E-Combretastatin and E-resveratrol structural modifications: Antimicrobial and cancer cell growth inhibitory β-E-nitrostyrenes

Article abstract of DOI:10.1016/j.bmc.2009.07.076

As part of a broad-based SAR investigation of E-resveratrol (strong sirtuin activator and antineoplastic) and the anticancer vascular-targeting combretastatin-type stilbenes, a series of twenty-three β-E-nitrostyrenes was synthesized in order to evaluate potential antineoplastic, antitubulin, and antimicrobial activities. The β-E-nitrostyrenes evaluated ranged from monosubstituted phenols to trimethoxy and 3-methoxy-4,5-methylenedioxy derivatives. Two of the β-nitrostyrenes were synthesized as water-soluble sodium phosphate derivatives (4t, 4v). All except four (4r, 4s, 4t, 4u) of the series significantly inhibited a minipanel of human cancer cell lines. All but eight led to an IC₅₀ of <10 μM for inhibition of tubulin polymerization, and all except three (4l, 4t, 4v) displayed antimicrobial activity.

Full text of DOI:10.1016/j.bmc.2009.07.076

Bioorganic & Medicinal Chemistry 17 (2009) 6606–6612
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
E-Combretastatin and E-resveratrol structural modifications: Antimicrobial
and cancer cell growth inhibitory b-E-nitrostyrenes q
a,
b
a
c
a
Robin K. Pettit a, , George R. Pettit , Ernest Hamel , Fiona Hogan , Bryan R. Moser , Sonja Wolf ,
*
*
Sandy Pon a, Jean-Charles Chapuis a, Jean M. Schmidt a
a Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, AZ 85287-1604, United States
b Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702-1201, United States
c United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 N University St, Peoria, IL 61604, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of a broad-based SAR investigation of E-resveratrol (strong sirtuin activator and antineoplastic)
and the anticancer vascular-targeting combretastatin-type stilbenes, a series of twenty-three b-E-nitro-
styrenes was synthesized in order to evaluate potential antineoplastic, antitubulin, and antimicrobial
activities. The b-E-nitrostyrenes evaluated ranged from monosubstituted phenols to trimethoxy and 3-
methoxy-4,5-methylenedioxy derivatives. Two of the b-nitrostyrenes were synthesized as water-soluble
sodium phosphate derivatives (4t, 4v). All except four (4r, 4s, 4t, 4u) of the series significantly inhibited a
Received 8 June 2009
Revised 29 July 2009
Accepted 30 July 2009
Available online 4 August 2009
Keywords:
Resveratrol
Combretastatin
b-E-Nitrostyrene
Anticancer agent
minipanel of human cancer cell lines. All but eight led to an IC50 of <10
merization, and all except three (4l, 4t, 4v) displayed antimicrobial activity.
Ó 2009 Published by Elsevier Ltd.
lM for inhibition of tubulin poly-
1. Introduction
the present investigation, we prepared and evaluated a series of
b-E-nitrostyrenes that represents the substitution of a trans nitro
group for one of the combretastatin A-series cis-olefin phenyl rings.
Initial focus on the more readily available b-E-nitrostyrenes was
also attractive owing to our early interest in SAR investigations
of the E-stilbene resveratrol (3),22,23 with its broad spectrum of
important biological properties22–25 that include antineoplastic,
anti-inflammatory, and antioxidant activities. Very importantly
for potential life extension, trans-stilbene 3 is a very strong activa-
tor of sirtuin enzymatic activity (which retards the aging pro-
cess).24 Interestingly, Z-combretastatin A-4 (1a) and E-resveratrol
(3) have recently been shown to share other important biological
properties such as activation of AMP-activated protein kinase,
which in turn has been shown to improve glucose tolerance in dia-
betic db/db mice.25
The diverse biological activities of b-nitrostyrenes have been
recognized for decades and include molluscicidal,26,27 insecti-
cidal,27 and antimicrobial properties.28–36 Human cancer cell line
growth inhibition has been demonstrated for certain b-nitrosty-
renes.37,38 The modes of action of these compounds include induc-
tion of apoptosis39,40 and inhibition of protein tyrosine kinases,41
phosphatases,42 and human telomerase.43 Inhibition of snake ve-
nom phospholipase was recently reported.44 While most b-nitro-
styrenes have been obtained synthetically, one exception is 3,4-
dihydroxy-b-E-nitrostyrene, isolated as a reddish-brown pigment
The A-1 to A-4 series of combretastatin Z-stilbenes that we iso-
lated from root bark of the South African tree Combretum caffrum
are now known for their remarkable biological activities, including
robust inhibition of cancer cell growth and tubulin polymeriza-
tion.2–5 Combretastatins A-1 (1a, CA1)6 and A-4 (1b, CA4),7 in par-
ticular, display powerful in vivo cancer vascular disrupting and
antiangiogenesis properties leading to potent reductions in tumor
blood flow.8–12 Both CA4 and CA1, along with their corresponding
phosphate prodrug salts (CA4P,13 1c; CA1P,14 1d) are of increasing
therapeutic interest.15–18 The CA1P and CA4P prodrugs are cur-
rently in Phase I and III human cancer clinical trials, respectively.
