Combretastatin dinitrogen-substituted stilbene analogues as tubulin-binding and vascular-disrupting agents

Article abstract of DOI:10.1021/np070377j

Several stilbenoid compounds having structural similarity to the combretastatin group of natural products and characterized by the incorporation of two nitrogen-bearing groups (amine, nitro, serinamide) have been prepared by chemical synthesis and evaluat

Full text of DOI:10.1021/np070377j

J. Nat. Prod. 2008, 71, 313–320
313
Combretastatin Dinitrogen-Substituted Stilbene Analogues as Tubulin-Binding and
Vascular-Disrupting Agents#
Rogelio Siles,† J. Freeland Ackley,† Mallinath B. Hadimani,† John J. Hall,† Benon E. Mugabe,† Rajsekhar Guddneppanavar,†
Keith A. Monk,† Jean-Charles Chapuis,§ George R. Pettit,§ David J. Chaplin, Klaus Edvardsen,‡ Mary Lynn Trawick,†
Charles M. Garner,† and Kevin G. Pinney*,†
Department of Chemistry and Biochemistry, Baylor UniVersity, One Bear Place #97348, Waco, Texas 76798-7348, Department of Cell and
Molecular Biology, UniVersity of Lund, BMC 112, 22184, Lund, Sweden, Cancer Research Institute and Department of Chemistry and
Biochemistry, Arizona State UniVersity, P.O. Box 872404, Tempe, Arizona 85287-2404, and Oxigene Inc., 230 Third AVenue, Waltham,
Massachusetts 02451
ReceiVed July 27, 2007
Several stilbenoid compounds having structural similarity to the combretastatin group of natural products and characterized
by the incorporation of two nitrogen-bearing groups (amine, nitro, serinamide) have been prepared by chemical synthesis
and evaluated in terms of biochemical and biological activity. The 2′,3′-diamino B-ring analogue 17 demonstrated
remarkable cytotoxicity against selected human cancer cell lines in vitro (average GI50 ) 13.9 nM) and also showed
good activity in regard to inhibition of tubulin assembly (IC50 ) 2.8 µM). In addition, a single dose (10 mg/kg) of
compound 17 caused a 40% tumor-selective blood flow shutdown in tumor-bearing SCID mice at 24 h, thus suggesting
the potential value of this compound and its corresponding salt formulations as new vascular-disrupting agents.
The African bush willow tree [Combretum caffrum Kuntze
(Combretaceae)] has proved to be an exceptionally rich source of
stilbenoid natural products initially isolated and characterized by
Pettit and colleagues.1,2 Two of these compounds, combretastatin
A4 (CA4)3 and combretastatin A1 (CA1),4 have pronounced activity
as inhibitors of tubulin assembly and, in appropriate phosphate
prodrug formulations (CA4P5,6 and CA1P,7 respectively), are
clinically relevant examples of potent vascular-disrupting agents
(VDAs).8,9 Small-molecule VDAs are characterized by their ability
to disrupt blood flow selectively in the tumor microenvironment,
resulting in further hypoxia and ultimately necrosis for certain tumor
types. CA4P is rapidly cleaved to CA4, which is a strong inhibitor
of tubulin assembly. Microtubule disruption induces cytoskeletal
rearrangements, leading to cell shape changes of endothelial cells
in tumor microvasculature that results in vessel occlusion.10–15
Structure–activity relationship (SAR) studies around the Z-
stilbenoid molecular template, inherent to a large combretastatin
group of compounds, resulted in the discovery of a CA4 analogue
substituted with an amine group at position 3′ of the B ring.16–20
This compound is a potent inhibitor of tubulin assembly and also
shows significant cytotoxicity against human cancer cell lines in
vitro.16,19 Formulated as a serinamide prodrug known as AVE8062,
this compound is currently in human clinical trials.16,21–23 Recently
we reported examination of a small molecular library of combre-
tastatins each substituted with one nitrogen entity (amine, nitro,
serinamide, etc.) and extended the SAR around this stilbenoid
molecular core by demonstrating that substitution at the 2′-position
of the B ring with an amino moiety results in a modified CA4
analogue with impressive biochemical and biological activity.24 The
current study delineates our recent efforts of molecular design and
synthesis along with biochemical and biological evaluation of
dinitrogen-substituted combretastatins. Of special significance is the
2′,3′-diaminocombretastatin analogue 17, which is the nitrogen
variant of the diphenol CA1.25 This molecule is especially
noteworthy since both CA4P and the corresponding nitrogen
analogue (AVE8062), along with the diphenol phosphate prodrug
(CA1P, also known as Oxi4503), are all currently in human clinical
trials.13,22,23,26,27 Details of the syntheses of these new dinitrogen
combretastatin analogues as well as preliminary biochemical and
biological results are presented herein.
Results and Discussion
Design and Synthesis. The synthetic strategy that allowed the
preparation of the combretastatin dinitrogen derivatives utilized a
Wittig reaction as a key step to form the requisite Z-stilbenoid.
Accordingly, the Z-stilbenoids were prepared by reacting 3,4,5-
trimethoxybenzylphosphonium bromide 2 (Scheme 1)19,28 with
commercially available 3,5-dinitro-4-methoxybenzaldehyde along
with aldehydes 6 and 7 (Scheme 2)24a,29 using NaH as the base to
generate the ylide (Scheme 3). The Z-alkenes were separated from
# Dedicated to Dr. G. Robert Pettit of Arizona State University for his
pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: (254) 710-4117.
Fax: (254) 710-4272. E-mail: Kevin_Pinney@baylor.edu.
† Baylor University.
‡ University of Lund.
§ Arizona State University.
Oxigene, Inc.
10.1021/np070377j CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/28/2008

314 Journal of Natural Products, 2008, Vol. 71, No. 3
Siles et al.
Scheme 1. Synthesis of 3,4,5-Trimethoxybenzyltriphenylphosphonium Bromide
Scheme 2. Synthesis of 4-Methoxy-2,5- and 2,3-Dinitrobenzaldehydes
Scheme 3. Synthesis of Z-CA1 Analogues
their corresponding E-isomers by flash chromatography to afford
stilbenes 8-10 in moderate yield.
positive charge on one of the vinylic carbons of isomer 15; thus
rendering the respective hydrogens more deshielded. The effect was
not seen in isomer 16 because the nitro group located at the 5′-
position does not allow a resonance structure that can be stabilized
by the carbon-carbon double bond. Additional spectroscopic
support for these assignments was obtained from DEPT 45, COSY,
and HETCOR NMR spectra of both isomers. Final confirmation
of the structure resulted from single-crystal X-ray crystallographic
analysis of monoamine 16 that indicated both the Z double bond
configuration as well as the correct regioisomeric assignment of
the amino group (Figure 1).32 In addition, X-ray crystallographic
analysis was carried out on compound 13, confirming the E double
bond configuration of this dinitro analogue (Figure 1).32
Successful reduction of both nitro groups of compounds 9 and
10 to their corresponding diamines 18 and 17, respectively, was
achieved using zinc powder in acetic acid (Scheme 3).33 It is
important to note that the 2′,5′-diamino analogue 18 partially
decomposed at room temperature from an initial purity level after
flash chromatography of approximately 95% (by NMR) to ap-
proximately 75%. However, the compound retains its original level
of purity if stored at freezer temperature (approximately -20 °C).
