en sta tin Dip h osp h a te (6f). To the
light brown foam in methanol (10 mL) was added lithium
hydroxide (0.049 g, 4.0 equiv) and the solution stirred for 12
h. The reaction mixture was concentrated and the residue
crystallized from water/acetone to yield an amphorous solid
(0.11 g, 74%) which was recrystallized from water/acetone: mp
174-176 °C (dec); LRFAB m/z 511 (M+), 505 (M+ - Li), 499
(M+ - 2Li), 493 (M+ - 3Li), 435, 413, 199; IR (Kbr, cm-1) νmax
3011, 2945, 2845, 1632, 1339, 1283, 1187, 1127, 1003; 1H NMR
(D2O, 300 MHz) δ 7.13 (2H, s, H2,6), 6.96 (1H, d, J ) 7.5 Hz,
H6′), 6.69 (1H, d, J ) 7.5 Hz, H5′), 3.77 (6H, s, OCH3 × 2), 3.76
(3H, s, OCH3), 3.75 (3H, s, OCH3). The solubility of lithium
hydroxyphenstatin diphosphate was found to be 25 mg/mL in
distilled water at 25 °C.
X-r ay Cr ystal Str u ctu r e Deter m in ation . Hydr oxyph en -
sta tin , 6a . A thick, plate-shaped X-ray sample (∼0.38 × 0.36
× 0.08 mm), grown from methanol solution, was mounted on
the tip of a glass fiber with Super-Glue. Data collection was
performed at 301 ( 1° K. Accurate cell dimensions were
determined by least-squares fitting of 25 carefully centered
reflections in the range of 35° < θ < 40° using Cu KR radiation.
Cr ysta l Da ta : C17H18O7, FW ) 334.31, monoclinic, space-
group Pc, a ) 10.423(2) Å, b ) 11.297(2) Å, c ) 14.173(2) Å, â
) 111.02(3)°, V ) 1557.8(5) Å3, Z ) 4, Fc ) 1.425 Mg/m3, µ(Cu
KR) ) 0.942 mm-1, λ ) 1.54178 Å.
All reflections corresponding to a complete quadrant (0 e h
e 12, 0 e k e 13, -16 e l e 15) were collected over the range
of 0 < 2θ < 130° using the ω/2θ scan technique. Three intensity
control reflections were also measured for every 60 min of
X-ray exposure time and showed a maximum variation of
-0.1% over the course of the collection. A total of 6255
reflections were collected. Subsequent statistical analysis of
the complete reflection data set using the XPREP23 program,
verified that the space group was Pc. After Lorentz and
polarization corrections, merging of equivalent reflections and
rejection of systematic absences, 2795 unique reflections re-
mained, of which 2492 were considered observed (Io > 2σ(Io))
and were used in the subsequent structure determination 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).24 Structure determi-
nation was readily accomplished with the direct-methods
program SHELXS.25 All non-hydrogen atom coordinates were
located in a routine run using default values in that program.
The remaining H atom coordinates were calculated at optimum
positions. All non-hydrogen atoms were refined anisotropically
in a full-matrix least-squares refinement using SHELXL.25 The
H atoms were included, their Uiso thermal parameters fixed
at either 1.2 or 1.5 (depending upon atom type) of the Uiso of
the atom to which they were attached and forced to ride that
atom. The final standard residual R1 value for 6a was 0.0661
for observed data and 0.0701 for all data. The goodness-of-fit
on F2 was 1.058. The corresponding Sheldrick R values were
wR2 of 0.1689 and 0.1771, respectively. A final difference Four-
ier map showed minimal residual electron density; the largest
difference peak and hole being 0.422 and -0.294 e/Å3, respec-
tively. Final bond distances and angles were all within expec-
ted and acceptable limits. Intermolecular hydrogen bonding
was observed between the carbonyl oxygen (O7) of one mole-
cule and the m-hydroxyl group (O10) of an adjacent molecule.
