Antineoplastic agents. 487. Synthesis and biological evaluation of the antineoplastic agent 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene and derived amino acid amides

Article abstract of DOI:10.1021/jm020204l

An efficient synthesis of 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene (1c) and hydrochloride (1d) is reported. The nitrostilbene intermediate 6a was obtained via a Wittig reaction using phosphonium salt 4 and 3-nitro-4-methoxybenzaldehyde 5. A o

Full text of DOI:10.1021/jm020204l

J . Med. Chem. 2003, 46, 525-531
525
An tin eop la stic Agen ts. 487. Syn th esis a n d Biologica l Eva lu a tion of th e
An tin eop la stic Agen t 3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-a m in o-Z-stilben e a n d
Der ived Am in o Acid Am id es
George R. Pettit,*,† Collin R. Anderson,† Delbert L. Herald,† M. Katherine J ung,‡ Debbie J . Lee,§
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, Science Applications International Corp.sFrederick, National Cancer Institute at Frederick, National
Institutes of Health, Frederick, Maryland 21702-1201, and Screening Technologies Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702-1201
Received May 13, 2002
An efficient synthesis of 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene (1c) and
hydrochloride (1d ) is reported. The nitrostilbene intermediate 6a was obtained via a Wittig
reaction using phosphonium salt 4 and 3-nitro-4-methoxybenzaldehyde 5. A one-step reduction
using zinc in acetic acid produced the synthetic objective amine 1c. The coupling of this amine
with various Fmoc amino acids, followed by cleavage of the R-amine protecting group, resulted
in a series of new cancer cell growth inhibitory amides. Amine 1c, hydrochloride 1d , glycine
amide 3b, and tyrosine amide 3f had the highest level (GI50 ) 10-2-10-3 µg/mL) of activity
against a panel of six human and one animal (P388) cancer cell lines. Amine 1c and its
hydrochloride 1d potently inhibited tubulin polymerization by binding at the colchicine site,
while the amides had little activity against purified tubulin. Nevertheless, most of the amides
caused a marked increase in the mitotic index of treated cells, indicating that tubulin was
their intracellular target.
In tr od u ction
adenocarcinoma cells.16 Both 2d and a serine derivative
2e, code name AC7700, have undergone further evalu-
ation.17,18 We have extended the early study of the
combretastatin A-4 3′-amino derivative to combretasta-
tin A-2 and now report the synthesis of 2,3-methylene-
dioxy-5,4′-dimethoxy-3′-amino-Z-stilbene (1c) and a se-
lection of amino acid amide derivatives (3a -g) for
purposes of an SAR investigation directed at locating
promising candidate(s) for preclinical development.
Combretastatin A-2 (1a , Chart 1) represents one of
the key antineoplastic and cancer vascular targeting
stilbenes,1,2 designated combretastatins A-1 to A-6, that
we isolated from the South African bush willow tree
Combretum caffrum.3 Initially we selected combretasta-
tin A-4 (2a ) for preclinical development, based on a
spectrum of promising biological properties that were
greatly enhanced by a structural modification to sodium
combretastatin A-4 phosphate prodrug (2b, CA4P).4,5
For example, 2b caused a 100-fold decrease in tumor
blood flow to the P22 carcinosarcoma in the rat while
causing no significant blood flow reduction in normal
heart, kidney, and small intestine.6 Other recent pre-
clinical findings include the antivascular and antitumor
effects produced by 2b against non-small-cell lung
cancer in vivo.7 Most importantly, the initial phase 1
human cancer clinical trials of 2b have been quite
successful.8-10 Our discovery of combretastatin A-411 has
prompted the synthesis ofmanystructuralvariations.12-14
One important variant replaces the 3′-hydroxyl of the
B ring with an amine (2c), which subsequently resulted
in a water-soluble hydrochloride salt (2d ) given the code
name AC-7739.15 In 1999, Ohsumi et al. published
interesting amino acid amide derivatives of 2c that
showed anticancer activity against murine colon 26
Resu lts a n d Discu ssion
Ch em istr y. The key precursor 3,4-methylenedioxy-
5,4′-dimethoxy-3′-nitro-Z-stilbene (6a ) was obtained
(4 f 6) by employing a Wittig reaction sequence
(Scheme 1). The required cis-stilbene was obtained in
a Z to E ratio of 1.4:1. Photochemical isomerization3,19
converted the trans-stilbene to the cis-stilbene isomer
in 62% yield. Reduction of nitrostilbene 6a with zinc in
acetic acid15 afforded amine 1c. Following its purifica-
tion by column chromatography, the 3,4-methylene-
dioxy-5,4′-dimethoxy-3′-amino-Z-stilbene crystallized (1:9
ethyl acetate/hexane) and was subjected to X-ray crystal
structure determination.
Treatment of amine 1c, in ethyl acetate, with 1 N HCl
in diethyl ether yielded the amine hydrochloride (1d ).
