Antineoplastic agents. 515. Synthesis of human cancer cell growth inhibitors derived from 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino- Z-stilbene

Article abstract of DOI:10.1021/np058033l

Further structure-activity relationship (SAR) exploration of 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene (1a) derivatives resulted in the efficient synthesis of tyrosine amide hydrochloride 9, two tyrosine amide phosphate prodrugs (3a and 6), an

Full text of DOI:10.1021/np058033l

J. Nat. Prod. 2005, 68, 1191-1197
1191
Antineoplastic Agents. 515. Synthesis of Human Cancer Cell Growth Inhibitors
Derived from 3,4-Methylenedioxy-5,4′-dimethoxy-3′-amino-Z-stilbene
George R. Pettit,*,† Collin R. Anderson,† Eric J. Gapud,‡ M. Katherine Jung,§ John C. Knight,†
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, 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, and SAIC-Frederick, Inc., Frederick, Maryland 21702
Received March 4, 2005
Further structure-activity relationship (SAR) exploration of 3,4-methylenedioxy-5,4′-dimethoxy-3′-amino-
Z-stilbene (1a) derivatives resulted in the efficient synthesis of tyrosine amide hydrochloride 9, two tyrosine
amide phosphate prodrugs (3a and 6), and sodium aspartate amide 11. Two additional cancer cell growth
inhibitors (14 and 16) were synthesized by employing peptide coupling between amine 1a and the Dap
unit of dolastatin 10 (4a) to yield amide 14 followed by Dov-Val-Dil (15) to yield peptide 16. The latter
represents a combination of stilbene 1a with the des-Doe tetrapeptide unit of the powerful tubulin assembly
inhibitor dolastatin 10. Peptide 16 was examined for potential binding to tubulin in the vinca and/or
colchicine regions and found to perform primarily as a relative of dolastatin 10. Amide 14 had
anticryptococcal and antibacterial activities.
Scheme 1
We recently completed syntheses of 3′-amino derivatives
(1a,b, 1d-j) of combretastatin A-2 (2c),1 encouraged by the
remarkablesuccessofthepotentcancervasculartargeting2-4
that occurs with combretastatin A-4 phosphate prodrug
(CA4P) 2h.5-10 Subsequently, we undertook the synthesis
and initial anticancer evaluation of two phosphate deriva-
tives (3a,b) of tyrosine stilbene amide 1i. Attachment of
the phosphate group at the 3′-phenol position has been
investigated in detail in the combretastatin A and B
series.1,11-13 In the present study we chose to use the 4′′-
hydroxyl group of tyrosine amide 1i for attachment of a
phosphate group (3a) as well as obtaining a diphosphoryl
derivative (3b) by phosphorylating tyrosine amide 1i at
both the amine14 and hydroxyl groups. A parallel important
objective of this research involved employing a tetrapeptide
segment of dolastatin 10 (4a) for bonding to the 3′-amino
group (1b). Dolastatin 10 (D-10, 4a), isolated15-17 from the
Indian Ocean sea hare Dolabella auricularia, has continued
to progress through preclinical18 and clinical development
as an impressive antineoplastic agent.19-22 Dolastatin 10
(4a) and various synthetic derivatives also have specific
antifungal activity against Cryptococcus neoformans.23,24
Previous SAR studies have confirmed that D-10 (4a)
inhibits microtubule assembly by binding to the â-tubulin
subunit near the vinca site. SAR studies have shown the
des-Doe tetrapeptide unit (4b, Dov-Val-Dil-Dap) to be
necessary for potent anticancer activity.25,26 We now also
report synthesis of the amide representing coupling of
amine 1a to Dov-Val-Dil-Dap (4b).
using bromotrimethylsilane to afford the free acid (3b),
which was subsequently converted to sodium salt 6 using
sodium methoxide in methanol.11,12 Phosphorylation solely
at the 4′′-hydroxyl position was accomplished via the
coupling of the 3′-amino stilbene 1a to O-tert-butyl-NR-Z-
L-Tyr using PyBroP as the coupling reagent to provide
amide 7 (Scheme 2).28 tert-Butyl removal using TFA in
DCM followed by phosphorylation afforded the dibenzyl
ester 8. Debenzylation at the ester and benzyloxycarbonyl
removal at the amine were achieved simultaneously with
the in situ generation of trimethylsilyl iodide (TMSI) from
the reaction of sodium iodide and chlorotrimethylsilane in
acetonitrile.29,30 This method resulted in the immediate
precipitation of the phosphate prodrug 3a. Surprisingly,
both phosphoric acids and the corresponding sodium salts
proved to be sparingly soluble in water. In retrospect, the
physical properties of 6 might correspond better to a
betaine structure arising from enolization of the amide
group in 3b with a concurrent shift of the adjacent
Results and Discussion
Our synthesis of the tyrosine stilbene amide 1i27 pre-
sented the opportunity to develop two additional phosphate
prodrugs in the combretastatin series. Accordingly, the 4′′-
phenol and R-amino groups were phosphorylated in one
step using dibenzyl phosphite (1i f 5) (Scheme 1). Deben-
zylation of the resulting phosphate diesters (5) was achieved
* To whom correspondence should be addressed. Phone: 480-965-3351.
Fax: 480-965-8558. E-mail: bpettit@asu.edu.
† Arizona State University.
‡ Division of Cancer Treatment and Diagnosis, National Cancer Institute
at Frederick.
§ SAIC-Frederick, Inc.
10.1021/np058033l CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/06/2005

1192 Journal of Natural Products, 2005, Vol. 68, No. 8
Pettit et al.
Figure 1.
Scheme 2
Scheme 3
Scheme 4
phosphate to give a phosphate ester (see Figure 1). This
structure would account for the trisodium salt formation
to give 6 and the lower aqueous solubility.