P ota ssiu m Hyd r oxyp h en sta tin Dip h osp h a te (6g). To
the light brown foam in methanol (10 mL) was added potas-
sium hydroxide (0.065 g, 4.0 equiv) in water (5 mL) and the
solution stirred for 12 h. The reaction mixture was concen-
trated and the yellow solid crystallized from water/acetone to
yield an amphorous solid (0.161 g, 86%) which was recrystal-
lized from water/acetone: mp 141-143 °C (dec); LRFAB m/z
647 (M+ - K), 545, 395, 333, 181; IR (Kbr, cm-1) νmax 3010,
2946, 2843, 1640, 1335, 1269, 1169, 1123, 988; 1H NMR (D2O,
300 MHz) δ 7.14 (2H, s, H2,6), 6.96 (1H, d, J ) 8.1 Hz, H6′),
6.69 (1H, d, 8.1 Hz, H5′), 3.77 (6H, s, OCH3 × 2), 3.76 (3H, s,
OCH3), 3.75 (3H, s, OCH3). The solubility of potassium
hydroxyphenstatin diphosphate was found to be >100 mg/mL
in distilled water at 25 °C.
2′,3′-O-Bis(ben zylp h osp h or yl)h yd r oxyp h en sta tin (6d ).
To a solution of hydroxyphenstatin 6a (4 g) in dry acetonitrile
(100 mL) and carbon tetrachloride (11.4 mL, 10 equiv) were
added DMAP (0.14 g, 0.1 equiv) and diisopropylethylamine (8.7
mL, 4.2 equiv). After cooling to -10 °C, dibenzyl phosphite
Ca lciu m Hyd r oxyp h en sta tin Dip h osp h a te (6h ). To the

Antineoplastic Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2737
Natural Products and their Interactions with Tubulin. Med. Res.
Rev. 1996, 16, 207-231. (c) Pettit, G. R.; Rhodes, M. R.; Herald,
D. L.; Chaplin, D. J .; Stratford, M.; Pettit, R. K.; Chapuis, J .-C.;
Oliva, D. Antineoplastic Agents 393. Synthesis of the trans-
isomer of Combretastatin A-4 Prodrug. Anti-Cancer Drug Design
1998, 13, 981-994. (d) Pettit, G. R.; Rhodes, M. R. Antineoplastic
Agents 389. New Syntheses of the Combretastatin A-4 Prodrug.
Anti-Cancer Drug Design 1998, 13, 183-191.
light brown foam in methanol (10 mL) was added calcium
acetate (0.102 g, 2.0 equiv) and the solution stirred for 12 h.
The reaction mixture was concentrated and the residue
crystallized from water/acetone to yield an amphorous solid
(0.159 g, 79%) which was recrystallized from water/acetone:
mp 186-188 °C (dec); LRFAB m/z 531 (M+), 493, 413, 395,
277; IR (KBr, cm-1) νmax 3011, 2940, 2847, 1638, 1337, 1296,
1
(5) Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.; Boyd,
M. R.; Hamel, E.; Pettit, R. K. Antineoplastic Agents 379.
Synthesis of Phenstatin Phosphate. J . Med. Chem. 1998, 41,
1688-1695.
(6) Chaplin, D. J .; Pettit, G. R.; Hill, S. A. Antivascular Approaches
to Solid Tumor Therapy: Evaluation of Combretastatin A-4
Phosphate. Anticancer Res. 1999, 19, 189-195.
1182, 1127, 964; H NMR (D2O, 300 MHz) δ 7.13 (1H, d, J )
8.4 Hz, H6′), 7.07 (2H, s, H2,6), 6.84 (1H, d, J ) 8.4 Hz, H5′),
3.82 (3H, s, OCH3), 3.76 (6H, s, OCH3 × 2), 3.75 (3H, s, OCH3);
The solubility of calcium hydroxyphenstatin diphosphate was
found to be <1 mg/mL in distilled water at 25 °C.