Conversion of monoamine 16 and diamines 14, 17, and 18 into
their corresponding hydrochloride salts proceeded smoothly with
a 4.0 N solution of hydrochloric acid in dioxane (Scheme 4).16,33
3′,5′-Diaminostilbene 14 was also converted to serinamide 24
Bromide 324a was hydrolyzed to benzyl alcohol 4,30 which upon
treatment with PCC, afforded the intermediate benzaldehyde 5.31
Nitration29 of 4-methoxy-2-nitrobenzaldehyde (5) afforded both 2,5-
and 2,3-dinitrobenzaldehydes (6 and 7, respectively),25 which were
separated by column chromatography. Yields of aldehydes 6 and
7 decreased considerably when the reaction was stirred for more
than 10 min, due to the formation of carboxylic acids, which
complicated product separation.
Reduction of the nitro groups on stilbenes 8 and 9 was carried
out using sodium dithionite (Scheme 3). Interestingly, when the
same number of molar equivalents of the reducing reagent was used
for both isomers, only stilbene 8 was successfully converted to its
corresponding diamine 14 (albeit in low yield), while stilbene 9
produced two isomeric monoamines, 15 and 16. The formation of
monoamine 15 resulted when the reduction took place at the 5′-
position of the B ring while the nitro group at the 2′-position
remained intact. Isomer 16 was formed when the 2′-nitro group
was selectively reduced. The regioisomeric assignments for 15 and
16 were initially determined by NMR. Significant differences in
the 1H and 13C NMR spectra for both isomers were noted,
particularly in the vinylic region, where the vinyl hydrogens of
isomer 15 appeared more downfield (δ 6.87 and 6.53 ppm) than
the respective hydrogens of isomer 16 (δ 6.63 and 6.32 ppm). This
was explained, in part, by resonance contributions that place a

Combretastatin Dinitrogen-Substituted Stilbene Analogues
Journal of Natural Products, 2008, Vol. 71, No. 3 315
Figure 1. Thermal ellipsoid plots at 50% probability for compounds 13 and 16.
Scheme 4. Synthesis of Mono- and Diamine Hydrochloride Salts
Scheme 5. Synthesis of 3′,5′-Diserinamide CA1 Derivative
(Scheme 5). While the 2′,5′-diamino analogue 18 is not entirely
stable at room temperature, its corresponding hydrochloride salt
22 is stable.
Biological Evaluation. A series of 15 new dinitrogen-
substituted combretastatin-type derivatives, each with two nitrogen
entities (amine, nitro, serinamide, etc.), have been evaluated for
their ability to inhibit tubulin assembly and for cytotoxicity against
human cancer cell lines. In addition, selected compounds were
evaluated for their ability to impair blood flow to tumors in SCID
mice.
A comparison of regioisomeric diaminocombretastatins 14 and
17, along with hydrochloride salts 19, 21, and 22, illustrates the
significant differences in terms of biological activity among the
3′,5′-, 2′,3′-, and 2′,5′-substitution patterns. The 2′,5′-diaminohy-
drochloride salt 22 demonstrated moderate activity against six
human cancer cell lines and had an IC50 for inhibition of tubulin
polymerization of 14.1 µM, while the 3′,5′-diamino compound 14
had more limited cancer cell cytotoxicity. Although 14 exhibited
some activity against the murine P388 lymphocytic leukemia cell
line, it showed no ability to inhibit tubulin polymerization. In
contrast, the 2′,3′-diamino analogue 17 demonstrated impressive
biological activity. For example, compound 17 inhibits tubulin
assembly with an IC50 value comparable to that of CA1. In addition,
compound 17 and its dihydrochloride salt 21 showed outstanding
cytotoxicity, with average GI50 values of 13.9 and 12.7 nM,
respectively (Table 1), against all six human cancer cell lines in
this study. This result was confirmed in the MTT assay (Table 3),
in which compound 17 demonstrated cytotoxicity in both 1 h (IC50
) 2.4 µM) and 5 day (IC50 ) 0.0043 µM) exposures. Furthermore,
compound 17 demonstrated significant in vivo bloodflow shutdown
in SCID mice at a dose of 10 mg/kg, which is intermediate between
the two clinically relevant compounds, CA4P and CA1P (Table
3).
Of the six dinitro-substituted stilbenoids 8-13, only compound
8 showed a significant ability to inhibit tubulin polymerization.
In this group, compound 8 was the most cytotoxic against the
six selected cell lines (Table 1). Despite their inability to inhibit
tubulin assembly, the Z-analogues 9 and 10 have significant

316 Journal of Natural Products, 2008, Vol. 71, No. 3
Siles et al.
Table 1. Inhibition of Microtubule Formation and Cytotoxicity against Six Cancer Cell Lines by Compounds 8-10, 14-22, and 24
GI50 (µM) (SRB assay)
tubulin inhibition
compound
R1
R2
OH
OH
NO2
H
NO2
NH2
H
R3
IC50 (µM)
BXPC-3
MCF-7
nab
na
0.41
2.3
2.3
1.5
2.6
2.6
0.0062
na
SF-268
NCI-H460
KM20L2
DU-145
CA4
CA1
8
H
H
H
1.2
1.9
7.4
>40
>40
>40
>40
31
2.8
>40
na
1.8
14.1
>40
0.39a
4.4a
0.79
3.6
2.1
1.5
2.7
3.2
0.0079
na
1.2
na
na
0.68
2.8
2.5
1.5
2.8
2.6
0.0083
na
0.0006a
0.74a
0.37
3.5
0.37
2.4
2.5
3.6
0.018
na
4.3
0.34a
0.061a
0.52
4.6
0.33
1.1
0.0008a
0.17a
0.26
5.5
0.2
2.1
3.3
3.6
0.018
na
3.5
OH
H
NO2
NO2
H
NH2
NH2
NO2
H
NH3Cl
NO2
H
NH3Cl
NH-Ser
9
NO2
NO2
H
NO2
NH2
NH2
H
NH3Cl
NH3Cl
NH3Cl
H
10
14
15
16
17
19
20
21
22
24
3.5
3.5
H
NH2
NH3Cl
H
NH3Cl
H
0.025
na
0.98
0.019
0.44
na
1.5
1.2
0.014
1.0
na
0.011
0.61
na
0.011
0.55
na
0.009
0.38
na
0.012
0.33
na
NH-Ser
a Ref 34 b na ) not analyzed in this study.