We further investigated the possibility of making a
soluble amide derivative first by making the hydrochloride
salt of 1i (Scheme 3) and second by coupling amine 1a to
aspartic acid and converting to the â-aspartate sodium salt
11 (Scheme 4). Amide formation was accomplished using
PyBroP followed by removal of the â-tert-butyl protecting
group in the presence of triethylsilane.31 The resulting
carboxylic acid was subsequently treated with sodium
methoxide in methanol, yielding sodium salt 11. Both the
hydrochloride salt 9 and the sodium salt 11 were es-
sentially insoluble (<0.1 mg/mL) in water. Although we
found earlier that the combretastatin A-2 phosphate pro-
drug (2d) has reduced aqueous solubility when compared
to its combretastatin A-1 (2b) and A-3 counterparts (2f),11,12
we were surprised to find that our new CA2 modifications
(1b, 3a, 6, 9, and 11) exhibited only sparing water solubil-
ity, presumably owing to the hydrophobicity created by the
stilbene. Interestingly, addition of the phosphate group to
the tyrosine amide 1i to yield 3a resulted in decreased
anticancer activity (Table 1). Since the cancer cell line
results obtained for each of the substances recorded in
Table 1 were obtained in solution, presumably the sparing

Human Cancer Cell Growth Inhibitors
Journal of Natural Products, 2005, Vol. 68, No. 8 1193
Table 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
CNS
lung-NSC
NCI-H460
colon
prostate
DU145
compound
MCF-7
SF-295
KM20L2
mean
0.013
1a
3a
5
0.00189
>10
>10
0.0071
>1
0.0047
>1
0.023
>1
0.0050
>1
0.022
>1
0.028
>1
6
0.270
0.380
0.133
0.0329
0.385
0.361
0.225
0.0263
<0.01
18.6
0.049
>10
1.3
0.36
>10
>1
0.44
>10
0.064
1.7
>1
0.41
>10
0.096
3.8
>1
1.8
>10
0.034
2.3
>1
0.39
>10
0.080
3.0
7
0.61
8
9
0.064
3.4
0.041
2.4
0.51
0.53
0.054
0.0031
0.059
2.4
0.33
0.70
0.11
10
11
13
14
16
0.38
0.25
0.75
0.062
0.025
0.30
0.52
0.083
0.033
0.19
1.9
0.26
0.020
0.33
0.38
0.070
0.032
0.58
0.23
0.048
Scheme 5
water solubility did not significantly influence the cell line
results. However, the reduced ability to hydrogen bond in
vitro could be important.
The des-Doe tetrapeptide unit (4b) of the potent anti-
cancer drug dolastatin 10 (D-10, 4a) isolated in 198415 was
chosen for coupling with amine 1a. By replacing the Doe
portion of D-10 with the amine 1a, we planned to synthe-
size a stilbene peptide with the possibility of binding to
the colchicine and/or vinca regions of tubulin. The tripep-
tide Dov-Val-Dil TFA salt (15) and Boc-Dap (12) were
obtained via published methods.32,33 PyBroP coupling of
amine 1a to Boc-Dap (12) followed by Boc deprotection with
TFA in DCM afforded compound 14 (Scheme 5). Further
coupling resulted in des-Doe D-10 stilbene amide 16. The
cancer cell inhibition activity of the potentially dual
functioning anticancer agent 16 was notable (Table 1), as
was the broad spectrum antimicrobial action of amide 14.
The latter substance (14) inhibited the growth of the
pathogenic fungus Cryptococcus neoformans ATCC 90112
(MIC ) 64 µg/mL), the pathogenic bacterium Neisseria
gonorrhoeae ATCC 49226 (MIC ) 16 µg/mL), and the
opportunistic bacteria Streptococcus pneumoniae ATCC
6303 (MIC ) 8-16 µg/mL) and Enterococcus faecalis ATCC
29212 (MIC ) 32-64 µg/mL). Prodrug 3a inhibited N.
gonorrhoeae (MIC ) 0.125 µg/mL) and E. faecalis (MIC )
16 µg/mL), and prodrug 6 inhibited N. gonorrhoeae (MIC
) 4 µg/mL). Stilbenes 8 and 13 inhibited E. faecalis (MIC
) 64 µg/mL) or C. neoformans (MIC ) 64 µg/mL), respec-
tively.
Previous work showed that D-10 (4a) is an exceptionally
cytotoxic antimitotic agent and that it inhibits tubulin
assembly by binding tightly in the vinca domain of tubulin
(its inhibition of vinblastine binding to tubulin, although
extensive, is noncompetitive).34,35 In contrast, while com-
bretastatin A-4 (2g) is quite potent for a colchicine site
drug, it is about 100-fold less cytotoxic than D-10 (4a), and
the stilbene inhibits tubulin assembly by binding avidly
to the colchicine site in a competitive fashion.36,37 These
findings were confirmed in the studies presented in Table
2. Note the similar inhibitory effects of 4a and 2g on
tubulin assembly, their contrasting effects on inhibition of
binding of radiolabeled vinblastine and colchicine, and the
greater than 100-fold enhanced inhibition of growth of the
MCF-7 cells by 4a relative to 2g.
ment of the Doe residue with a variety of aromatic
derivatives could restore some or all of the activity of D-10
(4a) in both the cytological and biochemical assays.23,24
SAR studies with structural manipulation of combret-
astatin A427,36 (2g) revealed that relatively minor effects
on cytotoxicity and tubulin interactions were observed
when a methylenedioxy bridge replaced two vicinal meth-
oxy groups in the A ring or an amino group replaced the
hydroxyl group in the B ring. These findings are demon-
strated again in Table 2, where amine hydrochloride 1b,
incorporating both these changes, is compared with CA4
2g. In addition, we found that a variety of amino acid
amides formed from amine 1a had sharply reduced activity
Previous SAR studies with D-1024 (4a) structural modi-
fications had shown that loss of its C-terminal unit Doe
had a significant effect on cell growth and a greater effect
on inhibition of ligand binding to tubulin than on inhibition
of tubulin assembly. Thus, Dov-Val-Dil-Dap (4b) is only
2-fold less active than 4a as an assembly inhibitor, but at
least 6-fold less active as an inhibitor of MCF-7 cell growth
(Table 2). These SAR studies had also shown that replace-

1194 Journal of Natural Products, 2005, Vol. 68, No. 8
Pettit et al.