In addition, CA4P is presently undergoing Phase II human trials
against age-related macular degeneration, the leading cause of
blindness.19,20
The structural features of CA4, and to a lesser extent, CA1, can
be easily modified, and we have been exploring the minimal struc-
tural modifications that allow retention of important biological ef-
fects. Illustrative of this was our discovery of phenstatin (2a) and
the subsequent development of its phosphate prodrug (2b).21 In
q
See Ref. 1.
* Corresponding author. Tel.: +1 480 965 3351; fax: +1 480 965 2747.
E-mail addresses: robin.pettit@asu.edu (R.K. Pettit), bpettit@asu.edu (G.R. Pettit).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.07.076

R. K. Pettit et al. / Bioorg. Med. Chem. 17 (2009) 6606–6612
6607
and ammonium acetate. Yields of the resulting yellow-colored b-
nitrostyrenes ranged from 70% to 99%. An improved synthetic pro-
cedure for the preparation of aldehyde 7 (from 6,47 Scheme 1) was
developed for obtaining the previously known b-E-nitrostyrene
4n.48 The synthetic route (Scheme 2) illustrated for obtaining the
new phosphate ester 4u and its sodium salt (4v) via 8 was also
used for preparing phosphates 4s and 4t.47 The aqueous solubility
of b-E-nitrostyrene sodium phosphates 4t and 4v should enhance
their utility in future in vivo studies.
Most of the b-nitrostyrenes synthesized in this study were ac-
tive against a variety of human cancer cell lines (Table 1). To our
knowledge, this is the first report of human cancer cell line growth
inhibition for these b-nitrostyrenes (with the exception of 4k,
which we reported49 in 2002). Sodium phosphate 4v but not 4t
was active as a cancer cell line inhibitor (in vitro). We would ex-
pect the phosphatase enzymes in vivo to cleave phosphate 4t to
the active phenol 4b. Because phosphate 4v does show activity,
the results suggest that instability in the phosphate ester bond
led to generation of the parent phenol, which is most likely the
active agent. The TBDMS derivative (4r) did not inhibit the murine
or human cancer cells. The cancer cell line results for these b-E-
from a saframycin-nonproducing mutant of Streptomyces lavendu-
lae.32 We have synthesized twenty-three b-E-nitrostyrenes (4a–v,
5), including new compounds 4r–v, with the goal of evaluating
inhibitory effects on the growth of human cancer cell lines, bacte-
ria, and fungi.
nitrostyrenes were relatively consistent (GI50 ca. 0.1
data we have obtained with a series of other E-stilbenes,50,51 as
compared to GI50 values of 0.01–0.001 g/ml for the corresponding
lg/mL) with
l
Z-isomers. Several (e.g., 4f, 4g, 4l) of the more active members of
the series will receive more detailed anticancer evaluation
in vivo. Interestingly, a SAR study of curacin A (9) from Lyngbya
majuscula led to discovery of the comparably active analog 1052
(tubulin inhibition IC50 about 1 lM, cancer cell growth inhibition
about 250 nM) bearing a cis-3,4,5-trimethoxystyrene unit reminis-
cent of combretastatin A-4 (1b) and colchicine (11).
We previously49 found nitrostyrene 4k to have good activity as
an inhibitor of tubulin polymerization53 (about 40% as potent as
combretastatin A-4), with relatively weak activity as an inhibitor
of colchicine binding to tubulin. The known reactivity of b-nitro-
styrenes with sulfhydryl groups34,42,54 was consistent with these
observations, since alkylation of cysteine-239 of b-tubulin results
in inhibition of assembly of tubulin.55,56 Inhibition of colchicine
binding by these alkylating compounds is more dependent on
the structure of the inhibitor. Thus, 2,4-dichlorobenzyl thiocyanate
(12),55 like 4k, inhibits tubulin assembly at much lower concentra-
tions than it inhibits colchicine binding, while T138067 (13)56 is a
much stronger inhibitor of colchicine binding (note its structural
analogy to combretastatin A-4 (1b)). Unexpectedly, nitrostyrene
4k did not cause mitotic arrest in the mammalian cells that were
treated with a cytotoxic concentration of the compound, as is typ-
ical of tubulin inhibitors.49 This suggested a different target was
responsible for the activity of nitrostyrene 4k in cells, in contrast
to the sulfhydryl-reactive compounds 1255 and 13,56 both of which
caused mitotic arrest.