Interestingly, this hydrochloride derivative was found
to be essentially insoluble in water. Conversion of amine
1c to a selection of Fmoc-amino acid amides was readily
accomplished using PyBroP as the coupling reagent
(Scheme 2).20 All Fmoc deprotection was performed
using tris(2-aminoethyl)amine.21 The acid-sensitive side
chain protecting groups on Cys, Ser, Trp, and Tyr were
removed with trifluoroactic acid in dichloromethane.
* To whom correspondence should be addressed. Phone: 480-965-
3351. Fax: 480-965-8558. E-mail: bpettit@asu.edu.
† Arizona State University.
‡ Science Applications International Corp.sFrederick, National
Cancer Institute at Frederick.
§ Division of Cancer Treatment and Diagnosis, National Cancer
Institute at Frederick.
10.1021/jm020204l CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/10/2003

526 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Pettit et al.
Ch a r t 1
compounds are summarized in Table 1. Amine 1c was
more active than the 3′-alcohol combretastatin A-2.3
This result mirrors combretastatin A-4 published data
reporting increased activity of amine 2c (IC50 ) 5.1 nM)
over phenol 2a (IC50 ) 18.0 nM) in a murine colon 26
adenocarcinoma cell study.15 Although Oshumi et al.
reported the serine derivative 2e of amine 2c to be the
most active among their amino acid derivatives of
combretastatin A-4,16 we found that our serine deriva-
tive 3d displayed only marginal activity. Against the
minipanel of cancer cell lines listed in Table 1, amine
1c, hydrochloride salt 1d , glycine amide 3b, and ty-
rosine amide 3f provided the strongest cancer cell line
growth inhibition, while the remaining amides were 1.5-
to 10-fold less active.
Because of the well-documented interactions of com-
bretastatins A-2 (1a ) and A-4 (2a ) with tubulin, the
newly synthesized analogues (1c,d , 3a -g, 6a ,b, and
7a -g) were examined for their effects on both tubulin
assembly and the binding of [3H]colchicine to tubulin
in comparison with the two natural products. Only
amine 1c and its hydrochloride salt 1d had significant,
and essentially identical, effects in either reaction (Table
2; only data for the more cytotoxic amides are shown
here; colchicine binding studies with 6a ,b and 7a ,c-g
were performed with the compounds at 100 µM). Con-
sistent with their overall cytotoxicity, the effects of 1c
and 1d on the tubulin-based reactions were between
those of the more potent combretastatin A-4 and the
less potent combretastatin A-2.
Because the cytotoxic activities of the amides 3a -g
in the human tumor cell lines and of 7b in the murine
P388 line were overall not that different from the
activities of 1c and 1d in these lines, we wondered
whether another cellular target might be involved in
their activity. As a preliminary approach to this ques-
tion, we evaluated these amides, the parent compounds
1c and 1d , and the combretastatins for their effects on
the mitotic index of MCF-7 cells. An increase in the
mitotic index generally represents an effect at the
tubulin level. As shown in Table 2, like the combret-
astatins (1a , 2a ) and the amine (1c) and its salt (1d ),
the amides 3a -g and 7b caused a marked rise in the
The trityl group on Cys was deprotected in the presence
of the triethylsilane.
Biologica l E va lu a t ion . The cancer cell growth
inhibitory activities of the protected amides and related
Sch em e 1a
a
Reagents and conditions: (a) n-BuLi, THF, Ar, 0 °C; (b) 3-nitro-4-methoxybenzaldehyde (5) in THF, Ar; (c) benzil, benzene, 254 nm
lamp, Ar; (d) zinc, acetic acid; (e) 1 M HCl in diethyl ether, ethyl acetate.

Antineoplastic Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 527
Ta ble 1. Human Cancer Cell Line GI50 (µg/mL) and Murine P388 Lymphocytic Leukemia Cell Line Inhibitory Activity ED50 (µg/mL)
of the 3′-Substituted Stilbenes
leukemia
P388
pancreas
BXPC-3
breast
MCF-7
CNS
SF-295
lung NSC
NCI-H460
colon
KM2OL2
prostate
DU145
compound
mean
0.39
6a
6b
1a
1b
1c
1d
3a
3b
3c
3d
3e
3f
0.227
>10.0
0.016
0.38
>10.0
0.014
2.1
0.36
>10.0
0.0042
0.045
0.0047
0.0042
0.050
0.014
0.044
0.017
0.035
0.035
0.037
0.33
4.6
0.35
>10.0
0.043
0.41
0.45
>10.0
0.47
0.64
>10.0
0.0054
0.053
0.0083
0.042
0.023
0.0082
0.037
0.059
1.7
0.049
0.042
0.0097
0.055
0.080
0.93
0.013
0.025
0.10
0.020
0.30
0.49
0.0250
0.00189
0.0388
0.332
0.0199
0.0585
0.172
0.0653
0.0475
0.306