Table 2. Inhibition of Microtubule Formation and Cytotoxicity against Six Cancer Cell Lines by Compounds 11-13
GI50 (µM) (SRB assay)
tubulin inhibition
compound
R1
R2
R3
IC50 (µM)
BXPC-3
MCF-7
SF-268
NCI-H460
KM20L2
DU-145
11
12
13
H
NO2
NO2
NO2
H
NO2
NO2
NO2
H
>40
>40
>40
>10
>10
>10
>10
2.5
>10
>10
1.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Table 3. Cytotoxicity and Blood Flow Reduction by Compounds 14, 16, 17, 19, and 20
in vivo blood flow shutdown (%)
P388
MTT (IC50 in vitro cytotoxicity) (µM)
compound
R1
R2
R3
ED50(µM)
10 mg/kg
100 mg/kg
1 h
5 days
CA4P
CA1P
14
16
17
19
20
H
OPO3Na2
OPO3Na2
NH2
H
NH2
H
H
NH2
NO2
H
0.0004a
<0.01a
5.2
10
70
3.1
0
40
0
88
99
4.6
0
-
6
0.8
0.0020
0.0046
>1.4
OPO3Na2
H
NH2
NH2
H
3.2
>44.8
37.7
2.4
>44.8
na
nab
>1.4
c
na
na
6.6
0.0043
>1.4
na
NH3Cl
H
NH3Cl
NO2
NH3Cl
0
13.7
a ref 34. b na ) not analyzed in this study. c SCID mice did not tolerate this dose level.
cytotoxicity, with compound 10 demonstrating submicromolar
activity against the NCI-H460, KM20L2, and DU-145 cell lines.
The E-isomers were inactive in these evaluation systems with
the exception of 12, which demonstrated moderate activity
against both the MCF-7 and SF-268 cell lines (Table 2). The
mixed amino/nitro-substituted compounds 15, 16, and 20, each
containing one amino or amine hydrochloride substitution and
one nitro substitution, showed comparable cytotoxicity toward
human cancer cell lines (Table 1).
analogue 17 with exceptional biological activity including vascular
disruption in a SCID mouse model. While substitution of the 5′-
position of the B ring is tolerated, diamino substitution at the 2′-
and 3′-positions is most effective. This result is particularly
noteworthy in that the 2′,3′-diamino analogue is capable of forming
the ortho di-imine species analogous to the ortho quinone derivative
of CA135 that is postulated to make a major contribution to the
anticancer activity of this vascular-disrupting agent. The most potent
compounds in this series, 17, 22, and 8, all have low IC50 values
for inhibition of tubulin polymerization, thus providing evidence
that a significant aspect of their activity is due to microtubule
disruption. The Z-series is much more active compared to the
corresponding E-isomers.
Conclusions
Expansion of our library of nitrogen-substituted combretastatin
analogues to the disubstituted series has extended the SAR of these
potent compounds and led to the discovery of the 2′,3′-diamino

Combretastatin Dinitrogen-Substituted Stilbene Analogues
Journal of Natural Products, 2008, Vol. 71, No. 3 317
Experimental Section
7.25 (1H, dd, J ) 8.7, 2.5 Hz, H-5), 3.98 (3H, s, C-4 OCH3); EIMS
m/z 181 (M+, 8), 151 (100), 134 (24), 106 (36), 63 (36).
General Experimental Procedures. 36 Reactions involving air- or
moisture-sensitive reagents were performed in oven-dried glassware
under inert atmospheric conditions (N2). Solvents used for chromatog-
raphy and reactions were purchased from commercial sources (e.g.,
Aldrich, Acros, Alfa Aesar) and used without further purification unless
indicated. Column chromatography was performed on Merck silica gel
60, 0.040–0.063 mm, 230–400 mesh ASTM. Precoated silica gel plates
(EM Science 60, F254, 250 µm and 2 mm) were used for both analytical
and preparative TLC. IR spectra were recorded either neat or as Nujol
mulls with a Genesis II FTIR spectrophotometer. The 1H and 13C NMR
4-Methoxy-2,5-dinitrobenzaldehyde (6)25,29 and 4-methoxy-2,3-
dinitrobenzaldehyde (7).25,29 At 0 °C, aldehyde 5 (1.53 g, 8.45 mmol)
was dissolved in concentrated sulfuric acid (25 mL), to which 7 mL of
a precooled (0 °C) mixture of fuming nitric acid (5.32 g, 84.5 mmol)
and concentrated sulfuric acid (5.29 g, 54.0 mmol) was slowly added.
After stirring for 10 min, the resulting solution was added dropwise
into ice–water (approximately 100 mL). After 2 h at 0 °C, the solution
was filtered and the solid was rinsed with ice–water (10 mL).
Purification by flash column chromatography (EtOAc/hexanes, 20:80)
yielded isomers 6 (0.80 g, 3.54 mmol, 42% yield) and 7 (0.89 g, 3.94
mmol, 47% yield).
1
spectra were obtained by using Bruker DPX (300 MHz for H, 121
MHz for 31P, and 75 MHz for 13C), Bruker AMX (360 MHz for H
1
Spectroscopic characterization of isomer 6: IR (CH2Cl2) νmax 3054,
and 90 MHz for 13C), and Varian (500 MHz for 1H, and 125 MHz for
13C) spectrometers in deuterated chloroform with 0.03% TMS as the
internal reference unless otherwise specified. Chemical shifts are
expressed in ppm (δ), coupling constants (J) are expressed in hertz
(Hz), and peaks are reported as broad (b), singlets (s), doublets (d),
triplets (t), quartets (q), or combinations of each. All 13C spectra reported
are proton decoupled. Additional COSY and HETCOR spectra were
performed on the same spectrometers to verify molecular structure.
Gas chromatography/mass spectrometry (Hewlett-Packard GCD system
with electron-impact ionization) was used to monitor selected reactions
and to characterize certain products.
1
2987, 2950, 2908, 2855, 1703, 1622, 1552 cm-1; H NMR (CDCl3,
300 MHz) δ 10.31 (1H, s, CHO), 8.43 (1H, s, H-6), 7.73 (1H, s, H-3),
4.14 (3H, s, C-4 OCH3); EIMS m/z 226 (M+, 4), 196 (12), 179 (16),
149 (56), 121 (100), 75 (40), 63 (32).
Spectroscopic characterization of isomer 7: IR (CH2Cl2) νmax 3097,
3054, 3005, 2987, 2953, 2900, 2858, 1709, 1612, 1564 cm-1; 1H NMR
(CDCl3, 300 MHz) δ 9.95 (1H, d, J ) 0.5 Hz, CHO), 8.15 (1H, d, J
) 8.9 Hz, H-6), 7.38 (1H, d, J ) 8.9 Hz, H-5), 4.08 (3H, s, C-4 OCH3);
EIMS m/z 132 (40), 120 (48), 103 (84), 75 (100).
General Procedure for the Synthesis of Z-Stilbenes. A suspension
of NaH (approximately 6.0 equiv) in anhydrous CH2Cl2 (approximately
0.7 M solution) at 0 °C under an inert (N2) atmosphere was stirred for
about 10 min, at which point 1.1 equiv of a previously prepared solution
of 3,4,5-trimethoxybenzylphosphonium bromide (2, approximately 0.1
M in CH2Cl2) was added dropwise. After stirring for 20 min, 1.0 equiv
of 4-methoxydinitrobenzaldehyde (0.1 M in CH2Cl2) was added, and
the mixture was stirred at room temperature for 3–7 h. At this point,
ice–water was slowly added until the hydrogen evolution stopped,
indicating that all of the NaH had reacted. The product was extracted
with CH2Cl2, washed twice with water and twice with brine, and dried
over Na2SO4. The requisite Z-stilbenes were separated from their
corresponding E-isomers by flash chromatography using the solvent
system specified for each alkene. Numbering for the combretastatin
analogues for spectroscopic analysis is as follows: the trimethoxy A
ring is numbered 1–6, and the B ring is numbered 1′-6′. The atoms of
the ethylene bridge are numbered 1a and 1a′, where 1a is bound to the
A ring and 1a′ is bound to the B ring.