2. Results and discussion
The key synthetic step for preparation of each b-nitrostyrene
was based on the McNulty et al.45 modification of the Henry reac-
tion,46 employing ultrasound to accelerate reaction of the aldehyde
precursor with nitromethane in the presence of glacial acetic acid
Scheme 1.

6608
R. K. Pettit et al. / Bioorg. Med. Chem. 17 (2009) 6606–6612
2.5 lM, which was the most active compound in the series and was
almost fourfold more active than its hydroxylated congener 4f.
Finally, the two phosphates were the least active compounds in
the series; the IC50 value of 4v was 18 lM, while 4t was completely
inactive. To summarize these structure–activity observations, the
interaction with tubulin is decreased by a meta- or para-hydroxyl
group and reduced further by phosphorylation of the compound,
but it can be enhanced by an appropriate bulky hydrophobic sub-
stituent. Specifically comparing the activities of the relatively inac-
tive compound 4f and its much more alkylated congener 4q, we
note that the latter in part recapitulates aspects of the structure
of the combretastatins, in that there is a two atom bridge between
the two phenyl rings. If this is a factor in the enhanced activity of
4q, then it would be worthwhile to prepare analogs with different
substituent patterns in both phenyl rings.
We investigated the antimicrobial activity of the b-nitrostyrene
series by two very different methods, both recommended proce-
dures of the Clinical and Laboratory Standards Institute (CLSI).57–59
All of the nitrostyrenes were evaluated against ten microorganisms
in solid-phase disk diffusion susceptibility assays. Compounds in
sufficient quantity were screened against the same microbes in the
liquid-phase broth microdilution assay (BMA). The BMA allows
determination of static versus cidal mechanism of action. Disk diffu-
sion assays demonstrated that most of the b-E-nitrostyrenes inhib-
ited growth of unicellular fungi and Gram-positive bacteria (Table
3). The potent activity of this series against the Gram-negative path-
ogen Neisseria gonorrhoeae is notable. The susceptibility of this bac-
terium to antibiotics bears more resemblance to Gram-positive than
Gram-negative bacteria, owing to short-chain lipopolysaccharides
in the outer membrane. The difference in activity of 3,4,5-trimeth-
oxy-b-nitrostyrene (4k) versus 2,4,6-trimethoxy-b-nitrostyrene
(4l) in disk diffusion assays was remarkable. While 4l was inactive,
4k showed broad-spectrum activity. In contrast, both compounds
inhibited human cancer cells (Table 1). The sodium phosphate
derivatives ( 4t, 4v) were not antimicrobial. The addition of
bis(benzyl)phosphoryl moieties (4s, 4u) resulted in the loss of anti-
fungal activity and the retention of anti-Gram-positive activity.
The antifungal activity of a subset of the b-nitrostyrenes was con-
firmed in BMAs (Table 4). Minimum fungicidal concentrations were
generally equal to or one- or twofold dilution higher than MICs, indi-
cating that these b-nitrostyrenes are fungicidal. This is an important
distinction from the fungistatic nature of most clinically useful anti-
fungals. Details of the in vitro antifungal action of nitrostyrene 4k
have been reported by us.49 With the exception of the exquisitely
sensitive human pathogen N. gonorrhoeae, antibacterial activity
was generally not demonstrated in BMAs. Currently, the BMA is
the most widely used method for antimicrobial susceptibility test-
ing, but whether the disk diffusion assay or the BMA more accurately
reflects in vivo conditions is a matter of some debate. Antibacterial
activity has been reported for compounds 4a,28 4b,28 4d,28,29 4e,35
4f,28,29,35 4g,35 4m,35 4o,29 and 4p,29,36 and antifungal activity for
compounds 4a,28 4b,28 4d,28,29 4f,28,29 4g,32 4h,32 4k,49 4o,29 4p,29
and 5,33 but the static versus cidal mechanism was not investigated,
small microbe panels were employed, and, with the exception of
one research group,35 CLSI protocols were not followed.
Scheme 2.