An tim icr obia l Su scep tibility Testin g. The new sub-
stances were screened against the bacteria Stenotrophomonas
maltophilia, Micrococcus luteus, Staphylococcus aureus, Es-
cherichia coli, Enterobacter cloacae, Enterococcus faecalis,
Streptococcus pneumoniae, and Neisseria gonorrhoeae and the
fungi Candida albicans and Cryptococcus neoformans, accord-
ing to established disk susceptibility testing protocols.26
Tu bu lin Assa ys. The tubulin polymerization and colchicine
binding assays were performed as described previously,12
except that Beckman DU7400/7500 spectrophotometers equipped
with “high performance” temperature controllers were used
in the former assay. Unlike the manual control possible with
the previously used Gilford spectrophotometers, the polymer-
ization assays required use of programs provided by MDB
analytical Associates, South Plainfield, NJ , since the Beckman
instruments are microprocessor controlled. The Beckman
instruments were unable to maintain 0 °C, and the lower
temperature in the assays fluctuated between 2 and 4 °C.
Temperature changes were, however, more rapid than in the
Gilford instruments with the jump from the lower temperature
to 30 °C taking about 20 s and the reverse jump about 100 s.
(7) Greene, T. W.; Wutz, P. G. M. Protective Groups in Organic
Synthesis. J . Wiley & Sons: New York, 1999; pp 27-33.
(8) Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as Ef-
fective Acylating Agents. Tetrahedron Lett. 1981, 22, 3815-3818.
(9) Collins, S.; Hang, Y. Alkyl and Aryllanthanum Triflates: New
Reagents for the Conversion of Tertiary Amides to Ketones.
Tetrahedron Lett. 1987, 28, 4391-4394.
(10) Maeda, H.; Okamoto, J .; Ohmori, H. Reactions of In Situ Formed
Acyl Tributylphosphonium Ions with Grignard Reagents as an
Effective Route to Ketones from Acid Chlorides. Tetrahedron
Lett. 1996, 37, 5381-5384.
(11) Cardellicchio, C.; Fiandanese, V.; Marchese, G.; Ronzini, L.
Functionalized Ketones by Iron Mediated Reaction of Grignard
Reagents with Acyl Chlorides. Tetrahedron Lett. 1987, 28, 2053-
2056.
(12) 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. Structure-activity Analysis of the
Interaction of Curacin A, the Potent Colchicine Site Antimitotic
Agent, with Tubulin and Effects of Analogues on the Growth of
MCF-7 Breast Cancer Cells. Mol. Pharmacol. 1998, 53, 62-76.
(13) Pettit, G. R.; Lippert, J . W., III, Herald, D. L.; Pettit, R. K.;
Hamel, E. 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. J . Nat. Prod. In press.
(14) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Isolation and Structure of the Strong Cell
Growth and Tubulin Inhibitor Combretastatin A-4. Experientia
1989, 45, 209-211.
(15) Pettit, G. R.; Freeman, S.; Simpson, M. J .; Thompson, M. A.;
Boyd, M. R.; Williams, M. D.; Pettit, G. R. III; Doubek, D. L.
Antineoplastic Agents 320. Synthesis of a Practical Pancratista-
tin Prodrug. Anticancer Drug Design 1995, 10, 243-250.
(16) Silverberg, L. J .; Dillon, J . L.; Vemishetti, P. A Simple, Rapid
and Efficient Protocol for the Selective Phosphorylation of
Phenols with Dibenzyl Phosphite. Tetrahedron Lett. 1996, 37,
771-774.
(17) Lambooy, J . P. The Syntheses, Paper Chromatography and
Substrate Specificity for Tyrosinase of 2,3-,2,4-,2,5-,2,6- and 3,5-
Dihydroxyphenylalanines. J . Am. Chem. Soc. 1954, 76, 133-
138.
(18) Simig, G.; Schlosser, M. The Synthesis of 6-Methoxy-7,8-meth-
ylenedioxyisoquinoline Derivatives. ACH-Models Chem. 1994,
131, 217-228.