Synthesis of Intermediates and Analogues. 3,4,5-Trimethoxy-
benzyl Bromide (1).28 At 0 °C and under a nitrogen atmosphere, a
solution of phosphorus tribromide (1.1 mL, 11.6 mmol) in CH2Cl2 (6.6
mL) was added to a well-stirred solution of 3,4,5-trimethoxybenzyl
alcohol (3.22 g, 15.8 mmol) in anhydrous CH2Cl2 (15 mL) at 0 °C.
The reaction mixture was stirred at 0 °C for 2 h and for an additional
4 h at room temperature. At this point, the reaction mixture was slowly
added to ice–water (100 mL) and neutralized with NaHCO3. The
product was extracted twice with CH2Cl2 from the aqueous phase, and
the combined organic phase was washed twice with water and brine
solution and dried over Na2SO4. The solvent was evaporated, yielding
a pale brown solid, which was purified by recrystallization (hexanes/
diethyl ether) to afford bromide 1 (3.15 g, 12.06 mmol, 77% yield):
1H NMR (CDCl3, 300 MHz) δ 6.62 (2H, s, H-2, H-6), 4.47 (2H, s,
benzylic CH2), 3.87 (6H, s, C-3, C-5 OCH3), 3.85 (3H, s, C-4 OCH3);
EIMS m/z 260/262 (M+, 1:1), 181 (base).
3,4,5-Trimethoxybenzyltriphenylphosphonium Bromide (2). 19
Bromide 1 (11.6 g, 44.5 mmol) and triphenylphosphine (11.8 g, 44.5
mmol) in CH2Cl2 (100 mL) were heated at reflux for 20 h, at which
point water was added and the product was extracted from the aqueous
phase with CH2Cl2. The organic phase was washed twice with water
and brine solution and dried over Na2SO4. The crude material was
triturated (Et2O) at 0 °C to afford phosphonium salt 2 (32.4 g, 61.9
mmol, 77% yield): 1H NMR (CDCl3, 300 MHz) δ 7.74 (9H, m, ArH),
7.60 (6H, m, ArH), 6.43 (2H, d, J ) 2.7 Hz, H-2, H-6), 5.43 (2H, d,
J ) 14.1 Hz, benzylic CH2), 3.74 (3H, s, C-4 OCH3), 3.48 (6H, s, C-3,
C-5 OCH3); 31P NMR (acetone-d6, 121 MHz) δ 23.37.
(Z)-2-(4′-Methoxy-3′,5′-dinitrophenyl)-1-(3,4,5-trimethoxyphenyl)-
ethene (8). Flash chromatography (EtOAc/hexanes, 10:90) led to the
product in a 45% yield: mp 112–115 °C; 1H NMR (CDCl3, 300 MHz)
δ 7.91 (2H, s, H-2′, H-6′), 6.77 (1H, d, J ) 12.1 Hz, H-1a), 6.44 (2H,
s, H-2, H-6), 6.43 (1H, d, J ) 12.0 Hz, H-1a′), 4.02 (3H, s, C-4′ OCH3),
3.86 (3H, s, C-4 OCH3), 3.74 (6H, s, C-3, C-5 OCH3); 13C NMR
(CDCl3, 75 MHz) δ 153.6 (C, C-3, C-5), 145.7 (C, C-4′), 145.1 (C,
C-1), 138.7 (C, C-4), 134.9 (C, C-3′, C-5′), 133.9 (C, C-1′), 130.4 (CH,
C-1a′), 128.9 (CH, C-2′, C-6′), 124.5 (CH, C-1a), 105.9 (CH, C-2, C-6),
64.9 (CH3, C-4 OCH3), 61.1 (CH3, C-4′ OCH3), 56.2 (CH3, C-3, C-5
OCH3); EIMS m/z 390 (M+, 100), 375 (36); anal. C 55.71%, H 4.69%,
N 6.85%, calcd for C18H18N2O8, C 55.39%, H 4.65%, N 7.18%.
4-Methoxy-2-nitrobenzyl Alcohol (4).30 Bromide 319 (0.0937 g,
0.38 mmol) was dissolved in acetone/water (6 mL, 2:1 ratio) and heated
at reflux for 26 h, at which point water was added and the product was
extracted with CH2Cl2. The organic phase was washed twice with water
and brine and dried over Na2SO4. After recrystallization (hexanes/
EtOAc), alcohol 4 (0.0635 g, 0.35 mmol, 91% yield) was obtained: 1H
NMR (CDCl3, 360 MHz) δ 7.60 (1H, d, J ) 2.7 Hz, H-3), 7.58 (1H,
d, J ) 8.6 Hz, H-6), 7.19 (1H, dd, J ) 8.6, 2.7 Hz, H-5), 4.86 (2H, d,
J ) 6.7 Hz, benzylic CH2), 3.88 (3H, s, C-4 OCH3), 2.53 (1H, t, J )
6.8 Hz, OH); 13C NMR (CDCl3, 75 MHz) δ 159.4 (C, C-4), 148.5 (C,
C-2), 131.6 (CH, C-6), 128.7 (C, C-1), 120.5 (CH, C-5), 109.7 (CH,
C-3), 62.4 (CH2, C-1 CH2OH), 55.9 (CH3, C-4 OCH3); EIMS m/z 183
(M+, 12), 165 (M+ - 18, 32), 135 (100), 106 (64), 77 (52).
(Z)-2-(4′-Methoxy-2′,5′-dinitrophenyl)-1-(3,4,5-trimethoxyphen-
yl)ethene (9). Flash chromatography (EtOAc/hexanes, 20:80) led to
the Z-isomer in a 52% yield: mp 90–92 °C; Rf 0.71 (EtOAc/hexanes,
50:50); 1H NMR (CDCl3, 300 MHz) δ 7.72 (1H, s, H-6′), 7.71 (1H, s,
H-3′), 6.74 (1H, d, J ) 12.0 Hz, H-1a′), 6.68 (1H, d, J ) 12.0 Hz,
H-1a), 6.29 (2H, s, H-2, H-6), 4.03 (3H, s, C-4′ OCH3,), 3.81 (3H, s,
C-4 OCH3), 3.66 (6H, s, C-3, C-5 OCH3); 13C NMR (acetone-d6, 75
MHz) δ 154.7 (C, C-3, C-5), 151.9 (C, C-4′), 150.9 (C, C-2′), 143.4
(C, C-5′), 140.3 (C, C-4), 135.4 (C, C-1), 132.9 (CH, C-1a′), 125.7
(CH, C-1a), 124.7 (CH, C-6′), 120.5 (C, C-1), 111.1 (CH, C-3′), 105.7
(CH, C-2, C-6), 60.7 (CH3, C-4 OCH3), 58.0 (CH3, C-4′ OCH3), 56.5
(CH3, C-3, C-5 OCH3); EIMS m/z 390 (M+, 60), 196 (92), 181 (100);
anal. C 55.32%, H 4.70%, N 7.00%, calcd for C18H18N2O8, C 55.39%,
H 4.65%, N 7.18%.