Table 2. Effects of Peptide 16 and Related Compounds on Tubulin Assembly, Binding of Colchicine and Vinblastine to Tubulin, and
the Growth and Mitotic Index of MCF-7 Breast Cancer Cells
inhibition of
inhibition of
colchicine binding
vinblastine binding
% inhibition ( SDa
% inhibition ( SDa
inhibition of
tubulin assembly
IC50 (µM) ( SDa
inhibition of growth
of MCF-7 breast cancer
cells IC50 (nM) ( SDa
mitotic index of
MCF-7 cells
% mitosesb
compound
2 µMc
50 µMc
10 µMc
80 µMc
16
1.3 ( 0.04
1.4 ( 0.03
3.4 ( 0.5d
0.70 ( 0.06
30 ( 2
5.2 ( 3
48 ( 3
16 ( 6
0
87 ( 2
68 ( 0.4
0
17 ( 7
36 ( 10
57
56
65
39
52
46
Dov-Val-Dil-Dap (4b)
1b
85 ( 3
13 ( 5
68 ( 30
dolastatin 10 (4a)
96 ( 0.7
0.083 ( 0.06
14 ( 2
4b + 1be
combretastatin
A-4 (2g)
1.8 ( 0.3
98 ( 0.4
0
0
11 ( 10
a SD, standard deviation. b The mitotic indices were determined following growth of the cells for 16 h at 10 times the IC50 concentration.
Without drug, 2.9% of the cells were in mitosis. c Inhibitor concentrations. d Compound 1a had a nearly identical IC50 value (4.3 ( 0.5
µM). e Equimolar concentrations of compounds 4b and 1b were present in all cell culture mixtures.
and O-tert-butyl-NR-Z-L-tyrosine were obtained from Calbio-
chem-Novabiochem Corporation (San Diego, CA). Diisopropyl-
ethylamine (DIPEA), anhydrous methanol, sodium methoxide,
triethylsilane (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 TLC using Analtech silica gel
GHLF Uniplates visualized under short-wave UV irradiation.
Solvent extracts of aqueous solutions were dried over anhy-
drous MgSO4 or Na2SO4. Where appropriate, the crude prod-
ucts were separated by column chromatography on flash (230-
400 mesh ASTM) silica from E. Merck, gravity (70-230 mesh
ASTM) silica from E. Merck, or Sephadex LH-20.
Melting points are uncorrected and were determined em-
ploying an Electrothermal 9100 apparatus. Optical rotations
were measured using a Perkin-Elmer 241 polarimeter. The
[R]D values are given in 10-1 deg cm2 g-1. The 1H and 13C NMR
spectra were recorded employing Varian Gemini 300, Varian
Unity 400, or Varian Unity 500 instruments using a deuter-
ated solvent and were referenced to either TMS or the solvent.
HRMS data were recorded with a JEOL LCmate mass
spectrometer. Elemental analyses were determined by Gal-
braith Laboratories, Inc., Knoxville, TN.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-[O,NR-di(bis-ben-
zylphosphoryl)-L-Tyr]-amido-Z-stilbene (5). To a stirred
solution of amine 1i27 (71 mg, 0.15 mmol) in acetonitrile (1
mL) at -10 °C in an acetone/ice bath, under Ar, was added
CCl4 (0.15 mL, 1.6 mmol, 11 equiv). Ten minutes later DIPEA
(0.12 mL, 0.66 mmol, 4.4 equiv) and 4-(dimethylamino)pyridine
(3.8 mg, 0.031 mmol, 0.21 equiv) were added, followed 1 min
later by the dropwise addition of dibenzyl phosphite (0.11 mL,
0.47 mmol, 3.1 equiv). After 1 h, the reaction was warmed to
room temperature and aqueous 0.5 M KH2PO4 was added. The
mixture was extracted with EtOAc (3 × 10 mL), and the
combined extracts were washed with brine (15 mL) and H2O
(15 mL). Upon drying and condensing in vacuo, the product
was separated by gravity column chromatography (4:1, DCM/
EtOAc) to afford a colorless oil 5 (97 mg, 65%): Rf 0.12 (4:1,
DCM/EtOAc); [R]25D -45.1° (c 0.72, CHCl3); P NMR (400 MHz,
CDCl3) δ 5.693, -8.113; 1H NMR (400 MHz, CDCl3) δ 2.89 (1H,
m, -CH2-), 3.18 (1H, m, -CH2-), 3.56 (3H, s, OCH3), 3.71
(3H, s, OCH3), 4.05 (1H, m, alpha H), 4.96 (8H, m, 4 × -CH2-
), 5.87 (2H, s, -OCH2O-), 6.39 (1H, d, J ) 12.0 Hz, vinyl H),
6.45 (3H, m, vinyl H, 2 × ArH), 6.59 (1H, d, J ) 8.4 Hz, ArH),
6.97 (1H, dd, J ) 8.4, 1.6 Hz, ArH), 6.99 (2H, d, J ) 8.4 Hz, 2
× ArH), 7.03 (2H, d, J ) 8.8 Hz, 2 × ArH), 7.29 (20 H, m, 20
× ArH), 8.27 (1H, d, J ) 1.6, ArH), 8.36 (1H, s); HRMS calcd
for C54H53N2O12P2 [M + H]+ 983.3074, found 983.3115; anal.
calcd for C54H52N2O12P2, C, 65.98; H, 5.33; N, 2.85; found, C,
65.51; H, 5.55; N, 2.84.
in the tubulin-based biochemical assays but retained the
amine’s cytotoxic activity.27 Since these compounds caused
a marked increase in the mitotic index of drug-treated cells,
the most reasonable explanation was that the amine 1a
was regenerated by either extracellular or intracellular
hydrolysis of the amides.