We examined most of the b-E-nitrostyrenes that we synthe-
sized for their effects on tubulin assembly (Table 2) in hope of find-
ing a more potent compound than nitrostyrene 4k, and for
structure–activity insight into the postulated covalent interaction
with tubulin. In the current experiments, nitrostyrene 4k again
showed inhibitory activity similar to that of combretastatin A-4
(1b, IC50 values of 4.5 and 2.1 lM, respectively). Most compounds
with small substituents on the phenyl ring (4a, 4c, 4d, 4g, 4h, 4i,
4m, 4n, and 4o) showed similar activity, with IC50 values ranging
from 3.9 to 7.4 lM. Either a meta-hydroxyl or para-hydroxyl group,
While it has been suggested that the nature of the substituents
on the phenyl ring plays a minor role in toxicity and that the b-
nitroethylene moiety is primarily responsible for the bioactivity,60
our results do not support this contention. b-nitrostyrenes have
been shown to interact with cysteine sulfhydryls54 and can inhibit
rat liver mitochondrial respiration.61 Gullner et al.34 found that
relative to the nitrovinyl group, generally caused a 50–100% loss in
activity. The IC50 values for these compounds (4b, 4e, 4f, 4j) ranged
from 8.2 to 10 lM (higher than those of their methylated congen-
three thiols, L-cysteine, glutathione and 2-mercaptoacetic acid,
ers 4d, 4g, and 4k). Bulky hydrophobic substituents on these hy-
droxyl groups did not seem to cause much further loss in activity
(compare 4p and 4s to 4b, 4n to 4f; also 4r and 5). The one excep-
tion to this generalization was compound 4q, with an IC50 value of
reacted readily with 27 antifungal b-nitrostyrenes (none are repre-
sented in the present study). As sulfhydryl-containing enzymes are
involved in metabolic and respiratory chain processes, it has been
proposed that this may be a general toxic mechanism for eukary-

R. K. Pettit et al. / Bioorg. Med. Chem. 17 (2009) 6606–6612
6609
Table 1
Murine P388 leukemia inhibitory activity [ED50 (lg/mL)] and human cancer cell line inhibitory activity [GI50 (lg/mL)] of b-E-nitrostyrenes
Compound
Leukemia
P388
Pancreas
BXPC-3
Breast
MCF-7
CNS
SF295
CNS
SF268
Lung-NSC
NCI-H460
Colon
KM20L2
Prostate
DU-145
Melanoma
SK-MEL-5
Ovary
OVCAR-3
Renal
A498
E-1b50
Z-1b23
E-323
Z-323
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
0.28
3.0 Â 10-4
4.49
24.4
3.5
1.5
0.51
1.7
3.9
0.39
3.3
15.5
1.1
1.6
1.6
1.8
NA
1.2
1.5
1.6
NA
1.5
1.4
NA
1.7
NA
1.2
NA
1.7
>10
>10
>10
>10
>10
1.4
NA
NA
3.9
NA
NA
NA
NA
NA
1.7
1.7
1.6
NA
0.37
NA
NA
NA
NA
1.3
0.45
NA
NA
NA
1.4
NA
>10
NA
NA
>10
NA
NA
NA
0.038
6.0 Â 10-4
3.6
13.2
0.95
1.7
1.7
1.7
NA
0.34
0.46
2
NA
0.38
1.1
0.32
2
NA
1.9
0.7
NA
0.34
13.1
22
0.037
8.0 Â 10-4
3.5
10.2
1
1.5
1.5
1.7
NA
0.38
1.4
1.5
NA
0.36
1
NA
1.8
NA
2.2
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.61
NA
NA
NA
0.34
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.48
NA
NA
NA
NA
0.42
0.42
NA
NA
NA
0.65
NA
>10
NA
NA
>10
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.36
NA
NA
NA
0.96
NA
NA
NA
NA
NA
NA
NA
>1.0 Â 10-2
4.1
5
1.2
NA
NA
NA
NA
NA
0.71
2.3
NA
0.54
NA
NA
1.9
NA
1.8
NA
14.8
0.47
0.48
0.48
0.54
NA
NA
0.53
1.8
NA
0.44
NA
NA
0.58
NA
1.6
1.5
1.5
1.5
NA
1.1
1.3
1.5
NA
1.1
1.3
0.38
1.8
NA
2.4
1.1
1.5
>10
>10
>10
>10
>10
1.4
NA
0.18
0.34
0.24
1.3
2.2
1.1
14
2
NA
3.8
1.1
NA
4p
4q
4r
4s
4t
4u
4v
6.2
NA
1.3
16.6
32.6
7
47.8
2.2
0.96
NA
>10
>10
NA
0.94
NA
>10
>10
NA
1.3
>10
>10
>10
>10
5.8
>10
>10
57.8
>10
4
5.7
3.7
4.3
0.4
4.2
1.9
5
1.6
1.3
otic cells.34 However; several of the b-E-nitrostyrenes were not ac-
tive in our biological screens, suggesting that an unsaturated
nitroolefinic bond is not necessarily toxic. We are currently inves-
tigating other cellular targets of the b-nitrostyrenes and plan to ex-
tend this study to a series of b-Z-nitrostyrenes.