(19) Brink, M. Bromo Derivatives of o-Vanillin and o-Bourbonal.
Chem. Abstr. 1968, 69, 76823.
(20) Cleaver, L.; Nimgirawath, S.; Ritchie, E.; Taylor, W. C. The
Alkaloids of elmerrilla papuana (Magnoliaceae): Structure and
Synthesis of Elmerrillicine. Aust. J . Chem. 1976, 29, 2003-2021.
(21) Friedman, D.; Ginsburg, D. Halogenation of Pyrogallol Trimethyl
Ether and Similar systems. J . Org. Chem. 1958, 23, 16-17.
(22) Harding, V. J . 2-Hydroxy-3:4:3′:4′:5′Pentamethoxybenzophenone
and its Derivatives. J . Chem. Soc. 1906, 89, 1665.
Ack n ow led gm en t. We are pleased to thank for
support of this research the Outstanding Investigator
Grant CA 44344-06-12 with the Division of Cancer
Treatment and Diagnosis, NCI, DHHS, the Arizona
Disease Control Research Commission, Gary L. and
Diane R. Tooker, Captain and Mrs. George M. Brereton,
the Caitlin Robb Foundation, Dr. J ohn C. Budzinski,
Robert and Sharon Witzer, Salley Schloegel, and the
Robert B. Dalton Endowment. For other assistance, we
thank Drs. Cherry L. Herald, Fiona Hogan, Michael D.
Williams, J ean M. Schmidt, David Molloy, and J ean-
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 crystallo-
graphic tables of atomic coordinates, bond lengths and angles,
and anisotropic thermal parameters for hydroxyphenstatin
(6a ). This material is available free of charge via the Internet
at http://pubs.acs.org.
Refer en ces
(1) For contribution 442, see: Pettit, G. R.; Lippert, J . W., III.; Boyd,
M. R.; Hamel, E.; Pettit, R. K. Antineoplastic Agents 442. The
Remarkable Antitubulin Assembly and Cancer Cell Growth
Inhibitor Dioxostatin. J . Med. Chem. Submitted.
(23) XPREP-The automatic space group determination program in
the SHELXTL. (see ref 25).
(2) (a) Morton, J . F. Major Medicinal Plants, C. G. Thomas,
Springfield, IL, 1997; p. 87-90. (b) Hartwell, J . L.; Schrecker,
A. W. Fortsch. Chem. Org. Nat. 1958, 83. (c) Pettit, G. R. The
Scientific Contributions of J onathan L. Hartwell, Ph.D. J . Nat.
Prod. 1995, 58, 359-364.
(3) (a) Cragg, G. M.; Newman, D. J . Discovery and development of
Antineoplastic Agents from Natural Sources. Cancer Investiga-
tion 1999, 17, 153-163. (b) Pettit, G. R.; Alkalay, D. S. Potential
Cancerocidal Agents I. The Aromatic System of Podophyllotoxin.
(Part A). J . Org. Chem. 1960, 25, 1363-1365. (c) Pettit, G. R.;
Baumann, M. F.; Rangammal, K. N. Antineoplastic Agents V.
The Aromatic System of Podophyllotoxin (Part B). J . Med.
Pharm. Chem. 1962, 5, 800-808.
(24) North, A. C. T.; Phillips, D. C.; Mathews, F. S. A Semiempirical
Method of Absorption Correction. Acta Crystallogr. 1968, A2,
351-359.
(25) 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 53719. This
package includes, among others: XPREP, an automatic space
group determination 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.
(26) National Committee for Clinical Laboratory Standards 1997.
Performance standards for antimicrobial disk susceptibility tests,
6th ed.; Approved Standard M2-A6. NCCLS: Wayne, PA, 1997.
(4) (a) Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R.
K.; Schmidt, J . M.; Hogan, F. Antineoplastic Agents 291.
Isolation and Synthesis of Combretastatins A-4, A-5, and A-6.
J . Med. Chem. 1995, 38, 1666-1672. (b) Hamel, E. Antimitotic
J M000045A