3.8
0.0071
0.075
0.20
0.025
0.20
0.027
0.044
0.0067
0.012
0.0050
0.0057
0.032
0.0042
0.044
0.042
0.053
0.012
0.064
0.022
0.035
0.055
0.012
0.034
0.019
0.018
0.022
0.019
0.028
0.0082
0.0046
0.0028
0.040
0.014
0.040
0.042
0.017
0.076
0.018
0.036
3g
Ta ble 2. Effects of Combretastatin A-2 Derivatives on Tubulin and on Mitosis in MCF-7 Breast Cancer Cells
inhibition of colchicine bindingb (% inhibition)
inhibition of
tubulin assemblya
(IC50 ( SD, µM)
2 µM
5 µM
20 µM
inhibitor
concentration
100 µM
inhibitor
concentration
mitotic
indexc
(% mitotic cells ( SD)
inhibitor
inhibitor
compound
concentration
concentration
combretastatin A-4
combretastatin A-2
2.1 ( 0.1
4.0 ( 0.2
3.1 ( 0.05
2.9 ( 0.05
>40
96
79
84
87
98
92
94
95
57 ( 6
57 ( 4
48 ( 5
47 ( 6
42 ( 4
52 ( 7
52 ( 4
35 ( 7
57 ( 12
44 ( 1
48 ( 1
56 ( 11
1c
1d
3a
3b
3c
3d
3e
3f
15
66
16
64
16
19
14
8
>40
31
22
>40
>40
>40
>40
3g
7b
>40
>40
a
Reaction mixtures contained 10 µM tubulin (1.0 mg/mL) and varying drug concentrations. A drug/tubulin preincubation in the absence
of GTP preceded the assembly reaction.24 Reaction mixtures contained tubulin at 1.0 µM, [3H]colchicine at 5.0 µM, and the inhibitory
b
compounds at the indicated concentrations.24 c MCF-7 cells were treated for 12 h with 10 times the GI50 concentrations shown in Table
2 except that the concentrations of combretastatins A-4 and A-2 and of 7b were 50 nM, 1.0 µM, and 1.0 µM, respectively. Cells with
condensed chromosomes were quantitated as mitotic cells. The mitotic index in untreated cells was 3.6%. See text for further details.
Sch em e 2a
mitotic index of the MCF-7 cells (data shown for drug
treatment for 12 h). The values obtained ranged from
35% to 57% mitotic cells, defined as those displaying
condensed chromosomes, compared with 4% in un-
treated cultures. This suggests either that there is a
markedly enhanced uptake of these amides by the cell
or that the amides are reconverted to 1c by an extra-
cellular or intracellular amidase.
All of the new substances were screened against the
bacteria Stenotrophomonas maltophilia, Micrococcus
luteus, Staphylococcus aureus, Escherichia coli, Entero-
bacter cloacae, Enterococcus faecalis, Streptococcus pneu-
moniae, Streptococcus pyogenes, Neisseria gonorrhoeae,
and the fungi Candida albicans and Cryptococcus
neoformans according to established broth microdilution
susceptibility assays.22,23 In broth microdilution assays,
glycine amide 3b exhibited the broadest antimicrobial
spectrum, targeting pathogenic yeasts and bacteria.
Con clu sion s
Present evidence indicates that the 3′-amino coun-
terpart (1c) of combretastatin A-2 and derived glycine
amide (3b) and tyrosine amide (3f) correspond to new
stilbenes with a very high level of inhibition against a
minipanel of cancer cell lines. The potential here was
further supported by the potent inhibition of tubulin
polymerization exhibited by amine 1c. Selected mem-
bers of the corresponding amides (3a -g), such as 3b
and 3f, are being further developed.
a
Reagents and conditions: (a) Fmoc-amino acid, PyBrOP,
DIPEA, Ar; (b) TAEA, DCM; (c) TFA, DCM; (d) TFA, triethylsilane,
DCM.

528 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Pettit et al.
mmol, 5.1 equiv). After flushing the reaction flask with Ar,
the reaction mixture was stirred overnight. The mixture was
then irradiated with a 254 nm UV lamp for 5 h. Upon removal
of the benzene in vacuo, the product was separated by gravity
column chromatography (4:1 hexane/ethyl acetate) to afford
unreacted starting material 6b (0.92 g, 32%) and the desired
Z-stilbene 6a (1.8 g, 62%).
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Ether refers to diethyl ether and
Ar to argon gas. All solvents were redistilled, and 3-nitro-4-
methoxybenzaldehyde was obtained from Alfa Aesar (Ward
Hill, MA). Bromo-tris-pyrrolidino-phosphonium hexafluoro-
phosphate (PyBroP), O-Boc-NR-Fmoc-L-tryptophan, O-tert-
butyl-NR-Fmoc-L-tyrosine, NR-Fmoc-glycine, and S-trityl-NR-
Fmoc-L-cysteine were obtained from Calbiochem-Novabiochem
Corporation (San Diego, CA). Acetic acid, N-butyllithium (2.5
M solution in hexanes), diisopropylethylamine (DIPEA), tri-
ethylsilane (TES), and trifluoroacetic acid (TFA) were obtained
from Acros Organics (Fisher Scientific, Pittsburgh, PA). All
other reagents were purchased from Sigma-Aldrich Chemical
Co. (Milwaukee, WI).