When the dolastatin 10-combretastatin amine hybrid 16
was evaluated, it was found to be active in all the
biochemical assays (Table 2), although its effect on the
binding of [3H]colchicine was relatively weak, concordant
with the weak activities observed previously with the
amino acid amides of 1a.27 In contrast, peptide 16 was
about half as active as D-10 (4a) as an inhibitor of both
tubulin assembly and [3H]vinblastine binding to tubulin.
We therefore conclude that at the biochemical level peptide
16 is acting primarily as a D-10 (4a) analogue.
The cytotoxic properties of peptide 16, however, are more
difficult to unravel. Using MCF-7 cells for a detailed
comparison, we found that peptide 16 was about twice as
active as Dov-Val-Dil-Dap (4b) and 4 times as active as
hydrochloride 1b, but only 1/200th as active as D-10 (4a).
Further, when 4b and 1b were mixed in equimolar
amounts, as would occur if 16 were completely hydrolyzed,
an IC50 value for inhibition of the growth of the MCF-7
cells was obtained that was almost identical to that
obtained with peptide 16 (14 vs 17 nM). Since the IC50
values obtained for 16, 4b, and 1b are in the same range,
it is impossible to determine whether the cytotoxicity of
16 derives from its acting primarily as a relatively weak
D-10 (4a) analogue or primarily as a relatively potent
combretastatin A-4 (2g) analogue. The similarity in IC50
values obtained with 16 and with the mixture of its
components 4b and 1b indicates that it, like the previously
studied amides,27 undergoes extracellular or intracellular
hydrolysis. Finally, the similarity in IC50 values for 16, 4b,
1b, and the 4b + 1b mixture indicates that there is little,
if any, synergistic in vitro cytotoxic effect obtained from
having 4b and 1b conjoined in a single molecule.
In conclusion, although initial attempts to synthesize a
very water soluble derivative from the 3,4-methylenedioxy-
5,4′-dimethoxy-3′-amido-Z-stilbene tyrosine amide and as-
partate amide series proved unrewarding and will require
a different approach, three new stilbene amides (9, 14, and
16) strongly inhibiting a minipanel of human cancer cell
lines were discovered. Most importantly, the preliminary
biological results from the potentially multitargeting com-
pounds (amide 14 and peptide 16) merit further investiga-
tion.
Sodium 3,4-Methylenedioxy-5,4′-dimethoxy-3′-(L-Tyr)-
amido-Z-stilbene 3′-O,NR-Diphosphate (6). To a stirred
solution of benzyl phosphate 5 (76 mg, 0.077 mmol) in DCM
(1 mL) at 0 °C, under Ar, was added bromotrimethylsilane (35
µL, 0.33 mmol, 4.3 equiv). The reaction mixture was stirred
for 30 min and concentrated under vacuum. The residue was
Experimental Section
Materials and Methods. Ether refers to diethyl ether and
Ar to argon gas. Bromotrispyrrolidinophosphonium hexafluo-
rophosphate (PyBroP), Oâ-tert-butyl-NR-Boc-L-aspartic acid,

Human Cancer Cell Growth Inhibitors
Journal of Natural Products, 2005, Vol. 68, No. 8 1195
dissolved in EtOH (1 mL), and NaOMe (20 mg, 0.36 mmol,
4.7 equiv) was added, yielding a precipitate that was collected
and washed with EtOAc and ether. The product (6) was
obtained as a colorless powder (52 mg, 95%); mp (dec) 188-
156.1, 149.0, 148.8, 143.3, 136.9, 134.2, 132.3, 129.7, 129.6,
128.9, 128.3, 127.7, 127.2, 127.0, 126.4, 122.8, 119.5, 118.9,
1
111.3, 106.8, 101.2, 99.5, 65.4, 57.0, 56.2, 36.4; H NMR (400
MHz, DMSO) δ 2.78 (2H, m, -CH2-), 3.06 (1H, m, -CH2-),
3.82 (3H, s, OCH3), 3.86 (3H, s, OCH3), 4.49 (1H, m, alpha H),
5.98 (2H, s, -OCH2O-), 6.89 (1H, d, J ) 12.8 Hz, vinyl H),
6.94 (1H, s, ArH), 7.07 (2H, m, vinyl H, ArH), 7.22 (1H, d, J )
6.4 Hz, ArH), 7.28 (2H, d, J ) 5.6, 2 × ArH), 7.32 (2H, d, J )
5.6, 2 × ArH), 7.74 (1H, d, J ) 6.4, ArH), 8.22 (1H, s), 9.25
(1H, s).