kee, WI). All solvents were redistilled. Ultrasound reactions were
carried out in a Branson 5520 ultrasound bath or 5510 ultrasonic
cleaner. Reactions were monitored by thin-layer chromatography
using Analtech silica gel GHLF Uniplates. Reactants and products
were visualized under long-wave (366 nm) and short-wave
(254 nm) UV irradiation. Solvent extracts of aqueous solutions
were dried over anhydrous magnesium sulfate. Where appropriate,
the crude products were purified by flash (230–400 Mesh ASTM) or
gravity (70–230 Mesh ASTM) silica gel (E. Merck) chromatography.
Melting points were measured with an electrothermal digital
melting point apparatus (Model IA 9200) and are uncorrected. All
1H NMR and 13C NMR spectra were recorded using a Varian Gemini
300 MHZ instrument with CDCl3 (TMS internal reference) or
CD3OD as solvent unless otherwise indicated. Elemental analyses
were determined by Galbraith Laboratories, Inc., Knoxville, TN.
All compounds reported gave acceptable combustion analytical re-
sults for carbon, hydrogen and nitrogen. Melting points and NMR
data were consistent with literature values for the following:
4a,62 4b,63 4c,63 4d,63 4e,35 4f,35 4g,35 4h,32 4i,64 4j,65 4k,66,67 4l,68
4m,35 4n,69 4o,70 4p,36 4q,71,72 and 5.33,73
3. Experimental section
3.1. General experimental procedures
Nitromethane was obtained from Acros Organics (Fisher
Scientific, Pittsburgh, PA). All other chemicals and reagents were
purchased from the Sigma–Aldrich Chemical Company (Milwau-
Table 2
Inhibitory effects of selected b-E-nitrostyrenes on tubulin polymerization
Compound
IC50
33
2.1 0.1
>40
(lM) SD
E-1b50
Z-1b
E-323
4a
7
4.4 0.5
10.0 0.5
7.4 0.7
5.7 0.4
8.2 0.3
9.3 0.6
6.0 0.9
3.9 0.7
5.4 0.5
8.5 0.5
4.5 0.2
5.3 0.5
5.6 0.08
5.5 0.6
11.0 0.7
2.5 0.02
12.0 0.8
10.0 1.0
>40
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4m
4n
4o
4p
4q
4r
3.2. General procedure for synthesis of the b-E-nitrostyrenes
(4a–s, 4u, 5)
To a solution of the benzaldehyde (1 equiv) in nitromethane
(>10 equiv) was added sequentially acetic acid (glacial, cat.) and
ammonium acetate (2.5 equiv). The mixture was then subjected to
ultrasound (sonication) for 2–12 h (reaction monitored by thin-
layer chromatography) at 22–25 °C. The crude mixture was ex-
tracted with dichloromethane. The combined extract was washed
(brine), dried, andconcentratedinvacuo. Purificationofthenitrosty-
rene was achieved by recrystallization (acetone–water) and/or silica
gel column chromatography (n-hexane–ethyl acetate as eluent).