4-Methoxy-2-nitrobenzaldehyde (5).31 A suspension of PCC (3.18
g, 14.7 mmol) and Celite (3.2 g) in anhydrous CH2Cl2 (30 mL) was
stirred at 0 °C under a nitrogen atmosphere for 30 min, at which point
a CH2Cl2 solution (20 mL) of alcohol 4 (1.80 g, 9.82 mmol) was added.
After stirring for 4.5 h at room temperature, ethyl ether (50 mL) was
added, and the solution was filtered through Florisil and washed with
ethyl ether (10 mL) followed by CH2Cl2 (10 mL). Purification by flash
chromatography (EtOAc/hexanes, 20:80) afforded aldehyde 5 (1.73 g,
(Z) + (E)-2-(4′-Methoxy-2′,3′-dinitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)ethene (10 and 13). Crystallization from CH2Cl2 at 4 °C
afforded a pure sample of the E-isomer 13: mp 226–228 °C; Rf 0.09
(EtOAc/hexanes, 50:50); 1H NMR (acetone-d6, 300 MHz) δ 8.18 (1H,
d, J ) 9.1 Hz, H-6′), 7.71 (1H, d, J ) 9.1 Hz, H-5′), 7.31 (1H, d, J )
16.1 Hz, H-1a′), 7.08 (1H, d, J ) 16.1 Hz, H-1a), 6.95 (2H, s, H-2,
H-6), 4.10 (3H, s, C-4′ OCH3), 3.86 (6H, s, C-3, C-5 OCH3), 3.75
1
9.55 mmol, 97% yield): H NMR (CDCl3, 300 MHz) δ 10.28 (1H, s,
CHO), 7.98 (1H, d, J ) 8.7 Hz, H-6), 7.51 (1H, d, J ) 2.5 Hz, H-3),

318 Journal of Natural Products, 2008, Vol. 71, No. 3
Siles et al.
(3H, s, C-4 OCH3); 13C NMR (CDCl3, 125 MHz) δ 153.6 (C, C-3,
C-5), 150.9 (C, C-4), 139.2 (C, C-2′), 134.8 (CH, C-6′), 131.4 (C, C-4),
129.9 (C, C-1′, C-3′), 124.2 (C, C-1′), 118.7 (CH, C-1a, C-1a′), 116.1
(CH, C-5′), 104.3 (CH, C-2, C-6), 61.0 (CH3, C-4 OCH3), 57.4 (CH3,
C-4′ OCH3), 56.5 (CH3, C-3, C-5 OCH3); anal. C 55.69%, H 4.58%,
N 7.07%, calcd for C18H18N2O8, C 55.39%, H 4.65%, N 7.18%. Single-
crystal X-ray diffraction further confirmed the E-configuration of 13.32
The filtrate was subjected to flash column chromatography (EtOAc/
hexanes, 50:50) to isolate a sample of the pure Z-isomer 10 in 51%
yield as a yellow powder: mp 146–148 °C; Rf 0.21 (EtOAc/hexanes,
50:50); 1H NMR (CDCl3, 300 MHz) δ 7.36 (1H, d, J ) 8.9 Hz, H-6′),
7.09 (1H, d, J ) 8.9 Hz, H-5′), 6.77 (1H, d, J ) 11.8 Hz, H-1a′), 6.49
(1H, d, J ) 11.8 Hz, H-1a), 6.30 (2H, s, H-2, H-6), 3.95 (3H, s, C-4′
OCH3), 3.82 (3H, s, C-4 OCH3), 3.69 (6H, s, C-3, C-5 OCH3); 13C
NMR (CDCl3, 75 MHz); δ 153.2 (C, C-3, C-5), 150.9 (C, C-4′), 143.1
(C, C-2′), 138.0 (CH, C-6′), 135.2 (C, C-4), 134.4 (C, C-1), 130.7 (C,
C-3′), 124.6 (CH, C-1a′), 121.6 (CH, C-1a), 115.9 (C, C-1′), 106.1
(CH, C-2, C-6, C-5′), 60.9 (CH3, C-4 OCH3), 57.3 (CH3, C-4′ OCH3),
56.0 (CH3, C-3, C-5 OCH3); anal. C 55.43%, H 4.58%, N 7.11%, calcd
for C18H18N2O8, C 55.39%, H 4.65%, N 7.18%.
General Procedure A for the Reduction of CA-1 Analogues. To
a round-bottomed flask containing approximately 1.0 equiv of Z-stilbene
in acetone/water (0.05 M, 2:1 ratio), heated to 50 °C (approximately
15 min to achieve solution), was added sodium dithionite (ap-
proximately 11.9 equiv). The reaction mixture was heated at reflux for
approximately 5 h, CH2Cl2 was added, and the organic phase was
washed three times with brine and dried over Na2SO4. The solvent was
evaporated at reduced pressure. The products were purified by flash
chromatography using the solvents specified.
filtered through Celite and the filtrate was concentrated at reduced
pressure. The desired diamine was purified by flash chromatography
(EtOAc/hexanes, 50:50).
(Z)-2-(2′,3′-Diamino-4′-methoxyphenyl)-1-(3,4,5-trimethoxyphen-
yl)ethene (17). The resulting brown-colored residue was subjected to
flash chromatography (EtOAc/hexanes, 50:50) to isolate the desired
diamine product as a brown oil in 43% yield: Rf 0.14 (EtOAc/hexanes,
50:50); IR (neat) νmax 3433, 3351, 3004, 2942, 2840, 2363, 2257, 1616,
1
1582, 1505, 1467, 1428 cm-1; H NMR (CDCl3, 300 MHz) δ 6.66
(1H, d, J ) 8.4 Hz, H-6′), 6.52 (1H, d, J ) 12.1 Hz, H-1a′), 6.49 (2H,
s, H-2, H-6), 6.48 (1H, d, J ) 12.1 Hz, H-1a), 6.38 (1H, d, J ) 8.4
Hz, H-5′), 3.82 (3H, s, C-4′ OCH3), 3.80 (3H, s, C-4 OCH3), 3.61 (6H,
s, C-3, C-5 OCH3), 3.41 (4H, s, NH2); 13C NMR (CDCl3, 75 MHz) δ
152.4 (C, C-3, C-5), 147.4 (C, C-4′), 137.0 (C, C-4), 132.7 (C, C-2′),
132.0 (C, C-1), 130.9 (CH, C-1a), 125.9 (CH, C-1a′), 123.0 (C, C-3′),
119.2 (CH, C-6′), 117.6 (C, C-1′), 105.7 (CH, C-2, C-6), 101.9 (C,
C-5′), 60.5 (CH3, C-4 OCH3), 55.6 (CH3, C-4′ OCH3), 55.5 (CH3, C-3,
C-5 OCH3); anal. C 65.19%, H 6.30%, N 7.82%, calcd for C18H22N2O4,
C 65.44%, H 6.71%, N 8.48%.