Reactions were monitored by thin-layer chromatography
using Analtech silica gel GHLF uniplates visualized under
long-wave and short-wave UV irradiation. Solvent extracts of
aqueous solutions were dried over anhydrous magnesium
sulfate. Where appropriate, the crude products were separated
by column chromatography, flash (230-400 mesh ASTM) or
gravity (70-230 mesh ASTM) silica from E. Merck.
Melting points are uncorrected and were determined on an
Electrothermal 9100 apparatus. Optical rotations were re-
corded using a Perkin-Elmer 241 polarimeter. The [R]D values
are given in 10-1 deg cm2 g-1. The IR spectra were obtained
using a Mattson Instruments 2020 Galaxy series FT-IR. The
1H NMR and 13C NMR spectra were recorded employing
Varian Gemini 300, Varian Unity 400, and Varian Unity 500
instruments using a deuterated solvent and were referenced
either to TMS or the solvent. HRMS data were recorded with
a J EOL LCmate mass spectrometer. Elemental analyses were
determined by Galbraith Laboratories, Inc., Knoxville, TN.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-n itr ostilben e, Z
a n d E Isom er s (6a a n d 6b). The phosphonium bromide 4
(20.1 g, 39.6 mmol) was placed in a flame-dried flask under
Ar and suspended in THF (300 mL). After being stirred for 45
min at room temperature, the solution was cooled to 0 °C and
n-butyllithium (15.9 mL, 39.8 mmol) was added. This resulted
in the reaction mixture turning deep-red. Stirring continued
for 4 h at room temperature. 3-Nitro-4-methoxybenzaldehyde
(5, 7.20 g, 3.97 mmol) was dissolved in THF (100 mL), and
the solution was added dropwise to the reacting mixture via
an addition funnel. The solution turned from deep-red to
yellow-green. After being stirred 18 h, the reaction mixture
was cooled to 0 °C. The reaction was terminated with ethyl
acetate, and the solution was filtered and concentrated under
vacuum to yield a dark-green oil. The product was separated
by gravity column chromatography on silica gel (4:1 hexane/
ethyl acetate) and recrystallized (hexane/acetone) to yield the
Z-stilbene 6a (4.09 g, 31%) as a yellow-green solid: mp 109-
110 °C; Rf ) 0.29 (4:1 hexane/ethyl acetate); 1H NMR (400
MHz, CDCl3) δ 3.78 (3H, s, OCH3), 3.94 (3H, s, OCH3), 5.96
(2H, s, -CH2-), 6.41 (3H, m, vinyl H, 2 × ArH), 6.54 (1H, d,
J ) 12.4 Hz, vinyl H), 6.94 (1H, d, J ) 8.4 Hz, ArH), 7.42 (1H,
dd, J ) 8.8, 2.0 Hz, ArH), 7.76 (1H, d, J ) 2.0 Hz, ArH); 13C
NMR (400 MHz, CDCl3) δ 151.7, 148.9, 143.6, 139.6, 134.9,
134.5, 131.1, 130.7, 129.7, 126.6, 126.0, 113.2, 108.5, 102.8,
101.5, 56.5, 56.5; HRMS calcd for C17H16NO6 [M + H]+
330.0978, found 330.0947. Anal. (C17H15NO6) C, H, N.