4s
4t
3.3. 3,4-Methylenedioxy-5-methoxybenzaldehyde (7)
4u
4v
5
10.0 0.2
18.0 2.0
15.0 2.0
To a stirred yellow mixture of pyridinium chlorochromate
(49.0 g, 228 mmol) and anhydrous sodium acetate (18.7 g,

6610
R. K. Pettit et al. / Bioorg. Med. Chem. 17 (2009) 6606–6612
Table 3
Antimicrobial activities [MIC (
l
g/disk)] of b -E-nitrostyrenes in the Disk Diffusion Assaya
Compound Candida
albicans
Cryptococcus Staphylococcus Streptococcus Enterococcus Micrococcus Stenotrophomonas Escherichia Enterobacter Neisseria
neoformans
aureus
pneumoniae
faecalis
luteus
maltophilia
coli
cloacae
gonorrhoeae
ATCC
90028
ATCC 90112 ATCC 29213
ATCC 6303
ATCC 29212 Presque Isle ATCC 13637
456
ATCC
25922
ATCC 13047 ATCC
49226
4a
1.56–
3.12
0.78–1.56
25–50
1.56–3.12
50–100
25–50
12.5–25
25–50
25–50
<0.78
4b
4c
12.5–25
1.56–
3.12
6.25–12.5
0.78–1.56
50–100
6.25–12.5
6.25–12.5
25–50
6.25–12.5
0.78–1.56
>100
>100
6.25–12.5
12.5–25
12.5–25
50–100
25–50
25–50
<0.78
0.78–1.56
4d
4e
4f
3.12–
6.25
3.12–
6.25
6.25–
12.5
6.25–
12.5
3.12–
6.25
0.78–
1.56
1.56–3.12
0.78–1.56
3.12–6.25
3.12–6.25
0.78–1.56
0.78–1.56
12.5–25
50–100
12.5–25
25–50
6.25–12.5
1.56–3.12
3.12–6.25
12.5–25
>100
50–100
>100
50–100
>100
12.5–25
25–50
50–100
>100
6.25–12.5
25–50
12.5–25
25–50
>100
0.78–1.56
<0.78
25–50
50–100
50–100
25–50
6.25–12.5
25–50
0.78–1.56
0.78–1.56
0.78–1.56
<0.78
4g
4h
4i
25–50
>100
6.25–12.5
<0.78
12.5–25
3.12–6.25
>100
>100
>100
6.25–12.5
1.56–3.12
25–50
12.5–25
>100
4j
4k
50–100
3.12–
6.25
25–50
3.12–6.25
>100
12.5–25
6.25–12.5
3.12–6.25
50–100
50–100
>100
25–50
50–100
>100
>100
>100
>100
>100
1.56–3.12
0.78–1.56
4l
4m
>100
1.56–
3.12
>100
0.78–1.56
>100
6.25–12.5
>100
0.78–1.56
>100
6.25–12.5
>100
50–100
>100
6.25–12.5
>100
6.25–12.5
>100
12.5–25
>100
<0.78
4n
4o
4p
1.56–
3.12
3.12–
6.25
6.25–
12.5
3.12–6.25
1.56–3.12
<6.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.56–3.12
0.78–1.56
<6.25
25–50
<6.25
12.5–25
<6.25
50–100
>100
50–100
>100
4q
4r
4s
4t
4u
4v
5
>100
>100
>100
>100
>100
>100
12.5–25
<0.78
6.25–12.5
3.12–6.25
3.12–6.25
>100
3.12–6.25
>100
12.5–25
3.12–6.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25–50
>100
>100
>100
>100
>100
>100
25–50
>100
>100
>100
>100
>100
>100
>100
<0.78
25–50
25–50
>100
>100
>100
3.12–6.25
6.25–12.5
>100
6.25–12.5
>100
1.56–3.12
6.25–12.5
>100
3.12–6.25
>100
3.12–6.25
0.78–1.56
>100
0.78–1.56
50–100
3.12–6.25
>100
12.5–25
6.25–12.5
>100
>100
50–100
a
E-1b: Micrococcus luteus MIC = 25–50 lg/disk; Z-1b: Neisseria gonorrhoeae MIC = 25–50 l
g/disk.50
228 mmol) in dichloromethane (1 L) at room temperature under Ar
was added in one portion 3,4-methylenedioxy-5-methoxybenzyl
alcohol (37.7 g, 208 mmol).47 After stirring for 6 h, the solution
was filtered and the filtrate concentrated in vacuo. The resultant
residue was adsorbed onto silica gel (gravity) and the crude mix-
ture separated by silica gel column chromatography (flash, 7:3
hexane–EtOAc), affording benzaldehyde 7 (31.0 g, 84%) as a color-
less powder: mp 133–134 °C (lit.74 mp 132–133 °C); 1H NMR
(300 MHz) 3.97 (3H, s, OCH3), 6.10 (2H, s, OCH2O), 7.06 (1H, s,
ArH), 7.14 (1H, s, ArH), 9.79 (1H, s, CHO).
(2H, s, OCH2Ar), 5.25 (2H, s, OCH2Ar), 7.37 (12H, m, ArH), 7.77 (1H,
s, ArH), 9.85 (1H, s, CHO).