(Z)-2-(2′,5′-Diamino-4′-methoxyphenyl)-1-(3,4,5-trimethoxyphen-
yl)ethene (18). Flash column chromatography led to the product in
1
61% yield: Rf 0.34 (EtOAc/hexanes, 50:50); H NMR (CDCl3, 300
MHz) δ 6.56 (1H, s, H-6′), 6.55 (2H, s, H-2, H-6), 6.46 (1H, d, J )
12.0 Hz, H-1a′), 6.41 (1H, d, J ) 12.1 Hz, H-1a), 6.25 (1H, s, H-3′),
3.82 (3H, s, C-4′ OCH3), 3.79 (3H, s, C-4 OCH3), 3.66 (6H, s, C-3,
C-5 OCH3), 3.31 (4H, s, NH2); 13C NMR (CDCl3, 75 MHz) δ 152.6
(C, C-3, C-5), 148.2 (C, C-4′), 137.2 (C, C-2′), 136.2 (C, C-4), 132.3
(C, C-1), 130.3 (CH, C-1a′), 128.1 (CH, C-1a), 126.0 (CH, C-5′), 116.1
(CH, C-6′), 115.7 (C, C-1′), 105.7 (CH, C-2, C-6), 99.8 (CH, C-3′),
60.8 (CH3, C-4 OCH3), 55.7 (CH3, C-3, C-5, OCH3), 55.4 (CH3, C-4′
OCH3). It is important to note that the 2′,5′-diamino analogue 18
partially decomposed at room temperature from an initial purity level
after flash chromatography of approximately 95% (by NMR) to
approximately 75%. However, the compound retains its original level
of purity if stored at -20 °C.
General Procedure for the Synthesis of Mono- and Diamine
Hydrochloride Salts. To 1.0 equiv of a well-stirred solution of a mono-
or diamino stilbene (0.02 M in CH2Cl2) was added 5 equiv of HCl
(4.0 N solution in dioxane), and the reaction mixture was stirred for
2–10 h at room temperature. At this point, the solvent was removed
under reduced pressure and the resulting oil or solid was purified as
described for each particular salt.
(Z)-2-(3′,5′-Diamino-4′-methoxyphenyl)-1-(3,4,5-trimethoxyphen-
yl)ethene (14). Flash chromatography (EtOAc/hexanes, 40:60) led to
1
a pure sample of diamine 14 in a 20% yield: mp 84–86 °C; H NMR
(CDCl3, 360 MHz) δ 6.56 (2H, s, H-2, H-6), 6.41 (1H, d, J ) 12.2
Hz, H-1a), 6.34 (1H, d, J ) 12.2 Hz, H-1a′), 6.14 (2H, s, H-2′, H-6′),
3.82 (3H, s, C-4′ OCH3), 3.72 (3H, s, C-4 OCH3), 3.70 (6H, s, C-3,
C-5 OCH3), 3.69 (4H, b, NH2); 13C NMR (CDCl3, 75 MHz) δ 152.6
(C, C-3, C-5), 139.7 (C, C-4′), 137.0 (C, C-3′, C-5′), 134.1 (C, C-4),
133.8 (CH, C-1), 132.6 (CH, C-1′), 130.2 (CH, C-1a), 129.0 (CH,
C-1a′), 106.7 (CH, C-2, C-6), 106.2 (CH, C-2′, C-6′), 60.9 (CH3, C-4
OCH3), 58.3 (CH3, C-3, C-5 OCH3), 55.8 (CH3, C-4′ OCH3); EIMS
m/z 330 (M+, 60), 315 (100); anal. C 65.49%, H 6.77%, N 8.40%,
calcd for C18H22N2O4, C 65.44%, H 6.71%, N 8.48%.
(Z)-2-(3′,5′-Diamine hydrochloride-4′-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)ethene (19). The solid was recrystallized (CH2Cl2/
MeOH) to obtain the product in 26% yield: mp 208–212 °C; 1H NMR
(CD3OD, 300 MHz) δ 7.08 (2H, s, H-2′, H-6′), 6.69 (1H, d, J ) 12.1
Hz, H-1a), 6.54 (1H, d, J ) 12.2 Hz, H-1a′), 6.51 (2H, s, H-2, H-6),
3.91 (3H, s, C-4 OCH3), 3.73 (3H, s, C-4′ OCH3), 3.69 (6H, s, C-3,
C-5 OCH3); anal. C 52.31%, H 5.96%, N 6.61%, calcd for C18H24Cl2-
N2O4-0.5 H2O, C 52.55%, H 6.11%, N 6.79%.
(Z)-2-(2′-Amine hydrochloride-4′-methoxy-5′-nitrophenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (20). The solid was filtered and rinsed
with ethyl ether (5 mL) to obtain the salt in 53% yield: 1H NMR
(CD3OD, 300 MHz) δ 7.78 (1H, s, H-6′), 6.74 (1H, s, H-3), 6.73 (1H,
d, J ) 12.0 Hz, H-1a), 6.55 (2H, s, H-2, H-6), 6.40 (1H, d, J ) 12.0
Hz, H-1a′), 3.92 (3H, s, C-4′ OCH3), 3.72 (3H, s, C-4 OCH3), 3.63
(6H, s, C-3, C-5 OCH3).
(Z)-2-(2′,3′-Diamine hydrochloride-4′-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)ethene (21). To the resulting oil was added
anhydrous methanol, and the product crystallized over a period of 3–4
days at -20 °C. The brown-colored crystalline solid that formed was
filtered, washed with methanol, and dried to afford the desired salt in
23% yield: mp 92 °C (dec); 1H NMR (CD3OD, 300 MHz) δ 7.05 (1H,
dd, J ) 8.5, 0.9 Hz, H-6′), 6.67 (1H, d, J ) 11.9 Hz, H-1a′), 6.52 (1H,
d, J ) 8.6 Hz, H-5′), 6.50 (2H, s, H-2, H-6), 6.44 (1H, d, J ) 11.9 Hz,
H-1a), 3.90 (3H, s, C-4′ OCH3), 3.70 (3H, s, C-4 OCH3), 3.60 (6H, s,
C-3, C-5 OCH3); 13C NMR (CD3OD, 75 MHz) δ 154.1 (C, C-3, C-5),
153.6 (C, C-4′), 138.6 (C, C-4), 133.9 (C, C-2′, CH, C-6′), 133.7 (C,
C-1′), 130.5 (C, C-1), 125.5 (CH, C-1a, C-1a′), 120.2 (C, C-3′), 107.4
(CH, C-2, C-6), 102.5 (CH, C-5′), 61.1 (CH3, C-4 OCH3), 56.8 (CH3,
C-4′ OCH3), 56.3 (CH3, C-3, C-5 OCH3).