190 °C; [R]24 -30.6° (c 0.72, DMSO); P NMR (400 MHz,
D
DMSO) δ 6.262, 0.585; 13C NMR (300 MHz, DMSO) δ 143.8,
132.3, 130.4, 130.2, 129.7, 129.4, 127.4, 127.2, 126.3, 122.1,
119.7, 116.5, 115.1, 111.0, 106.8, 101.2, 99.5, 56.6, 56.2, 55.9;
1H NMR (400 MHz, DMSO) δ 2.63 (1H, m, -CH2-), 3.10 (1H,
m, -CH2-), 3.30 (1H, d, J ) 8.8 Hz), 3.56 (1H, m), 3.86 (3H,
s, OCH3), 3.87 (3H, s, OCH3), 6.00 (2H, s, -OCH2O-), 6.55
(1H, d, J ) 0.8 Hz, ArH), 6.99 (8H, m, 2 × vinyl H, 6 × ArH),
7.11 (1H, d, J ) 8.4 Hz, ArH), 7.23 (1H, dd, J ) 6.8, 1.6 Hz,
ArH), 10.12 (1H, br s); anal. calcd for C26H25N2Na3O12P2‚3H2O,
C, 42.06; H, 4.21; N, 3.77; found, C, 41.68; H, 4.65; N, 3.45.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O-tert-butyl-NR-
Z-L-Tyr)-amido-Z-stilbene (7). To a stirred mixture of amine
1a (0.19 g, 0.63 mmol), O-tert-butyl-NR-Z-L-Tyr (0.45 g, 1.2
mmol, 1.9 equiv), and PyBroP (0.57 g, 1.2 mmol, 1.9 equiv) in
DCM (2 mL) at 0 °C under Ar was added DIPEA (0.55 mL,
3.2 mmol, 5.1 equiv). The reaction mixture was stirred for 45
min at room temperature and concentrated under vacuum. The
product was obtained by gravity column chromatography (4:
1, DCM/EtOAc) as a colorless oil (7, 0.38 g, 93%); Rf 0.74 (4:1,
DCM/EtOAc); [R]24D +1.9° (c 1.04, CHCl3); 1H NMR (300 MHz,
CDCl3) δ 1.30 (9H, s, tBu), 3.05 (2H, m, -CH2-), 3.63 (3H, s,
OCH3), 3.68 (3H, s, OCH3), 4.47 (1H, m, alpha H), 5.05 (2H,
d, J ) 2.4 Hz, -CH2-), 5.60 (1H, br), 5.85 (2H, s, -OCH2O-),
6.33 (1H, d, J ) 12.0 Hz, vinyl H), 6.40 (3H, m, vinyl H, 2 ×
ArH), 6.59 (1H, d, J ) 9.0 Hz, ArH), 6.83 (2H, m, ArH), 6.91
(1H, dd, J ) 8.1, 1.5 Hz, ArH), 7.05 (2H, d, J ) 8.4 Hz, 2 ×
ArH), 7.26 (5H, m, 5 × ArH), 7.92 (1H, s), 8.22 (1H, d, J ) 2.1
Hz, ArH); HRMS calcd for C38H41O8N2 [M + H]+ 653.2863,
found 653.2876; anal. calcd for C38H40N2O8, C, 69.92; H, 6.18;
N, 4.29; found, C, 69.45; H, 6.37; N, 4.21.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(NR-L-Tyr)-amido-
Z-stilbene Hydrochloride (9). To a stirred solution of amine
1i (27 mg, mmol) in ethyl acetate (1 mL) was added ethereal
HCl (1 M) in excess. A white solid immediately formed, the
solvent was removed in vacuo, and the resulting solid was
recrystallized from ethanol/ethyl acetate to yield an off-white
powder (9, 29 mg, quantitative): mp 169-170.5 °C; [R]25D 82.5°
1
(c 0.73, CH3OH); H NMR (300 MHz, CDCl3) δ 3.07 (2H, m,
-CH2-), 3.70 (3H, s, OCH3), 3.79 (3H, s, OCH3), 4.27 (1H, m,
alpha H), 5.93 (2H, s, -OCH2O-), 6.37 (1H, d, J ) 1.2 Hz,
ArH), 6.42 (1H, d, J ) 12.0 Hz, vinyl H), 6.46 (1H, d, J ) 12.6
Hz, vinyl H), 6.49 (1H, d, J ) 1.2 Hz, ArH), 6.75 (2H, d, J )
8.4 Hz, 2 × ArH), 6.90 (1H, d, J ) 8.1 Hz, ArH), 7.03 (1H, dd,
J ) 8.1, 2.1 Hz, ArH), 7.09 (2H, d, J ) 8.7 Hz, 2 × ArH), 7.92
(1H, d, J ) 1.5 Hz, ArH); anal. calcd for C26H26ClN2O6, C,
59.32; H, 5.75; N, 5.33; found, C, 59.53; H, 5.56; H, 5.26.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(Oâ-tert-butyl-
NR-Boc-L-Asp)-amido-Z-stilbene (10). To a stirred mixture
of amine 1a (0.13 g, 0.43 mmol), Oâ-tert-butyl-NR-Boc-L-Asp
(0.22 g, 0.76 mmol, 1.8 equiv), and PyBroP (0.35 g, 0.76 mmol,
1.8 equiv) in DCM (3 mL) at 0 °C under Ar was added DIPEA
(0.21 mL, 1.2 mmol, 2.8 equiv). The reaction mixture was
stirred for 75 min at room temperature, and the solvent was
removed in vacuo. The product was obtained by flash column
chromatography (1:1, n-hexane/acetone) as an oil (10, 0.22 g,
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O-bisbenzylphos-
phoryl-NR-Z-L-Tyr)-amido-Z-stilbene (8). To a stirred solu-
tion of amide 7 (0.26 g, 0.40 mmol) in DCM (2 mL) was added
TFA (2 mL). The reaction mixture was stirred for 20 min, the
solution was concentrated under vacuum, and the free phenol
obtained by gravity column chromatography (4:1, DCM/EtOAc)
was placed in acetonitrile (4 mL) under Ar. The mixture was
cooled to -10 °C, and CCl4 (0.21 mL, 2.1 mmol, 5.3 equiv) was
added. Ten minutes later DIPEA (0.16 mL, 0.89 mmol, 2.2
equiv) and 4-(dimethylamino)pyridine (5 mg, 0.041 mmol, 0.10
equiv) were added, followed 1 min later by the dropwise
addition of dibenzyl phosphite (0.15 mL, 0.65 mmol, 1.6 equiv).