The E-stilbene 6b was isolated from the aforementioned
column and recrystallized (hexane/acetone) as an orange solid
(2.90 g, 22%): mp 165.5-167 °C; Rf ) 0.18 (4:1 hexane/ethyl
acetate); 1H NMR (500 MHz, CDCl3) δ 3.92 (3H, s, OCH3), 3.94
(3H, s, OCH3), 5.97 (2H, s, -CH2-), 6.62 (1H, s, ArH), 6.70
(1H, s, ArH), 6.81 (1H, d, J ) 16 Hz, vinyl H), 6.90 (1H, d, J
) 16 Hz, vinyl H), 7.03 (1H, d, J ) 9.0 Hz, ArH), 7.58 (1H, dd,
J ) 9.0, 2.0 Hz, ArH), 7.92 (1H, d, J ) 2.0 Hz, ArH); 13C NMR
(500 MHz, CDCl3) δ 152.0, 149.3, 143.6, 139.7, 135.4, 131.7,
131.6, 130.3, 129.2, 124.4, 122.9, 113.7, 107.1, 101.6, 99.8, 56.6,
56.6; HRMS calcd for C17H16NO6 [M + H]+ 330.0978, found
330.0869. Anal. (C17H15NO6) C, H, N.
3,4-Me t h yle n e d ioxy-5,4′-d im e t h oxy-3′-a m in o-Z-st il-
ben e (1c). To a stirred solution of nitrostilbene 6a (1.4 g, 4.3
mmol) in acetic acid (350 mL) was added zinc dust (60 g, <10
µm diameter). After 1.5 h, the solution was filtered under
vacuum through Celite and the filtrate was concentrataed
under vacuum. The product was separated by flash column
chromatography (4:1 hexane/ethyl acetate) and recrystallized
(∼9:1 hexane/ethyl acetate) to afford colorless crystals of 1c
(1.0 g, 77%): mp 93.5-94.5 °C; Rf ) 0.17 (4:1 hexane/ethyl
acetate); 1H NMR (300 MHz, CDCl3) δ 3.75 (3H, s, OCH3), 3.84
(3H, s, OCH3), 4.25-4.45 (2H, br, NH2), 5.93 (2H, s, -CH2-),
6.34 (1H, d, J ) 12.0 Hz, vinyl H), 6.41 (1H, d, J ) 12.0 Hz,
vinyl H), 6.48 (1H, s, ArH), 6.51 (1H, s, ArH), 6.68 (2H, m, 2
× ArH), 6.72 (1H, s, ArH); 13C NMR (400 MHz, CDCl3) δ 148.5,
146.6, 143.2, 135.5, 134.1, 132.0, 130.0, 129.6, 129.2, 119.5,
115.4, 110.1, 108.3, 103.0, 101.3, 56.3, 55.4; HRMS calcd for
C
17H18NO4 [M + H]+ 300.1236, found 330.1250. Anal. (C17H17
NO4) C, H, N.
-
3,4-Me t h yle n e d ioxy-5,4′-d im e t h oxy-3′-a m in o-Z-st il-
ben e Hyd r och lor id e (1d ). To a stirred solution of amine 1c
(40 mg, 0.13 mmol) in ethyl acetate (1 mL) was added ethereal
HC1 (1 M) in excess. A white solid immediately formed, and
this was collected and washed with ethyl acetate followed by
ether to yield a colorless powder 1d (45 mg, quantitative): mp
179.5-181 °C. Anal. (C17H18NO4Cl) C, H, N.
X-r a y Cr ysta l Str u ctu r e Deter m in a tion of Am in e 1c.
A single plate-shaped X-ray sample (∼0.40 mm × 0.10 mm ×
0.10 mm) of 1c was obtained by cleavage from a pale-yellow
crystalline cluster grown from a hexane/ethyl acetate solution
and was mounted on the tip of a glass fiber. Initial cell
constants were calculated from reflections collected from three
sets of 60 frames at 298(2) K on a Bruker 6000 diffractometer.
Cell parameters indicated a monoclinic space group. Subse-
quent data collection, using 15 s scans/frame and 0.396° steps
in ω, was conducted in such a manner to completely survey a
complete hemisphere of reflections. This resulted in >93%
coverage of the total reflections possible to a resolution of 0.83
Å. A total of 7211 reflections were harvested from the total
data collection, and final cell constants were calculated from
a set of 332 strong, unique reflections from these data.
Subsequent statistical analysis of the complete reflection
data set using the XPREP25 program indicated that the space
group was P21. Crystal data for C17H17N1O4: a ) 11.5714(3)
Å, b ) 5.3425(2) Å, c ) 12.5632(3) Å, â ) 105.605(1)°,
V ) 748.03(4) Å3, λ(Cu KR) ) 1.541 78 Å, µ(Cu KR) ) 0.783
mm-1, Pc ) 1.329 g cm-3 for Z ) 2 and Mr ) 299.32, F(000) )
316.
After data reduction and merging of equivalent reflections
and rejection of systematic absences, 2310 unique observed
reflections remained (Rint ) 0.1867) and these were used in
the subsequent structure solution and refinement. An absorp-
tion correction was applied to the data with SADBS.26 Direct
methods structure determination and refinement were ac-
complished with the SHELXTL NT, version V5.1025 suite of
programs. All non-hydrogen atoms for amine 1c were located
using the default settings of that program. Hydrogen atom
coordinates were calculated at optimum positions and forced
to ride the atom to which they were attached. Anisotropic
refinement of the model shown in Figure 1 resulted in a final
residual value of 0.0778 for the observed data (0.0878 for all
data). The difference Fourier map showed insignificant re-
sidual electron density, the largest difference peak and hole
being +0.324 and -0.286 e/Å3, respectively. Final bond
distances and angles were all within acceptable limits.
P h otoch em ica l Isom er iza tion of E-Stilben e 6b to Z-
Stilben e (6a ). To a stirred solution of the E-stilbene 6b (2.9
g, 8.8 mmol) in benzene (550 mL) was added benzil (9.5 g, 45
Unless otherwise noted, the following general procedure was
employed for synthesis of the Fmoc-protected amino acid
amides of amine 1c.

Antineoplastic Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 529
ArH), 8.33 (1H, d, J ) 1.5 Hz, ArH), 9.90 (1H, s); 13C NMR
(500 MHz, CDCl3) δ 170.9, 148.5, 147.6, 143.3, 134.2, 131.9,
130.0, 129.4, 128.8, 127.1, 124.3, 120.4, 109.7, 108.4, 103.0,
101.3, 56.5, 56.3, 55.8, 38.2. Anal. (C20H22N2O5S) C, H, N.