(Z)-2-(5′-Amino-4′-methoxy-2′-nitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)ethene (15) and (Z)-2-(2′-Amino-4′-methoxy-5′-nitrophen-
yl)-1-(3,4,5-trimethoxyphenyl)ethene (16). Purification by flash chro-
matography (EtOAc/hexanes, 20:80) afforded pure amines 15 (4%
yield) and 16 (27% yield, mp 139–141 °C). Spectroscopic characteriza-
1
tion of isomer 15: H NMR (CDCl3, 300 MHz) δ 7.70 (1H, s, H-3′),
6.87 (1H, d, J ) 12.0 Hz, H-1a′), 6.53 (1H, d, J ) 12.0 Hz, H-1a),
6.46 (1H, d, J ) 0.6 Hz, H-6′), 6.31 (2H, s, H-2, H-6), 4.36 (2H, b,
NH2), 3.92 (3H, s, C-4′ OCH3), 3.79 (3H, s, C-4 OCH3), 3.62 (6H, s,
C-3, C-5 OCH3); 13C NMR (CDCl3, 75 MHz) δ 152.8 (C, C-3, C-5),
145.0 (C, C-4′), 142.2 (C, C-5′), 137.5 (C, C-2′), 137.4 (C, C-4), 131.8
(C, C-1), 130.1 (CH, C-1a′), 129.5 (CH, C-1a), 127.9 (C, C-1′), 114.6
(CH, C-6′), 107.0 (CH, C-3′), 106.4 (CH, C-2, C-6), 60.9 (CH3, C-4
OCH3), 56.0 (CH3, C-4′ OCH3), 55.9 (CH3, C-3, C-5 OCH3); anal. C
59.51%, H 5.65%, N 7.17%, calcd for C18H20N2O6, C 59.99%, H 5.59%,
N 7.77%.
Spectroscopic characterization of isomer 16: 1H NMR (CDCl3, 300
MHz) δ 7.97 (1H, s, H-6′), 6.63 (1H, d, J ) 12.0 Hz, H-1a), 6.47 (2H,
s, H-2, H-6), 6.32 (1H, d, J ) 12.0 Hz, H-1a′), 6.20 (1H, s, H-3′), 4.38
(2H, b, NH2), 3.91 (3H, s, C-4′ OCH3), 3.81 (3H, s, C-4 OCH3), 3.65
(6H, s, C-3, C-5 OCH3); 13C NMR (CDCl3, 75 MHz) δ 155.5 (C, C-4′),
153.0 (C, C-3, C-5), 150.3 (CH, C-2′), 138.1 (C, C-4), 133.5 (C, C-1),
131.3 (CH, C-1a), 129.7 (CH, C-1a′), 129.3 (C, C-5′), 122.7 (CH, C-6′),
114.5 (C, C-1′), 105.9 (CH, C-2, C-6), 97.4 (CH, C-3′), 60.9 (CH3,
C-4 OCH3), 56.4 (CH3, C-4′ OCH3), 55.9 (CH3, C-3, C-5 OCH3); anal.
C 60.65%, H 5.88%, N 7.26%, calcd for C18H20N2O6, C 59.99%, H
5.59%, N 7.77%. Single-crystal X-ray diffraction further confirmed the
Z-configuration of 16.32
General Procedure B for the Reduction of CA-1 Analogues. To
a well-stirred solution of Z-stilbene (1.0 equiv, 0.03 M in glacial acetic
acid) was added zinc powder (221 equiv), and the resulting suspension
was stirred for 2 h at room temperature. At this point, the solution was
(Z)-2-(2′,5′-Diamine hydrochloride-4′-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)ethene (22). The salt was recrystallized from
anhydrous methanol. The resultant white solid was filtered, washed

Combretastatin Dinitrogen-Substituted Stilbene Analogues
Journal of Natural Products, 2008, Vol. 71, No. 3 319
with methanol, and dried to give the desired salt in 27% yield: 1H NMR
(CD3OD, 300 MHz) δ 7.27 (1H, s, H-6′), 7.26 (1H, s, H-3′), 6.89 (1H,
d, J ) 11.9 Hz, H-1a′), 6.56 (1H, d, J ) 11.8 Hz, H-1a), 6.47 (2H, s,
H-2, H-6), 4.03 (3H, s, C-4′ OCH3), 3.71 (3H, s, C-4 OCH3), 3.63
(6H, s, C-3, C-5 OCH3); 13C NMR (CD3OD, 75 MHz) δ 154.5 (CH,
C-3, C-5), 154.2 (C, C-4′), 139.2 (C, C-4), 136.4 (C, C-1), 133.8 (C,
C-2′), 132.5 (CH, C-6′), 127.3 (C, C-1′), 125.7 (CH, C-1a′), 122.5 (CH,
C-1a), 120.2 (CH, C-5′), 108.2 (C, C-3′), 107.7 (CH, C-2, C-6), 61.1
(CH3, C-4 OCH3), 57.5 (CH3, C-4′ OCH3), 56.5 (CH3, C-3, C-5 OCH3);
anal. C 53.24%, H 6.02%, N 6.88%, calcd for C18H24Cl2N2O4, C
53.61%, H 6.00%, N 6.95%.
untreated control group provided the relative cytotoxicity, reflecting
the loss of cell viability as MTT reduction decreased. Heart endothe-
lioma cells (MHEC5-T) from mice were exposed to serial dilutions of
the reported compounds, and cell viability was determined after
incubation at 37 °C at 1 h and at 5 days by the MTT method, yielding
the drug concentration that reduced cell viability by 50% of the control
(IC50).
Blood Flow Reduction. In vivo experiments were performed in the
MHEC5-T tumor model established by the injection of cultured
MHEC5-T cells into the right flank of SCID mice.15 When the
established tumor reached the size of 300 mm3 (a mass without the
development of necrosis), mice were injected ip with doses of the
various compounds at 100 or 10 mg/kg. At 24 h after injection, the
animals were injected in the tail vein with 0.25 mL of diluted
FluoSphere beads (1:6 in physiological saline). The mice were sacrificed
3 min thereafter, and cryosections at a thickness of 8 µm were removed
from the tumor, heart, liver, spleen, and kidney. Three control animals
were tested for blood flow reduction in tumor and control tissues only
after being injected with the vehicle without any reduction in blood
flow. These cryosections were directly examined under a fluorescent
microscope, providing a blue fluorescence from the injected microbeads.
The results were quantified from three sections of three tumors in each
group and in each section, recording more than 70% of the area using
a microscopic digital camera at 100× magnification. The computer
program Stage Pro (Media Cybernetics, Bethesda, MD) was used to
control the picture recording, and image analysis was performed using
Image Plus software (Media Cybernetics).
(Z)-2-(3′,5′-Diamino-4′-methoxyphenyl)-1-(3,4,5-trimethoxyphen-
yl)ethane-Fmoc-L-serinamide (23). A solution, at room temperature,
of compound 14 (0.4407 g, 1.34 mmol), DCCI (0.6680 g, 3.21 mmol),
FMOC (Ac) serine amino acid (1.196 g, 3.21 mmol), and HOBt ·H2O
(0.501 g, 3.21 mmol), in anhydrous DMF (5 mL), was stirred for 4 h.