After 2 h the reaction mixture was warmed to room temper-
ature, and aqueous 0.5 M KH2PO4 (16 mL) added. The same
procedure given for phosphate 5 was followed to obtain the
product 8 as a colorless oil (0.24 g, 71%); Rf 0.60 (4:1, DCM/
EtOAc); [R]23D -5.1° (c 0.25, CHCl3); P NMR (400 MHz, CDCl3)
δ -8.095; 1H NMR (MHz, CDCl3) δ 3.08 (2H, m, -CH2-), 3.66
(1H, s, OCH3), 3.72 (1H, s, OCH3), 4.51 (1H, m, alpha H), 5.08
(2H, d, J ) 0.9 Hz, -CH2-), 5.11 (4H, m, 2 × -CH2-), 5.44
(1H, br d, J ) 7.2 Hz), 5.90 (2H, s, -OCH2O-), 6.39 (1H, d, J
) 12.0 Hz, vinyl H), 6.46 (3H, m, vinyl H, 2 × ArH), 6.62 (1H,
d, J ) 8.1 Hz, ArH), 6.98 (1H, dd, J ) 8.4, 1.8 Hz, ArH), 7.04
(2H, d, J ) 8.1 Hz, 2 × ArH), 7.14 (2H, d, J ) 8.1 Hz, 2 ×
ArH), 7.31 (15H, m, 15 × ArH), 7.90 (1H, s), 8.24 (1H, d, J )
2.4 Hz, ArH); HRMS calcd for C48H46N2O11P [M + H]+
857.2840, found 857.2853; anal. calcd for C48H45N2O11P, C,
67.28; H, 5.29; N, 3.27; found, C, 66.89; H, 5.27; N, 3.17.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(O-phosphoryl-
NR-L-Tyr)-amido-Z-stilbene (3a). To a stirred solution of
benzyl ester 8 (98 mg, 0.11 mmol) in acetonitrile under Ar was
added sodium iodide (60 mg, 0.40 mmol, 3.6 equiv), followed
by chlorotrimethylsilane (51 µL, 0.41 mmol, 3.7 equiv). A white
precipitate formed while the reaction mixture was stirred for
20 min. Aqueous (1%) sodium thiosulfate (0.5 mL) was added
to the mixture before the precipitate was collected and washed
with ethyl acetate, H2O, and acetone. The product was
obtained as a colorless amorphous solid (3a, 35 mg, 58%): mp
88%): Rf 0.64 (1:1, n-hexane/acetone); [R]24 -13.0° (c 1.42,
D
1
CHCl3); H NMR (300 MHz, CDCl3) δ 1.44 (9H, s, tBu), 1.49
(9H, s, tBu), 2.88 (2H, m, -CH2-), 3.73 (3H, s, OCH3), 3.84
(3H, s, OCH3), 4.59 (1H, m, alpha H), 5.81 (1H, br), 5.92 (2H,
s, -OCH2O-), 6.38 (1H, d, J ) 12.6 Hz, vinyl H), 6.44 (1H, s,
ArH), 6.46 (1H, d, J ) 12.6 Hz, vinyl H), 6.47 (1H, s, ArH),
6.70 (1H, d, J ) 8.1 Hz, ArH), 6.97 (1H, dd, J ) 8.4, 1.8 Hz,
ArH), 8.28 (1H, d, J ) 2.4 Hz, ArH), 8.76 (1H, s); HRMS calcd
for C30H39N2O9 [M + H]+ 571.2655, found 571.2617; anal. calcd
for C30H38N2O9, C, 63.14; H, 6.71; N, 4.91; found, C, 62.64; H,
7.00; N, 4.89.
Sodium 3,4-Methylenedioxy-5,4′-dimethoxy-3′-(NR-L-
Asp)-amido-Z-stilbene (11). To tert-butyl ester 10 (0.13 g,
0.23 mmol) in DCM (1.5 mL) was added a mixture of TFA (0.71
mL, 9.6 mmol, 42 equiv) and TES (0.30 mL, 1.8 mmol, 7.8
equiv), and stirring was continued for 4 h under Ar. The
solvents were removed in vacuo, and the TFA salt, obtained
by Sephadex LH-20 column chromatography (solvent, MeOH),
was placed in methanol (1 mL). Sodium methoxide (0.22 mg,
0.41 mmol, 1.8 equiv) was added to the reaction mixture, and
the white precipitate that formed was collected and reprecipi-
tated from DCM/CH3OH to give the product 11 as a colorless
solid (62 mg, 62%): mp 168-170 °C; [R]25D +14.0° (c 1.18, CH3-
OH); 1H NMR (300 MHz, CD3OD) δ 2.62 (1H, m, -CH2-), 2.77
(1H, m, -CH2-), 2.78 (2H, br), 3.69 (3H, s, OCH3), 3.87 (3H,
s, OCH3), 4.29 (1H, m, alpha H), 5.87 (2H, s, -OCH2O-) 6.36
(1H, d, J ) 1.2 Hz, ArH), 6.40 (1H, d, J ) 12.0 Hz, vinyl H),
6.45 (1H, d, J ) 12.3 Hz, vinyl H), 6.50 (1H, d, J ) 0.9 Hz,
ArH), 6.91 (1H, d, J ) 8.4 Hz, ArH), 7.02 (1H, dd, J ) 8.4, 1.8
Hz, ArH), 7.99 (1H, d, J ) 2.4 Hz, ArH), 8.52 (1H, s); anal.
calcd for C21H21N2NaO7‚H2O, C, 55.51; H, 5.10; N, 6.16; found,
C, 55.81; H, 5.70; N, 6.16.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(NR-Boc-L-Dap)-
amido-Z-stilbene (13). To a stirred mixture of amine 1a (51
mg, 0.17 mmol), NR-Boc-L-Dap 12 (52 mg, 0.18 mmol, 1.1
equiv),32,33 and PyBroP (87 mg, 0.19 mmol, 1.1 equiv) at 0 °C
under Ar was added DIPEA (65 µL, 0.37 mmol, 2.2 equiv). The
reaction mixture was stirred for 1.5 h at room temperature.