3,4-Meth ylen edioxy-5,4′-d im eth oxy-3′-(Nr-Fm oc-L-Gly)-
a m id o-Z-stilben e (7b). Following the general procedure, to
NR-Fmoc-Gly (82 mg, 0.28 mmol, 1.9 equiv) in DCM (1 mL) at
0 °C under Ar was added DIPEA (0.075 mL, 0.43 mmol, 2.9
equiv). Next, PyBroP (0.123 g, 0.26 mmol, 1.8 equiv) and amine
1c (44 mg, 0.15 mmol) were added. The product was crystal-
lized from hexanes/ethyl acetate (75 mg, 88%): mp 67-69 °C
(dec); Rf ) 0.40 (8:1 DCM/ethyl acetate). Anal. (C34H30N2O7)
C, H, N.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(L-Gly)a m id o-Z-
stilben e (3b). Following the general procedure, amide 7b (75
mg, 0.13 mmol) in chloroform (6 mL) was treated with TAEA
(0.4 mL, 2.7 mmol, 21 equiv). After the reaction mixture was
stirred for 15 min, additional TAEA (0.4 mL, 2.7 mmol, 21
equiv) was added. The product was obtained as a colorless oil
3b (38 mg, 83%): Rf ) 0.38 (9:1 DCM/CH3OH); HRMS calcd
for C19H21N2O5 [M + H]+ 357.1450, found 357.1489. Anal.
(C19H20N2O5‚0.5H2O) C, H. N: calcd 7.67; found 8.19.
3,4-Meth ylen edioxy-5,4′-dim eth oxy-3′-(Nr-Fm oc-L-P h e)-
a m id o-Z-stilben e (7c). Amine 1c (0.16 g, 0.53 mmol) was
mixed with NR-Fmoc-Phe (0.31 g, 0.80 mmol, 1.5 equiv) in
DCM (3 mL), DIPEA (0.23 mL, 1.3 mmol, 2.5 equiv), and
PyBroP (0.36 g, 0.77 mmol, 1.5 equiv). After 40 min, a white
precipitate was collected and recrystallized from DCM/ethyl
acetate to afford the product as a colorless solid (7c, 270 mg,
F igu r e 1. ORTEP molecular structure of amine (1c) with the
numbering scheme and thermal ellipsoids drawn at the 40%
probability level.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(S-Tr t-Nr-F m oc-
L-Cys)a m id o-Z-stilben e (7a ). To a stirred mixture of amine
1c (57 mg, 0.19 mmol), S-Trt-NR-Fmoc-L-Cys (0.15 g, 0.26
mmol, 1.4 equiv), and PyBroP (127 mg, 0.27 mmol, 1.4 equiv)
in DCM (1 mL) at 0 °C under Ar was added DIPEA (0.075
mL, 0.43 mmol, 2.3 equiv). The reaction mixture was stirred
for 1 h at room temperature. Ethyl acetate was added, and
the solvents were removed in vacuo, leaving a white foam. The
product was obtained by flash column chromatography (8:1
DCM/ethyl acetate) as a white foam 7a (0.14 g, 85%): Rf )
0.81 (8:1 DCM/ethyl acetate); [R]22D -11.5° (c 0.87, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 2.71 (1H, m), 2.80 (1H, m), 3.71 (3H,
s, OCH3), 3.72 (3H, s, OCH3), 3.93 (1H, m), 4.21 (1H, t, J )
7.0 Hz, Fmoc), 4.38 (2H, d, J ) 7.0, Fmoc), 5.88 (2H, s,
-CH2-), 6.39 (1H, d, J ) 12.0 Hz, vinyl H), 6.44 (1H, s, ArH),
6.44 (1H, d, J ) 12.5 Hz, vinyl H), 6.47 (1H, s, ArH), 6.66 (1H,
d, J ) 9.0 Hz, ArH), 6.98 (1H, dd, J ) 8.0, 1.5 Hz, ArH), 7.27
(2H, m, Fmoc), 7.36 (2H, m, Fmoc), 7.58 (2H, d, J ) 6.5 Hz,
75%): mp 85-87 °C; Rf ) 0.42 (2:1 hexane/ethyl acetate); [R]25
D
-29.4° (c 1.00, CHCl3). Anal. (C41H36N2O7‚0.5H2O) C, H, N.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(L-P h e)a m id o-Z-
stilben e (3c). Cleavage of amide 7c (0.12 g, 0.18 mmol) was
achieved in chloroform (3 mL) and TAEA (0.60 mL, 4.0 mmol,
22 equiv). After 20 min, DCM (12 mL) was added to the
reaction mixture and the solvent was successively washed with
brine (4 mL) and phosphate buffer (pH 5.5, 2 × 6 mL). The
organic phase was concentrated under vacuum, and the
residue was subjected to gravity column chromatography (1:1
hexane/DCM followed by 95:5 DCM/CH3OH) to yield a color-
less oil 3c (74 mg, 94%): Rf ) 0.76 (95:5 DCM/CH3OH); [R]25
-67.0° (c 1.00, CHCl3). Anal. (C26H26N2O5) C, H, N.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(O-Bu t-Nr-F m oc-
L-Ser )a m id o-Z-stilben e (7d ). To a stirred mixture of 1c (0.13
g, 0.43 mmol) and O-But-NR-Fmoc-Ser (0.25 g, 0.66 mmol, 1.5
equiv) in DCM (2 mL) at 0 °C under Ar was added DIPEA
(0.180 mL, 1.0 mml, 2.3 equiv) followed by PyBroP (0.29 g,
0.63 mmol, 1.5 equiv). The mixture was stirred at room
temperature for 1 h. It was then washed with aqueous citric
acid (10% by weight), dried with magnesium sulfate, and
concentrated under vacuum. DCM and ethyl acetate were
added to the residue, and the resulting off-white precipitate
was collected. The solvents were removed in vacuo, and the
residue was subjected to gravity column chromatography
(1:1, DCM/ethyl acetate) to yield a pale-yellow oil. The product
was precipitated from hexanes/ethyl acetate to afford a color-
less powder 7d (0.21 g, 73%): mp 107.5-109.5 °C; Rf ) 0.80
Fmoc), 7.74 (2H, m, Fmoc), 8.23 (1H, d, J ) 2.0 Hz, ArH); 13
C
NMR (500 MHz, CDCl3) δ 167.8, 155.8, 148.5, 147.1, 147.1,
144.3, 143.7, 143.6, 143.3, 141.3, 134.3, 131.8, 130.0, 129.5,
129.3, 129.0, 128.1, 127.9, 127.8, 127.1, 126.9, 125.0, 124.6,
120.8, 120.7, 120.0, 120.0, 109.6, 108.4, 103.0, 101.3, 67.4, 67.1,
56.3, 55.7, 53.4, 47.1, 33.9. Anal. (C54H46N2O7S) C, H, N.