3.5. General procedure for synthesis of the b-E-nitrostyrene
sodium phosphates (4t, 4v)
To a stirred solution of the bis(benzyl)phosphate (1 equiv) in
dichloromethane (>10 equiv) at 0 °C under Ar was added (drop-
wise slowly) bromotrimethylsilane (2.1 equiv). After 1 h, methanol
(>10 equiv) was added, and the solution was stirred an additional
15 min and then concentrated in vacuo to afford the phosphoric
acid intermediate as a yellow solid. The phosphoric acid was
immediately dissolved in ethanol (>10 equiv), and sodium methox-
ide (2.1 equiv) was added at 0 °C. After stirring for 6–12 h, the mix-
ture was concentrated in vacuo to afford a yellow-orange solid,
which was dissolved in water and extracted with ethyl acetate
(2Â). The aqueous phase was concentrated by freeze-drying
(12 h) to afford the desired disodium nitrostyrene phosphate. Com-
pound 4t: 1H NMR (CD3OD, 300 MHz) d 7.35 (2H, J = 9.0 Hz), 7.59
(2H, d, J = 9.0 Hz), 7.80 (1H, d, J = 13.5 Hz), 8.05 (1H, d, J = 13.5 Hz).
3.4. 3-O-Bis(benzyl)phosphoryl-4-methoxybenzaldehyde (8)
To a stirred solution of isovanillin (5.0 g, 33 mmol) in acetonitrile
(50 mL) at À10 °C under Ar was added (dropwise) carbon tetrachlo-
ride (16.2 ml, 168 mmol). After 10 min, diisopropylethylamine
(12.0 mL, 69 mmol) and 4-dimethylaminopyridine (cat.) were
added. Five minutes later dibenzyl phosphite (10.9 mL, 49 mmol)
was added (dropwise slowly). The reaction mixture was then equil-
ibrated to room temperature under Ar while stirring. After 3 h, the
mixture was cooled to 0 °C, and KH2PO4 (25 mL, 0.5 M) was added
to terminate the phosphorylation. The mixture was washed with
brine (20 mL), and the residue was concentrated in vacuo and ad-
sorbed onto silica gel (gravity). The crude mixture was separated
by silica gel chromatography (flash, 1:1 hexane–EtOAc) to afford
bis(benzyl) phosphate 8 (12.6 g, 93%) as a colorless solid: mp 67–
68 °C; 1H NMR (acetone-d6, 300 MHz) d 3.96 (3H, s, OCH3), 5.22
3.6. Cancer cell line testing
Human cancer cell growth inhibition was assessed in duplicate
using the sulforhodamine B assay as previously described.75 Cells
in 5% fetal calf serum/RPMI-1640 were inoculated into 96-well
plates and incubated for 24 h at 37 °C with 5% CO2, and 10-fold

R. K. Pettit et al. / Bioorg. Med. Chem. 17 (2009) 6606–6612
6611
Table 4
Antimicrobial Activities [MIC (
l
g/mL)/MFC (
l
g/mL) or MIC (
l
g/mL)/MBC (
l
g/mL)] of b-E-nitrostyrenes in the Broth Microdilution Assaya
Stenotrophomonas Escherichia Enterobacter Neisseria
Compound Candida Cryptococcus Staphylococcus Streptococcus Enterococcus Micrococcus
albicans neoformans
aureus
ATCC
29213
pneumoniae
ATCC
6303
faecalis
ATCC
29212
luteus
Presque Isle ATCC
456
maltophilia
coli
ATCC
25922
cloacae
ATCC
13047
gonorrhoeae
ATCC
49226
ATCC
ATCC
90028
90112
13637
4a
4b
4c
4d
4e
4f
4g
4h
4j
4k
4m
4n
4q
5
16/32
16/16
8/16
8/32
8/8
>64
>64
>64
64/>64
>64
>64
>64
>64
>64
>64
>64
32/>64
>64
4/>16
64/>64
32/>64
64/>64
64/>64
>64
64/>64
>64
>64
>64
64/>64
>64
>64
>64
>64
>64
>64
>64
64/>64
>64
>64
64/>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64/>64
>64
4/>16
64/>64
32/>64
>64
32/64
>64
>64
>64
>64
>64
>64
>64
64/>64
>64
64/64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64/>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2/2
4/4
2/2
2/2
4/4
4/4
4/4
2/2
4/4
4/8
1/2
>64
1/2
8/8
8/16
16/16
32/32
16/16
16/16
16/16
64/64
16/16
8/16
16/32
16/16
16/16
16/16
>64
32/32
8/16
>64
>64
64/>64
64/>64
>64
16/32
>64
64/>64
>64
>64
64/>64
>64
>64
64/>64
32/64
16/32
>64
32/64
16/32
64/64
64/>64
>64
a
E-3: Neisseria gonorrhoeae MIC = 16–32 l
g/mL.23
dilutions of the test compounds were added. After a 48-h incuba-
tion, plates were fixed with trichloroacetic acid, washed, stained
with sulforhodamine B, and read with an automated microtiter
plate reader. Inhibition of mouse leukemia P388 cells76 was as-
sayed in Fisher’s medium containing 10% horse serum. After 24 h
of incubation, test compounds were added for another 48 h, and
cells were counted with a Z1 Coulter particle counter. P388 assays
were run in duplicate.