DCM (10 mL) was added, and the mixture was washed with
aqueous citric acid (10% by wt, 10 mL). The organic layer was
(dec) 175-177 °C; [R]25 -6.3° (c 0.35, DMSO); P NMR (400
D
MHz, DMSO) δ -2.043; 13C NMR (400 MHz, DMSO) δ 170.6,

1196 Journal of Natural Products, 2005, Vol. 68, No. 8
Pettit et al.
dried with MgSO4 and concentrated in vacuo, and the residue
was subjected to gravity column chromatography (8:1, DCM/
EtOAc), resulting in the product 13 as a colorless oil (40 mg,
41%): Rf 0.24 (8:1, DCM/EtOAc); [R]24D -48.5° (c 1.2, CHCl3);
13C NMR (500 MHz, CDCl3) δ 148.5, 146.9, 143.3, 134.2, 131.9,
130.1, 129.5, 128.8, 127.6, 123.8, 120.8, 109.6, 108.5, 103.0,
101.3, 58.7, 56.3, 55.8, 28.5; 1H NMR (300 MHz, CDCl3) δ 1.30
(3H, d, J ) 6.9 Hz, CH3), 1.46 (9H, s, Boc), 1.72 (1H, m, Pro),
1.90 (3H, m, Pro), 2.62 (1H, m), 3.23 (1H, m), 3.44 (1H, m),
3.49 (3H, s, OCH3), 3.58 (1H, m), 3.74 (3H, s, OCH3) 3.85 (3H,
s, OCH3), 3.90 (1H, m), 5.92 (2H, s, -OCH2-), 6.38 (1H, d, J
) 12.3 Hz, vinyl H), 6.45 (1H, s, ArH), 6.46 (1H, d, J ) 12.0
Hz, vinyl H), 6.47 (1H, s, ArH), 6.69 (1H, d, J ) 8.7, ArH),
6.96 (1H, dd, J ) 8.4, 1.8 Hz, ArH), 8.30 (1H, d, J ) 2.4, ArH),
8.41 (1H, br); HRMS calcd for C31H41N2O8 [M + H]+ 569.2863,
found 569.2884; anal. calcd for C31H40N2O8, C, 65.48; H, 7.09;
N, 4.93; found, C, 64.92; H, 7.42; N, 4.97.
tubulin, 5 µM [3H]vinblastine, potential inhibitor as indicated,
0.5 mM MgCl2, 0.1 M 4-morpholineethanesulfonate (1.0 M
stock solution adjusted to pH 6.9 with NaOH), and 4% (v/v)
dimethyl sulfoxide. Incubation (30 min) and centrifugal gel
filtration were performed at room temperature (20-22 °C).
Cytotoxicity assays were performed by the sulforhodamine
B method, in which inhibition of formation of cellular protein
is measured.42 The mitotic index of MCF-7 breast cancer cells
was determined as described previously,27 except that cells
were incubated in the presence of drug for 16 h prior to
addition of the DNA stain.
Antimicrobial Evaluation. Compounds were screened in
broth microdilution assays according to National Committee
for Clinical Laboratory Standards.43,44 Assays were repeated
at least twice on separate days.
Acknowledgment. We are pleased to thank the following
for financial assistance: Outstanding Investigator Grant
CA44344-05-12 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 Caitlin Robb Foundation; Dr. Alec D. Keith and
Mrs. Kay M. Keith; the J. W. Kieckhefer Foundation; the
Margaret T. Morris Foundation; Rod J. and Hazel V. McMul-
lin; Gary L. and Diane Tooker; Polly J. Trautman; John and
Edie Reyno; Dr. John C. Budzinski; the Ladies Auxiliary to
the Veterans of Foreign Wars; and the Robert B. Dalton
Endowment Fund. In addition, we would like to thank Dr. G.
Boland for conducting some preliminary experiments. Other
very helpful assistance was provided by Drs. F. Hogan, J. M.
Schmidt, and J.-C. Chapuis, and by Ms. B. Fakoury and Mr.
M. J. Dodson. This work was supported in part by NCI,
National Institutes of Health Contract NO1-CO-12400.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(NR-L-Dap)-amido-
Z-stilbene TFA Salt (14). To a stirred solution of amide 13
(0.15 g, 0.26 mmol) in DCM (0.65 mL) at 0 °C was added TFA
(0.20 mL, 2.6 mmol, 10 equiv). The reaction mixture was
stirred for 20 min at room temperature. The product was
obtained by gravity column chromatography (20:1, DCM/
MeOH) as an oil (14, 69 mg, 46%): Rf 0.54 (8:1, DCM/MeOH);
[R]24 -61.9° (c 0.88, CHCl3); 13C NMR (300 MHz, CDCl3) δ
D
170.5, 148.1, 146.9, 142.8, 131.3, 129.6, 128.6, 126.3, 124.3,
120.5, 109.3, 108.1, 102.4, 100.8, 100.3, 80.2, 60.5, 58.6, 55.8,
55.3, 44.8, 41.5, 24.1, 23.4, 12.1; 1H NMR (300 MHz, CDCl3) δ
1.27 (3H, d, J ) 6.9, CH3), 1.98 (4H, m), 2.94 (1H, m), 3.33
(2H, m), 3.57 (3H, s, OCH3), 3.73 (1H, s, OCH3), 3.78 (1H, m),
3.85 (3H, s, OCH3), 3.89 (1H, m), 5.91 (2H, s, -OCH2O-), 6.38
(1H, d, J ) 12.6, vinyl H), 6.43 (3H, m, vinyl H, 2 × ArH),
6.73 (1H, d, J ) 9.0, ArH), 7.00 (1H, dd, J ) 8.4, 1.8 Hz, ArH),
8.15 (1H, d, J ) 1.8 Hz, ArH), 8.35 (1H, s); HRMS calcd for
C26H33N2O6 [M + H]+ 469.2339, found 469.2374; anal. calcd
for C30H34F6N2O10‚2H2O, C, 49.18; H, 5.23; N, 3.82; found, C,
49.48; H, 5.23; N, 4.09.