Synthesis of 3′-L-Cys-amide-Z-stilbene 3a provides the
general procedure (except for use of trifluoroacetic acid (TFA)
and triethylsilane (TES) with Cys Trt cleavage) for cleavage
of the Fmoc-amino acid protecting group
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(L-Cys)a m id o-Z-
stilben e (3a ). To a stirred solution of 3′-L-Cys-amido-Z-
stilbene (7a , 42 mg, 0.048 mmol) in DCM (1 mL) was added
TES (0.5 mL) and TFA (1.5 mL). The reaction mixture was
stirred for 20 min, and the solution was concentrated under
vacuum. The residue was dissolved in DCM (1 mL), and tris-
(2-aminoethyl)amine (TAEA, 0.5 mL) was added. Fifteen
minutes later, DCM (10 mL) was added and the mixture was
washed with brine (10 mL). The organic solvent was removed
in vacuo to yield an oil that was subjected to gravity column
chromatography (8:1 DCM/ethyl acetate followed by 9:1 DCM/
CH3OH). Product 3a was obtained as a colorless foam (5.0 mg,
(1:1 DCM/ethyl acetate); [R]24 -3.4° (c 1.01, CHCl3). Anal.
D
(C39H40N2O8) C, H, N.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(L-Ser )a m id o-Z-
stilben e (3d ). To a stirred solution of 7d (110 mg, 0.16 mmol)
in DCM (1 mL) at 0 °C under Ar was added trifluoracetic acid
(1 mL). The mixture immediately turned magenta. After being
stirred for 1 h, the solution was concentrated under vacuum.
The residue was taken up in DCM (2 mL). TAEA (1 mL) was
added, and stirring continued for 10 min. DCM (15 mL) was
added, and the solution was washed successively with brine
(6 mL) and phosphate buffer (pH 5.5, 15 mL). After back-
extracting the phosphate buffer with DCM, the organic
extracts were concentrated under vacuum and subjected to
gravity column chromatography (1:1 DCM/ethyl acetate fol-
lowed by 95:5 DCM/CH3OH) to afford an oil 3d (25 mg, 41%):
26%): Rf ) 0.87 (9:1 DCM/CH3OH); [R]28 -97.3° (c 0.44,
D
CHCl3); 1H NMR (300 MHz, CDCl3) δ 2.90 (1H, m), 3.36 (1H,
m), 3.72 (3H, s, OCH3), 3.83 (1H, m), 3.86 (3H, s, OCH3), 5.91
(2H, s, -CH2-), 6.38 (1H, d, J ) 12.3 Hz, vinyl H), 6.45 (1H,
s, ArH), 6.47 (1H, s, ArH), 6.48 (1H, d, J ) 12.6 Hz, vinyl H),
6.72 (1H, d, J ) 8.7 Hz, ArH), 6.98 (1H, dd, J ) 8.7, 1.8 Hz,
Rf ) 0.33 (95:5 DCM/CH3OH); [R]26 -8.6° (c 0.70, CHCl3).
D
Anal. (C20H22N2O6‚0.5H2O) C, H, N.

530 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Pettit et al.
3,4-Meth ylen edioxy-5,4′-dim eth oxy-3′-(N-Boc-Nr-Fm oc-
L-Tr p )a m id o-Z-stilben e (7e). Following the general method,
amine 1c (51 mg, 0.17 mmol), NR-Fmoc-Trp(Boc) (0.13 g, 0.25
mmol, 1.5 equiv), PyBroP (0.12 g, 0.26 mmol, 1.5 equiv) in
DCM (1 mL), and DIPEA (0.075 mL, 0.43 mmol, 2.5 equiv)
were used to obtain amide 7e as a colorless solid (0.13 g, 93%,
reprecipitated from hexanes/ethyl acetate): mp 118-120 °C;
Rf ) 0.71 (8:1 DCM/ethyl acetate); [R]22D -22.8° (c 0.65, CHCl3).