3.10. Broth microdilution susceptibility testing of yeasts
A subset of the b-E-nitrostyrenes was screened against yeasts
by BMAs according to the CLSI.59 Isolated yeast colonies were sus-
pended and diluted as recommended to yield final inocula ranging
from 0.5 to 2.5 Â 103 CFU/mL. Tests were performed in sterile 96-
well plates containing twofold dilutions of the test compounds in
0.165 M morpholinepropanesulfonic acid buffered RPMI 1640
medium (pH 7.0). One well was left drug-free (but contained an
equivalent volume of DMSO) for a turbidity control. Plates were
incubated without agitation at 35 °C. MICs were determined at
48 h for Candida and at 72 h for Cryptococcus. The MIC was defined
as the lowest drug concentration that inhibited all visible growth
of the test organism (optically clear).
3.7. Tubulin polymerization evaluations
The tubulin assembly studies were performed as described
previously.51,53
3.8. Disk diffusion susceptibility assays
3.11. Minimum bactericidal and minimum fungicidal
concentrations
Antimicrobial activity was assayed by the CLSI disk susceptibil-
ity test.57 The b-nitrostyrenes were reconstituted in a small volume
of sterile dimethylsulfoxide (DMSO) prior to susceptibility experi-
ments. Mueller–Hinton agar supplemented with 5% sheep blood
was used for Streptococcus pneumoniae, gonococcal typing agar
for N. gonorrhoeae, and Mueller–Hinton agar for all other bacteria.
Candida albicans was tested on Sabouraud dextrose agar (SDA), and
Cryptococcus neoformans on yeast morphology agar (YM). The min-
imum inhibitory concentration (MIC) was defined as the lowest
drug concentration resulting in a clear zone of growth inhibition.
Minimum bactericidal and fungicidal concentrations (MBCs,
MFCs) were determined by subculturing 100 lL from each well
with no visible growth in the MIC broth microdilution series onto
drug-free plates. The plates were incubated for 24 h (bacteria and
C. albicans) or 48 h (C. neoformans), and the MBC and MFC were de-
fined as the lowest drug concentration that resulted in P99.9%
reduction in the initial inoculum.
Disclaimer
3.9. Broth microdilution susceptibility testing of bacteria
Product names are necessary to report factually on available
data; however, the USDA neither guarantees nor warrants the stan-
dard of the product, and the use of the name by USDA implies no
approval of the product to the exclusion of others that may also
be suitable.
The antibacterial activity of a subset of the b-nitrostyrenes was
evaluated by the CLSI BMA.58 The b-E-nitrostyrenes were reconsti-
tuted in a small volume of sterile DMSO and diluted in the appro-
priate media prior to susceptibility experiments. Isolated colonies
from overnight cultures were suspended and diluted as recom-
mended to yield final inocula of approximately 5 Â 105 CFU/mL.
Tests were performed in sterile 96-well plates containing twofold
dilutions of the test compounds in gonococcal typing broth (Neis-
seria), Mueller–Hinton II (MHII) (cation-adjusted) broth containing
3% lysed horse blood (Streptococcus), or MHII broth (all other bac-
teria). One well was left drug-free (but contained an equivalent
volume of DMSO) for a turbidity control. Plates were incubated
without agitation at 37 °C with 5% CO2 (Neisseria) or at 35 °C (all
other bacteria). MICs were determined at 24 h. The MIC was de-
fined as the lowest drug concentration that inhibited all visible
growth of the test organism (optically clear).
Acknowledgments
The very necessary financial support was provided by Out-
standing Investigator Grant CA44344-8-12, Grant RO1 CA90441-
01-03, and Grant 5RO1 CA 090441-07 awarded to GRP by the Divi-
sion of Cancer Treatment and Diagnosis, National Cancer Institute
DHHS; the Arizona Disease Control Research Commission; the Rob-
ert B. Dalton Endowment Fund; and the Caitlin Robb Foundation.
For other helpful assistance, we thank Drs. Michael D. Williams
and Mathew D. Minardi, as well as Lee Williams.

6612
R. K. Pettit et al. / Bioorg. Med. Chem. 17 (2009) 6606–6612
35. Milhazes, N.; Calheiros, R.; Marquez, M. P. M.; Garrido, J.; Cordeiro, M. N. D. S.;
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