References and Notes
(1) Pettit, G. R.; Moser, B. R.; Boyd, M. R.; Schmidt, J. M.; Pettit, R. K.;
Chapuis, J. C. Anti-Cancer Drug Des. 2002, 16, 185-194.
(2) Kanthou, C.; Tozer, G. M. Blood 2002, 99, 2060-2069.
(3) Beauregard, D. A.; Pedley, R. B.; Hill, S. A. Brindle, K. M. NMR in
Biomed. 2002, 15, 99-105.
(4) Eikesdal, H. P.; Landuyt, W.; Dahl, O. Cancer Lett. 2002, 178, 209-
217.
(5) Li, L.; Rojiani, A. M.; Siemann, D. W. Acta Oncol. 2002, 41, 91-97.
(6) Boehle, A. S.; Sipos, B.; Kliche, U.; Kalthoff, H.; Dohrmann, P. Ann.
Thoracic Surg. 2001, 71, 1657-1665.
(7) Dowlati, A.; Robertson, K.; Cooney, M.; Petros, W. P.; Stratford, M.;
Jesberger, J.; Rafie, N.; Overmoyer, B.; Makkar, V.; Stambler, B.;
Taylor, A.; Waas, J.; Lewin, J. S.; McCrae, K. R.; Remick, S. C. A
Cancer Res. 2002, 62, 3408-3416.
3,4-Methylenedioxy-5,4′-dimethoxy-3′-(Dov-Val-Dil-
Dap)-amido-Z-stilbene (16). To a stirred mixture of amide
14 (41 mg, 0.070 mmol), Dov-Val-Dil TFA salt 15 (62 mg, 0.11
mmol, 1.6 equiv), and PyBroP (50 mg, 0.11 mmol, 1.6 equiv)
in DCM (0.5 mL) at 0 °C under Ar was added DIPEA (91 µL,
0.52 mmol, 7.4 equiv). The reaction mixture was stirred 14 h
at room temperature, and the solvent was removed in vacuo.
The product was obtained by gravity column chromatography
(1:1, n-hexane/acetone) as a colorless oil (16, 30 mg, 48%): Rf
0.34 (1:1, n-hexane/acetone); [R]23 -62° (c 1.05, CHCl3); 1H
D
NMR (300 MHz, CDCl3) δ 0.79 (3H, t, J ) 6.9 Hz, CH3), 0.91
(3H, d, J ) 6.6 Hz, CH3), 0.94 (3H, d, J ) 6.6 Hz), 0.94 (3H, d,
J ) 6.3 Hz, CH3), 1.00 (3H, d, J ) 6.6 Hz, CH3), 1.01 (3H, d,
J ) 6.3 Hz, CH3), 1.32 (3H, d, J ) 6.9 Hz, CH3), 1.35 (2H, m),
1.80 (1H, m), 2.0 (3H, m), 2.31 (6H, s, 2 × NCH3), 2.34 (4H,
m), 2.62 (1H, m), 3.00 (3H, s, NCH3), 3.05 (1H, m) 3.08 (1H,
s), 3.20 (1H, m), 3.29 (1H, m), 3.32 (3H, OCH3), 3.34 (1H, m),
3.44 (1H, m), 3.45 (3H, s, OCH3), 3.72 (3H, s. OCH3), 3.84 (3H,
s, OCH3), 4.06 (1H, m), 4.12 (1H, m), 4.20 (1H, m), 4.79 (1H,
m), 5.90 (2H, s, -OCH2O-), 6.36 (1H, d, J ) 12.0 Hz, vinyl
H), 6.44 (3H, m, vinyl H, 2 × ArH), 6.68 (1H, d, J ) 8.7 Hz,
ArH), 6.94 (1H, dd, J ) 8.1, 1.5 Hz, ArH), 8.27 (1H, d, J ) 1.8
Hz), 8.33 (1H, s); HRMS calcd for C48H74N5O10 [M + H]+
880.5435, found 880.5426; anal. calcd for C48H73N5O10, C,
65.50; H, 8.36; N, 7.96; found, C, 65.42; H, 8.74; N, 7.59.
Tubulin and Cancer Cell Procedures. Bovine brain
tubulin was prepared38 and the tubulin assembly39 and colchi-
cine binding40 assays were performed as described previously.
In the assembly assay, reaction mixtures contained 1.0 mg/
mL (10 µM) tubulin and varying concentrations of potential
inhibitors. In the colchicine binding assay reaction mixtures
contained 0.1 mg/mL (1.0 µM) tubulin, 5.0 µM [3H]colchicine,
and potential inhibitor as indicated. The vinblastine binding
assay was performed as described previously for GTP binding41
by centrifugal gel filtration, except that a Beckman Allegra
6KR centrifuge equipped with a GH-3.8A swinging bucket
rotor was used and the syringe-columns were centrifuged at
2000 rpm. Reaction mixtures contained 0.5 mg/mL (5.0 µM)
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