Anal. (C48H45N3O9) N. C, H: calcd 71.36, 5.61; found 71.84,
6.03.
containing 8% formaldehyde, 50 mM 1,4-piperazineethane-
sulfonate (pH 6.9 with NaOH), 5 mM MgC12, and 5% dimethyl
sulfoxide for 45 min. The slide was washed twice with
phosphate-buffered saline (pH 7.4), and the DNA was fluo-
rescently labeled with 2.5 µM 4′,6-diamidino-2-phenylindole.
Coverslips were mounted on the Citifluor AF1 antifade agent
obtained from Marivac, Ltd. The slides were examined with a
Nikon E800 epifluorescence microscope equipped with an
appropriate filter, and cells with condensed chromosomes were
quantitated.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(L-Tr p )a m id o-Z-
stilben e (3e). Amide 7e (90 mg, 0.11 mmol), DCM (0.2 mL),
and TFA (1.8 mL) were mixed and stirred for 1 h at 0 °C and
then condensed in vacuo. By use of the general Fmoc depro-
tection procedure, the residue was taken up in DCM (2 mL),
heated with TAEA (1 mL), and stirred for 20 min to yield an
Ack n ow led gm en t. We are pleased to thank the
following for financial assistance: Division of Cancer
Treatment, National Cancer Institute, DHHS, for award-
ing an Outstanding Investigator Grant CA44344-05-12;
the Arizona Disease Control Research Commission; the
Caitlin Robb Foundation; Diane Cummings Halle; Rod
J . and Hazel V. McMullin; Gary L. and Diane Tooker;
Lottie Flugel; Polly J . Trautman; J ohn and Edit Reyno;
Dr. J ohn C. Budzinski; the Ladies Auxiliary to the
Veterans of Foreign Wars; and the Robert B. Dalton
Endowment Fund. In addition, we thank Dr. Gerald
Boland for conducting some preliminary experiments.
Other very helpful assistance was provided by Drs.
Fiona Hogan, J ohn C. Knight, J ean M. Schmidt, J ean-
Charles Chapuis, Ms. Bridget Fakoury, and Mr. Michael
J . Dodson. This work was supported in part by NCI,
National Institutes of Health Contract NO1-CO-1240.
oil (3e, 11 mg, 20%): Rf ) 0.31 (8:1 DCM/ethyl acetate); [R]29
D
-87.5° (c 0.44, CHCl3); HRMS calcd for C28H28N3O5 [M + H]+
486.2029, found 486.2008. Anal. (C28H27N3O5‚H2O) H, N. C:
calcd 66.79; found 66.31.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(O-Bu t-Nr-F m oc-
L-Tyr )a m id o-Z-stilben e (7f). Amine 1c (0.13 g, 0.43 mmol),
O-But-NR-Fmoc-Tyr (0.29 g, 0.63 mmol, 1.5 equiv), DCM (2
mL), DIPEA (0.180 mL, 1.0 mmol, 2.3 equiv), and PyBroP (0.29
g, 0.63 mmol, 1.5 equiv) were mixed to yield stilbene 7f. The
product was isolated by gravity column chromatography (1:1
DCM/ethyl acetate) as a slightly yellow oil. Stilbene 7f was
then precipitated from hexanes/ethyl acetate as a colorless
powder (7f, 0.29 g, 91%): mp 95-97 °C; Rf ) 0.78 (1:1 DCM/
ethyl acetate); [R]24D -9.2° (c 1.15, CHCl3). Anal. (C45H44N2O8)
C, H, N.
3,4-Meth ylen ed ioxy-5,4′-d im eth oxy-3′-(L-Tyr )a m id o-Z-
stilben e (3f). To a stirred solution of 7f (0.63 g, 0.85 mmol)
in DCM (4 mL) was added TFA (4 mL). The mixture was
stirred for 20 min. Solvent was removed under vacuum, and
the mixture was subjected to gravity column chromatography
(4:1 DCM/ethyl acetate). The phenol obtained was dissolved
in DCM (4.5 mL) to which TAEA (2.7 mL) was added. The
mixture was stirred for 20 min, washed with brine, and dried
with magnesium sulfate. Solvent was removed under vacuum,
and the product was purified by gravity column chromatog-
raphy (4:1 DCM/ethyl acetate followed by 9:1 DCM/CH3OH).
The product was obtained as a colorless oil (3f, 0.35 g, 90%):
Su p p or tin g In for m a tion Ava ila ble: X-ray supporting
material for amine 1c, murine P388 lymphocytic leukemia cell
line ED50 (µg/mL) evaluation of NR-Fmoc-amino acid amides
7a -g, and antimicrobial activities of protected amides and
related compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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D
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0.26 (4:1 DCM/ethyl acetate); [R]25 -39.5° (c 0.43, CHCl3). Anal.
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