At this point, EtOAc was added and the solution was filtered. The filtrate
was washed five times with water and twice with brine and then dried
over Na2SO4. The solvent was removed under reduced pressure, and
the product was purified by flash chromatography (EtOAc/hexanes, 50:
50) to afford the FMOC-L-serinamide 23 (0.2518 g, 0.24 mmol, 19%
1
yield): H NMR (CDCl3, 300 MHz) δ 8.44 (1H, b, C-3′ NH), 7.75
(4H, d, J ) 7.6 Hz, FMOC H-4), 7.57 (4H, d, J ) 7.5 Hz, FMOC
H-1), 7.39 (4H, t, J ) 7.5 Hz, FMOC H-3), 7.31 (4H, t, J ) 7.2 Hz,
FMOC H-2), 6.52 (2H, s, H-2, H-6), 6.50 (1H, s, H-2′), 6.49 (1H, s,
H-6′), 6.46 (1H, d, J ) 12.5 Hz, H-1a′), 6.41 (1H, d, J ) 12.3 Hz,
H-1a), 5.70 (1H, b, C-5′ NH), 4.36 (12H, m), 3.82 (3H, s, C-4′ OCH3),
3.68 (6H, s, C-3, C-5 OCH3), 3.64 (3H, s, C-4 OCH3), 2.7 (2H, b,
COCHNH), 2.09 (3H, s, CH3CO2), 2.03 (3H, s, CH3CO2).
SRB Assay.41 Inhibition of human cancer cell growth was assessed
using the National Cancer Institute’s standard sulforhodamine B assay,
as previously described.41 Briefly, cells in a 5% fetal bovine serum/
RPMI1640 medium solution were inoculated in 96-well plates and
incubated for 24 h. Serial dilutions of the compounds were then added.
After 48 h, the plates were fixed with trichloroacetic acid, stained with
sulforhodamine B, and read with an automated microplate reader. A
growth inhibition of 50% (GI50 or the drug concentration causing a
50% reduction in the net protein increase) was calculated from optical
density data with Immunosoft software.
(Z)-2-(3′,5′-Diamino-4′-methoxyphenyl)-1-(3,4,5-trimethoxyphen-
yl)ethane-L-serinamide (24). Fmoc-L-serinamide 23 (0.131 g, 0.226
mmol) dissolved in CH2Cl2/MeOH (6 mL, 1:1 ratio) along with a
solution of 2 N sodium hydroxide (0.53 mL) were stirred at room
temperature for 3.5 h. At this point, CH2Cl2 was added and the organic
phase was washed once with water and twice with brine and dried with
Na2SO4. The solvent was removed under reduced pressure. Purification
by normal-phase preparative TLC (CH2Cl2/MeOH, 95:5) afforded
P388 Assay. Cell growth inhibition (ED50) of the murine P388
lymphocytic leukemia cell line was determined as previously de-
scribed.42
1
serinamide 24 (19.1 mg, 0.04 mmol, 16% yield): H NMR (CDCl3,
360 MHz) δ 9.88 (1H, b, C-3′ NH), 7.66 (1H, d, J ) 1.6 Hz, C-5′
NH), 6.52 (2H, s, C-2, C-6), 6.46 (1H, s, C-2′), 6.47 (1H, s, C-6′),
6.45 (1H, d, J ) 12.5 Hz, C-1a′), 6.39 (1H, d, J ) 12.3 Hz, C-1a),
3.98 (1H, dd, J ) 10.7, 5.0 Hz, COCHNH2), 3.81 (3H, s, C-4′ OCH3),
3.77 (4H, m, CH2OH), 3.73 (3H, s, C-4 OCH3), 3.68 (6H, s, C-3, C-5
OCH3), 3.62 (1H, t, J ) 4.5 Hz, COCH), 2.40 (4H, b, CHNH2CH2OH);
13C NMR (CDCl3, 90 MHz) δ 171.6 (C, CdO), 152.7 (C, CdO), 139.4
(C, C-3, C-5), 137.1 (C, C-4′), 135.7 (C, C-1), 134.2 (C, C-1′), 132.5
(CH, C-1a′), 131.1 (CH, C-1a), 129.9 (C, C-3′), 129.6 (C, C-5′), 111.9
(CH, C-2′), 111.2 (CH, C-6′), 106.2 (CH, C-2, C-6), 65.0 (CH2,
CH2OH), 60.9 (CH3, C-4 OCH3), 59.4 (CH3, C-4′ OCH3), 56.6 (CH,
CHNH2), 55.9 (CH3, C-3, C-5 OCH3).
Acknowledgment. The authors are grateful to Oxigene Inc. (Walth-
am, MA, grants to K.E., M.L.T., C.M.G., and K.G.P.), The Welch
Foundation (grant no. AA-1278 to K.G.P. and AA-1395 to C.M.G.),
grant (to G.R.P.) R-01 CA-90441 from the Division of Cancer
Treatment and Diagnosis, NCI, DHHS for generous financial support
of this project, and the National Science Foundation for funding both
the 500 MHz NMR spectrometer (award CHE-0420802) and the Bruker
X8 APEX diffractometer (grant CHE-0321214). The authors are grateful
to F. Hung-Low and Dr. K. K. Klausmeyer for their valuable assistance
with the X-ray crystallographic structures. The authors also thank H&B
Packing (Waco, TX) for providing calf brain and Mr. L. Williams for
other assistance.
32
X-ray Crystallographic Analysis of Compounds 13 and 16.
Crystallographic data were collected on crystals with dimensions
0.25 × 0.29 × 0.33 mm3 for 13 and 0.119 × 0.137 × 0.242 mm3 for
16. Data were collected at 110 K on a Bruker X8 Apex using Mo KR
radiation (λ ) 0.71073 Å). The structure was solved by direct methods
after correction of the data using SADABS.37 Crystallographic data
and refinement details for the complexes mentioned herein are found
in the Supporting Information (Table 4 and Table 5). The thermal
ellipsoid plots at 50% probability for compounds 13 and 16 are
displayed in Figure 1. All data were processed using the Bruker AXS
SHELXTL software, version 6.10.38 All hydrogen atoms were placed
in calculated positions for 13. For 16 the amine and ethene hydrogens
were located in the difference map and refined isotropically; all other
hydrogen atoms were placed in calculated positions. For both 13 and
16 all non-hydrogen atoms were refined anisotropically. The absolute
structure of 16 could not be determined reliably.
Biological Assays. Tubulin Polymerization Assay. Tubulin was
purified from calf brain according to the method of Hamel and Lin.24a,39
Polymerization was followed turbidimetrically at 350 nm. IC50 values
of the various analogues were determined from the data using nonlinear
regression analysis with Prism software (GraphPad) 3.02 version.
MTT Assay. The MTT cell proliferation assay was used to quantify
cell viability, measuring cell survival and proliferation spectrophoto-
metrically.40 Comparison of the cells treated with the drug to an
Supporting Information Available: 1H NMR spectra for com-
pounds 16-21 and 24 along with X-ray crystallographic data for
compounds 13 and 16. This material is available free of charge on the
Internet at http://pubs.acs.org.
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NP070377J